The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops The Molecular and Physiological Basis of Nut...

Author: Malcolm J. Hawkesford | Peter Barraclough

80 downloads 588 Views 7MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops Edited by

Malcolm J. Hawkesford Peter Barraclough

A John Wiley & Sons, Inc., Publication

This edition first published 2011 © 2011 by John Wiley & Sons, Inc. Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical and Medical business with Blackwell Publishing. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 2121 State Avenue, Ames, Iowa 50014-8300, USA The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 9600 Garsington Road, Oxford, OX4 2DQ, UK For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Blackwell Publishing, provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payments has been arranged. The fee codes for users of the Transactional Reporting Service are ISBN-13: 978-0-8138-1992-1/2011. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Disclaimer The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation warranties of fitness for a particular purpose. No warranty may be created or extended by sales or promotional materials. The advice and strategies contained herein may not be suitable for every situation. This work is sold with the understanding that the publisher is not engaged in rendering legal, accounting, or other professional services. If professional assistance is required, the services of a competent professional person should be sought. Neither the publisher nor the author shall be liable for damages arising herefrom. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data The molecular and physiological basis of nutrient use efficiency in crops / edited by Malcolm J. Hawkesford, Peter Barraclough. p. cm. Includes bibliographical references and index. ISBN-13: 978-0-8138-1992-1 (hard cover : alk. paper) ISBN-10: 0-8138-1992-X (hard cover : alk. paper) 1. Crops—Nutrition. 2. Crops—Nutrition—Molecular aspects. 3. Crops—Nutrition—Physiology. 4. Plant nutrients. 5. Crop yields. I. Hawkesford, Malcolm J. II. Barraclough, Peter (Peter B.) SB112.5.M65 2011 631.8–dc23 2011019195 A catalogue record for this book is available from the British Library. This book is published in the following electronic formats: ePDF 9780470960677; Wiley Online Library 9780470960707; ePub 9780470960684; Mobi 9780470960691 Set in 10/12 pt Times by Toppan Best-set Premedia Limited The cover includes a photograph of the Broadbalk field at Rothamsted Experimental Station in southern England in 2003. The Broadbalk wheat experiment was established in 1842 and is the oldest continuous agricultural experiment in the world. The aim was to compare the efficacy of new synthetic inorganic fertilisers with organic manures and to rank plant nutrients for their effects on yield. The effects of nutrient deficiencies can clearly be seen in the photograph. Yields and soil and plant analyses have been recorded and samples archived since the start. The experiment is continually updated to reflect modern farming practice. Short-straw wheat is now grown continuously and in rotation, with and without crop protection chemicals, on plots having no nutrient inputs, plots with established mineral deficiencies, and plots testing rates and forms N including organic and inorganic sources. It has been used to quantify nitrogen balances in crop production including losses by leaching and de-nitrification. More recently it has been used to examine the impact of nutrition on wheat grain quality (Godfrey et al., 2010, Journal of Agricultural Food Chemistry 58, 3012–3021), and for the identification of nutrient-related genes through grain transcriptome analysis (for examples, Lu et al., 2005 Proceedings of the Royal Society, Series B 272, 1901–1908). Photograph taken by Richard F. Wallis Photography and used by permission of Rothamsted Research. 1

2011

Contents

Preface

vii

Contributors

ix

Part I:

3

Chapter 1

Generic Aspects of Crop Nutrition An Overview of Nutrient Use Efficiency and Strategies for Crop Improvement Malcolm J. Hawkesford

5

Chapter 2

Crop Root Systems and Nutrient Uptake from Soils Peter J. Gregory

21

Chapter 3

The Role of the Rhizosphere in Nutrient Use Efficiency in Crops Petra Marschner

47

Chapter 4

Optimizing Canopy Physiology Traits to Improve the Nutrient Utilization Efficiency of Crops M. John Foulkes and Erik H. Murchie

Chapter 5

Senescence and Nutrient Remobilization in Crop Plants Per L. Gregersen

Chapter 6

Effects of Nitrogen and Sulfur Nutrition on Grain Composition and Properties of Wheat and Related Cereals Peter R. Shewry

Part II:

Nitrogen as a Key Driver of Production

65 83

103

121

Chapter 7

Genetic Improvement of Nutrient Use Efficiency in Wheat Jacques Le Gouis

123

Chapter 8

The Molecular Genetics of Nitrogen Use Efficiency in Crops Bertrand Hirel and Peter J. Lea

139 v

vi

CONTENTS

Chapter 9

Biotechnological Approaches to Improving Nitrogen Use Efficiency in Plants: Alanine Aminotransferase as a Case Study Allen G. Good and Perrin H. Beatty

Chapter 10

Transporters Involved in Nitrogen Uptake and Movement Anthony J. Miller and Nick Chapman

Chapter 11

Crop Improvement for Nitrogen Use Efficiency in Irrigated Lowland Rice Shaobing Peng

Part III:

Other Critical Macro- and Micronutrients

Chapter 12

Phosphorus as a Critical Macronutrient Carroll P. Vance

Chapter 13

Uptake, Distribution, and Physiological Functions of Potassium, Calcium, and Magnesium Frans J.M. Maathuis and Dorina Podar

Chapter 14

Sulfur Nutrition in Crop Plants Luit J. De Kok, Ineke Stulen, and Malcolm J. Hawkesford

Chapter 15

Iron Nutrition and Implications for Biomass Production and the Nutritional Quality of Plant Products Jean-François Briat

Chapter 16

Zinc in Soils and Crop Nutrition Behzad Sadeghzadeh and Zed Rengel

Chapter 17

Overview of the Acquisition and Utilization of Boron, Chlorine, Copper, Manganese, Molybdenum, and Nickel by Plants and Prospects for Improvement of Micronutrient Use Efficiency Patrick H. Brown and Elias Bassil

Part IV:

Specialized Case Studies

165 193

211

227 229

265 295

311 335

377

429

Chapter 18

Drought and Implications for Nutrition Eric Ober and Martin A.J. Parry

Chapter 19

Salt Resistance of Crop Plants: Physiological Characterization of a Multigenic Trait Sven Schubert

443

Legumes and Nitrogen Fixation: Physiological, Molecular, Evolutionary Perspectives, and Applications Muthusubramanian Venkateshwaran and Jean-Michel Ané

457

Chapter 20

Index

431

491

Preface

Achieving global food security is a major challenge for plant biology. Crop improvement including efficient nutrient use is required to meet increasing demands to feed the world population with sustainable agricultural systems. This volume both summarizes the current state of knowledge and anticipates directions of future research and prospects for crop improvement in the area of efficient use of nutrients. An overview describing the principles and scope of the problems relating to efficient nutrient use is provided, with invited experts contributing specialist chapters. These are divided into four sections, covering generic aspects of crop nutrition, a focus on nitrogen as the key driver of production, contributions on all the other major nutrients, and finally a section containing specialist topics including abiotic stresses and legume nitrogen fixation. Substantial advances have been made recently at the molecular and genetic levels in terms of understanding plant function. In parallel, increases in nutrient use efficiency have arisen through successful breeding for yield and through better agronomic prac-

tices. The aim of this volume is to place current molecular studies in the context of the important agronomic trait of efficient nutrient use, bridging academic advances to practical application. Understanding the molecular basis of nutrient use efficiency is fundamental for targeting improvements in this complex trait. Selecting for yield has improved nitrogen use efficiency specifically, but in recent years selection has mostly been at high inputs. There is increasing pressure to decrease inputs while maintaining or improving productivity. In addition, in many agricultural systems, inputs are low or nonexistent and require germplasm quite different from intensively managed systems. There is a real concern that alleles for efficient scavenging of nutrients may have been lost in breeding programs for some crops. Worldwide there are many plant nutrition issues that impact on yield and food security, and in all cases, increased basic knowledge will contribute to solutions. It is essential to consider the problem at all levels, and place molecular knowledge, often detailed at the cellular level, into the context of whole-plant physiology and even in terms of crops in the vii

viii

PREFACE

agro-ecosystem. This volume attempts to synthesize the complexity of the underpinning mechanisms, alongside an appreciation of the systems in which they operate. As such, it is hoped that it will be useful to scientists and students involved in plant nutrition research as well as to plant breeders and biotechnologists

who are responsible for delivery of new germplasm. Malcolm J. Hawkesford Peter Barraclough Rothamsted Research May 2011

Contributors

Jean-Michel Ané Department of Agronomy University of Wisconsin Madison Madison, WI

Patrick H. Brown Department of Plant Sciences University of California-Davis Davis, CA

Peter Barraclough Rothamsted Research Harpenden, Hertfordshire UK

Nick Chapman Rothamsted Research Harpenden, Hertfordshire UK

Elias Bassil Department of Plant Sciences University of California-Davis Davis, CA

Luit J. De Kok Centre for Ecological and Evolutionary Studies University of Groningen Groningen The Netherlands

Perrin H. Beatty Department of Biological Sciences University of Alberta Edmonton, Alberta Canada Jean-François Briat Centre National de la Recherche Scientifique Université Montpellier 2 Montpellier France

M. John Foulkes Division of Plant and Crop Sciences University of Nottingham School of Biosciences Leicestershire UK Allen G. Good Department of Biological Sciences University of Alberta Edmonton, Alberta Canada ix

x

CONTRIBUTORS

Per L. Gregersen Department of Genetics and Biotechnology Aarhus University Denmark Peter J. Gregory East Malling Research East Malling, Kent UK and Centre for Food Security University of Reading Reading UK Malcolm J. Hawkesford Rothamsted Research Harpenden Hertfordshire UK Bertrand Hirel Département Adaptation des Plantes à leur Environnement Institut National de la Recherche Agronomique Route de Saint-Cyr Versailles France Jacques Le Gouis INRA / UBP UMR 1095 Génétique, Diversité et Ecophysiologie des Céréales Clermont-Ferrand France and Diversité et Ecophysiologie des Céréales Université Blaise Pascal Aubière France

Peter J. Lea Lancaster Environment Centre Lancaster University Lancaster UK Frans J.M. Maathuis Biology Department Area 9 University of York York UK Petra Marschner School of Agriculture, Food and Wine The University of Adelaide Adelaide Australia Anthony J. Miller Department of Disease and Stress Biology John Innes Centre Norwich Research Park Colney, Norwich UK Erik H. Murchie Division of Plant and Crop Sciences University of Nottingham Leicestershire UK Eric Ober Rothamsted Research Harpenden Hertfordshire UK Martin A.J. Parry Rothamsted Research Harpenden Hertfordshire UK

CONTRIBUTORS

Shaobing Peng Crop Physiology and Production Center College of Plant Science and Technology Huazhong Agricultural University Wuhan, Hubei P.R. China

Peter R. Shewry Rothamsted Research Harpenden Hertfordshire UK

Dorina Podar Department of Experimental Biology Faculty of Biology-Geology Babes-Bolyai University Cluj-Napoca Romania

Ineke Stulen Laboratory of Plant Physiology Centre for Ecological and Evolutionary Studies (CEES) University of Groningen Groningen The Netherlands

Zed Rengel Soil Science and Plant Nutrition School of Earth and Environment, University of Western Australia Crawley WA Australia

Carroll P. Vance USDA/Agricultural Research Service Plant Science Research Unit Agronomy and Plant Genetics University of Minnesota St. Paul, MN

Behzad Sadeghzadeh Dryland Agricultural Research Institute (DARI) Maragheh Iran

Muthusubramanian Venkateshwaran Department of Agronomy University of Wisconsin Madison Madison, WI

Sven Schubert Institute of Plant Nutrition Interdisciplinary Research Center for Environmental Research, (IFZ) Justus-Liebig-Universität Giessen Germany

xi

Part I

Generic Aspects of Crop Nutrition

Chapter 1

An Overview of Nutrient Use Efficiency and Strategies for Crop Improvement Malcolm J. Hawkesford

Abstract

Introduction

Understanding the molecular basis of crop nutrient use efficiency is a prerequisite for genetic improvement aimed at maximizing yield and minimizing inputs. Plant breeding has been hugely successful at developing high yielding varieties, albeit often with high nutrient requirements. In addition, substantial progress has been made in improving nutrient use efficiency in terms of agronomic practice. Recent developments in genetic and genomic resources, combined with existing physiological and biochemical knowledge, should facilitate substantial further genetic improvements targeted at yield increase and efficient resource utilization. However, this is not a trivial task, given the complexity of the plant systems involved: The traits need to be defined and resolved into specific processes, and appropriate genetic targets need to be identified. This overview considers some issues relating to likely traits, the potential molecular basis of the traits, and potential routes for improvement.

The major challenge facing plant biology is to improve crop production to feed an expanding world population. This is against a background of pressure on agricultural land use and climate change having negative impacts on growing conditions and limiting geographic regions for agriculture (Parry and Hawkesford, 2010). The adverse effects of agriculture, and specifically fertilizer use, include damage to the environment, a large carbon footprint for the manufacture and use of agrochemicals, and the utilization of nonrenewable resources. One solution is to increase the area of land for agriculture, as well as increasing production while maintaining the current rate of inputs; however, this is predicted to have substantial negative impacts on the environment (Tilman et al., 2001; Tilman et al., 2002), is unsustainable in terms of phosphate use, and would have a huge economic footprint in terms of energy demands for nitrogenous fertilizer production. The challenge is to increase yield, decrease inputs, and improve resistance to

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 5

6

NUTRIENT USE EFFICIENCY IN CROPS

abiotic and biotic stresses. Improving crop nutrient use efficiency ideally requires an understanding of the whole system, from the macro- (agro-ecosystem) to the molecular level. While acknowledging the critical contribution of agronomy to improving efficient nutrient use, particularly in classically inefficient systems, there is a point at which crop genetic improvement becomes essential for further improvement. This may be achieved by conventional breeding, as indeed has been the case to the present day with ever increasing yields, albeit often in parallel with increased nutrient demands, with marker-assisted breeding utilizing genetic information derived from basic plant science, and by the utilization of this same information to produce genetically modified crops. Nutrients, along with light, temperature, and water, are critical determinants of crop production, but fertilizers are costly inputs and inappropriate overuse can have many ecologically damaging effects, making efficient use of fertilizers a major issue for agriculture. For example, excessive nitrogen use results in a major fraction of anthropogenic nitrous oxide and methane emissions, which contribute substantially to climate change, and inefficient nutrient uptake may result in pollution of inland and coastal waters by leaching and runoff. Worldwide, it has been estimated that nitrogen use efficiency (NUE) for cereal production is only 33% (nitrogen removed in grain as a percentage of that applied) (Raun and Johnson, 1999). Both agronomic practice and plant breeding have a responsibility to optimize efficient nutrient use, particularly nitrogen, in crop systems. Furthermore, crop improvements to anticipate changing patterns of rainfall and temperature must include an anticipation of nutritional demands influenced by changing cropping systems and crop ideotypes. Optimal plant growth demands a balanced nutrient supply, with a deficiency of any individual essential nutrient having a

detrimental effect on production (law of the minimum). Some nutrients are required at high levels (the macronutrients nitrogen, phosphorus, potassium, and sulfur; see Chapters 8–14), while others are only required at low levels (the micronutrients: iron, zinc, magnesium, etc., see Chapters 15, 16, and 17). In some cases, excess or luxury accumulation of nutrients in plant organs is an issue, negatively impacting on crop growth or quality for the consumer. Agricultural production systems have a range of demands for nutrients; low input as compared with intensive highly managed systems will have different issues and the solutions will be specific for each system. Solutions for efficient fertilizer capture and conversion to biomass or yield in high-input agriculture will be quite different from targets in extensive, organic, or low-input agriculture. In recent years, the emphasis has alternated from a primary objective of improving yields (a target that has always been present in all but the most productive environments) to minimizing impacts on the environment (especially in intensively cropped systems), and back to yield in order to achieve global food security. In the 21st century, sustainable food production has become a major issue with a growing world population, negative impacts of climate change, and demands on land use. To optimize progress on crop improvement, an understanding of nutrient use efficiency from the agronomic or agro-ecosystem level down to the molecular level (the genes involved and their regulation) is required. Substantial progress is being made on the functions and regulation of genes and proteins; molecular data are usually interpreted at the cellular level; however, it is essential that this understanding is placed at the organ, plant, and whole-crop levels. Targets for improvement need to take into account the different agricultural systems, crop physiology and yield components, and the demands

AN OVERVIEW OF NUTRIENT USE EFFICIENCY

of the consumer. Safe, sustainable, and secure food, feed, fiber, and fuel production will demand optimized genetic material including the trait of nutrient use efficiency. Yield and fertilizers: the need for crop improvement

7

in more recent years, there have been incrementally smaller improvements in theoretically achievable yields. In some cases, theoretical yields may not be achievable due to limiting fertilizer application. Theoretical, record, and average (wheat) yields are approximately 18, 16, 8 (in the United Kingdom), and 3 t ha−1 (worldwide), respectively.

Meeting the yield potential Plant breeding has resulted in considerable increases in yield for many crops (for example: maize [Evans and Fischer, 1999], wheat [Ortiz-Monasterio et al., 1997; Brancourt-Hulmel et al., 2003], sugar cane [Robinson et al., 2007]), with the principal target being resistance to biotic and abiotic stresses, as well as for yield itself. Yield is a good measure of nutrient use efficiency, especially as related to nitrogen (Barraclough et al., 2010): The definition of NUE is grain or total biomass (depending on crop) yield divided by available nutrient (see Table 1.1). Theoretical or best yields are seldom achieved in practice as production field conditions are seldom as ideal as breeding plots, which have optimum inputs and agronomy (Fig. 1.1A). This discrepancy is inevitable as the “yield gap” is usually reported on a national level and will encompass growth of varieties in a range of conditions and environments (Austin, 1999; Fischer and Edmeades, 2010). Including traits for resistance to as many stress factors as possible, for example, drought (Chapter 18) and salinity stress (Chapter 19) in breeding programs, will narrow this gap but is unlikely ever to close it. Breeding for resistance to stress, as well as yield, is a key target for crop improvement (Araus et al., 2008). A greater issue is the observed plateau of yield improvements, probably due to abiotic and biotic stress. Initial large improvements in wheat yields were brought about by introduction of dwarfing genes and the consequent improvement in (grain) harvest index (HI). However,

Nutrient response curves As already stated, most plant breeding is performed under “ideal” conditions, which usually include high fertilizer inputs. Nutrient use efficiency has seldom been a key target; however, yield and NUE are closely related at a given fertilizer input. Nutrient use efficiency is the product of both uptake and utilization efficiencies (see the next sections for fuller descriptions), and therefore selecting for yield effectively selects for the combination of these two very separate traits. Small improvements, or even negative trends in one trait (most likely acquisition or uptake), may be hidden by gains in the other (in this case, utilization efficiency). Therefore, selecting for yield alone may not select for optimal nutrient acquisition characteristics, especially at reduced inputs or in environments with specific nutrient deficiencies. It is obvious that maximum acquisition is determined by availability; however, the efficiency of scavenging mechanisms will have a substantial impact on acquisition, although this may not be sustainable in the long term in any single location if nutrients are being mined and not replaced. Traits that will contribute to efficient acquisition are mainly root-associated properties for which there are immense practical difficulties for assessment. A simple approach is to determine overall nutrient capture (nutrient uptake), which integrates the separate features of root architecture and function contributing to this trait (see below for details on the dissection of this trait),

8 Term Fertilizer use efficiency Nutrient (usually nitrogen) use efficiency Nutrient (usually nitrogen) uptake efficiency Nutrient (usually nitrogen) utilization efficiency Harvest index

Nutrient (usually nitrogen) harvest index

Nitrogenagronomic efficiency Apparent recovery efficiency

Abbreviation

FUE

NUE

NUpE

NUtE

HI

NHI

NAE

ARE

kg kg−1

NUp/nitrogen applied as fertilizer

Yield with fertilizer minus yield without nitrogen applied

Fraction of nutrient harvested fraction (e.g., grain)/nutrient in total aboveground biomass (e.g., grain + straw)

Mass of harvested fraction (e.g., grain)/ total aboveground biomass (e.g., grain + straw)

Improvement in yield for fertilizer applied

kg kg−1 kg kg−1

Measure of partitioning of nutrient to harvested fraction of biomass

fraction

Measure of partitioning of yield to harvested fraction of biomass

Also NAPE (agrophysiological efficiency)

kg kg−1

NUp/Nav (soil + fertilizer)

fraction

Also NPE (physiological efficiency)

kg kg−1

NUpE × NUtE = yield/N available

Yield/NUp

% of applied fertilizer recovered by crop NUE is closely related to yield at a given nutrient input

%

(NUp/N applied) × 100

Notes

Unit

Formula

Table 1.1. Some definitions of NUE, mostly used with respect to nitrogen

AN OVERVIEW OF NUTRIENT USE EFFICIENCY

A yield

theoretical achieved

year to present B

Variety 1

yield

Variety 2

9

tilization with any limiting nutrient will improve yield. The tendency for yields to reach a plateau as shown for nitrogen in Figure 1.1B are indicative of secondary limiting factors being present, which may be other nutrients, or may be due to constraints with conversion to harvestable biomass (limiting photosynthesis). A consequence of this plateauing is decreased NUE, specifically attributable to the nitrogen utilization efficiency component (see below). Yield quality conundrum

increasing nitrogen fertilizer

protein content

C

high medium

fertilizer application

low

yield Schematic yield trends and relationships with nutrient inputs. (A) Yield improvements in recent years (second half of the 20th century onward), showing theoretical yield improvements delivered by breeders under ideal growing conditions and those typically achieved on farms. (B) Possible theoretical nitrogen response curves for two varieties, where variety 1 outperforms variety 2 (increasing nitrogen inputs further often results in a down trend of this curve) at all nitrogen inputs. (C) The inverse relationship between yield and nutrient content, in this case nitrogen content expressed as protein content, at three fertilizer levels. Fig. 1.1.

but this is not ideal as selection for the component traits is not achieved. There is little data to indicate whether best uptake performance as selected at high inputs equates to best performance at low inputs. Increasing inputs of nitrogen fertilizer will, in the absence of other limiting nutrients or environmental constraints, result in increasing yield (Fig. 1.1B). Similarly, fer-

In many crops, including grain crops, yield is determined by photosynthetic carbohydrate production and storage. As a consequence, as yield is increased, other nutritional components are often diluted as is seen for the protein content in grain (Fig. 1.1C; Monaghan et al., 2001). Similarly, reduced micronutrient concentrations in grain will occur as a result of dilution with starch. Outliers to the regression relationship between yield and quality shown in Figure 1.1C at any defined nutrient (nitrogen in this case) input must have particularly efficient acquisition and/or partitioning mechanisms. It has been suggested that postanthesis nitrogen uptake is an important contributory mechanism (Monaghan et al., 2001; Kichey et al., 2007; Bogard et al., 2010), although the molecular and genetic basis for this has not been determined. It may be assumed that deep rooting systems, which have access to untapped nutrient reserves at depth in the soil profile, may be important, along with mechanisms to ensure that such nutrients that are taken up are preferentially allocated to the harvested sink material. The diversity of inputs to cropping systems Agricultural systems span a wide range of inputs from none (organic), through extensive (low input), to intensively farmed (high-input)

10

NUTRIENT USE EFFICIENCY IN CROPS

systems. These different situations present quite different challenges in terms of the ideal germplasm required. Targeting improvement of nutrient use efficiency needs to take into account these contrasting agro-ecosystems, the associated agronomic practices, and end-product requirements (e.g., bread vs. feed wheat). In some cases, it is clear that substantial improvements in NUE can be achieved through improved agronomic practice alone. Genetic improvements are likely to be small and incremental by comparison. In low and no input systems, with ever more nutrient capture-efficient varieties, there is a danger of “mining” reserves, leaving land completely unproductive; here the target for genetic improvement needs to be low-nutrient-requiring genotypes combined with improved agronomy to supply minimal nutrition. In many extensive systems, where fertilizers are applied at seed sowing, improved early capture is a critical phenotype. In intensive, high-input systems, conversion to biomass is the principal concern, along with minimization of losses from the system from overfertilization or inappropriate application. Nutrient use efficiency: critical processes, definitions, mechanisms, and targets for improvement Within the broad concept of nutrient use efficiency and depending on the nutrient in question, many definitions of efficiency are possible (see Table 1.1). In this work, depending on the nutrient involved, authors use their own definitions, but in all cases these are clearly stated. Table 1.1 lists many of the common definitions of efficient nutrient and fertilizer use. As already indicated, efficient use of any nutrient comprises two fundamental aspects: acquisition efficiency and utilization efficiency (see also Fig. 1.2). For the target of improving crop nutrient use

efficiency, it is important to separate the individual processes and identify the respective genes involved, monitoring improvements with the appropriate physiological measures. Resolving NUE into two component traits, nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE), is a first step to resolving the complexity, and subsequently each of these traits can be subdivided into many specific physiology traits, each of which are complex traits in itself, the result of networks of biochemical pathways, encoded by multiple genes and subject to complex regulatory processes (Figs. 1.2 and 1.3; Nikiforova et al., 2005; Gojon et al., 2009). Acquisition efficiency and root architecture Nutrient capture (NUpE) is essentially a root trait, although to be fully expressed, it also requires adequate sinks for temporary storage or final deposition of the nutrients. Efficient acquisition will depend first on root architecture (see Chapter 2), root functions in terms of transporters and exudates(Chapter 10), and often the presence of symbiotic associations such as mycorrhiza (Chapters 3 and 12). As indicated in Figure 1.2, early root establishment is essential for scavenging soil nutrients prior to the application of fertilizer, or alternatively to capture fertilizer applied at the time of sowing. Nutrients will be immediately available in the soil solution, and further availability will be depend on mineralization of organic matter and release from sparingly soluble soil minerals (oxides, clays, etc.). High activity of the high-affinity transporter systems required for uptake into root cells, expressed in the plasma membranes of cells of roots, root tips, root hairs, or in associated organisms (mycorrizha), will be important in this situation as diffusion of nutrients through soil is the rate-limiting factor. In addition, a

AN OVERVIEW OF NUTRIENT USE EFFICIENCY

11

Yield: photosynth etic activity, canopy architecture, canopy longevity, and senescence

Canopy establishment and photosynthesis in seedling

Canopy development and setting of yield potential

Nutrient export, patritioning, HI, and NHI

Early root establishment

Nutrients and water

Architecture/ proliferation/activity: •shallow roots for intercepting fertilizers •deep roots for accessing deep resources

Parameters influencing components of nutrient use efficiency at the crop plant level. Both nutrients and water acquisition depend on root architecture and function. The developing canopy will determine yield; however, for grain crops, nutrient (particularly nitrogen) redistribution from the canopy to the grain (essential for efficient nutrient use and for quality attributes) will negatively affect photosynthesis and limit yield.

Fig. 1.2.

well-developed shallow root system will be ideal for intercepting further applications of fertilizer. Deeper roots assume importance with the depletion of surface nutrients, as water near the surface becomes limiting and restricts uptake of nutrients, or in the case of high water supply, for the interception of nutrients that would be potentially leached from the soil profile. In some cases, local proliferation of roots in response to nutrient supply is observed (Drew and Saker, 1978), controlled by specific transcription factors (Zhang and Forde, 1998; Forde 2002). An alternative approach to enhancing capture mechanisms (root architecture and

function) to improve acquisition is to enhance mechanisms for increasing bioavailability of nutrients (e.g., for phosphorus by acid secretions, see Chapters 3 and 12), or to inhibit nitrification losses by the secretion of bio-inhibitors of this process (Subbarao et al., 2007a,b). Acquisition efficiency and nutrient transporter systems A key step in mineral nutrient acquisition is the initial transmembrane transport step. In many cases, for any individual nutrient, there are gene families encoding multiple

12

NUTRIENT USE EFFICIENCY IN CROPS

nutritional availability/ demand

allosteric effectors

ions/metabolites

pathways

metabolism

sensors

posttranscriptional/ posttranslational regulation

gene expression for structural components: transporters, assimilatory pathway, root architecture

transcriptional regulators Fig. 1.3. Simple pathway linking supply and demand of nutrients to regulation of gene expression, as envisioned

at the cellular level. Complex pathways with multiple components link demands for and availability of nutrients for plant growth, mediated by changes in metabolism, usually as a result of changes in gene expression, but in some cases at the level of enzyme/pathway activity (allosteric regulation).

homologs. In Arabidopsis, for example, there are two gene families for nitrate transporters, NRT1 and NRT2, with 53 and 7 members, respectively (see Chapter 10), a gene family of 14 sulfate transporters (Chapter 14 and Hawkesford, 2003) and 9 members of the phosphate transporter family pht1 (Chapter 12 and Smith et al., 2003). While in most cases there are families specific for a single nutrient, there are instances of nonspecificity: Sulfate transporters effectively transport selenate (Shinmachi et al., 2010) and molybdate (Tomatsu et al., 2007; Baxter et al., 2008; Fitzpatrick et al., 2008; Shinmachi et al., 2010). While there is some potential redundancy of function with these large gene families, it has become apparent that there is tissue, developmental, and even membrane specificity with regard to expression patterns. Functionally, there are usually both high and low affinities for the substrate ions, depending on functional requirements: In relation to primary uptake into root cells, the most common functionality is for high-affinity uptake, as required for effec-

tive acquisition from soil solutions with low concentrations of ions. Patterns of expression within the root are often complex to effectively transfer the respective ions from the soil solution to the vasculature for transfer to the shoot material. In some instances, vacuolar storage may also play an important part (Kataoka et al., 2004). Many studies have focused on the impacts of nutrient limitation on patterns of transporter expression and the contribution to overall nutrient use efficiency strategies of plants in limiting nutrient availability (Buchner et al., 2004, 2010). For phosphate and sulfate, there is an apparent de-repression system controlling gene expression, facilitating increased expression when nutrient demand exceeds availability (Smith, 2002; Hawkesford and De Kok, 2006). For nitrate, the pattern is more complex, with some transporters induced and others repressed, depending on the presence of nitrate and the nutritional status of the plant (see Chapter 10). The transporters play essential roles, contributing to nutrient use efficiency, for the

AN OVERVIEW OF NUTRIENT USE EFFICIENCY

most part extremely effectively scavenging nutrients from the soil (potentially present at low concentrations), and particularly in conjunction with effective root proliferation. As targets for improvement of NUE, sophisticated strategies are likely to be important. Modifications to the selectivity (Rogers et al., 2000) may enhance preferential uptake of beneficial ions and exclude toxic ions. Overriding negative feedback mechanisms may facilitate luxury uptake, but appropriate sinks or temporary storage would also be required (see below). One approach that apparently overrides limits on nitrogen uptake is the overexpression of alanine amino transferase in root exodermal tissues, thus channeling nitrogen away from metabolites involved in negative feedback (see Chapter 9). In some instances, enhancing remobilization and optimizing partitioning to harvested organs may require optimization of transporter expression. Metabolic responses to nutrient availability Plant responses to nutrient availability are complex and involve changes in pathway fluxes, in activity of pathway enzymes mediated by posttranslation modifications and/or changes in substrate/inhibitor ratios (allosteric effects), as well as changes in expression of genes encoding the pathway enzymes and many additional proteins (Fig. 1.3). The challenge for the plant is to optimize growth and development given the available nutrient inputs. Matching availability to demand may entail many regulatory steps and sensory mechanisms. It is essential to understand these networks before intervention through transgenesis or molecular breeding. For the most part, our knowledge of these regulatory loops is restricted in plants (Gojon et al., 2009). Nutrient use efficiency, although simply divided into uptake and utilization, encompasses all processes of plant growth and

13

development, and all aspects of metabolism. Potential targets for nutrient use efficiency improvement are therefore diverse. Obvious targets in, for example, nitrogen metabolism (see Chapters 8 and 9) include genes of the assimilatory pathway. Glutamine synthetase has been a specific target for transgenic approaches (Chapter 8), as it is not only involved in primary assimilation but also has a role in efficient recycling of ammonia during senescence processes (Kichey et al., 2006). Generally, results of single-gene manipulation have been disappointing, in part because metabolic pathways form networks that have a great plasticity in responding to perturbations, whether due to gene targeting or environmental fluctuations, for example, in nutrient supply (Wasaki et al., 2003; Palenchar et al., 2004; Nikiforova et al., 2005). Typically, nutrient uptake is balanced by nutritional requirement for growth, and a coordination of pathway expression and activity is seen (Prosser et al., 2001; Hawkesford and De Kok, 2006; Howarth et al., 2008; Gojon et al., 2009) and excess uptake of nutrient is avoided. Excess accumulation of some ions does occur but only to the point at which available storage pools are saturated (for example, nitrate accumulation in vacuoles); this is a strategy to aid with fluctuating supplies of nutrients but is not helpful when one nutrient becomes permanently limiting. Utilization efficiency Efficiency of utilization may be defined as biomass production (predominantly fixed carbon) as a function of nutrient taken up (see Table 1.1). This is most often applied for nitrogen, as total canopy nitrogen content reflects the extent of photosynthetically active biomass, as the greatest proportion of the total nitrogen content in this tissue is a major component of proteins involved in photosynthesis. The effectiveness of this capacity in producing

14

NUTRIENT USE EFFICIENCY IN CROPS

harvestable biomass is defined by NUtE. The key attributes that will enhance NUtE are photosynthetic activity, canopy size, longevity, and sink organ capacity (Fig. 1.2). Photosynthetic activity includes the ability to intercept light, which is clearly linked to canopy architecture and the light harvesting complex density, as well as the biochemistry of the carbon fixation processes, particularly Rubisco, for efficient fixation of carbon dioxide (Parry et al., 2003). An alternative and radical solution is to engineer C4 photosynthesis, which is up to 50% more efficient than C3 photosynthesis, into C3 plants such as rice (Hibberd et al., 2008). Attributes of canopy development and architecture include rapid establishment, followed by proliferation and eventual canopy closure (full coverage of the ground), and then effective architecture to intercept radiant light. Depending on the harvestable product, which may be the canopy itself, or it may be biomass derived from this, for example, woody stem or generative material such as seed, the canopy must be photosynthetically active for as long as possible. Delaying senescence and prolonging the period of photosynthesis results in increased carbon fixation (see Chapters 4 and 5). However, the complexity of processes involved in leaf senescence is highlighted by transcriptome analysis, emphasizing the difficulty in manipulating this process to enhance yield (Gregersen and Holm, 2007). As a target, this process has huge potential for crop improvement, as by definition for a fixed amount of nutrient (nitrogen) taken up, the more carbon that is fixed, the better the NUtE (for a full discussion on the physiology associated with these traits, see Chapter 4). Sinks An important attribute for uptake efficiency is having adequate sinks to store acquired

nutrients, whether nitrogen or minor but important nutritional components including Fe, Zn, and Se. Adequate sinks will prevent negative feedback regulation on the initial acquisition/assimilatory processes and should provide important remobilizable storage that can be accessed should supply be limiting as well as during production of harvested organs such as seed. Sinks may be subcellular, for example, vacuoles, may be chemical such as nitrogen stores in protein, or may be defined at the organ level, for example, stems (see Chapter 4). Attempts have been made to engineer both metabolism and (Zhu and Galili, 2003) protein sinks to enhance nutritional quality with high methionine, cysteine, or lysine content (Tabe and Higgins, 1998; Nikiforova et al., 2002; see also Chapter 6). As already indicated, one explanation for the remarkable improvement in NUE seen by the overexpression of alanine aminotransferase is that alanine is a local metabolic sink for nitrogen that does not have negative feedback effects on uptake, unlike glutamate (Chapter 9). HI and partitioning of nutrients By definition, measures of nutrient use efficiency will be optimum if HI is high and nutrients are partitioned to the harvested material. This ignores the impact at the whole ecosystem level, and there may be merit in not harvesting some nutrients but allowing them to be recycled within the field; this might particularly apply to phosphorus; it does, however, assume that leaching losses will be minimal between crops. In many instances, partitioning to the cropped organ is preferable, for example, nitrogen in the case of grain protein (Chapter 6) and minerals for human nutrition (Chapters 14, 16, and 17). As indicated, a major improvement in yields and NUE was obtained with the introduction of dwarfing genes (into wheat and rice), minimizing the nonhar-

AN OVERVIEW OF NUTRIENT USE EFFICIENCY

vested fraction of wheat and rice. Although there are efforts to extend the repertoire of dwarfing genes, which may have additional benefits (Ellis et al., 2005), overall, as the HI for many crops has already been optimized, there is likely little benefit from further manipulation of the HI. For those crops for which this is not the case, improving the HI is a high priority. While nutrient harvest index (NHI) for nitrogen is usually high in cereals, this is not the case for all minerals in all crops. Even in wheat, different minerals are partitioned with varied efficiency: selenium and molybdenum were shown to be differentially partitioned to grain (Shinmachi et al., 2010). In Brassica napus (oil seed rape, Colza), sulfur is very inefficiently partitioned to the seed (Blake-Kalff et al., 1998). Manipulating senescence to produce stay-green phenotypes, thus enhancing yield may have a detrimental effect on the HI and NHI. In this case, additional selection for late but rapid nutrient remobilization would be required. Conversely, enhancing the rate of senescence by the introduction of a NAC transcription factor increased remobilization and enhanced grain protein as well as zinc and iron content (Uauy et al., 2006; Waters et al., 2009). However, increasing the onset and rate of senescence may have a negative effect on yield. Many nutrient deficiencies lead to changes in biomass allocation between roots and shoots, generally increasing the root : shoot ratio (Hermans et al., 2006). This is an adaptive strategy, facilitating the ability to scavenge for nutrients. In nutrientpoor environments, this would be an advantageous trait; however, this does need to be balanced with production of harvestable material. Little is known about the signaling pathways involved; however, the signals may be linked to imbalances in nutrient accumulation in shoot tissues. Signals from the shoots to the roots (Forde, 2002) may be

15

hormonal (Signora et al., 2001) or may be metabolites, for example, carbohydrates (Hermans et al., 2006). Ultimately, partitioning to the cropped organ is of most importance in agricultural production. Strategies and approaches for the genetic improvement of NUE traits There is considerable pressure to improve fertilizer use efficiency (see above: economic, environmental, nonrenewable resource use), and this has been traditionally achieved by agronomic practice and breeding for yield in specific environments and agronomic systems. In the case of nitrogen, breeding for yield is equivalent to breeding for nitrogen utilization efficiency at any given nitrogen input, and there has been considerable progress in improving yields and, therefore, the NUtE component of NUE. Generally, wheat varieties responding well at high inputs also respond well at low inputs (Ortiz-Monasterio et al., 1997; Barraclough et al., 2010). However, there is a strong case for selection at varied inputs and for seeking new and untested germplasm to find new alleles for greater NUtE efficiency. However, acquisition efficiency has been much less specifically selected for, in part due to the difficulties of phenotyping roots. Here, selection at low inputs is vital. Additionally, it will certainly be necessary to introduce wider germplasm pools (landraces, wild relatives) into screening programs, as alleles for high efficiency of acquisition will have almost certainly been lost from the gene pool without the selection pressure for high acquisition efficiency. Many new technologies for gene discovery (microarrays, deep sequencing tilling transformation SNP detection) are now available and may be combined with established breeding approaches (breeding, quantitative trait loci [QTLs], germplasm

16

NUTRIENT USE EFFICIENCY IN CROPS

screening). In combination with the identification of new traits, technologies for introducing these into modern breeding lines are required (synthetic polyploids, alien introgression, gene transformation; Able et al., 2007). As already mentioned and cautioned by others, single-gene introductions, perhaps through crop transgenesis, are often not successful, particularly when gene selection is from only preexisting biochemical knowledge (Sinclair et al., 2004), almost certainly due to a lack of appreciation of the complexity of the systems being manipulated. Figure 1.4 outlines an approach for genetic improvement, beginning with a statement of the need to precisely define the trait of interest. Ideally, in order to effectively target the trait, this needs to be resolved to the smallest subcomponent, encoded by just a few genes. After trait prioritization, assessment of variation is required. This may be either natural variation, or variation induced by mutation or by crossing an examination of mapping populations, and will provide material directly for commercial breeding. Variation may also be used to aid in the identification of the target genes. Transcriptome approaches will indicate genes coexpressed with traits of interest; however, these candidates are usually very numerous (Wang et al., 2000, 2001, 2003, 2004; Lu et al., 2005; Gregersen and Holm, 2007). Examination of occurrence across diverse germplasm and expression patterns under multiple conditions will narrow these candidate lists to a few key genes worth further investigation. Definitive implication in crop improvement with respect to NUE may require transgenesis in the crop of interest. Such genetically modified crops may be the end product, or the genes may be used as “perfect” markers for screening other natural populations, avoiding the need for transgenesis. Critically, genes identified by such a route may be

•

Trait de convolution and prioritization

•

Assessment of variation

•

•

–

Provision of materials for breeding

–

Aid gene discovery

Identification of genes/markers –

Target appropriate tissues

–

Correlation with traits

–

Mapping populations

Breeding or biotechnology

Approaches to finding novel target genes and crop improvement. Germplasm identified with appropriate traits is of direct value for crop improvement. Identified genes will facilitate breeding either as markers or in transgenic approaches. Fig. 1.4.

more robust than selection based on biochemical pathways alone. Prospects The main targets for improving NUE have been outlined and a case for a thorough understanding of the underlying molecular process has been made. The major targets are improving nutrient capture and interception to avoid losses, modifying requirements (reducing if possible) and enhancing utilization efficiency by generally improving carbon fixation and yield. An ideotype of an idealized set of traits for nutrient use efficiency can be defined and will be crop specific (Foulkes et al., 2009). Furthermore, such ideotypes will be specific to different environments and cropping systems.

AN OVERVIEW OF NUTRIENT USE EFFICIENCY

Nutrient use efficiency in its broadest sense indicates how effectively a plant is able to capture and utilize nutrients to produce biomass. It is most usually specified for nitrogen as this is a main driver for production. However, healthy and productive crop growth requires a balanced nutrition including several macronutrients and many micronutrients. Irrespective of the quantity needed, all are essential and any limitation will impact on plant growth and crop yields. In almost all cases, the nutrient in question must be obtained from the pedosphere and therefore uptake processes dependent on architecture and functioning of the roots are critical. Subsequent to this, partitioning within the plant is a vital prerequisite to efficient utilization of the element as part of the plant’s growth and developmental cycle. Independent but simultaneous selection for both of these traits must be performed. A radical and alternative solution to providing nitrogen fertilizer would be the transfer of nitrogen fixation capacity, or the ability to form the required symbioses, to nonlegume crops (see Chapter 20). NUE is an essential component of crop production, and irrespective of the agronomic system, low-input or intense, efficient utilization of valuable resources will be essential for future sustainable food production. NUE is a complex trait that can be broken down into subtraits, all of which are also complex in nature. Few instances can be expected where single genes or a single locus will have a huge benefit; dwarfing genes were an exception. Modern tools and resources available to plant scientists and the agronomy and breeding communities should aid further improvements in NUE and hence crop production. Great variability exists in the extent to which individual crops have been optimized in relation to NUE, and while large improvements may be anticipated for some crops, for the major world grain crops such as wheat maize and rice,

17

smaller incremental improvements are likely. The prospect of step changes in primary production by engineering the photosynthetic process itself will require additional concomitant improvements in nutrient acquisition efficiency. Acknowledgments Rothamsted Research is an institute of the Biotechnology and Biological Sciences Research Council of the United Kingdom. The author ’s research is also supported by the Biotechnology and Biological Sciences Research Council (BB/G022437/1 and BB/ C514066/1) and the Department of Environment, Food and Rural Affairs (WGIN project IF0146). References Able, J.A., Langridge, P., & Milligan, A.S. (2007) Capturing diversity in the cereals: many options but little promiscuity. Trends in Plant Science 12, 71–79. Araus, J.L., Slafer, G.A., Royo, C., et al. (2008) Breeding for yield potential and stress adaptation in cereals. Critical Reviews in Plant Sciences 27, 377–412. Austin, R.B. (1999) Yield of wheat in the United Kingdom: recent advances and prospects. Crop Science 39, 1604–1610. Barraclough, P.B., Howarth, J.R., Jones, J., et al. (2010) Nitrogen efficiency of wheat: genotypic and environmental variation and prospects for improvement. European Journal of Agronomy 33, 1–11. Baxter, I., Muthukumar, B., Park, H.C., et al. (2008) Variation in molybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter (MOT1). PLoS Genetics 4, e1000004. Blake-Kalff, M.M.A., Harrison, K.R., Hawkesford, M.J., et al. (1998) Distribution of sulfur within oilseed rape leaves in response to sulfur deficiency during vegetative growth. Plant Physiology 118, 1337–1344. Bogard, M., Allard, V., Brancourt-Hulmel, M., et al. (2010) Deviation from the grain protein concentration–grain yield negative relationship is highly correlated to post-anthesis N uptake in

18

NUTRIENT USE EFFICIENCY IN CROPS

winter wheat. Journal of Experimental Botany 61, 4303–4312. Brancourt-Hulmel, M., Doussinault, G., Lecomte, C., et al. (2003) Genetic improvement of agronomic traits of winter wheat cultivars released in France from 1946 to 1992. Crop Science 43, 37–45. Buchner, P., Takahashi, H., & Hawkesford, M.J. (2004) Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport. Journal of Experimental Botany 55, 1765–1773. Buchner, P., Parmar, S., Kriegel, A., et al. (2010) The sulfate transporter family in wheat: tissue-specific gene expression in relation to nutrition. Molecular Plant 3, 374–389. Drew, M.C. & Saker, L.R. (1978) Nutrient supply and growth of seminal root-system in barley. 3. Compensatory increases in growth of lateral roots, and in rates of phosphate uptake, in response to a localized supply of phosphate. Journal of Experimental Botany 29, 435–451. Ellis, M.H., Rebetzke, G.J., Azanza, F., et al. (2005) Molecular mapping of gibberellin-responsive dwarfing genes in bread wheat. Theoretical and Applied Genetics 111, 423–430. Evans, L.T. & Fischer, R.A. (1999) Yield potential: its definition, measurement, and significance. Crop Science 39, 1544–1551. Fischer, R.A.T. & Edmeades, G.O. (2010) Breeding and cereal yield progress. Crop Science 50, S85–S98. Fitzpatrick, K.L., Tyerman, S.D., & Kaiser, B.N. (2008) Molybdate transport through the plant sulfate transporter SHST1. FEBS Letters 582, 1508–1513. Forde, B.G. (2002) Local and long-range signaling pathways regulating plant responses to nitrate. Annual Review of Plant Biology 53, 203–224. Foulkes, M.J., Hawkesford, M.J., Barraclough, P.B., et al. (2009) Identifying traits to improve the nitrogen economy of wheat: recent advances and future prospects. Field Crops Research 114, 329–342. Gojon, A., Nacry, P., & Davidian, J.C. (2009) Root uptake regulation: a central process for NPS homeostasis in plants. Current Opinion in Plant Biology 12, 328–338. Gregersen, P.L. & Holm, P.B. (2007) Transcriptome analysis of senescence in the flag leaf of wheat (Triticum aestivum L. Plant Biotechnology Journal 5, 192–206. Hawkesford, M.J. (2003) Transporter gene families in plants: the sulphate transporter gene family— redundancy or specialization? Physiologia Plantarum 117, 155–163. Hawkesford, M.J. & De Kok, L.J. (2006) Managing sulphur metabolism in plants. Plant, Cell & Environment 29, 382–395.

Hermans, C., Hammond, J.P., White, P.J., et al. (2006) How do plants respond to nutrient shortage by biomass allocation? Trends in Plant Science 11, 610–617. Hibberd, J.M., Sheehy, J.E., & Langdale, J.A. (2008) Using C-4 photosynthesis to increase the yield of rice—rationale and feasibility. Current Opinion in Plant Biology 11, 228–231. Howarth, J.R., Parmar, S., Jones, J., et al. (2008) Coordinated expression of amino acid metabolism in response to N and S deficiency during wheat grain filling. Journal of Experimental Botany 59, 3675–3689. Kataoka, T., Watanabe-Takahashi, A., Hayashi, N., et al. (2004) Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis. The Plant Cell 16, 2693–2704. Kichey, T., Heumez, E., Pocholle, D., et al. (2006) Combined agronomic and physiological aspects of nitrogen management in wheat highlight a central role for glutamine synthetase. The New Phytologist 169, 265–278. Kichey, T., Hirel, B., Heumez, E., et al. (2007) In winter wheat (Triticum aestivum L.), post-anthesis nitrogen uptake and remobilisation to the grain correlates with agronomic traits and nitrogen physiological markers. Field Crops Research 102, 22–32. Lu, C.G., Hawkesford, M.J., Barraclough, P.B., et al. (2005) Markedly different gene expression in wheat grown with organic or inorganic fertilizer. Proceedings of the Royal Society B-Biological Sciences 272, 1901–1908. Monaghan, J.M., Snape, J.W., Chojecki, A.J.S., et al. (2001) The use of grain protein deviation for identifying wheat cultivars with high grain protein concentration and yield. Euphytica 122, 309–317. Nikiforova, V., Kempa, S., Zeh, M., et al. (2002) Engineering of cysteine and methionine biosynthesis in potato. Amino Acids 22, 259–278. Nikiforova, V.J., Kopka, J., Tolstikov, V., et al. (2005) Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants. Plant Physiology 138, 304–318. Ortiz-Monasterio, J.I., Sayre, K.D., Rajaram, S., et al. (1997) Genetic progress in wheat yield and nitrogen use efficiency under four nitrogen rates. Crop Science 37, 898–904. Palenchar, P.M., Kouranov, A., Lejay, L.V., et al. (2004) Genome-wide patterns of carbon and nitrogen regulation of gene expression validate the combined carbon and nitrogen (CN)-signaling hypothesis in plants. Genome Biology 5, R91.

AN OVERVIEW OF NUTRIENT USE EFFICIENCY

Parry, M.A.J. & Hawkesford, M.J. (2010) Genetic approaches to reduce greenhouse gas emissions: increasing carbon capture and decreasing environmental impact. In: Climate Change and Crop Production (ed. M.P. Reynolds), pp. 139–150. CABI, Wallingford, U.K. Parry, M.A.J., Andralojc, P.J., Mitchell, R.A.C., et al. (2003) Manipulation of Rubisco: the amount, activity, function and regulation. Journal of Experimental Botany 54, 1321–1333. Prosser, I.M., Purves, J.V., Saker, L.R., et al. (2001) Rapid disruption of nitrogen metabolism and nitrate transport in spinach plants deprived of sulphate. Journal of Experimental Botany 52, 113–121. Raun, W.R. & Johnson, G.V. (1999) Improving nitrogen use efficiency for cereal production. Agronomy Journal 91, 357–363. Robinson, N., Fletcher, A., Whan, A., et al. (2007) Sugarcane genotypes differ in internal nitrogen use efficiency. Functional Plant Biology 34, 1122–1129. Rogers, E.E., Eide, D.J., & Guerinot, M.L. (2000) Altered selectivity in an Arabidopsis metal transporter. Proceedings of the National Academy of Sciences of the United States of America 97, 12356–12360. Shinmachi, F., Buchner, P., Stroud, J.L., et al. (2010) Influence of sulfur deficiency on the expression of specific sulfate transporters and the distribution of sulfur, selenium, and molybdenum in wheat. Plant Physiology 153, 327–336. Signora, L., De Smet, I., Foyer, C.H., et al. (2001) ABA plays a central role in mediating the regulatory effects of nitrate on root branching in Arabidopsis. The Plant Journal 28, 655–662. Sinclair, T.R., Purcell, L.C., & Sneller, C.H. (2004) Crop transformation and the challenge to increase yield potential. Trends in Plant Science 9, 70–75. Smith, F.W. (2002) The phosphate uptake mechanism. Plant and Soil 245, 105–114. Smith, F.W., Mudge, S.R., Rae, A.L., et al. (2003) Phosphate transport in plants. Plant and Soil 248, 71–83. Subbarao, G.V., Rondon, M., Ito, O., et al. (2007a) Biological nitrification inhibition (BNI)—is it a widespread phenomenon? Plant and Soil 294, 5–18. Subbarao, G.V., Tomohiro, B., Masahiro, K., et al. (2007b) Can biological nitrification inhibition (BNI) genes from perennial Leymus racemosus (Triticeae) combat nitrification in wheat farming? Plant and Soil 299, 55–64. Tabe, L. & Higgins, T.J.V. (1998) Engineering plant protein composition for improved nutrition. Trends in Plant Science 3, 282–286.

19

Tilman, D., Fargione, J., Wolff, B., et al. (2001) Forecasting agriculturally driven global environmental change. Science 292, 281–284. Tilman, D., Cassman, K.G., Matson, P.A., et al. (2002) Agricultural sustainability and intensive production practices. Nature 418, 671–677. Tomatsu, H., Takano, J., Takahashi, H., et al. (2007) An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil. Proceedings of the National Academy of Sciences of the United States of America 104, 18807–18812. Uauy, C., Distelfeld, A., Fahima, T., et al. (2006) A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314, 1298–1301. Wang, Y.H., Garvin, D.F., & Kochian, L.V. (2001) Nitrate-induced genes in tomato roots. Array analysis reveals novel genes that may play a role in nitrogen nutrition. Plant Physiology 127, 345–359. Wang, R.C., Guegler, K., LaBrie, S.T., et al. (2000) Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate. Plant Cell 12, 1491–1509. Wang, R.C., Okamoto, M., Xing, X.J., et al. (2003) Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiology 132, 556–567. Wang, R.C., Tischner, R., Gutierrez, R.A., et al. (2004) Genomic analysis of the nitrate response using a nitrate reductase-null mutant of Arabidopsis. Plant Physiology 136, 2512–2522. Wasaki, J., Yonetani, R., Kuroda, S., et al. (2003) Transcriptomic analysis of metabolic changes by phosphorus stress in rice plant roots. Plant, Cell & Environment 26, 1515–1523. Waters, B.M., Uauy, C., Dubcovsky, J., et al. (2009) Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain. Journal of Experimental Botany 60, 4263–4274. Zhang, H. & Forde, B.G. (1998) An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279, 407–409. Zhu, X.H. & Galili, G. (2003) Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds. Plant Cell 15, 845–853.

Chapter 2

Crop Root Systems and Nutrient Uptake from Soils Peter J. Gregory

Abstract Root systems have evolved to allow thorough exploration of the soil for water and nutrients, and to modify soil properties so that scarce nutrients can be acquired. The maximum depth of rooting of crops (average 2.1 m) is less than that of other types of vegetation and inﬂuenced by genetic, cultural, and environmental factors. Downward progression of the rooting front follows a sigmoidal pattern with time, and roots are frequently distributed logarithmically in the soil, with the highest concentrations at the surface (typically 5–10 cm root cm−3 soil for temperate cereals and 1–2 cm cm−3 for other crops at 0–0.1 m). Acquisition of nutrients, especially immobile nutrients such as phosphorus, is dependent on root architecture (especially branching), associations with microorganisms (especially mycorrhizal fungi), and modiﬁcations to the bioavailability of the nutrient in the rhizosphere (see, for example, Chapters 3 and 12). Several studies have demonstrated the importance of root architecture for phosphorus acquisition in low-

phosphorus soils, and markers associated with quantitative trait locus (QTL) are emerging that should facilitate breeding of improved genotypes. Suitable placement of fertilizers to complement root architecture and the ontology of the crop is already widely practiced, and the ability to manipulate and engineer rhizosphere properties to exploit nutrients more effectively is emerging rapidly as an area of essential research endeavor if the challenge of increasing production while using resources more efﬁciently is to be realized. Introduction This chapter is about root systems growing in soil. Much useful information has been learned from the model plant Arabidopsis about the genes and regulation of individual root growth (e.g., Ubeda-Tomás et al., 2008; Péret et al., 2009; Brun et al., 2010), but for crops it is the functioning of the whole system that is important. Measurements of such systems are difﬁcult and timeconsuming, with a variety of approaches used to gain basic information about depth,

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 21

22

NUTRIENT USE EFFICIENCY IN CROPS

distribution, and various architectural features (see Smit et al., 1994 and Gregory, 2006b for examples). This brief overview focuses on the features of crop root systems contributing to the ability to acquire nutrients. It draws on plant science as well as agricultural and ecological studies to demonstrate how our improved understanding of root interactions with soils to facilitate nutrient acquisition is paving the way for improved genotypes and management techniques to use nutrients more efﬁciently in crop production. Exploration of the soil Roots have evolved to explore the soil for water and nutrients; roots both respond to the edaphic environment and modify its properties to secure resources. The depth to which roots are able to grow has many implications for the hydrological balance and biogeochemical cycling of ecosystems by extracting nutrients for growth and preventing the leaching of nutrients to water courses. Depth and distribution of crop root systems The depth of rooting and the distribution of roots in the soil proﬁle are affected by genetic and environmental factors. On deep soils, the maximum depth of rooting of crops is typically less than that of forests or grasslands. For example, Canadell et al. (1996) summarized 290 observations of maximum rooting depth of 253 woody and herbaceous species (23 crop studies) from the major terrestrial biomes and found that while the average maximum rooting depth for crops was 2.1 ± 0.2 m, those for temperate deciduous and tropical evergreen forests were 2.6 ± 0.2 m and 7.3 ± 2.8 m, respectively, and those for temperate and tropical grasslands were 2.6 ± 0.2 m and 15.0 ± 5.4 m, respectively. Only tundra and

boreal forests had shallower maximum rooting depths (0.5 ± 0.1 m and 2.0 ± 0.3 m, respectively). Comparisons of crops grown on the same sites with deep soils demonstrate that the maximum depth of rooting is genetically determined and differs between species grown under identical conditions. For example, lupin (Lupinus spp.), pea, and wheat grown on deep sands (entisols) at different sites in Western Australia had signiﬁcantly different (P < 0.001) maximum rooting depths (Hamblin and Hamblin, 1985). Rooting depths averaged 0.65 m for peas, 1.13 m for wheats, and 1.9 m for lupins. Similarly, Greenwood et al. (1982) grew a range of vegetables on a sandy loam at Wellesbourne, United Kingdom, and found that while onion and lettuce roots were conﬁned to the upper 0.65 m, pea rooted to 0.75 m, broadbean to 0.85 m, and turnip, parsnip, and cauliﬂower to >0.85 m. Merrill et al. (2002) also found differences between eight crops grown at different sites (soil predominantly a silt loam) over three seasons in North Dakota, USA, with average maximum rooting depths of 1.0 m in common bean, soybean, and pea; about 1.15 m in crambe (Crambe abysinnica); 1.3 m in spring wheat and canola (Brassica rapa); 1.45 m in sunﬂower; and 1.6 m in safﬂower. In practice, however, both cultural and environmental conditions often play an important role in determining rooting depth. Comparison of autumn- and spring-sown wheat crops on a sandy loam in Denmark over three seasons showed that autumn sowing gave consistently deeper root systems (2.2 m) than spring sowing (1.1 m; Thorup-Kristensen et al., 2009). This occurred because the downward extension of the rooting front of the winter and spring wheat varieties used was similar (1.3 mm °C day−1), but the period of root growth was longer for the winter crop. In

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

23

rapid downward extension depending on the crop and growing conditions. Gregory et al. (1978) found that the rate of downward growth of a winter wheat crop grown in the United Kingdom averaged 6 mm day−1 during the winter and 18 mm d−1 between early April and early June, when temperatures were much warmer. The rate of downward progression of the rooting front differs between crops with crops such as leeks and common velvet grass extending at 0.07 cm °C day−1, while others such as cereals and fodder radish were much faster (0.18– 0.26 cm °C day−1; Smit and Groenwold, 2005). For a temperature of 15°C, this corresponds to a root front extending downward at about 1–4 cm day−1. The Borg and Grimes (1986) analysis of data from 48 crop species in 135 ﬁeld studies found that while the increase in rooting depth with time followed a sigmoidal pattern, the ﬁnal depth achieved, as well as the time required to achieve it, depended on the crop species and environmental conditions. The rooting depth (RD) could be estimated from RDmax (the maximum

regions with a Mediterranean climate, the depth of rooting is frequently determined by the annual depth of rewetting by rainfall and varies with both site and season. For example, on a deep vertisol at Jindiress, northern Syria, the depths of rooting and of water extraction of barley and chickpea crops were similar at 1.2 m (Gregory and Brown, 1989). Similarly, lupin (Lupinus angustifolius) and wheat crops planted on a duplex (sand over clay) soil in Western Australia both rooted to 0.8 m because of physical impediments to growth in both the sand and clay layers, and because of the limited depth of wetting by rain (Dracup et al., 1992). Rates of downward progression of the rooting front typically vary during the growing season and the front often shows a sigmoidal pattern (Borg and Grimes, 1986). Figure 2.1 gives some examples of the change of rooting depth with time for soybean, sunﬂower, and winter wheat crops. Downward rates of root extension are typically 10–40 mm day−1 during the phase of

0 20 40 60

Root depth (cm)

80 100 120 140 160 180 200 220 240 260 280 0

20

40

60

80

100

120

140

160

180

200

220

240

Days after sowing Change in rooting depth with time for soybean (•), sunﬂower (䊏, data for two crops), and winter wheat (䉱). See Gregory (2006b) for details of origin of the data. Reproduced with permission from P.J. Gregory, Plant Roots: Growth, Activity and Interaction with Soils; Wiley-Blackwell Publishing, 2006. Fig. 2.1.

24

NUTRIENT USE EFFICIENCY IN CROPS

Root length (cm cm–3) 0.01 0

0.1

1

10

20 40

Depth (cm)

60 80 100 120 140 160 180

Distribution of root length with depth in the soil proﬁle for maturing crops of cauliﬂower (•), oilseed rape (䊏), winter wheat (䉱), and sugar beet (䊊). Linear regressions have been drawn for the distributions of cauliﬂower and winter wheat roots. See Gregory (2006b) for details of origin of the data. Reproduced with permission from Gregory, Plant Roots: Growth, Activity and Interaction with Soils; Wiley-Blackwell Publishing, 2006. Fig. 2.2.

rooting depth) and tr (the relative time elapsed between sowing and maturity) using: RD = RDmax [0.5 + 0.5 sin(3.03 t r − 1.47)]. (2.1) In practice, while the length of the cropping cycle is usually easy to estimate, selecting RDmax requires some local knowledge. Roots are not distributed evenly throughout the soil proﬁle, and the length in layers within the soil proﬁle is normally expressed in terms of a root length per unit volume of soil (Lv often with units of cm root cm−3 soil), sometimes referred to as a root length density. Typical values of Lv in the upper 0.1 m of soil are about 20 cm cm−3 in grasses, 5–10 cm cm−3 in temperate cereal crops, and 1–2 cm cm−3 in other crops (Gregory, 2006a). In many, but not all, studies, it has been found that roots are distributed in the soil

such that their length and mass decrease exponentially with depth (Gerwitz and Page, 1974; Schenk and Jackson, 2002). Figure 2.2 shows that the distribution of roots of some crops (e.g., cauliﬂower and winter wheat) is well described by such a relation, but for others (e.g., rape and sugar beet), while this relation can be found in the surface layers, there is a tendency for values of Lv in deeper soil layers to be almost constant (see also Smit and Groenwold, 2005 for fodder radish). Whether this is strictly a property of the crop or a result of an interaction between the crop and soil properties remains to be established. Typically the gradient of the relationship between Ln Lv and depth changes rapidly during the early part of the growing season as the crop is establishing (see Gregory 1994 for examples). For example, in a winter wheat crop grown at Sutton Bonington in the United Kingdom, the gradient decreased from 0.077 cm−1 in

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

mid-January to 0.038 cm−1 shortly before anthesis in June (Gregory, 1994). King et al. (2003) found that after early season changes in the relationship, the gradient was fairly stable during the main phase of growth, suggesting that during the major period of growth, the relative extension of roots proceeds at a similar rate at all depths. Major soil limitations to growth As indicated in the previous section, the development and growth of a crop’s root system is profoundly affected by the properties of the soil and especially by its strength and chemical properties (see Gregory, 2006b for a fuller account). Soils must have sufﬁcient mechanical strength to provide anchorage for the plant but also contain a system of pores containing water and air, which are essential for plant growth. Dense regions of the soil may limit root growth because they offer a large mechanical resistance to root growth and/or restrict the supply of oxygen to roots. Almost all roots growing through soil experience some degree of mechanical impedance, and if continuous pores of appropriate size do not already exist, then the root tip region must exert sufﬁcient force to deform the soil. Typically, the elongation rate of individual roots decreases almost linearly with increase in mechanical strength over a range of about 0.05–0.2 MPa to 2–2.5 MPa (Taylor and Ratliff, 1969; Kirkegaard et al., 1992; Bengough et al., 2006). The ability of a root to deform soil during elongation inﬂuences the mechanical resistance to root penetration and results in changes to soil porosity close to the root. Young (1998) summarized results from several studies showing changes in bulk density up to 4–5 mm away from the root surface, while Bruand et al. (1996) found that bulk density increased to 1.8 Mg m−3 at the root–soil interface compared with 1.54 Mg m−3 at distances of greater than

25

about 0.8 mm. Analysis of the displacement of individual particles close to a maize root grown in sand found density increases of up to 30% adjacent to growing root tips, with an approximately exponential variation in particle displacement as a function of distance from the root surface (Vollsnes et al., 2010). Local variation in sand density was associated with the frictional properties of the root cap, and capless mutant roots that shed neither mucilage nor border cells had zones of greater density in front of the root tip, whereas intact wild-type roots deformed the soil more radially, with density increases generally conﬁned to the ﬂanks of the root. Bengough et al. (1997) suggested that cell wall properties were more important than turgor in regulating the elongation rate of roots and that both properties were inﬂuenced by stress history. Relatively little is known about the properties of cell walls of roots compared with those of leaves and stems, but Croser et al. (2000) demonstrated that reduced cell extension in mechanically impeded pea roots was associated with a loosening of cell walls in the radial direction and a stiffening of walls in the axial direction, although there was no change in turgor pressure. There are two clear responses of roots to mechanical impedance: slowing of the rate of extension and an increase in root diameter immediately behind the root tip. The strength of most soils increases as they dry, so that shortage of soil water and hard soils are commonly interlinked. Whalley et al. (2007) determined that the strength (Q) of a number of UK and Canadian soils could be described using the product of matric potential (ψ) and degree of saturation (S) together with bulk density (ρ) by: log10 Q = 0.35 log10 ( I ψ S I ) + 0.93 ρ + 1.26. (2.2) This relation demonstrates that even wet soils can be strong and that dense soils can

26

NUTRIENT USE EFFICIENCY IN CROPS

become too strong for roots to penetrate at quite high matric potentials. For example, for a silt, Whitmore and Whalley (2009) showed that a penetrometer pressure of 2 MPa was achieved at a matric potential of about −2000 kPa when the soil was loosely packed at a bulk density of 1.2 Mg m−3, but that the same strength was achieved at only about −60 kPa when more densely packed to a bulk density of 1.6 Mg m−3. Roots also respond to their chemical environment, especially to pH, aluminum, and salinity (see Chapters 3 and 19); responses to localized enhanced nutrient supplies will be covered in the next section. Almost 50% of all nonirrigated soils are acidic and are very common in the tropics where high rainfall has leached soluble bases. As pH falls to less than 5–5.5, aluminum materials become soluble and root growth responds negatively to both increased H+ and Al3+ concentrations. In saline soils, both Na+ and Cl− can inhibit growth. The effects of acidity and salinity on the growth of root systems have been extensively studied in Australia, where both soil conditions are common. For example, in southeastern Australia, varying combinations of salinity, sodicity, alkalinity, and boron toxicity in subsoils restrict the depth of rooting and thereby limit the access to water, a crucial yield-determining resource (Sadras et al., 2003; Adcock et al., 2007). Root system architecture Root architecture, the spatial conﬁguration of a root system in the soil, results from the production of new root apical meristems and a combination of extension and initiation of lateral branches in the proximity of root apices. Most dicotyledons have an allorhizic system consisting of a primary (tap) root and lateral roots, whereas monocotyledons have a secondary homorhizic system consisting of multiple axes and associated lateral roots.

Studies of root architecture do not usually include ﬁne details such as root hairs, but describe the entire root system of an individual plant (Lynch, 1995) and encapsulate both the topology (a description of how individual roots are connected through branching) and the distribution (the presence of roots in a spatial framework) of roots. Root system architecture is complex and varies between and within plant species, but drawings of excavated root systems (e.g., Kutschera, 1960) allow some broad generalizations to be made about the shape of the system and the spatial orientation and distribution of roots. Nearly all such drawings show that, with the exception of the taproot, which grows almost vertically throughout, most other root axes grow initially at some angle relative to the vertical but gradually become more vertically orientated. A large number of signals, both within and outside the plant, affect the root system and its ﬁnal architecture, particularly via the initiation and development of lateral roots (Nibau et al., 2008). Recent studies with Arabidopsis have elucidated some of the processes and genes controlling the production and growth of primary and lateral roots (Osmont et al., 2007). Nibau et al. (2008) identify four key stages in lateral root formation: (1) stimulation and de-differentiation of pericycle founder cells; (2) cell cycle reentry and asymmetrical cell division to give a lateral root primordium (LRP); (3) LRP emergence through the outer layers of the primary root via cell expansion; and (4) activation of the lateral root meristem and elongation of the new lateral root. Lateral root development is very dependent on auxin and auxin transport (Reed et al., 1998; Casimiro et al., 2003) and constitutes a common signal that integrates all stages of the developmental process (Péret et al., 2009). Other hormones such as ethylene and abscisic acid, together with nutrients such as nitrate and phosphate, also affect lateral development,

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

27

Fig. 2.3. Geometric simulation modeling of bean root systems that vary in basal root angle but are otherwise identical in length and branching. The variation illustrated is present among different genotypes of Phaseolus vulgaris and has been shown to be inﬂuenced by soil phosphorus availability. The scale on the right is from 0 to 40 cm. Reproduced with permission from Ge et al., Plant and Soil; Springer-Verlag, 2000.

but there is still some way to go in understanding the integration of these multiple signals. In contrast to auxin control of root development, root elongation is primarily regulated via gibberellins (GAs; Swarup et al., 2005). Ubeda-Tomás et al. (2008) demonstrated that the endodermis was the main GA-responsive tissue regulating root growth, and that the rate of elongation of the endodermis limited the rates of elongation of other tissues, and hence of the whole root. Root architecture is important because it inﬂuences the ability of plants to access nutrients and water (Lynch, 1995). This has been demonstrated in a program of work undertaken with beans (Phaseolus vulgaris). Bonser et al. (1996) found that in soils low in phosphorus the gravitropic sensitivity of both the taproot and the basal roots was decreased, resulting in a shallower root system. They hypothesized that the shallower root system was a positive adaptive response to low soil phosphorus availability,

which concentrated roots in the surface soil layers, where soil phosphorus availability was highest, and also reduced the spatial competition for phosphorus among roots of the same plant. Ge et al. (2000) tested this hypothesis by modeling root growth and phosphorus acquisition by plants with nine contrasting root systems in which the root angle of the basal roots was varied but the root length and degree of branching were kept constant (Fig. 2.3). Irrespective of the distribution of phosphorus in the soil, shallower root systems acquired more phosphorus per unit of carbon than deeper root systems, and in soils with higher phosphorus availability in the surface layers, the hypothesis was substantiated with shallower root systems acquiring more phosphorus than deeper root systems (Ge et al., 2000). Subsequent simulation studies have demonstrated that roots borne by the underground stem (termed adventitious roots) are also important in phosphorus acquisition, especially when phosphorus is preferentially located in the upper soil,

28

NUTRIENT USE EFFICIENCY IN CROPS

despite the accompanying reduction in growth of the tap and lateral roots (Walk et al., 2006). The effects of root architecture extend beyond phosphorus acquisition and also inﬂuence losses from soil erosion (Henry et al., 2010). In a ﬁeld study on a low-phosphorus soil in Costa Rica, Henry et al. (2010) found that genotypes of common bean with a shallow roots trait acquired more phosphorus, had greater shoot biomass, and thereby lost less phosphorus via soil erosion. They concluded that selection of root traits such as shallowness, when combined with integrated nutrient management, was a promising strategy for increasing both productivity and sustainability. The structure and dynamics of root system architecture are complex, and architectural models have been developed to take account of space and the biophysical interactions between roots and their environment. Simple spatial models of root distribution have been available for many years (e.g., Gerwitz and Page, 1974; Fig. 2.2), but more recently more complex architectural models (Dunbabin et al., 2003; Pagès et al., 2004; Pierret et al., 2007) have emerged that reproduce the developmental processes of root apical meristems to construct virtual root architectures. Single roots are assembled incrementally through the growth of a set of virtual apical meristems whose activity is determined at each time step of the simulation. Usually, the same morphogenetic rules are used to deﬁne the behavior of sets of meristems, and complex architectures arise as emergent properties of these simple rules (Prusinkiewicz, 2004). Other mathematical approaches, however, offer alternatives to architectural models and may more easily interact with soil mechanics and transport models that use partial differential equations to describe soil strength, and diffusion and mass ﬂow of nutrients in soil (Dupuy et al., 2010a).

Analytical models incorporating exact or approximate solutions to growth equations can be obtained in the form of mathematical functions that provide insight into the development of the root system as a whole. They also have the added advantage in that they can be parameterized relatively easily from ﬁeld data. For example, Dupuy et al. (2010b) observed barley roots in large soil bins and found that root meristems propagated like waves through the soil and that the morphology of the waves was a function of speciﬁc root developmental processes linked to properties such as gravitropism and frequency of branching. The use of continuous variables to aggregate root morphological properties also facilitates the coupling of growth with environmental and physical properties such as soil heterogeneity (de Willigen et al., 2002). Finally, representing root systems as continua allows more efﬁcient computational models to be developed (Dupuy et al., 2010a). Accessing and capturing nutrients Capture of mobile and immobile nutrients Even in root systems with complex architecture, roots are only in direct contact with a small proportion of the nutrients in the soil solution. This means that for a plant to access nutrients, either the nutrients must move from the bulk soil to the root surface or the plant must extend its inﬂuence into the soil. There is an extensive literature on the movement of nutrients to roots growing in soils via the processes of mass ﬂow and diffusion (Tinker and Nye, 2000; Gregory, 2006b). Mass ﬂow (convection) occurs as a result of transpiration, and dissolved ions are carried to the root surface. Because the

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

membranes of the root are highly selective about what they allow to enter, concentration gradients of different strengths and directions are established around roots; ions will diffuse toward the root if they are taken up faster than they are carried to the root surface by mass ﬂow and away from the root if the converse pertains. A consequence of this differential movement of ions in the soil solution is that the zones of competition for different ions around roots vary (Bray, 1954). The concentration of ions in the soil solution varies widely, but in soils used for crops it is typically about 1 mM for Ca2+, 0.1–1 mM for Mg2+, K+, and Na+, and 0.01–0.001 mM for phosphate. Phosphate is strongly adsorbed by many soils, ensuring that its concentration in solution is low, while nitrate concentration, although generally higher than that of phosphate, is very dependent on microbial and root activities. The supply of nutrients to several crops by mass ﬂow has been estimated from the product of the quantity of water taken up and the concentration of ions in the soil solution and shows that, in general, mass ﬂow will transport more than sufﬁcient sulfur, calcium, sodium, and magnesium to the root surface, signiﬁcant but insufﬁcient quantities of potassium and nitrogen, and insufﬁcient phosphorus (maize, Barber et al., 1963; leek, Brewster and Tinker, 1970; winter wheat, Gregory et al., 1979). The concentration of micronutrients in solution is highly dependent on pH and there are few experimental results, so generalizations are impossible. Such measurements demonstrate that unadsorbed nutrients such as nitrate can be regarded as mobile, while adsorbed nutrients with low concentrations in solution supplied principally to the root surface by diffusion are immobile. It follows that the zone of competition between roots for a mobile nutrient such as nitrate will be much larger than

29

that for an immobile nutrient such as phosphate. The more strongly the ion is adsorbed by the soil and the drier the soil, the slower the diffusion of the ion to the root surface. Typically, in moist soil, an ion such as nitrate will move 4 mm in 1 day, while potassium will move 1.3 mm, but phosphate only 0.04 mm. These distances are reduced substantially as the soil dries so that nitrate, for example, will move by only 0.2 mm in a soil at a permanent wilting point and movement of phosphate will effectively cease. These distances give some indication of the quantities of nutrients that are potentially available to plants by diffusion and of the difﬁculty of acquiring phosphorus relative to other nutrients. In contrast, a molecule of gas will move about 800 mm in a day in air and about 8 mm in solution (Tinker and Nye, 2000). While much attention has been paid to the ability of root systems to capture nutrients in pursuit of crop production, there is increasing emphasis on the role of root system architecture in capturing nutrients, such as nitrate, that might otherwise leach from the soil proﬁle into water courses. Dunbabin et al. (2003) have shown the role that root architecture may play in this regard and the importance of quickly producing a high density of roots in the top soil on the sandy Australian soils that they studied. Similarly, the growth of deep rooting arable and cover crops over winter has been shown to reduce nitrate leaching on deep soils in northern Europe (ThorupKristensen et al., 2009; Pedersen et al., 2010). In many parts of the world mixed cropping, intercropping, and agroforestry associations rely on differences in root architecture to overcome spatial competition and ensure spatial complementarity of root systems in their quest for nutrients (van Noordwijk et al., 1996).

30

NUTRIENT USE EFFICIENCY IN CROPS

Response of roots to localized nutrient supplies Nutrients are rarely, if ever, uniformly distributed in soils, and the response of roots to the heterogeneity of their distribution has been of considerable interest. A common, though not universal, response of roots to a nutrient-rich patch is root proliferation with suppression elsewhere. For example, Drew and coworkers undertook a series of experiments with barley grown in solution culture and sand irrigated with nutrient solution, with part of seminal axis 1 exposed to either higher or lower concentrations of nutrients than the remainder of the axis (Drew et al., 1973; Drew, 1975; Drew and Saker, 1975 and 1978). When exposed to a localized high concentration of nutrient, the root responded by increasing the number and length of ﬁrstand second-order laterals with phosphate, NH4+, and NO3−, but not K+. The reason for the lack of response to localized potassium is uncertain (see Drew, 1975 for possible explanations). In other plants, different responses are obtained, and in a review by Robinson (1994) one third of the studies showed little or no response of roots to localized nutrient supply. Hodge (2009) concluded that roots are very adaptable and plastic in their response to their environment so that “rules of response” and variation in strategy among plant species that can be applied in different environments are still absent. Studies with Arabidopsis seedlings are starting to reveal the mechanisms by which nutrient availability affects root architecture. For NO3−, increased elongation rates of laterals in the zone of enhanced concentration were due to enhanced meristematic activity and appeared to arise from a direct signal from the NO3− ion rather than from a product of NO3− metabolism (a mutant with low nitrate reductase activity showed a similar response to NO3−-rich zones; Zhang and

Forde, 1998). Zhang and Forde (1998) suggested that cells in the lateral root tips have a NO3− sensor and a signal transduction pathway to convert the NO3− signal into a growth response. Remans et al. (2006) concluded that the nitrate transporter NRT1.1, expressed in root tips, is a key component of this nitrate-sensing system, enabling the plant to detect and exploit nitrate-rich patches. Two genes have been identiﬁed as playing a role in the signal transduction pathway: (1) ANR1 is a NO3−-regulated member of the MADS-box family of transcription factors (Zhang and Forde, 1998); (2) AXR4 is an auxin-sensitive gene that may be involved because of the failure of an axr4 mutant to respond to localized NO3− (Zhang et al., 1999; Forde, 2002). More recently, studies have demonstrated an intricate N regulatory network at the root tip that coordinates changes in root growth rate and architecture to external and internal supplies of nitrogen (Forde and Walch-Liu, 2009). In Arabidopsis grown on agar, distinct mechanisms for sensing and responding to nitrate and glutamate (an amino acid representative of the major form of soluble organic nitrogen in soils) have been identiﬁed. The major effect of nitrate is to stimulate the growth of lateral roots, while that of glutamate is to slow growth of the primary root and stimulate branching near the root tip. Whether such responses to soluble organic nitrogen are replicated in soil is still to be determined. While a role for auxin in plant development and in lateral root development is undisputed, its role in nutrient signal transduction pathways is still to be established unequivocally. For example, while Williamson et al. (2001) demonstrated that responses to localized phosphate indicated a response to internal phosphate concentration, there was no indication from the auxin mutants axr1, aux1, and axr4 that auxin played any role in the response. In contrast, Al-Ghazi et al. (2003) concluded that auxin

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

signaling was involved in the response of the root system architecture of Arabidopsis to phosphate deprivation, and López-Bucio et al. (2003) concluded that the responses of root architecture to nutrients can be modiﬁed by several plant growth regulators such as auxins, cytokinins, and ethylene, so that nutritional effects on root development may be mediated by changes in hormone synthesis, transport, or sensitivity. Many effects of nutrients on root growth may also be linked to changes in carbon supply, a feature that is only now being quantitatively modeled (Brun et al., 2010). Several workers have attempted to assess the signiﬁcance of root proliferation for nutrient acquisition. Robinson (1996) used Drew’s (1975) results to calculate the potential exploitation of individual nutrients and showed that the production of additional lateral roots was highly beneﬁcial in the exploitation of locally available phosphorus, principally because of its low diffusion coefﬁcient. For nitrate, however, the necessity for lateral roots, let alone proliferation of laterals, was not demonstrated, and their growth appeared to be superﬂuous for effecting nitrate capture. Indeed, it has frequently been difﬁcult to link quantitatively the proliferation response directly to strategies for nutrient acquisition (Hodge, 2009). Hodge (2004) reviewed three experiments in which root proliferation of wheat and two grass species were measured in response to nitrogen-enriched organic patches. A relationship between root proliferation in, and nitrogen capture from, the patches was not found in any experiment, but in experiments where two different plant species were grown together and allowed to explore a common enriched patch, there was a direct relation between root proliferation in, and nitrogen capture from, the patch. Hodge et al. (1999) and Robinson et al. (1999) concluded that root proliferation was important for nitrogen capture when plants are in inter-

31

speciﬁc competition for organic patches containing a ﬁnite supply of mixed nitrogen sources, but when any one of these factors was not present, then the importance of root proliferation for nitrogen capture was less obvious. Whether roots proliferate depends on the plant demand for the nutrient, the mobility of the nutrient within the plant, and the concentration in the patch relative to background (Hodge, 2004). The substantial plasticity of plant responses to heterogenous nutrient supplies opens the possibility of developing crops with enhanced capacity to capture nutrients. Dunbabin et al. (2001a,b) examined growth responses of Lupinus angustifolius (dominant tap root and lateral system) and Lupinus pilosis (minor tap root and well-developed laterals) to nitrate supplied either uniformly or split between the upper and lower root system. In both species, increased root proliferation in the high nitrate zone was accompanied by decreased root growth in the low nitrate zone to give about the same total growth as the uniform low nitrate treatment. However, while L. angustifolius increased its rate of nitrate uptake from parts of the root system supplied locally with high nitrate, L. pilosis did not and only used its increased root growth to exploit nitrate patches. From this range of responses, it may be possible to select a lupin type with an enhanced ability to capture nitrate from the soil proﬁle. In practice, of course, plants have multiple resource constraints to contend with (e.g., heterogeneously distributed phosphorus and soil water) and will try to optimize their investment in roots. Modeling of these responses is at an early stage. Ho et al. (2004) investigated this possible optimization for beans grown under different combinations of water and phosphorus availability and found that the basal root angle was shallower for localized shallow phosphorus, and deeper for localized deep water than that

32

NUTRIENT USE EFFICIENCY IN CROPS

obtained in the case of uniformly distributed water and phosphorus. When phosphorus was concentrated in the surface and water located deep, the optimal basal root angle depended on the relative rates of change with depth in the values ascribed to the available resources. Root-microbe–soil interactions that inﬂuence nutrient availability Roots and their associated microorganisms play a crucial role in modifying soils and in the availability of nutrients (see also Chapter 3). About 80% of all higher plant species form mycorrhizal symbioses, with the arbuscular mycorrhizal (AM) association being the most common among the seven different types of mycorrhizal symbioses (Smith and Read, 2008; Brundrett, 2009). The symbiosis evolved early in the evolution of land plants and facilitated the uptake of poorly soluble ions such as phosphate and zinc. Plants beneﬁt from the AM fungi because these acquire nutrients that would otherwise be inaccessible for reasons of distance from the root, location in pores that are too small for roots to enter, or occurrence in forms unavailable to plants but not fungi; in return, the fungi beneﬁt from a supply of C from photosynthesis (Lambers et al., 2009). The AM hyphae may also be more effective than roots in competing with other soil microbes for phosphorus and may also have a higher afﬁnity for phosphorus than roots (Smith and Read, 2008). The nutritional beneﬁts of AM fungi to plants grown on soils low in phosphorus are well documented (Smith and Read, 2008), although many questions about their functioning remain. For example, Hodge (2009) identiﬁes the importance of AM fungal foraging, the nutrients captured, the differences that occur among AM fungal species, and the consequences for different plants linked together in a common network as unresolved

questions. Transfer of nutrients between the fungus and the host is also still not well understood (Tinker and Nye, 2000; Smith and Read, 2008). Ryan et al. (2003) measured the concentrations of phosphorus, potassium, and magnesium in the hyphae and young arbuscules of indigenous AM of ﬁeld- and glasshouse-grown plants of subterranean clover, white clover, leek, and pea, and concluded that nutrient transfer to the host and carbon transfer to the fungus occurred in both young arbuscules and intercellular hyphae. They suggested that magnesium and potassium ions are probably the balancing cations for phosphorus transfer. Different mycorrhizal fungi do not all behave in identical manner (for example, some fungi grow only close to a root while others extend to considerable distance), with the result that different fungal–plant combinations are functionally diverse. Smith et al. (2003, 2004) grew combinations of ﬂax, medic, and tomato with the AM fungi Glomus caledonium, Glomus intraradices, and Gigaspora rosea in pots of equal mixtures of sand and irradiated soil with root + hyphal, and hyphal-only compartments. After 6 weeks, the dry weight of ﬂax was increased by all fungi relative to a nonmycorrhizal control, while medic increased with the two Glomus spp. but not Gigaspora rosea, and tomato showed no positive responses with any species. There was no simple relation between plant growth and external hyphal growth in the soil, and the contributions of the AM fungi to phosphorus content of the plants varied signiﬁcantly (Table 2.1). After 6 weeks of growth, G. intraradices contributed almost 100% of the phosphorus taken up by ﬂax, 80% by tomato, and only 60% by medic. The contribution of G. caledonium was 25–40% across all plants, with only small contributions of Gi. rosea throughout. Smith et al. (2004) concluded that mycorrhizal uptake of phosphorus could replace uptake by roots and root

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

Table 2.1. Contribution (%) of the mycorrhizal pathway to phosphorus uptake in plants of ﬂax, medic, and tomato after 6 weeks when colonized by either Glomus caledonium or Glomus intraradices. The standard error is in brackets

Flax Medic Tomato

G. caledonium

G. intraradices

42 (3) 38 (5) 25 (8)

94 (15) 62 (5) 83 (12)

Data from Smith et al. (2004).

hairs even in plants such as tomato, which showed no growth response to AM colonization; therefore, lack of a growth response to an AM fungus does not mean that the fungus makes no contribution to phosphorus uptake. Much work with AM fungi has been performed with young plants grown in media that are not natural soils, where nutrients may be in uncommon forms and without the microbes that would normally be part of the mineralization processes, raising questions as to the transferability of results to ﬁeldgrown plants. In managed production systems where fertilizers and manures are applied, the carbon cost of maintaining the fungus may exceed the beneﬁts of its activities in phosphorus uptake, and there has been little evidence to date for a beneﬁcial role of AM fungi in improving productivity (Ryan et al., 2002). Ryan and Graham (2002) reviewed literature of ﬁeld-based studies and found that even when phosphorus availability was low, and AM colonization levels were high (as on some long-term organic farms), there was no obvious net beneﬁt from the symbiosis. They concluded that AM fungi “do not play a vital role in the nutrition and growth of plants in many production-orientated agricultural systems” (p. 263). Rhizodeposition of carbon compounds from roots provides a source of energy to a wide variety of soil microorganisms and results in rhizosphere soil having different chemical, physical, and biological properties

33

to the bulk soil (Jones et al., 2009). These deposits affect nutrient availability in multiple ways. For example, surfactants present in root mucilage can affect phosphorus adsorption to increase the amount of phosphorus in solution and thereby increase availability to the plant; they also reduced net rates of ammonium consumption and nitrate production in soil (Read et al., 2003). Rhizodeposits may also stimulate microbial growth, promoting a wide range of effects beneﬁcial to plant growth (Lambers et al., 2009). Exploiting genotypic variation in root properties to improve nutrient capture Case studies with genotypes As detailed throughout this book, there is much interest in exploiting genotypic differences in the uptake of nutrients (especially of nitrogen and phosphorus), and particularly in improving the efﬁciency with which resources are used. Kirk et al. (1998) summarized the root factors contributing to phosphorus uptake efﬁciency as: 1. Root geometry—differences in root length and its distribution in soils, root hair length, and density, root diameter, and so on. 2. Mycorrhizal effects—differences in the extent or rate of infection, or species of mycorrhizal fungus. 3. Solubilization effects—differences in phosphorus solubility close to the root surface arising from changed soil chemical conditions. This is a complex set of properties that produce interrelated effects on internal, physiological efﬁciency and the efﬁciency of recovery (see Ladha et al., 2005 for deﬁnitions). The interactions arise mainly

34

NUTRIENT USE EFFICIENCY IN CROPS

Table 2.2. Root growth parameters for four genotypes of common bean grown in containers of an oxisol for 14 days. Values are the mean of four replicates with the standard error shown in brackets

Genotype −1

Total root dry weight (g plant ) Total root length (m plant−1) Number of basal roots Relative total root growth rate (day−1) Relative total root elongation rate (day−1)

Tostado

Porrillo Sintetico

Carioca

HAB 229

0.38 (0.03) 65.9 (23.9) 252 (14) 0.20 (0.01) 0.48 (0.03)

0.23 (0.05) 23.9 (2.6) 171 (22) 0.20 (0.04) 0.38 (0.03)

0.27 (0.04) 35.1 (5.0) 271 (39) 0.18 (0.01) 0.40 (0.03)

0.28 (0.01) 49.6 (15.6) 216 (44) 0.25 (0.01) 0.42 (0.02)

Data from Lynch and van Beem (1993).

because any additional nutrients provided by externally efﬁcient roots may also stimulate root growth. For example, model simulations of rice by Wissuwa (2003) showed that small changes (22%) in root diameter or internal efﬁciency had large effects (threefold) on phosphorus uptake. The same result could be achieved by a 33% increase in root external efﬁciency, but only 10% of the threefold increase in phosphorus uptake was directly attributable to the direct effect of increased external root efﬁciency, with 90% due to enhanced root growth as a consequence of higher phosphorus uptake per unit of root. Wissuwa (2003) concluded that large genotypic differences in phosphorus uptake from phosphorus-deﬁcient soils can result from small differences in tolerance mechanisms and that these small changes will be difﬁcult to detect as changes in recovery efﬁciency because they are likely to be overshadowed by the effects on root growth. The importance of different root architecture in response to soil conditions, or as a consequence of genotypic differences in root growth, for nutrient uptake is starting to emerge. Lynch and van Beem (1993) grew four genotypes of common bean representing distinct shoot growth habits (erect determinate, erect indeterminate, prostrate indeterminate, and climbing) in containers of an oxisol and measured a range of root

parameters up to 14 days after planting. Table 2.2 shows that there were signiﬁcant differences between the genotypes after 14 days in root length and mass, number of roots arising from the base of the hypocotyls (basal roots), and root growth and root elongation rates. The phosphorus-efﬁcient genotype Tostado, which grows well in highly acidic, infertile soils in Rwanda, had the most vigorous seedling root system, which was highly branched and had numerous basal roots, whereas the landrace Porrillo sintetico, which grows well on fertile soils in South America, had a smaller, less branched root system. Such results demonstrate that substantial genetic variability exists for root traits that determine the relative distribution of roots in different soil layers, and thereby inﬂuence the acquisition of resources. Liao et al. (2004) exploited this variation by crossing deep-rooted and shallow-rooted genotypes of bean to obtain recombinant inbred lines, and found that lines with the highest phosphorus acquisition efﬁciency had shallower root systems. At least two factors are believed to contribute to this greater phosphorus efﬁciency of shallower root systems compared with deeper root systems: ﬁrst, spatial coincidence of root and resource; and second, lower intra-plant inter-root competition. The latter is an important consideration because at typical planting densities of crops, inter-

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

root competition has been found to be more important in determining the efﬁciency of phosphorus uptake than root competition between plants (Rubio et al., 2001). Signiﬁcant correlations between root architectural features and phosphorus uptake and phosphorus use efﬁciency have also been found in a range of other dicotyledonous species. For example, Hammond et al. (2009) found that in a wide range (355) of Brassica oleracea L. accessions, many measures of phosphorus use efﬁciency were correlated with root development and architecture, especially with lateral root number, length, and growth rate. Physiological phosphorus use efﬁciency varied four- to ﬁvefold in a range of commercial genotypes, suggesting that there is potential to breed more efﬁcient cultivars. Similarly Ao et al. (2010) employed two soybean genotypes together with their 88 recombinant inbred lines and found that phosphorus use efﬁciency was signiﬁcantly correlated with root length, surface area, root width, and root depth. These correlations together with the high broad-sense heritability values of the root traits suggest the feasibility of screening phosphorusefﬁcient genotypes through selection of simple root traits in the ﬁeld. In cereals, there is also ample evidence of genetic variation in root architecture (e.g., Chloupek et al., 2006; Manschadi, 2008; Hargreaves et al., 2009), although there has been relatively little work so far to relate these features to nutrient use. Wojciechowski et al. (2009) explored the effects of semidwarﬁng and dwarﬁng alleles on root growth of young wheat seedling grown in a range of media. No signiﬁcant differences in root length were found between semi-dwarﬁng lines and the control lines, but the dwarﬁng lines had signiﬁcant effects on early root growth although no effect on early shoot growth. These results suggest a direct effect of dwarﬁng genes on root growth during

35

seedling establishment, although the mechanism for this is uncertain but probably involving the gibberellic acid pathway (Wojciechowski et al., 2009). This study also found that root length of the dwarf lines was signiﬁcantly increased relative to the control lines when grown in gel but decreased when grown in soil, thereby raising questions about the relationship between results obtained in gel-based experiments and ﬁeld performance in soils. Similar concerns were evident in the studies of Hargreaves et al. (2009) with genotypes of barley. Quantitative Trait Locus (QTL) studies The development of DNA-based molecular markers has opened up opportunities for identifying the genetic factors (QTL) underpinning root traits inﬂuencing nutrient acquisition and efﬁciency. For example, the tolerance of rice to phosphorus deﬁciency was investigated by Wissuwa et al. (1998) using 98 backcross inbred lines derived from a cross of the traditional Indian indica variety Kasalath, which is tolerant of phosphorus deﬁciency with the modern Japanese japonica variety Nipponbare, which does not yield well under low phosphorus conditions. Three QTL explained 45% of the variation in dry weight and four QTL explained 55% of the variation in phosphorus uptake. For both traits, the QTL linked to marker C443 on chromosome 12 had a major effect, and this was conﬁrmed in a complementary study of tiller numbers under conditions of phosphorus deﬁciency. One of the minor QTL associated with tolerance to phosphorus deﬁciency coincided with a locus found in another study to account for 10% of the variation in total root number. Subsequent transference of the major QTL linked to marker C443 on chromosome 12 into Nipponbare by three backcrosses resulted in an improved line that

36

NUTRIENT USE EFFICIENCY IN CROPS

increased phosphorus uptake by 170% and grain yield by 230% compared with Nipponbare when grown in the ﬁeld on a low-phosphorus andosol (Wissuwa and Ae, 2001). This approach meant that it was possible to combine the high phosphorus uptake of the traditional variety Kasalath with the high harvest index of the modern variety to produce a plant that yielded 21% more grain under low phosphorus conditions, even though it produced only 58% of the shoot weight and 68% of the root weight of Kasalath. As described, earlier, shoot-borne (adventitious) roots play a signiﬁcant role in the phosphorus nutrition of bean. Ochoa et al. (2006) screened a population of recombinant inbred lines of P. vulgaris L. under high and low phosphorus conditions in glasshouse and ﬁeld conditions, and found that 19 QTLs accounted for 19–61% of the total phenotypic variation for adventitious root traits in the ﬁeld and 128–39% under glasshouse conditions. Under low phosphorus conditions in the ﬁeld, two major QTL located on linkage groups B2 and B9 accounted for 61% of the observed phenotypic variation. The identiﬁcation of such QTL suggests that the use of markers for speciﬁc adventitious root traits might be possible to assist in breeding beans for lowphosphorus soils (Ochoa et al., 2006). Identifying appropriate traits and markers to assist the breeding of nutrient-efﬁcient genotypes is emerging as a major challenge in the development of more sustainable cropping systems (Foulkes et al., 2009). However, it is important that the screening for such markers is undertaken in a number of conditions because there may be a large interaction between the genes/markers identiﬁed and the environment. George et al. (2011) found signiﬁcant variation in the phosphorus nutrition of spring and winter barley genotypes, but the variation was different between crops grown with conventional

plow and minimum tillage. Results of an association mapping exercise showed that the associations between phosphorus use and speciﬁc markers were different in the two cultivation systems, implying that for multimechanistic traits such as phosphorus use efﬁciency, robust markers will emerge only after tests under different climatic and agronomic conditions. Management to optimize capture by root systems A major means of achieving efﬁcient nutrient use is to ensure that the availability of individual nutrients is matched to crop demand in time and space. Management options that can increase the efﬁciency of nutrient use from fertilizers focus on a combination of increasing fertilizer use during the growing season when the fertilizer is applied, and decreasing fertilizer losses so that the recovery of any residual fertilizer by subsequent crops can be maximized. Placement of fertilizers For mobile nutrients such as nitrate, proximity to a root is less crucial for uptake by the plant than that of an immobile nutrient such as phosphate, so that placement techniques for phosphorus fertilizers have generally been more widely investigated than those for nitrogen. Field experiments on fertilizer placement have typically compared applications as a band in, or near, the row at planting with applications broadcast on the soil surface and then incorporated. While phosphorus applications may be drilled with the seed in the same row, there is much work that demonstrates that nitrogen and potassium fertilizers applied in this manner will damage the seed and/or seedling and reduce germination (Cooke, 1982). The advantages of fertilizer placement vary depending on the fertilizer, crop, soil,

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

and climate factors, so that prediction of effects is imprecise at present. Banded phosphorus typically gives higher yields than broadcast phosphorus on soils low in phosphorus at low rates of application, with the yield advantage decreasing and eventually reversing as both native soil phosphorus and/or amount of phosphorus applied increase. For example, Welch et al. (1966) found that banded phosphorus gave greater yields of maize than broadcast phosphorus at application rates <30 kg phosphorus/ha on two of three soils that had least phosphorus, while at higher application rates, broadcasting gave greater yields also on two of the three soils. In widely planted crops such as maize, a treatment intermediate between broadcasting and banding near the seed, strip-application has been found by some to be advantageous presumably because this technique optimizes phosphorus availability by simultaneously reducing adsorption while increasing proximity to the young root system (Barber, 1984). It is possible that phosphorus application enhanced root growth in the strip, thereby increasing uptake. Root contact with phosphorus fertilizer is very important for the utilization of banded phosphorus, and can have signiﬁcant effects on the phosphorus nutrition of young plants (Eghball and Sander, 1989). Sander and Eghball (1999) grew winter wheat on soils of low to very low phosphorus status and found that while two methods of banding phosphorus (application with a knife together with anhydrous ammonia before sowing, and application with the seed at sowing) performed similarly when seed was planted at the optimum sowing date, application at sowing was superior when sowing was delayed. They suggested that the difference in results arose because the application at sowing allowed quicker root access to the fertilizer phosphorus in autumn stimulating tillering at the later planting dates and

37

thereby increasing the number of ears and grain yield at maturity. The effects of placing fertilizers on crop growth are often discernable in young crops but may not persist until maturity. For example, Mallarino et al. (1999) showed that banding of phosphorus fertilizer promoted the early growth of maize (and increased phosphorus uptake) overcoming the combination of low soil temperature and increased soil strength that may be responsible for the poor early growth of maize in no-till systems compared with conventional tillage. However, the positive effects on early growth did not signiﬁcantly increase grain yield compared with broadcast phosphorus fertilizer. Rhizosphere engineering Our greater knowledge of roots and their interactions with microorganisms has led to interest in the possibilities of manipulating them to beneﬁt plant health and productivity (Ryan et al., 2009a; Gregory et al., 2011). Temporary changes in rhizosphere properties are induced by applications of fertilizers, irrigation, and other inputs, with changes in pH (see above) altering rhizosphere chemistry and the composition of microbial communities. Plant breeders have introgressed favorable rhizosphere traits into breeding programs, often unwittingly, and there is interest in improving the identiﬁcation of traits that might improve on this process. For example, resistance of several cereals to aluminum toxicity is often associated with the release of organic anions, especially malate, into the rhizosphere (Ryan et al., 1995). Efﬂux of organic anions protects crops from aluminum toxicity by chelating Al3+ ions and preventing their interaction with root apices (Ma et al., 2001). Several of the genes controlling these exudates have been identiﬁed and their expression modiﬁed by genetic engineering (de la

38

NUTRIENT USE EFFICIENCY IN CROPS

Fuente et al., 1997; Sasaki et al., 2004). The ALMT and MATE gene families encode membrane-bound proteins that facilitate the efﬂux of malate and citrates from plant cells offering possibilities for manipulating organic anion efﬂux from roots (Delhaize et al., 2004; Wang et al., 2007; Ryan et al., 2009b). Some of these genes (e.g., TaALMT1), while constitutively expressed, require activation by Al3+ ions so that there practical application is limited to acid soils in which the concentration of Al3+ is sufﬁcient to activate the protein. Grain yield of transgenic barley modiﬁed with TaALMT1 was twice that of nontransformed barley when grown on acid soil (pH 4.1) and 80% of that of limed controls, and was also more efﬁcient at taking up phosphorus (Delhaize et al., 2009). Moreover, in the absence of Al3+, shoot mass and grain yield were unaffected by the expression of TaALMT1 in barley (Delhaize et al., 2009). The potential for modifying the root soil interface to increase the availability of nutrients, especially phosphorus, has been reviewed by several workers (e.g., Hinsinger, 2001). Phosphatase enzymes are produced by many bacteria and fungi in soil and break down organically bound phosphorus into inorganic forms that can be more readily taken up by microorganisms and plants. The release from higher plants is generally limited, although some plants, typically growing in low phosphorus soils, do release phosphatase (George et al., 2002a,b; Li et al., 1997). For example, George et al. (2002a,b) found that Tithonia diversifolia and Crotalaria grahmiana had enhanced levels of phosphatase activity in their rhizospheres compared with maize and that there was depletion of organic phosphorus that was subsequently identiﬁed as orthophosphate monoesters. Extracellular secretion of phosphatases from roots is correlated with the ability of plants to obtain phosphorus from organic phosphorus sources when they

were grown under sterile conditions (Richardson et al., 2000; George et al., 2008). Wheat and a range of pasture species were able to utilize phosphorus from various monoester (e.g., glucose 6 phosphate) and diester (e.g., ribonucleic acid) forms but show limited capacity to acquire phosphorus directly from myo-inositol hexakisphosphate (Richardson et al., 2000; George et al., 2008), despite inositol phosphates being the most abundant form of organic phosphorus in many soils. Such studies demonstrate the vital role that rhizosphere microorganisms make to facilitate the utilization of organic phosphorus, and it is evident that mineralization of organic phosphorus occurs in the rhizosphere and can make an important contribution to the orthophosphate requirement of plants. George et al. (2008) screened a range of wheat lines for variation in the activity of root-associated phosphatases toward different organic phosphorus substrates; however, while some relationships were identiﬁed between the different activities and the ability of the plants to utilize speciﬁc organic phosphorus substrates in vitro, no clear relationships were found for the growth and phosphorus nutrition of the plants when grown in a range of soils (George et al., 2008). This suggests either that variability in phosphatase activities has little signiﬁcance in the phosphorus nutrition of soil-grown plants or, more likely, that any beneﬁt from hydrolysis of organic phosphorus by either plant or microbialderived phosphatases was common to all genotypes. Genetic modiﬁcation to enhance the extracellular production of phosphatases by roots has shown only limited beneﬁts to phosphorus nutrition because of adsorption of the enzyme by soil solids (George et al., 2005a). For example, although George et al. (2004) showed that incorporation of the phyA gene into ﬁve lines of subterranean clover increased exudation of extracellular

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

39

Table 2.3. Phosphorus accumulation (μg phosphorus plant−1) for transformed Nicotiana tabacum lines that variously express phyA from either Aspergillus niger (phy[An]) or Peniophora lycii (phy A[An]) and are compared with the transformed vector control

Cattle

Pig

Phosphorus Phosphorus accumulation s.e. Response accumulation Vector 549 control Asperigillus 442 niger phyA Peniphora 401 lycii phyA

63

s.e.

338

32.3

Hen Phosphorus Response accumulation s.e. Response 182

44

91

0.8

670

35

2.0

754

82

4.1

64

0.7

452

74

1.3

462

52

2.5

Plants were grown for 25 days in topsoil (0–10 cm) collected from either Benvie fertilized with cattle manure, Tayport fertilized with pig manure, or Benvie fertilized with hen manure; all sites were in northeastern Scotland. Values represent the mean of ﬁve replicates with standard errors (s.e.) of mean. Where signiﬁcant differences occurred, x-fold responses of the mean of the different phyA constructs compared with the controls are also presented (from Gregory et al., 2011).

phytase by an average of 77-fold, and uptake of phosphorus from phytate by 1.3 to 3.6fold compared with controls when plants were grown in agar, only one of them showed increased shoot biomass or phosphorus uptake when grown in soil. George et al. (2005a) demonstrated that phytase activity in solution was lost within 10 min when phytase was added to three soils, because of rapid sorption of phytase onto the solid phase, although there was less sorption to soil collected from the rhizosphere of transgenic subterranean clover plants expressing phyA, suggesting that the rhizosphere may maintain phytase activity in solution possibly by altering pH. Rapid sorption of phytase by soils may, then, limit the ability of roots to acquire phosphorus from soil phytate. Under certain circumstances, plants exuding phytase have shown relatively large growth and phosphorus uptake responses. Plants that express fungal phytase genes, and have enhanced root phytase activity, have the ability to accumulate up to 70%

more phosphorus than controls following recent application of inorganic phosphorus (George et al., 2005b). This is thought to reﬂect the relative availability of recently immobilized organic phosphorus following addition of inorganic phosphorus fertilizer (George et al., 2006). Moreover, a very large 1.3 to 4.1-fold increase in phosphorus uptake over the control plants was found when soils were supplemented with monogastric animal manures (Gregory et al., 2011; Table 2.3). This contrasted with the lack of phosphorus uptake response when the same plants were grown in soils supplemented with ruminant manure. This lack of response was attributed to the relatively small content of inositol phosphate found in ruminant guts, which have naturally occurring phytase present (He et al., 2007). Gregory et al. (2011) suggested that the integration of transgenic plants expressing phytase genes into agricultural systems that rely on monogastric animal manure for fertilization may have beneﬁts to both the economic and environmental sustainability of these enterprises.

40

NUTRIENT USE EFFICIENCY IN CROPS

Gregory et al. (2011) suggested that although much is now known about individual aspects of rhizosphere chemistry and biology, knowledge of how these properties interact with soil particles to drive the physical and chemical genesis of the rhizosphere is largely speculative. Future research needs to move from reporting “What is there?” to “Why is it there?” This would allow the identiﬁcation of speciﬁc plant or microbial traits that promote rhizosphere formation and the development of appropriate technologies to exploit these traits for the beneﬁt of crop nutrition. Because of the paucity of mechanistic understanding, relatively few aspects of root : soil interactions have yet been quantitatively modeled, although Watt et al. (2006) have pioneered modeling of the spatial and temporal dynamics of root colonization by soil bacteria, and provided a semi-quantitative framework for linking root colonization to root growth dynamics and relevant scales of organism movement and multiplication. Tantalizing glimpses of the beneﬁts that might be gained from such manipulation for crop nutrition are starting to emerge (Ryan et al., 2009a), but, at present, we are quite a long way from being able to quantitatively manipulate or engineer the rhizosphere to reliably beneﬁt crop growth. References Adcock, D., McNeill, A.M., McDonald, G.K., & Armstrong, R.D. (2007) Subsoil constraints to crop production on neutral and alkaline soils in southeastern Australia: a review of current knowledge and management strategies. Australian Journal of Experimental Agriculture 47, 1245–1261. Al-Ghazi, Y., Muller, B., Pinloche, S., et al. (2003) Temporal responses of Arabidopsis root architecture to phosphate starvation: evidence for the involvement of auxin signalling. Plant, Cell & Environment 26, 1053–1068. Ao, J., Fu, J., Tian, J., Yan, X., & Liao, H. (2010) Genetic variability for root morph-architecture traits and root growth dynamics as related to

phosphorus efﬁciency in soybean. Functional Plant Biology 37, 304–312. Barber, S.A. (1984) Soil Nutrient Bioavailability: A Mechanistic Approach. Wiley Interscience, New York. Barber, S.A., Walker, J.M., & Vasey, E.H. (1963) Mechanisms for the movement of plant nutrients from the soil and fertiliser to the plant root. The Journal of Agriculture and Food Chemistry 11, 204–207. Bengough, A.G., Croser, C., & Pritchard, J. (1997) A biophysical analysis of root growth under mechanical stress. Plant and Soil 189, 155–164. Bengough, A.G., Bransby, M.F., Hans, J., McKenna, S.J., Roberts, T.J., & Valentine, T.A. (2006) Root responses to soil physical conditions; growth dynamics from ﬁeld to cell. Journal of Experimental Botany 57, 437–447. Bonser, A.M., Lynch, J.P., & Snapp, S. (1996) Effect of phosphorus deﬁciency on growth angle of basal roots in Phaseolus vulgaris. The New Phytologist 132, 281–288. Borg, H. & Grimes, D.W. (1986) Depth development of roots with time: an empirical description. Transactions of the American Society of Agricultural Engineers 29, 194–197. Bray, R.H. (1954) A nutrient mobility concept of soilplant relationships. Soil Science 78, 9–22. Brewster, J.L. & Tinker, P.B. (1970) Nutrient cation ﬂow in soil around plant roots. Soil Science Society of America Proceedings 34, 421–426. Bruand, A., Cousin, I., Nicoullaud, B., Duval, O., & Begon, J.C. (1996) Backscattered electron scanning images of soil porosity for analyzing soil compaction around roots. Soil Science Society of America Journal 60, 895–901. Brun, F., Richard-Molard, C., Pagès, L., Chelle, M., & Ney, B. (2010) To what extent may changes in the root system architecture of Arabidopsis thaliana grown under contrasted homogenous nitrogen regimes be explained by changes in carbon supply? A modelling approach. Journal of Experimental Botany 61, 2157–2169. Brundrett, M.C. (2009) Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conﬂicting information and developing reliable means of diagnosis. Plant and Soil 320, 37–77. Canadell, J., Jackson, R.B., Ehleringer, J.R., Mooney, H.A., Sala, O.E., & Schulze, E.D. (1996) Maximum rooting depth of vegetation types at the global scale. Oecologia 108, 583–595. Casimiro, I., Beeckman, T., Graham, N., et al. (2003) Dissecting Arabidopsis lateral root development. Trends in Plant Science 8, 165–171.

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

Chloupek, O., Forster, B.P., & Thomas, W.T.B. (2006) The effect of semi-dwarf genes on root system size in ﬁeld-grown barley. Theoretical and Applied Genetics 112, 779–786. Cooke, G.W. (1982) Fertilizing for Maximum Yield, 3rd ed. Granada, London. Croser, C., Bengough, A.G., & Pritchard, J. (2000) The effect of mechanical impedance on root growth in pea (Pisum sativum). II. Cell expansion and wall rheology during recovery. Physiologia Plantarum 109, 150–159. de la Fuente, J.M., Ramirez-Rodriguez, V., CabreraPonce, J.L., & Herrera-Estrella, L. (1997) Aluminium tolerance intransgenic plants by alteration of citrate synthesis. Science 276, 1566–1568. de Willigen, P., Heinen, M., Mollier, A., & van Noordwijk, M. (2002) Two-dimensional growth of a root system modelled as a diffusion process. I. Analytical solutions. Plant and Soil 240, 225–234. Delhaize, E., Ryan, P.R., Hebb, D.M., Yamamoto, Y., Sasaki, T., & Matsumoto, H. (2004) Engineering high-level aluminium tolerance in barley with the ALMT1 gene. Proceedings of the National Academy of Science of the United States of America 101, 15249–15254. Delhaize, E., Taylor, P., Hocking, P.J., Simpson, R.J., Ryan, P.R., & Richardson, A.E. (2009) Transgenic barley (Hordeum vulgare L.) expressing the wheat alumunium resistance gene (TaALMT1) shows enhanced phosphorus nutrition and grain production when grown on an acid soil. Plant Biotechnology Journal 7, 391–400. Dracup, M., Belford, R.K., & Gregory, P.J. (1992) Constraints to root growth of wheat and lupin crops in duplex soils. Australian Journal of Experimental Agriculture 32, 947–961. Drew, M.C. (1975) Comparison of the effects of a localized supply of phosphate, nitrate, ammonium and potassium on the growth of the seminal root system, and the shoot, in barley. New Phytologist 75, 479–490. Drew, M.C. & Saker, L.R. (1975) Nutrient supply and the growth of the seminal root system in barley. II. Localized, compensatory increases in lateral root growth and rates of nitrate uptake when nitrate supply is restricted to only part of the root system. Journal of Experimental Botany 26, 79–90. Drew, M.C. & Saker, L.R. (1978) Nutrient supply and the growth of the seminal root system in barley. III. Compensatory increases in growth of lateral roots, and in rates of phosphate uptake, in response to a localized supply of phosphate. Journal of Experimental Botany 29, 435–451. Drew, M.C., Saker, L.R., & Ashley, T.W. (1973) Nutrient supply and the growth of the seminal root

41

system in barley. I. The effect of nitrate concentration on the growth of axes and laterals. Journal of Experimental Botany 24, 1189–1202. Dunbabin, V., Rengel, Z., & Diggle, A. (2001a) The root growth response to heterogeneous nitrate supply differs for Lupinus angustifolius and Lupinus pilosis. Australian Journal of Agricultural Research 52, 495–503. Dunbabin, V., Rengel, Z., & Diggle, A. (2001b) Lupinus angustifolius has a plastic uptake response to hetergeneously supplied nitrate while Lupinus pilosis does not. Australian Journal of Agricultural Research 52, 505–512. Dunbabin, V., Diggle, A., & Rengel, Z. (2003) Is there an optimal root architecture for nitrate capture in leaching environments? Plant, Cell & Environment 26, 835–844. Dupuy, L., Gregory, P.J., & Bengough, A.G. (2010a) Root growth models: towards a new generation of continuous approaches. Journal of Experimental Botany 61, 2131–2143. Dupuy, L., Vignes, M., McKenzie, B.M., & White, P.J. (2010b) The dynamics of root meristem distribution in the soil. Plant, Cell & Environment 33, 358–369. Eghball, B. & Sander, D.H. (1989) Distance and distribution effects of phosphorus fertilizer on corn. Soil Science Society of America Journal 53, 282–287. Forde, B.G. (2002) Local and long-range signalling pathways regulating plant responses to nitrate. Annual Review of Plant Biology 53, 203–224. Forde, B.G. & Walch-Liu, P. (2009) Nitrate and glutamate as environmental cues for behavioural responses in plant roots. Plant, Cell & Environment 32, 682–693. Foulkes, M.J., Hawkesford, M.J., Barraclough, P.B., et al. (2009) Identifying traits to improve the nitrogen economy of wheat: recent advances and future prospects. Field Crops Research 114, 329–342. Ge, Z., Rubio, G., & Lynch, J.P. (2000) The importance of root gravitropism for inter-root competition and phosphorus acquisition efﬁciency: results from a geometric simulation model. Plant and Soil 218, 159–171. George, T.S., Gregory, P.J., Robinson, J.S., & Buresh, R.J. (2002a) Changes in phosphorus concentrations and pH in the rhizosphere of some agroforestry and crop species. Plant and Soil 246, 65–73. George, T.S., Gregory, P.J., Robinson, J.S., Buresh, R.J., & Jama, B. (2002b) Utilisation of soil organic P by agroforestry and crop species in the ﬁeld, western Kenya. Plant and Soil 246, 53–63.

42

NUTRIENT USE EFFICIENCY IN CROPS

George, T.S., Richardson, A.E., Hadobas, P.A., & Simpson, R.J. (2004) Characterisation of transgenic Trifolium subterraneum L. which expresses phyA and releases extracellular phytase: growth and phosphorus nutrition in laboratory media and soil. Plant, Cell & Environment 27, 1351–1361. George, T.S., Richardson, A.E., & Simpson, R.J. (2005a) Behaviour of plant-derived extracellular phytase upon addition to soil. Soil Biology & Biochemistry 37, 977–988. George, T.S., Simpson, R.J., Hadobas, P.A., & Richardson, A.E. (2005b) Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition in plants grown in amended soil. Plant Biotechnology Journal 3, 129–140. George, T.S., Turner, B.L., Gregory, P.J., & Richardson, A.E. (2006) Depletion of organic phosphorus from oxisols in relation to phosphatase activities in the rhizosphere. European Journal of Soil Science 57, 47–57. George, T.S., Gregory, P.J., Hocking, P.J., & Richardson, A.E. (2008) Variation in root-associated phosphatase activities in wheat contributes to the utilisation of organic P substrates in vitro, but does not effectively predict P-nutrition in different soils. Environmental and Experimental Botany 64, 239–249. George, T.S., Brown, L.K., Newton, A.C., et al. (2011) Impact of soil tillage on the robustness of the genetic component of variation in phosphorus (P) use efﬁciency in barley (Hordeum vulgare L). Plant and Soil 339, 113–123. Gerwitz, A. & Page, E.R. (1974) An empirical mathematical model to describe plant root systems. Journal of Applied Ecology 11, 773–782. Greenwood, D.J., Gerwitz, A., Stone, D.A., & Barnes, A. (1982) Root development of vegetable crops. Plant and Soil 68, 75–96. Gregory, P.J. (1994) Root growth and activity. In: Physiology and Determination of Crop Yield (eds. K.J. Boote, J.M. Bennett, T.R. Sinclair & G.M. Paulsen), pp. 65–93, American Society of Agronomy Inc., Madison, WI. Gregory, P.J. (2006a) Roots, rhizosphere and soil: the route to a better understanding of soil science? European Journal of Soil Science 57, 2–12. Gregory, P.J. (2006b) Plant Roots: Growth, Activity and Interaction with Soils. Blackwell Publishing, Oxford. Gregory, P.J. & Brown, S.C. (1989) Root growth, water use and yield of crops in dry environments: what characteristics are desirable? Aspects of Applied Biology 22, 235–243. Gregory, P.J., McGowan, M., Biscoe, P.V., & Hunter, B. (1978) Water relations of winter wheat. 1. Growth of the root system. Journal of Agricultural Science, Cambridge 91, 91–102.

Gregory, P.J., Crawford, D.V., & McGowan, M. (1979) Nutrient relations of winter wheat. 2. Movement of nutrients to the root and their uptake. Journal of Agricultural Science, Cambridge 93, 495–504. Gregory, P.J., Bengough, A.G., George, T.S., & Hallett, P.D. (2011) Rhizosphere engineering by plants: quantifying soil-root interactions. In: Enhancing Understanding and Quantiﬁcation of Soil-Root Growth Interactions (eds. C. Timlin & L. Ahuja), Advances in Modeling Agricultural Systems series. American Society of Agronomy, Madison, WI. In Press. Hamblin, A.P. & Hamblin, J. (1985) Root characteristics of some temperate legume species and varieties on deep, free-draining Entisols. Australian Journal of Agricultural Research 36, 63–72. Hammond, J.P., Broadley, M.R., et al. (2009) Shoot yield drives phosphorus use efﬁciency in Brassica oleracea and correlates with root architecture traits. Journal of Experimental Botany 60, 1953–1968. Hargreaves, C.E., Gregory, P.J., & Bengough, A.G. (2009) Measuring root traits in barley (Hordeum vulgare ssp. vulgare and ssp. spontaneum) seedlings using gel chambers, soil sacs and X-ray microtomography. Plant and Soil 316, 285–297. He, Z., Cade-Menun, B.J., Toor, G.S., Fortuna, A.-M., Honeycutt, C.W., & Sims, J.T. (2007) Comparison of phosphorus forms in wet and dried animal manures by solution phosphorus-31 nuclear magnetic resonance spectroscopy and enzymatic hydrolysis. Journal of Environmental Quality 36, 1086–1095. Henry, A., Chaves, N.F., Kleinman, P.J.A., & Lynch, J.P. (2010) Will nutrient-efﬁcient genotypes mine the soil? Effects of genetic differences in root architecture in common bean (Phaseolus vulgaris L.) on soil phosphorus depletion in a low-input agroecosystem in Central America. Field Crops Research 115, 67–78. Hinsinger, P. (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant and Soil 237, 173–195. Ho, M.D., McCannon, B.C., & Lynch, J.P. (2004) Optimization modeling of plant root architecture for water and phosphorus acquisition. Journal of Theoretical Biology 226, 331–340. Hodge, A. (2004) The plastic plant: root responses to heterogeneous supplies of nutrients. New Phytologist 162, 9–24. Hodge, A. (2009) Root decisions. Plant, Cell & Environment 32, 628–640. Hodge, A., Robinson, D., Grifﬁths, B.S., & Fitter, A.H. (1999) Why plants bother: root proliferation results in increased nitrogen capture from an organic patch

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

when two grasses compete. Plant, Cell & Environment 22, 811–820. Jones, D.L., Nguyen, C., & Finlay, R.D. (2009) Carbon ﬂow in the rhizosphere: carbon trading at the soilroot interface. Plant and Soil 321, 5–33. King, J., Gay, A., Sylvester-Bradley, R., et al. (2003) Modelling cereal root systems for water and nitrogen capture: towards an economic optimum. Annals of Botany 91, 383–390. Kirk, G.J.D., George, T., Courtois, B., & Senadhira, D. (1998) Opportunities to improve phosphorus efﬁciency and soil fertility in rainfed lowland and upland rice ecosystems. Field Crops Research 56, 73–92. Kirkegaard, J.A., So, H.B., & Troedson, R.J. (1992) The effect of soil strength on the growth of pigeonpea radicles and seedlings. Plant and Soil 140, 65–74. Kutschera, L. (1960) Wurzelatlas mitteleuropäischer Ackerunkräuter und Kulturpﬂanzen. DLG-VerlagsGMBH, Frankfurt, Germany. Ladha, J.K., Pathak, H., Krupnik, T.J., Six, J., & van Kessel, C. (2005) Efﬁciency of fertilizer nitrogen in cereal production: retrospects and prospects. Advances in Agronomy 87, 85–156. Lambers, H., Mougel, C., Jaillard, B., & Hinsinger, P. (2009) Plant-microbe-soil interactions in the rhizosphere: an evolutionary perspective. Plant and Soil 321, 83–115. Li, M., Osaki, M., Rao, I.M., & Tadano, T. (1997) Secretion of phytase from the roots of several plant species under phosphorus-deﬁcient conditions. Plant and Soil 195, 161–169. Liao, H., Yan, X., Rubio, G., Beebe, S.E., Blair, M.W., & Lynch, J.P. (2004) Genetic mapping of basal root gravitropism and phosphorus acquisition efﬁciency in common bean. Functional Plant Biology 31, 959–970. López-Bucio, J., Cruz-Ramírez, A., & Herrera-Estrella, L. (2003) The role of nutrient availability in regulating root architecture. Current Opinion in Plant Biology 6, 280–287. Lynch, J.P. (1995) Root architecture and plant productivity. Plant Physiology 109, 7–13. Lynch, J.P. & van Beem, J.J. (1993) Growth and architecture of seedling roots of common bean genotypes. Crop Science 33, 1253–1257. Ma, J.F., Ryan, P.R., & Delhaize, E. (2001) Aluminium tolerance in plants and the complexing role of organic acids. Trends in Plant Science 6, 273–278. Mallarino, A.P., Bordoli, J.M., & Borges, R. (1999) Phosphorus and potassium placement effects on early growth and nutrient uptake of no-till corn and relationships with grain yield. Agronomy Journal 91, 37–45.

43

Manschadi, A.M. (2008) Genotypic variation in seedling root architectural traits and implications for drought adaptation in wheat (Triticum aestivum L). Plant and Soil 303, 115–129. Merrill, S.D., Tanaka, D.L., & Hanson, J.D. (2002) Root length growth of eight crop species in Haplustoll soils. Soil Science Society of America Journal 66, 913–923. Nibau, C., Gibbs, D.J., & Coates, J.C. (2008) Branching out in new directions: the control of root architecture by lateral root formation. New Phytologist 179, 595–614. Ochoa, I.E., Blair, W., & Lynch, J.P. (2006) QTL analysis of adventitious root formation in common bean under contrasting phosphorus availability. Crop Science 46, 1609–1621. Osmont, K.S., Sibout, R., & Hardtke, C.S. (2007) Hidden branches: developments in root system architecture. Annual Review of Plant Biology 58, 93–113. Pagès, L., Vercambre, G., Drouet, J.-L., Lecompte, F., Collet, C., & Le Bot, J. (2004) Root Typ: a generic model to depict and analyse the root system architecture. Plant and Soil 258, 103–119. Pedersen, A., Zhang, K., Thorup-Kristensen, K., & Jensen, L.S. (2010) Modelling diverse root density dynamics and deep nitrogen uptake—a simple approach. Plant and Soil 326, 493–510. Péret, B., de Rybel, B., Casimiro, I., et al. (2009) Arabidopsis lateral root development: an emerging story. Trends in Plant Science 14, 399–408. Pierret, A., Doussan, C., Capowiez, Y., Bastardie, F., & Pagès, L. (2007) Root functional architecture: a framework for modeling the interplay between roots and soil. Vadose Zone Journal 6, 269–281. Prusinkiewicz, P. (2004) Modeling plant growth and development. Current Opinion in Plant Biology 7, 79–83. Read, D.B., Bengough, A.G., Gregory, P.J., et al. (2003) Plant roots release phospholipid surfactants that modify the physical and chemical properties of soil. New Phytologist 157, 315–326. Reed, R.C., Brady, S.R., & Muday, G.K. (1998) Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis. Plant Physiology 118, 1369–1378. Remans, T., Nacry, P., Pervent, M., et al. (2006) The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches. Proceedings of the National Academy of Science 103, 19206–19211. Richardson, A.E., Hadobas, P.A., & Hayes, J.E. (2000) Phosphomonoesterase and phytase activities of wheat (Triticum aestivum L.) roots and utilisation of organic phosphorus substrates by seedlings

44

NUTRIENT USE EFFICIENCY IN CROPS

grown in sterile culture. Plant, Cell & Environment 23, 397–405. Robinson, D. (1994) The responses of plants to nonuniform supplies of nutrients. New Phytologist 127, 635–674. Robinson, D. (1996) Resource capture by localized root proliferation: why do plants bother? Annals of Botany 77, 179–185. Robinson, D., Hodge, A., Grifﬁths, B.S., & Fitter, A.H. (1999) Plant root proliferation in nitrogen-rich patches confers competitive advantage. Proceedings of the Royal Society of London B266, 431–435. Rubio, G., Walk, T., Ge, Z., Yan, X., Liao, H., & Lynch, J.P. (2001) Root gravitropism and below-ground competition among neighbouring plants: a modelling approach. Annals of Botany 88, 929–940. Ryan, P.R., Delhaize, E., & Randall, P.J. (1995) Characterisation of Al-stimulated efﬂux of malate from the apices of Al-tolerant wheat roots. Planta 196, 103–111. Ryan, M.H. & Graham, J.H. (2002) Is there a role for arbuscular mycorrhizal fungi in production agriculture? Plant and Soil 244, 263–271. Ryan, M.H., Norton, R.M., Kirkegaard, J.A., McCormick, K.M., Knights, S.E., & Angus, J.F. (2002) Increasing mycorrhizal colonisation does not improve growth and nutrition of wheat on Vertosols in south-eastern Australia. Australian Journal of Agricultural Research 53, 1173–1181. Ryan, M.H., McCully, M.E., & Huang, C.X. (2003) Location and quantiﬁcation of phosphorus and other elements in fully hydrated, soil-grown arbuscular mycorrhizas: a cryo-analytical scanning electron microscopy study. New Phytologist 160, 429–441. Ryan, P.R., Dessaux, Y., Thomashow, L.S., & Weller, D.M. (2009a) Rhizosphere engineering and management for sustainable agriculture. Plant and Soil 321, 363–383. Ryan, P.R., Raman, H., Gupta, S., Horst, W.J., & Delhaize, E. (2009b) A second mechanism for aluminium resistance in wheat relies on the constitutive efﬂux of citrate from roots. Plant Physiology 149, 340–351. Sadras, V., Baldock, J., Roget, D., & Rodriguez, D. (2003) Measuring and modelling yield and water budget components of wheat crops in coarsetextured soils with chemical constraints. Field Crops Research 84, 241–260. Sander, D.H. & Eghball, B. (1999) Planting date and phosphorus fertilizer placement effects on winter wheat. Agronomy Journal 91, 707–712. Sasaki, T., Yamamoto, Y., Ezaki, B., et al. (2004) A wheat gene encoding an aluminium-activated malate transporter. Plant Journal 37, 645–653.

Schenk, H.J. & Jackson, R.B. (2002) The global biogeography of roots. Ecological Monographs 72, 311–328. Smit, A.L. & Groenwold, J. (2005) Root characteristics of selected ﬁeld crops: data from the Wageningen Rhizolab (1990–2002). Plant and Soil 272, 365–384. Smit, A.L., Groenwold, J., & Vos, J. (1994) The Wageningen Rhizolab—a facility to study soilroot-shoot-atmosphere interactions in crops. II. Methods of root observations. Plant and Soil 161, 289–298. Smith, S.E. & Read, D.J. (2008) Mycorrhizal Symbiosis, 3rd ed. Elsevier, Amsterdam. Smith, S.E., Smith, F.A., & Jakobsen, I. (2003) Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses. Plant Physiology 133, 16–20. Smith, S.E., Smith, F.A., & Jakobsen, I. (2004) Functional diversity in arbuscular mycorrhizal (AM) symbioses: the contribution of the mycorrhizal P uptake pathway is not correlated with mycorrhizal responses in growth or total P uptake. New Phytologist 162, 511–524. Swarup, R., Kramer, E.M., Perry, P., et al. (2005) Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal. Nature Cell Biology 7, 1057–1065. Taylor, H.M. & Ratliff, L.F. (1969) Root elongation rates of cotton and peanuts as a function of soil strength and soil water content. Soil Science 108, 113–119. Thorup-Kristensen, K., Cortasa, M.S., & Loges, R. (2009) Winter wheat roots grow twice as deep as spring wheat roots, is this important for N uptake and N leaching losses? Plant and Soil 322, 101–114. Tinker, P.B. & Nye, P.H. (2000) Solute Movement in the Rhizosphere. Oxford University Press, Oxford. Ubeda-Tomás, S., Swarup, R., Coates, J., et al. (2008) Root growth in Arabidopsis requires gibberellin/ DELLA signalling in the endodermis. Nature Cell Biology 10, 625–628. van Noordwijk, M., Lawson, G., Soumaré, A., Groot, J.J.R., & Hairiah, K. (1996) Root distribution of trees and crops: competition and/or complementarity. In: Tree-Crop Interactions: A Physiological Approach (eds. C.K. Ong & P. Huxley), pp. 319– 364, CAB International, Wallingford, UK. Vollsnes, A., Futsaether, C., & Bengough, A.G. (2010) Rhizosphere particle movement around mutant maize roots using timelapse imaging and particle image velocimetry. European Journal of Soil Science 61, 926–939. Walk, T.C., Jaramillo, R., & Lynch, J.P. (2006) Architectural tradeoffs between adventitious and

CROP ROOT SYSTEMS AND NUTRIENT UPTAKE FROM SOILS

basal roots for phosphorus acquisition. Plant and Soil 279, 347–366. Wang, J., Raman, H., Zhou, M., et al. (2007) Highresolution mapping of Alp, the aluminium tolerance locus in barley (Hordeum vulgare L.), identiﬁes a candidate gene controlling tolerance. Theoretical and Applied Genetics 115, 265–276. Watt, M., Silk, W.K., & Passioura, J.B. (2006) Rates of root and organism growth, soil conditions, and temporal and spatial development of the rhizosphere. Annals of Botany 97, 839–855. Welch, L.F., Mulvaney, D.L., Boone, L.V., McKibben, G.E., & Pendleton, J.W. (1966) Relative efﬁciency of broadcast versus band phosphorus for corn. Agronomy Journal 58, 283–287. Whalley, W.R., To, J., Kay, B., & Whitmore, A.P. (2007) Predicting the penetrometer resistance of soil. Geoderma 137, 370–377. Whitmore, A.P. & Whalley, W.R. (2009) Physical effects of soil drying on roots and crop growth. Journal of Experimental Botany 60, 2845–2857. Williamson, L.C., Ribrioux, S., Fitter, A.H., & Leyser, O. (2001) Phosphate availability regulates root system architecture in Arabidopsis. Plant Physiology 126, 875–882. Wissuwa, M. (2003) How do plants achieve tolerance to phosphorus deﬁciency? Small causes with big effects. Plant Physiology 133, 1947–1958.

45

Wissuwa, M. & Ae, N. (2001) Genotypic variation for tolerance to phosphorus deﬁciency in rice and the potential for its exploitation in rice improvement. Plant Breeding 120, 43–48. Wissuwa, M., Yano, M., & Ae, N. (1998) Mapping of QTLs for phosphorus-deﬁciency tolerance in rice (Oryza sativa L.). Theoretical and Applied Genetics 97, 777–783. Wojciechowski, T., Gooding, M.J., Ramsay, L., & Gregory, P.J. (2009) The effects of dwarﬁng genes on seedling root growth of wheat. Journal of Experimental Botany 60, 2565–2573. Young, I.M. (1998) Biophysical interactions at the rootsoil interface: a review. Journal of Agricultural Science, Cambridge 130, 1–7. Zhang, H.M. & Forde, B.G. (1998) An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279, 407–409. Zhang, H., Jennings, A., Barlow, P.W., & Forde, B.G. (1999) Dual pathways for regulation of root branching by nitrate. Proceedings of the National Academy of Science 96, 6529–6534.

Chapter 3

The Role of the Rhizosphere in Nutrient Use Efficiency in Crops Petra Marschner

Abstract The rhizosphere plays a pivotal role in nutrient use efficiency in crops because it is the interface between roots and the soil; nutrient availability in the rhizosphere determines plant uptake. Plants can alter nutrient availability in the rhizosphere directly by releasing nutrient-mobilizing compounds or changing the pH. Indirectly, plants affect nutrient availability by changing the activity and community composition of rhizosphere microorganisms. Rhizosphere microorganisms can increase nutrient availability by mobilizing nutrients and stimulating root growth; however, they can also decrease nutrient availability via decomposition of nutrient-mobilizing compounds released by the roots and immobilization of nutrients in the microbial biomass. Introduction Apart from their role as organs for anchorage in soil, soil exploration, and uptake of water and nutrients, plant roots can modify the physicochemical and biological condi-

tions in the surrounding soil that will affect nutrient availability and thus nutrient use efficiency. The German phytopathologist Lorenz Hiltner (1904) recognized that the interface between soil matrix, plant roots, and soil microorganisms, which directly or indirectly are influenced by the activity of plant roots, plays a critical role in plant growth, and called this interface the rhizosphere (Hiltner, 1904). The rhizosphere extends up to a few millimeters from the root surface. Root-induced physicochemical changes in the rhizosphere are major determinants for plant availability of nutrients in soils, either directly or via their effects on rhizosphere microorganisms. The changes induced by roots are influenced by a wide range of factors including plant genotype, nutritional status, rainfall, temperature, soil type, and root age (Neumann and Roemheld, 2000; Neumann, 2007). The availability of nutrients in the rhizosphere is controlled by the combined effects of soil properties, plant characteristics, and the interactions of plant roots with microorganisms and the surrounding soil (Bowen, 1991).

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 47

48

NUTRIENT USE EFFICIENCY IN CROPS

Physicochemical properties of the rhizosphere Soil structure in the rhizosphere The soil structure in the rhizosphere is usually more stable than the surrounding soil because high-molecular-weight exudates from active roots and microorganisms act as a glue that holds soil particles together. Additionally, root hairs and hyphae of mycorrhizal and saprophytic fungi enmesh soil particles and aggregates (Tisdall, 1991; Amellal et al., 1998; Andrade et al., 1998). The high-molecular-weight exudates represent an easily available nutrient source for microorganisms and are therefore rapidly decomposed. Thus, their effect is transient, being present only around living roots where exudation exceeds decomposition. On the other hand, fungal hyphae and roots can enhance structural stability even when they are dead. Greater structural stability can increase nutrient use efficiency by improved aeration and therefore oxygen supply for nutrient uptake by the roots and for microbial activity. Higher microbial activity can lead to increased nutrient transformations and availability.

Nutrient transport in the rhizosphere Plant roots take up inorganic nutrients as dissolved ions from the soil solution. Only a very small proportion of the nutrient demand of plants can be met by the amount of nutrients in the immediate vicinity of the roots. Less than 2% of plant’s requirement for major nutrients such as nitrogen, potassium, and phosphorus is met by interception (Barber, 1995). Hence, nutrient movement from the surrounding soil is essential for supplying sufficient nutrients to plants. Nutrient movement to the roots can occur via mass flow and diffusion. The nutrient concentration in the rhizosphere is deter-

mined by the ratio of nutrient movement to the roots and uptake of the nutrients by the roots. Nutrient accumulation is the result of nutrient movement to the roots exceeding nutrient uptake, whereas nutrient depletion occurs when the rate of movement to the roots is smaller than nutrient uptake. Mass flow is induced by the uptake of water by roots and the resulting gradient in water potential between rhizosphere and the surrounding, moister soil. It is the most important transport mechanism for nutrients with high concentrations in the soil solution such as nitrate. Up to 80% of the nitrogen demand of maize can be met by mass flow (Barber, 1995). For nutrients with low concentrations in the soil solution, mass flow provides only a small proportion of the plant demand; in maize, only 18% of potassium and 5% of phosphorus demand are supplied by mass flow (Barber, 1995). The concentrations of phosphorus and potassium in the soil solution are low because they react with soil surfaces and, in the case of phosphorus, form poorly soluble compounds with calcium, iron, or aluminum. Compared with transport by mass flow, uptake of these nutrients by the roots is high; thus, they are depleted around the roots; that is, their concentration in the rhizosphere is lower than in the bulk soil. The concentration gradient between the low concentration at the root surface and the higher concentration in the surrounding soil results in diffusion (Hendriks et al., 1981; Jungk and Claassen, 1986). However, diffusion of potassium and particularly phosphorus in soils is very slow, causing a widening of the depletion zone around the roots. In most soils, diffusion of phosphorus to the root surface is too slow to meet plant demand (Jungk and Claassen, 1986). To ensure adequate phosphorus uptake, plants have to increase the soil volume explored by growing long root hairs or new roots, or increase the

THE ROLE OF THE RHIZOSPHERE IN NUTRIENT USE EFFICIENCY IN CROPS

availability of phosphorus in the rhizosphere by solubilization or mineralization. Water availability in the rhizosphere If water uptake by the plants is greater than mass flow, that is, at high transpiration rates, water availability in the rhizosphere may be lower than in the surrounding soil, particularly in semi-arid or arid regions with hot and dry summers. This may not only limit water uptake by the plants but will also have negative effects on nutrient uptake, as mass flow is only possible in the presence of water and the rate of diffusion decreases when the water films around aggregates become thinner and the diffusion pathway becomes more tortuous (Barber, 1995). As mentioned above, roots and rhizosphere microorganisms produce polysaccharides; these can bind water and thereby slow the drying of soils (Amellal et al., 1998). However, these polysaccharides are unlikely to be important in climates where soils are dry for long periods. Some deep-rooted plants that are adapted to climates with long dry seasons can increase the water availability in the rhizosphere of roots in the dry top soil by hydraulic lift (Horton and Hart, 1998). In dry periods, the top soil dries out and plants rely on deeper roots for water uptake. However, the nutrient concentration in the deeper soil horizons is usually lower than in the top soil; therefore, these roots cannot supply the plants with sufficient nutrients. Nutrient uptake from the roots in the nutrient-richer top soil is limited because of the low soil water content. During the day, water taken up by deep roots is primarily transported to the shoots. However, during the night, when the stomata are closed, water taken up by the deep roots is transported to the roots in the top soil and released into the rhizosphere, termed hydraulic lift (Vetterlein and Marschner, 1993; Dawson, 1997; Espeleta

49

et al., 2004). The amount of water released into the rhizosphere is small but thought to be sufficient to allow diffusion of nutrients to the root surface and possibly also increase microbial activity. The higher water content in the rhizosphere is transient; as soon as the stomata open after dawn, water taken up by the roots is transported to the shoots because of their high transpiration rates. Nevertheless, hydraulic lift is an adaptation of plants in arid climates to increase nutrient use efficiency (Dawson, 1997; Wan et al., 2000). Redox potential in the rhizosphere Roots and rhizosphere microorganisms can change the redox potential in their immediate vicinity (Fischer et al., 1989; Alcantara et al., 1991). This can have strong effects on nutrient availability and thus nutrient use efficiency. Gas diffusion is very slow in water. Thus, in wet or submerged soils, oxygen consumed by roots and microorganisms is not replenished, leading to decreasing redox potential (Flessa and Fischer, 1992; Frenzel et al., 1999; Liesack et al., 2000). Under reducing conditions, iron and manganese availability is high and may reach toxic concentrations. Many plant species, particularly those adapted to submerged conditions such as low-land rice, can change root histology by forming aerenchyma in poorly aerated soils. Aerenchyma are a system of voids in the roots that transport oxygen from the shoots to the roots, some of which is released into the rhizosphere (Luxmore et al., 1970). This increases the redox potential and leads to the oxidation of iron and manganese on the root surface (Flessa and Fischer, 1992; Frenzel et al., 1999). Since oxidized iron and manganese are poorly soluble, this prevents uptake of toxic amounts of iron and manganese by the roots. Moreover, the increased oxygen supply in the rhizosphere also affects the activity of microorganisms and decomposition rates. The rate of aerobic

50

NUTRIENT USE EFFICIENCY IN CROPS

decomposition is greater than that of anaerobic decomposition (McGill, 2007). Thus, the release of oxygen by the roots allows aerobic rhizosphere microorganisms to remain active, which leads to greater nutrient availability. In aerobic soils, iron and manganese availability is low. Plants and microorganisms increase iron and manganese availability by increasing the plasmalemma-bound reductase activity (Uren, 1981; Roemheld and Marschner, 1983; Alcantara et al., 1991). Reduction of Fe3+ to Fe2+ or Mn4+ to Mn2+ increases their solubility and thus uptake by roots and microorganisms. However, this mechanism is only effective for interception of roots or microorganisms with Fe3+ or Mn3+ and is not sufficient to ensure adequate uptake of iron and manganese. Iron and manganese solubility is further increased by acidification of the rhizosphere and release of chelating compounds by roots and microorganisms such as organic acid anions, siderophores, or phenolics (for further details see below). Rhizosphere pH Soil pH strongly affects nutrient availability and therefore nutrient efficiency of plants. Plant roots can change the pH in the rhizosphere by release of CO2 and release or uptake of protons (Marschner, 1995). Respiration of roots and microorganisms can decrease the rhizosphere pH. Respired CO2 rapidly forms the weak acid H2CO3 (pK = 6.36). Thus, except for the most acidic soils, where H2CO3 remains undissociated, respiration will result in a pH decrease in the rhizosphere. Greater changes in the rhizosphere pH are induced by the balance between anion and cation uptake of the roots. Cells have to maintain charge balance in the cytoplasm; therefore, an excess of cations entering the root cells is counterbalanced by a net efflux

of protons and thus rhizosphere acidification, while alkalization occurs in case of influx of excess anions over cations (Roemheld, 1986; Marschner, 1995). Alkalization is the result of anion–proton cotransport into the cells or net release of OH–. This rule applies to all nutrients, but the form of nitrogen that is taken up by the roots, as ammonium (cation) or nitrate (anion) has the greatest effect on rhizosphere pH because nitrogen is taken up in large amounts by plants (Roemheld, 1986). Changes in rhizosphere pH have profound effects on nutrient availability. In neutral to alkaline soils, rhizosphere acidification can increase availability of phosphorus, iron, and other metals. Hence, rhizosphere acidification is an adaptive response of many plant species to both iron and phosphorus deficiency (Gardner and Parbery, 1982; Brancadoro et al., 1995; Neumann and Roemheld, 2000). Rhizosphere acidification as a result of ammonium supply leads to improved phosphorus acquisition by plants, compared with plants fed nitrate (Gahoonia and Nielsen, 1992; Gahoonia, 1993; Hinsinger and Gilkes, 1996). Acidification of acidic soils may also increase phosphorus availability by increasing the solubility of iron and aluminum, but this may also result in aluminum toxicity. On the other hand, an increased pH in the rhizosphere of plants growing in acidic soils could reduce aluminum toxicity. Changes in soil pH as a result of differential cation and anion uptake by microorganism are probably small due to the low nutrient uptake of individual cells compared with that of roots. Nevertheless, pH changes on the surface of biofilms that are formed on the surface of roots could affect nutrient availability to plants (Costerton et al., 1994; Briones et al., 2003). A greater effect of microorganisms on soil pH is due to nutrient transformations, namely nitrogen mineralization. Since microbial activity in the rhi-

THE ROLE OF THE RHIZOSPHERE IN NUTRIENT USE EFFICIENCY IN CROPS

zosphere is high, the rate of nitrogen mineralization is also increased compared with the bulk soil (Parkin et al., 2002; De Angelis et al., 2008). Ammonification (transformation of amino acids to ammonium) increases the pH, whereas nitrification decreases the pH (Xu et al., 2006). Thus, whereas ammonium uptake by roots decreases the pH, ammonification increases it; that is, pH changes induced by nitrogen mineralization may counteract pH changes that result from differential cation and anion uptake by roots. However, the fact that rhizosphere acidification with ammonium supply and alkalinization with nitrate supply can be shown in soil-grown plants (Roemheld, 1986) suggests that root-induced changes dominate the pH in the rhizosphere. It should be noted that a pH decrease in the rhizosphere with ammonium fertilization may be limited to conditions in which inorganic nitrogen is supplied as ammonium with a nitrification inhibitor because in many soils ammonium is rapidly transformed into nitrate. Carbon availability in the rhizosphere Rhizodeposition includes lysates of sloughed-off cells and tissues as a result of root turnover and root exudates released from intact root cells. Root exudates may be lost from cells by diffusion or actively released for nutrient mobilization, detoxification, plant–microbial signaling, and defense reactions (Neumann, 2007). Some exudates, such as amino acids, can be taken up again by the roots, thus minimizing nitrogen loss (Jones and Darrah, 1994a). In higher plants, a substantial proportion of carbon fixed during photosynthesis (20– 60%) can be translocated below ground (Grayston et al., 1996; Kuzyakov and Domanski, 2000; Neumann, 2007), corresponding to 800–4500 kg carbon ha−1 year−1

51

(Lynch and Whipps, 1990; Kuzyakov and Domanski, 2000). Depending on root activity, 15–60% of this carbon fraction is released as CO2 (Merbach et al., 1999). Root exudates may comprise 2–10% of the net fixed carbon in soil-grown plants (Jones et al., 2004; Neumann, 2007). The high concentration of easily degradable substrates from root exudates leads to a proliferation of microorganisms in the rhizosphere (Rouatt and Katznelson, 1961; Foster, 1986). Root exudation is greatest at the root tip (Marschner, 1995; Neumann, 2007), where the microbial density is low (Schoenwitz and Ziegler, 1989). With increasing distance from the root tip, exudation generally decreases, whereas microbial density increases. Thus, the region of greatest release of root exudates and the region of highest microbial population density are spatially separated. Clearly, roots have a strong effect on physical, chemical, and biological properties of the rhizosphere. However, in soil-grown plants, nutrient availability in the rhizosphere is not simply a function of rootinduced changes. The interactions between plants and microorganisms are pivotal for nutrient availability in the vicinity of the roots; hence, without detailed knowledge of these interactions, it will not be possible to understand the role of the rhizosphere in nutrient use efficiency. In the next section, rhizosphere processes and interactions for nitrogen, phosphorus, iron, and manganese will be outlined. Nutrient use efficiency and availability in the rhizosphere as the result of interactions between roots and microorganisms Nitrogen use efficiency Nitrogen fixation by legumes in association with Rhizobium plays an important role in

52

NUTRIENT USE EFFICIENCY IN CROPS

nitrogen uptake efficiency of legumes and other crops following legumes in rotation. However, this symbiosis will not be discussed here because Rhizobia are only transient members of the rhizosphere microbial community; their main activity occurs in the nodules (see Chapter 20). In nonlegumes, exudates can serve as an energy source for associative N2 fixers; for example, the density of free-living nitrogen fixers such as Azospirillum sp. is higher in the rhizosphere than in the bulk soil (Assmus et al., 1995). However, the rate of N2 fixation is low compared with symbiotic N2 fixation because associative bacteria have to compete with other rhizosphere microorganisms for exudates, whereas symbiotic N2 fixers within structures such as nodules are spatially separated from rhizosphere microorganisms, and directly supplied with photosynthates (Marschner, 1995). The relevance of associative N2 fixation for nitrogen nutrition of plants is unclear but may be important in nutrientpoor soils. A special case is endophytic N2 fixation in sugar cane by Azospirillum sp. The bacteria enter the roots of sugar cane and colonize the vascular system of roots and stems, where they are thought to fix substantial amounts of N2 (Boddey et al., 2003). Nitrogen mineralization, that is, the conversion of organic nitrogen into ammonium and nitrate, is microbially mediated. It is important for plant nitrogen supply even in fertilized soils because more than 90% of soil nitrogen is organic (Stevenson and Cole, 1999). Nitrogen mineralization is higher in the rhizosphere than in the bulk soil because of the release of exudates, which are easily decomposable substrates compared with native soil organic matter (Parkin et al., 2002). Denitrification rates may also be higher in the rhizosphere due to respiration by roots and microbial biomass, which creates anaerobic microsites in the rhizosphere (Qian et al., 1997).

Ammonia-oxidizing bacteria have been shown to form biofilms on the root surface of rice (Briones et al., 2003). These bacteria, being in intimate contact with roots, could play an important role in the nitrogen nutrition of plants as nitrate produced in the biofilms could be taken up directly by roots. However, microorganisms may also decrease nitrogen availability to plants by immobilization of nitrogen in the microbial biomass (Bakken, 1990; Corbeels et al., 2003; Moritsuka et al., 2004). Microorganisms have a low C : N ratio (<15) and thus require substantial amounts of nitrogen. The C : N ratio of plant material is higher, ranging from 20 in immature legume residues to more than 100 in mature wheat straw (Stevenson and Cole, 1999). Decomposition of high C/N residues leads to net nitrogen immobilization; that is, nitrogen uptake into the microbial biomass exceeds nitrogen mineralization (Corbeels et al., 2003). Immobilization of nutrients is transient: When carbon availability decreases, part of the microbial biomass dies, releasing nutrients. Nevertheless, net nitrogen immobilization can temporarily reduce nitrogen availability to plants (Bakken, 1990; Trinsoutrot et al., 2000). Root exudates are low in nitrogen since sugars represent the majority of root exudates and amino acids and other nitrogen-containing compounds make up only a small fraction (Neumann, 2007). Hence, net nitrogen immobilization may be dominating in root zones with high root exudation rates and high microbial growth rates. Thus, nitrogen use efficiency could be enhanced by attracting associative N2 fixers and possibly by releasing exudates that can be utilized only by them since this may increase their competitiveness in the rhizosphere. Endophytic colonization by N2 fixers such as Azospirillum would also increase nitrogen use efficiency. Regarding the use of nitrogen from the soil, nitrogen use efficiency could be enhanced by increasing

THE ROLE OF THE RHIZOSPHERE IN NUTRIENT USE EFFICIENCY IN CROPS

microbial activity and thus nitrogen mineralization. However, this would have to be accompanied by stimulating microbial biomass turnover to enhance release of immobilized nitrogen. Phosphorus use efficiency Although the total amount of phosphorus in the soil may be high, phosphorus is mainly present in forms that are unavailable to plants and microorganisms; with only a very small proportion being immediately available (Stevenson and Cole, 1999; Richardson, 2001). Differential phosphorus use efficiency can be due to differences in uptake and/or internal utilization (see Chapter 12). Here only phosphorus use efficiency related to uptake will be considered. Due to the poor mobility of phosphorus in soil compared with phosphorus uptake rates, available phosphorus is rapidly depleted around roots until the concentration of available phosphorus at the root surface reaches Cmin, that is, the minimum phosphorus concentration at which root phosphorus transporters can take up phosphorus into the cells (Hendriks et al., 1981; Jungk and Claassen, 1986). Plants may increase phosphorus uptake from the soil by altering root morphology or physiology. Morphological changes include increased root length, which allows plants to access a greater soil volume, that is, growing into soil not yet depleted of available phosphorus (Foehse et al., 1988). The volume of the depletion zone around roots can be increased by enhanced root hair growth (Hendriks et al., 1981). For example, clover varieties with longer root hairs grew better than varieties with short root hairs in soils with low phosphorus availability (Caradus, 1981). Hyphae of mycorrhizal fungi also grow beyond the depletion zone of the roots and increase the soil volume accessible to plants (Li et al., 1991).

53

Physiological changes such as release of protons, organic acid anions, or phenolics and phosphatase enzymes increase the availability of phosphorus in the rhizosphere. Proton release, that is, acidification, can increase solubility of calcium phosphates in alkaline soils and Fe/Al phosphates in acidic soils. Organic acid anions increase phosphorus availability by two mechanisms: anion (ligand) exchange and solubilization of iron and aluminum (Gerke and Hermann, 1992). Due to the high cytoplasmic pH, organic acids are released as anions (deprotonated); therefore, they do not decrease the soil pH (Jones, 1998). The organic acid anions compete with phosphate anions for binding sites, thus releasing phosphorus into the soil solution (Gerke, 1993, 1994). A large proportion of phosphorus in soils is bound to humic-Fe/Al complexes and to amorphous Fe/Al oxides (Gerke and Hermann, 1992). Organic acid anions form water-soluble complexes with iron and aluminum, thereby decreasing the free iron and aluminum ion concentration in the rhizosphere soil solution. This leads to increased solubilization of Fe3+ or Al3+ and thus release of phosphorus bound to Al/Fe oxides or phosphorus bound to clays and organic matter via Fe/Al bridges (Gerke et al., 2000). Phenolics released by roots can also increase phosphorus availability (Hu et al., 2005; Weisskopf et al., 2006). A large proportion of total soil phosphorus can be in an organic form, up to 80% in soils with high organic matter content, but only about 30% in agricultural soils in Australia (Richardson, 2001; Buenemann, person. comm.). Hence, release of phosphatase enzymes by roots and microorganisms that mineralize organic phosphorus can be an important mechanism for increasing phosphorus uptake by plants (Richardson, 2001). The effectiveness of these exudates in phosphorus mobilization depends on the pH

54

NUTRIENT USE EFFICIENCY IN CROPS

buffering capacity of the soil and decomposition rate and/or sorption of organic acid anions and phosphatases to soil particles (Jones, 1998; Jones and Brassington, 1998; George et al., 2007). Cluster roots of Proteaceae and white lupin are considered prime examples for efficient phosphorus mobilization in the rhizosphere and thus phosphorus use efficiency. Cluster root density increases with decreasing phosphorus availability (Neumann et al., 2000). Cluster roots are characterized by a large number of closely spaced, tertiary lateral rootlets with limited growth (3–5 mmlong, 50–1000 rootlets cm−1 root axis) which are densely covered with root hairs (Watt and Evans, 1999a,b). They are formed within a few days, then remain active for 4–5 days before activity decreases and they senesce (Watt and Evans, 1999b). During their active phase, cluster roots increase phosphorus availability by a number of strategies: high organic acid anion exudation rates, particularly citrate (Neumann et al., 2000; Kania et al., 2003), increased activity of plasma membrane H+-ATPase for proton extrusion (Neumann et al., 1999; Kania et al., 2003), increased production and exudation of phenolics (Dinkelaker et al., 1995; Neumann et al., 1999; Weisskopf et al., 2006), and increased release of acid phosphatases (Gilbert et al., 1999; Neumann et al., 2000). Interestingly, the release of phenolics can also inhibit microbial activity, that is, decomposition of phosphorusmobilizing exudates, rendering root-released phosphorus-mobilizing compounds more effective (Weisskopf et al., 2006). Rhizosphere microorganisms can enhance or decrease plant phosphorus availability (Marschner, 2008). Plant phosphorus uptake can be increased by (1) phosphorus solubilization and mineralization (Banik and Dey, 1983; Kumar and Narula, 1999; Whitelaw et al., 1999) and (2) enhanced root growth or mycorrhizal colonization (Azcon-Aguilar

et al., 1986; Martin et al., 1989; Frey-Klett et al., 1997). Availability of phosphorus to plants is decreased by (1) immobilization of phosphorus in the microbial biomass (McLaughlin et al., 1988; Oberson et al., 2001), (2) decomposition of phosphorusmobilizing compounds released by the roots (Jones and Darrah, 1994b; Jones, 1998), and (3) inhibition of root growth or mycorrhizal colonization (Garret, 1948; Skou, 1981; Duponnois and Garbaye, 1991). The importance of rhizosphere microorganisms for phosphorus uptake may vary with plant species. In a comparison of Poaceae species with differential phosphorus efficiencies, microbial biomass phosphorus in the rhizosphere was positively correlated with plant phosphorus uptake, indicating that phosphorus stored in the microbial biomass can act as a slow release phosphorus source for phosphorus-efficient grasses (Marschner et al., 2006). On the other hand, microbial biomass phosphorus in the rhizosphere was not correlated with plant phosphorus uptake in brassicas with differential phosphorus efficiencies grown in the same soil as the Poaceae (Marschner et al., 2007). In the brassicas, plant phosphorus uptake was positively correlated with root length and available phosphorus in the rhizosphere, suggesting that plant properties such as the ability to produce an extensive root system and phosphorus mobilization in the rhizosphere were the key factors in phosphorus acquisition. In both Poaceae and brassicas, the microbial community composition in the rhizosphere did not differ consistently between phosphorus-efficient and phosphorus-inefficient cultivars. Hence, microbial community composition does not seem to play a role in phosphorus efficiency of Poaceae and brassicas. This would need to be confirmed in further studies; however, the ease at which microorganisms that are able to mobilize phosphorus can be isolated from soil suggests that this capacity is wide-

THE ROLE OF THE RHIZOSPHERE IN NUTRIENT USE EFFICIENCY IN CROPS

spread (Reyes et al., 1999; Marschner et al., 2002). Therefore, changes in microbial community composition may not affect the ability of the community to mobilize phosphorus. Hence, with regard to the rhizosphere, phosphorus efficiency in plants would be due to increased soil volume exploited (extensive root system, longer root hairs, mycorrhizal colonization) and/or changes in phosphorus availability in the rhizosphere by (1) pH change, (2) release of phosphorusmobilizing compounds (organic acid anions, phenolics, phosphatases), (3) inhibition of microbial decomposition of root-derived phosphorus-mobilizing compounds, and/or (4) increased microbial mobilization of phosphorus accompanied by a high turnover of the microbial biomass. Since the capacity to mobilize phosphorus is widespread among soil microorganisms, the potential for increasing phosphorus uptake efficiency by inoculating plants with phosphorus solubilizers would seem rather limited, except in soils where the native microflora has been severely reduced, for example, by fumigation. Iron use efficiency The total iron content in soil typically ranges from 2% to 4%, but in aerated soils its availability is limited by the very low solubility of Fe(III) hydrolysis species and the slow dissolution of iron minerals. Plants have two different strategies for responding to iron deficiency (Marschner, 1995). Strategy I plants (dicotyledons and nongraminaceous monocotyledons) release protons and organic acid anions to increase iron availability. Release of protons mobilizes iron because the solubility of Fe(III) salts increases with decreasing pH. Organic acid anions can complex Fe3+, and these soluble complexes can diffuse to the root surface. Iron solubility is also increased by increased reducing capacity of the roots, converting

55

the poorly soluble Fe3+ into more soluble Fe2+. The reductase on the cell surface will also release iron from Fe3+ chelators. Other responses to iron deficiency in Strategy I plants can include changes in root morphology and histology, for example, root tip swelling, increased root branching, more root hairs, and rhizodermal transfer cell formation (Marschner, 1995). In contrast to dicotyledonous plants, Strategy II plants (Poaceae) release phytosiderophores, which are nonproteinogenic amino acid derivatives such as mugineic acid (Takagi, 1976; Marschner, 1995), mainly released at the root tip for a few hours after the onset of light (Neumann and Roemheld, 2000). These chelates preferentially bind iron but can also bind zinc or copper. Iron is taken up in the chelated form as iron– phytosiderophore (Roemheld, 1991; Von Wiren et al., 1993). Under severe iron deficiency, phytosiderophores may represent 50–90% of the exudates released at the root tip (Fan et al., 1997). Under iron deficiency stress, microorganisms also release organic acid anions or siderophores that chelate Fe3+. The chelated Fe3+ is reduced either outside the cell envelope or within the cell (Neilands, 1984). Bacterial siderophores are usually poor sources of iron for monocotyledonous and dicotyledonous plants (Bar-Ness et al., 1992; Crowley et al., 1992; Walter et al., 1994). In some cases, microbial siderophores have alleviated iron deficiency-induced chlorosis in dicotyledons, but only at high siderophore concentrations in hydroponics (Jurkevitch et al., 1988; Wang et al., 1993; Yehuda et al., 2000; Sharma et al., 2003). Although the localized production of siderophores at microsites of high microbial activity could result in high concentrations of siderophores in the rhizosphere, their contribution to plant iron nutrition is unclear. The interactions between different iron chelators, and thus the competition between

56

NUTRIENT USE EFFICIENCY IN CROPS

organisms for iron, depend on the affinity of the chelators toward iron and their relative concentrations. Compared with phytosiderophores, bacterial siderophores such as pyoverdine have a much higher affinity toward iron (Yehuda et al., 1996). Thus, if siderophores and phytosiderophores are present at similar concentrations, iron is preferentially bound to the microbial siderophores, which may even remove iron from the iron–phytosiderophore complex. However, rhizoferrin from the fungus Rhizopus arrhizus has only a slightly higher affinity toward iron than phytosiderophores and has been shown to be a good source of iron for barley, probably because of the exchange of iron from rhizoferrin to the phytosiderophore (Yehuda et al., 1996). Microorganisms can further affect iron uptake by plants by rapidly decomposing organic acid anions and phytosiderophores (Von Wiren et al., 1995; Jones, 1998), using them as energy and nutrient source. Moreover, plant-derived iron–phytosiderophore or iron– citrate complexes appear to be a good source of iron for bacteria, depressing the release of their own siderophores to take advantage of the iron that has been mobilized by the plant chelators (Marschner and Crowley, 1998). Thus, rhizosphere microorganisms may negatively affect iron uptake by roots because they (1) can use iron bound to plant-derived chelators, (2) decompose plant-derived chelators, and (3) produce chelators with a higher affinity for iron than plant chelators. However, the distribution of iron among different chelators is dependent not only on the affinity of the chelators toward iron, but also on their relative concentrations (Yehuda et al., 1996). The diurnal rhythm of phytosiderophore release by grasses (Neumann and Roemheld, 2000) results in a high concentration of phytosiderophores just behind the root tip at certain times of the day, such that phytosiderophores may even remove iron from bacterial siderophores (Crowley and

Gries, 1994), particularly because the density of microorganisms at the root tip is low (Darrah, 1991). Even if a proportion of the phytosiderophores is decomposed by microorganisms (Von Wiren et al., 1995), the concentration remaining is likely to be sufficient to mobilize sufficient amounts of iron. Thus, rhizosphere properties that could increase iron efficiency include increased iron reduction capacity of root cells, release of iron-mobilizing compounds (phytosiderophores, organic acid anions), particularly immediately behind the root tip, and/or stimulation of release of microbial siderophores. The latter could directly increase iron availability to plants if the microbial siderophores could be utilized by the plants. It could also increase iron availability indirectly by reducing the need of microorganisms to utilize iron chelated by plant-derived compounds. Manganese use efficiency Millions of hectares of arable land worldwide are deficient in manganese (Welch, 1995). Only the reduced form of manganese (Mn2+) is available to plants, while its oxidized form (Mn4+) is unavailable. Hence, reduction increases manganese availability, whereas oxidation decreases it. Oxidation is biological, whereas reduction can be either biological or chemical (Ghiorse, 1988). Crop plant genotypes differ in sensitivity toward manganese deficiency, and rhizosphere microorganisms may play an important role in these genotypic differences (Rengel, 1997; Marschner et al., 2003). Under manganese-deficient conditions, the density of manganese reducers is higher in the rhizosphere of manganese-efficient than manganese-inefficient wheat (Rengel et al., 1996) and oat genotypes (Timonin, 1946). Interestingly, the Take-All fungus Gaeumannomyces graminis var. tritici (Ggt) is a strong manganese oxidizer and thus decreases manganese availability. The fungus

THE ROLE OF THE RHIZOSPHERE IN NUTRIENT USE EFFICIENCY IN CROPS

is inhibited by high manganese availability (Wilhelm et al., 1990); hence, the growth of Ggt can be decreased by manganese reducers (Marschner et al., 1991). Differential manganese efficiency in cereals may also affect their susceptibility to Ggt. Increasing manganese fertilization decreases Take-All infection in wheat, which has been explained by two factors (Rengel et al., 1993): (1) increased defense reaction by roots to Take-All infection, and (2) inhibition of the growth of the Take-All fungus by high manganese concentrations in soil. The increased defense reaction in plants that have adequate manganese status is related to the role of manganese in the synthesis of phenolic compounds and lignin, which form a mechanical barrier in roots, preventing penetration by the pathogen into cells, thereby inhibiting or limiting infection. Manganese-deficient roots have lower lignin contents and are more susceptible to Ggt (Wilhelm et al., 1990). Hence, manganese-efficient plants would have a high capacity to reduce manganese at the root surface, stimulate manganese reducers, and inhibit manganese oxidizers in the rhizosphere. This would not only increase manganese uptake but also decrease the susceptibility to root pathogens. Nutrient uptake and root zone In the above discussion, the rhizosphere was considered as a whole. However, root physiology and rhizosphere properties vary along the root axis (Marschner et al., 2011). The zone immediately behind the root tip is the site of highest root exudation (Neumann and Roemheld, 2000; Neumann, 2007) and nutrient uptake (Haeussling et al., 1988; Colmer and Bloom, 1998; Fang et al., 2007), whereas microbial density is low (Schoenwitz and Ziegler, 1989). With increasing distance from the tip, exudate release and nutrient uptake rates decrease while microbial density increases. Root exudation and nutri-

57

ent uptake may show a second peak in the zone of lateral root emergence because lateral roots break through the Casparian strip that surrounds the stele, but this peak is lower than that just behind the root tip. These differences in exudation, nutrient uptake capacity, and microbial density along the root axis may be important for nutrient use efficiency. Immediately behind the root tip, where exudation rates are high and microbial density in the rhizosphere is relatively low, root exudates can mobilize nutrients without strong competition from microorganisms or substantial decomposition of exudates by microorganisms. The high rate of exudation just behind the root tip stimulates the growth of rhizosphere microorganisms in the proximal elongation zone and the root hair zone, accompanied by strong nutrient mobilization. However, most of the mobilized nutrients will be taken up by microorganisms, resulting in net immobilization in the microbial biomass. Behind the root hair zone, where root exudation is lower, nutrient mobilization may equal immobilization and some of the nutrients mobilized by root exudates can be taken up by the plant. Moreover, plants may inhibit microbial activity and thus competitiveness by decreasing the rhizosphere pH or releasing phenolic compounds (Weisskopf et al., 2006). In the mature root zones where cortex cells senesce, the lack of easily decomposable carbon sources results in lower microbial growth rates and thus lower nutrient demand as well as death of microorganisms. Additionally, the turnover of the microbial biomass in the mature root zones is enhanced due to predation by nematodes and protozoa (Moore et al., 2003; Bonkovski, 2004). Hence, nutrients from the microbial biomass are likely to become available to the plant. However, plant uptake of the nutrients released from the microbial biomass may be relatively small because of the low capacity of root tissues in mature root zones to

58

NUTRIENT USE EFFICIENCY IN CROPS

take up nutrients (Haeussling et al., 1988; Colmer and Bloom, 1998; Fang et al., 2007). According to this model, nutrientefficient plants would be characterized by (1) high release rates of nutrient-mobilizing exudates immediately behind the root tip, (2) moderate to high exudate release between the root tip and the zone of lateral root emergence to enhance mobilization of nutrients in the rhizosphere but also immobilization in the microbial biomass, (3) low rates of exudation in the mature root zones to enhance nutrient release from the biomass coupled with high nutrient uptake capacity of the mature root zones to enable uptake of the released nutrients. It should be noted that this model remains to be verified. Modern techniques such as reporter genes and microarrays to measure microbial activity and nutrient uptake combined with measurement of expression of genes involved in nutrient uptake by the roots will help test the model. Such measurements could be accompanied by tracer studies with frequent sampling as well as nanoscale secondary ion mass spectroscopy (nano-SIMS) (Herrmann et al., 2007) to determine the nutrient flux of between root cells and rhizosphere microorganisms (Marschner et al., 2011). Table 3.1.

Conclusion Chemical, physical, and biological processes in the rhizosphere play a key role in nutrient uptake by plants. Plants change the conditions in the rhizosphere by taking up water and nutrients, releasing exudates as well as by affecting the growth and activity of microorganisms (Table 3.1). Nutrient efficiency studies often include measurements of root growth and root physiology but rarely take into account the role of rhizosphere microorganisms or the differential conditions along the root axis. This overview suggests that rhizosphere microorganisms are highly competitive compared with plants and could therefore reduce nutrient uptake by roots. Rhizosphere microorganisms decompose nutrient-mobilizing root exudates, immobilize nutrients in the microbial biomass, and may decrease root growth. On the other hand, microorganisms are pivotal for mineralization of nutrients, N2 fixation, and some may stimulate root growth (Table 3.2). The proposed model suggests that the interactions between roots and rhizosphere microrganisms may vary along the root axis. Clearly, for a more comprehensive understanding of the rhizosphere in relation to nutrient efficiency, microscale studies in different root zones are required. With the

Plant induced changes in the rhizosphere that may increase nutrient availability

Plant-Induced Change

Effect on Nutrient Availability

Root hair growth

Increased extent of phosphorus and potassium depletion zone around roots Increased nutrient movement to roots via diffusion and mass flow, increased microbial mineralization/solubilization Improved soil structural stability → increased aeration, water transport Increased availability of phosphorus and metals Increased availability of phosphorus and metals Increased availability of iron Increased microbial mineralization/solubilization

Release of water (hydraulic lift) Release of polysaccharides pH change Release of organic acid anions Release of phytosiderophores Release of sugars and other easily available compounds Release of signaling compounds

Attraction of beneficial microorganisms (mycorrhizal fungi, N2 fixers), repellence of pathogens or deleterious microorganisms

THE ROLE OF THE RHIZOSPHERE IN NUTRIENT USE EFFICIENCY IN CROPS

Table 3.2.

59

Effects of rhizosphere microorganisms on nutrient availability to plants

Increase of Nutrient Availability

Decrease of Nutrient Availability

Nutrient mineralization Release of polysaccharides

Decomposition of nutrient-mobilizing root exudates Ligand exchange to microbial complexes with poor availability to plants Utilization of nutrients mobilized by root exudates and nutrient immobilization in the microbial biomass (competition) Inhibition of root growth/mycorrhizal colonization

Release of organic acid anions

Release of siderophores Release of growth stimulators (roots, mycorrhizal fungi) pH change Nutrient release from microbial biomass

advent of molecular techniques such as in situ fluorescent labeling, reporter gene expression, and nano-SIMS, such studies will be possible and may generate novel insights that can be used for breeding nutrient-efficient crops. References Alcantara, E., de la Guardia, M.D., & Romera, F.J. (1991) Plasmalemma redox activity and H+ extrusion in roots of Fe-deficient cucumber plants. Plant Physiology 96, 1034–1037. Amellal, N., Burtin, G., Bartoli, F., et al. (1998) Colonization of wheat roots by an exopolysaccharideproducing Pantoea agglomerans strain and its effect on rhizosphere soil aggregation. Applied and Environmental Microbiology 64, 3740–3747. Andrade, G., Mihara, K.L., Linderman, R.G., et al. (1998) Soil aggregation status and rhizobacteria in the mycorrhizosphere. Plant and Soil 202, 89–96. Assmus, B., Hutzler, P., Kirchhof, G., et al. (1995) In situ localization of Azospirillum brasiliense in the rhizosphere of wheat with fluorescently labeled, r-RNA-targeted oligonucleotide probes and scanning confocal laser microscopy. Applied and Environmental Microbiology 61, 1013–1019. Azcon-Aguilar, C., Diaz, R.R.M., & Barea, J.M. (1986) Effect of soil micro-organisms on spore germination and growth of the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. Transactions of the British Mycological Society 86, 337–340. Bakken, L.R. (1990) Microbial growth and immobilization/mineralization of N in the rhizosphere. Symbiosis 9, 37–41. Banik, S. & Dey, B.K. (1983) Phosphate-solubilizing potentiality of the microorganisms capable of utiliz-

ing al phosphate as a sole phosphorus source. Zentralblatt fuer Mikrobiologie 138, 17–23. Barber, S.A. (1995) Soil Nutrient Bioavailability : A Mechanistic Approach. Wiley, New York. Bar-Ness, E., Hadar, Y., Chen, Y., et al. (1992) Shortterm effects of rhizosphere microorganisms on Fe uptake from microbial siderophores by maize and oat. Plant Physiology 100, 451–456. Boddey, R.M., Uruquiaga, S., Alves, B.J.R., et al. (2003) Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant and Soil 252, 139–149. Bonkovski, M. (2004) Protozoa and plant growth: the microbial loop in soil revisited. New Phytologist 162, 617–631. Bowen, G.D. (1991) Microbial dynamics in the rhizosphere : possible strategies in managing rhizosphere populations. In: The Rhizosphere and Plant Growth (eds. D.L. Kleister & P.B. Cregan), pp. 25–32, Kluwer Academic Publishers, Dordrecht. Brancadoro, L., Rabotti, G., Scienza, A., et al. (1995) Mechanisms of Fe-efficiency in roots of Vitis spp. in response to iron deficiency stress. Plant and Soil 171, 229–234. Briones, A.M., Okabe, S., Umemiya, Y., et al. (2003) Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars. Plant and Soil 250, 335–248. Caradus, J.R. (1981) Effect of root hair length on white clover growth over a range of soil P levels. New Zealand Journal of Agricultural Research 24, 353–358. Colmer, T.D. & Bloom, A.J. (1998) A comparison of NH4+ and NO3− net fluxes along roots of rice and maize. Plant, Cell & Environment 21, 240–246. Corbeels, M., O’Connell, A.M., Mendham, D.S., et al. (2003) Nitrogen release from eucalyptus leaves and

60

NUTRIENT USE EFFICIENCY IN CROPS

legume residues as influenced by their biochemical quality and degree of contact with soil. Plant and Soil 250, 15–28. Costerton, J.W., Lewandowski, Z., De Beer, D., et al. (1994) Biofilms, the customized microniche. Journal of Bacteriology 176, 2137–2142. Crowley, D.E. & Gries, D. (1994) Modeling of iron availability in the plant rhizosphere. In: Biochemistry of Metal Micronutrients in the Rhizosphere (eds. J.A. Manthey, D.E. Crowley & D.G. Luster), pp. 199–224, Lewis Publishers, Boca Raton, FL. Crowley, D.E., Roemheld, V., Marschner, H., et al. (1992) Root-microbial effects on plant iron uptake from siderophores and phytosiderophores. Plant and Soil 142, 1–7. Darrah, P.R. (1991) Models of the rhizosphere. I. Microbial population dynamics around a root releasing soluble and insoluble carbon. Plant and Soil 133, 187–199. Dawson, T.E. (1997) Water loss from tree roots influences soil water and nutrient status and plant performance. In: Radical Biology: Advances and Perspectives on the Function of Plant Roots (eds. H.E. Flores, J.M. Lynch & D.M. Eissenstat), pp. 235–250, American Society of Plant Physiology Press, Rockville, MD. De Angelis, K.M., Lindow, S.E., & Firestone, M.K. (2008) Bacterial quorum sensing and nitrogen cycling in rhizosphere soil. FEMS Microbial Ecology 66, 197–207. Dinkelaker, B., Hengeler, C., & Marschner, H. (1995) Distribution and function of proteoid roots and other root clusters. Botanica Acta 108, 183–200. Duponnois, R. & Garbaye, J. (1991) Mycorrhization helper bacteria associated with the Douglas firLaccaria laccata symbiosis: effects in aseptic and in glasshouse conditions. Annales des Sciences Forestières 48, 239–251. Espeleta, J.F., West, J.B., & Donovan, L.A. (2004) Species-specific patterns of hydraulic lift in cooccurring adult trees and grasses in a sandhill community. Oecologia 138, 341–349. Fan, T.W.M., Lane, A.N., Pedler, J., et al. (1997) Comprehensive analysis of organic ligands in whole root exudates using nuclear magnetic resonance and gas chromatography-mass spectroscopy. Analytical Biochemistry 251, 57–68. Fang, Y., Babourina, O., Rengel, Z., et al. (2007) Spatial distribution of ammonium and nitrate fluxes along roots of wetland plants. Plant Science 173, 240–246. Fischer, W.R., Flessa, H., & Schaller, G. (1989) pH values and redox potentials in microsites of the rhizosphere. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde 152, 191–195.

Flessa, H. & Fischer, W.R. (1992) Plant-induced changes in the redox potentials of rice rhizospheres. Plant and Soil 143, 55–60. Foehse, D., Claassen, N., & Jungk, A. (1988) Phosphorus efficiency in plants. I. External and internal P requirement and P uptake efficiency of different plant species. Plant and Soil 110, 101–109. Foster, R.C. (1986) The ultrastructure of the rhizoplane and rhizosphere. Annual Review of Phytopathology 24, 211–234. Frenzel, P., Bosse, U., & Janssen, P.H. (1999) Rice roots and methanogenesis in a paddy soil: ferric iron as an alternative electron acceptor in the rooted soil. Soil Biology & Biochemistry 31, 421–430. Frey-Klett, P., Pierrat, J.C., & Garbaye, J. (1997) Location and survival of mycorrhiza helper Pseudomonas fluorescens during establishment of ectomycorrhizal symbiosis between Laccaria bicolor and Douglas fir. Applied and Environmental Microbiology 63, 139–144. Gahoonia, T.S. (1993) Influence of root-induced pH on the solubility of soil al in the rhizosphere. Plant and Soil 149, 289–291. Gahoonia, T.S. & Nielsen, N.E. (1992) Control of pH at the soil-root interface. Plant and Soil 140, 49–54. Gardner, W.K. & Parbery, D.G. (1982) The acquisition of phosphorus by Lupinus albus L. I. Some characteristics of the soil/root interface. Plant and Soil 68, 19–32. Garret, S.D. (1948) Soil conditions and the take-all disease of wheat. Interaction between host plant nutrition, disease escape and disease resistance. The Annals of Applied Biology 35, 14–17. George, T.S., Simpson, R.J., Gregory, P.J., et al. (2007) Differential interaction of Aspergillus niger and Peniophora lycii phytases with soil particles affects the hydrolysis of inositol phosphates. Soil Biology and Biochemistry 39, 793–803. Gerke, J. (1993) Solubilization of Fe (III) from humicFe complexes, humic/Fe- oxide mixtures and from poorly ordered Fe-oxide by organic acids— consequences for P adsorption. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde 156, 253–257. Gerke, J. (1994) Kinetics of soil phosphate desorption as affected by citric acid. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde 157, 17–22. Gerke, J. & Hermann, R. (1992) Adsorption of orthophosphate to humic-Fe-complexes and to amorphous Fe-oxide. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde 155, 233–236. Gerke, J., Beissner, L., & Roemer, W. (2000) The quantitative effect of chemical phosphate mobilization

THE ROLE OF THE RHIZOSPHERE IN NUTRIENT USE EFFICIENCY IN CROPS

by carboxylate anions on P uptake by a single root. I. The basic concept and determination of soil parameters. Journal of Plant Nutrition and Soil Science 163, 207–212. Ghiorse, W.C. (1988) The biology of manganese transforming microorganisms in soils. In: Manganese in Soils and Plants (ed. R.D. Graham), pp. 75–85, Kluwer Academic Publishers, Dordrecht. Gilbert, G.A., Knight, J.D., Vance, C.P., et al. (1999) Acid phosphatase activity in phosphorus-deficient white lupin roots. Plant, Cell & Environment 22, 801–810. Grayston, S.J., Vaughan, D., & Jones, D. (1996) Rhizosphere carbon flow in trees, in comparison with annual plants: the importance of root exudation and its impact on microbial activity and nutrient availability. Applied Soil Ecology 5, 29–56. Haeussling, M., Jorns, C.A., Lehmbecker, G., et al. (1988) Ion and water uptake in relation to root development in Norway spruce (Picea abies (L.) Karst). Journal of Plant Physiology 133, 486–491. Hendriks, L., Claassen, N., & Jungk, A. (1981) Phosphatverarmung des wurzelnahen Bodens und P-Aufnahme von Mais und Raps. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde 144, 486–499. Herrmann, A.M., Ritz, K., Nunan, N., et al. (2007) Nano-scale secondary ion mass spectrometry—a new analytical tool in biogeochemistry and soil ecology: a review article. Soil Biology and Biochemistry 39, 1835–1850. Hiltner, L. (1904) Über neuere Erfahrungen und Probleme der Bodenbakteriologie unter besonderer Berücksichtigung der Gründüngung und Brache. Arbeiten der Deutschen Landwirtschaftlichen Gesellschaft 98, 59–78. Hinsinger, P. & Gilkes, R.J. (1996) Mobilization of phosphate from phosphate rock and alumina-sorbed phosphate by the roots of ryegrass and clover as related to rhizosphere pH. European Journal of Soil Science 47, 533–544. Horton, J.L. & Hart, S.C. (1998) Hydraulic lift: a potentially important ecosystem process. Tree 13, 232–235. Hu, H., Tang, C., & Rengel, Z. (2005) Role of phenolics and organic acids in phosphorus mobilization in calcareous and acidic soils. Journal of Plant Nutrition 28, 1427–1439. Jones, D.L. (1998) Organic acids in the rhizosphere—a critical review. Plant and Soil 205, 25–44. Jones, D.L. & Brassington, D.S. (1998) Sorption of organic acids in acid soils and its implications in the rhizosphere. European Journal of Soil Science 49, 447–455.

61

Jones, D.L. & Darrah, P.R. (1994a) Amino-acid influx at the soil-root interface of Zea mays L. and its implications in the rhizosphere. Plant and Soil 163, 1–12. Jones, D.L. & Darrah, P.R. (1994b) Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant and Soil 166, 247–257. Jones, D.L., Hodge, A., & Kuzyakov, Y. (2004) Plant and mycorrhizal regulation of rhizodeposition. New Phytologist 163, 459–480. Jungk, A. & Claassen, N. (1986) Availability of phosphate and potassium as the result of interactions between root and soil in the rhizosphere. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde 149, 411–427. Jurkevitch, E., Hadar, Y., & Chen, Y. (1988) Involvement of bacterial siderophores in the remedy of limeinduced chlorosis in peanut. Soil Science Society of America Journal 52, 1032–1037. Kania, A., Langlade, N., Martinoia, E., et al. (2003) Phosphorus deficiency-induced modifications in citrate catabolism and in cytosolic pH as related to citrate exudation in cluster roots. Plant and Soil 248, 117–127. Kumar, V. & Narula, N. (1999) Solubilization of inorganic phosphates and growth emergance of wheat as affected by Azotobacter chroococcum mutants. Biology and Fertility of Soils 28, 301–305. Kuzyakov, Y. & Domanski, G. (2000) Carbon input by plants into soils. A review. Journal of Plant Nutrition and Soil Science 163, 421–431. Li, X.L., George, E., & Marschner, H. (1991) Extension of the phosphorus depletion zone in VA-mycorrhizal white clover in a calcareous soil. Plant and Soil 136, 41–48. Liesack, W., Schnell, S., & Revsbech, N.P. (2000) Microbiology of flooded rice paddies. FEMS Microbiology Reviews 24, 625–645. Luxmore, R.J., Stolzy, L.H., & Letey, J. (1970) Oxygen diffusion in the soil plant system. Agronomy Journal 62, 322–332. Lynch, J.M. & Whipps, J.M. (1990) Substrate flow in the rhizosphere. Plant and Soil 129, 1–10. Marschner, H. (1995) Mineral Nutrition of Higher Plants. Academic Press, London. Marschner, P. (2008) The role of rhizosphere microorganisms in relation to P uptake by plants. In: The Ecophysiology of Plant-Phosphorus Interactions (eds. P.J. White & J.P. Hammond), pp. 165–176, Springer, Heidelberg. Marschner, P., Ascher, J.S., & Graham, R.D. (1991) Effect of manganese-reducing rhizosphere bacteria on the growth of Gaeumannomyces graminis var. tritici and on manganese uptake by wheat (Triticum aestivum L.). Biology and Fertility of Soils 12, 33–38.

62

NUTRIENT USE EFFICIENCY IN CROPS

Marschner, P. & Crowley, D.E. (1998) Phytosiderophore decrease iron stress and pyoverdine production of Pseudomonas fluorescens Pf-5 (pvd-inaZ). Soil Biology and Biochemistry 30, 1275–1280. Marschner, P., Marino, W., & Lieberei, R. (2002) Seasonal effect of microorganisms in the rhizosphere of two tropical plants in a polyculture agroforestry system in Central Amazonia, Brazil. Biology and Fertility of Soils 35, 68–71. Marschner, P., Fu, Q., & Rengel, Z. (2003) Manganese availability and microbial populations in the rhizosphere of wheat genotypes differing in tolerance to Mn deficiency. Journal of Plant Nutrition and Soil Science 166, 712–718. Marschner, P., Solaiman, M.Z., & Rengel, Z. (2006) Rhizosphere properties of Poaceae genotypes under P-limiting conditions. Plant and Soil 283, 11–24. Marschner, P., Solaiman, M.Z., & Rengel, Z. (2007) Brassica genotypes differ in growth, phosphorus uptake and rhizosphere properties under P-limiting conditions. Soil Biology and Biochemistry 39, 87–98. Marschner, P., Crowley, D., & Rengel, Z. (2011) Rhizosphere interactions between microorganisms and plants govern iron and phosphorus acquisition along the root axis—model and research methods. Soil Biology & Biochemistry 43, 883–894. Martin, P., Glatzle, A., Kolb, W., et al. (1989) N2-fixing bacteria in the rhizosphere: quantification and hormonal effects on root development. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde 152, 237–245. McGill, W.B. (2007) The physiology and biochemistry of soil organisms. In: Soil Microbiology, Ecology, and Biochemistry (ed. E.A. Paul), pp. 231–256, Elsevier, Amsterdam. McLaughlin, M.J., Alston, A.M., & Martin, J.K. (1988) Phosphorus cycling in wheat-pasture rotations. II. The role of the microbial biomass in phosphorus cycling. Australian Journal of Soil Research 26, 333–342. Merbach, W., Mirus, E., Knof, G., et al. (1999) Release of carbon and nitrogen compounds by plant roots and their possible ecological importance. Journal of Plant Nutrtion and Soil Science 162, 373–383. Moore, J.C., McCann, K., Setaelae, H., et al. (2003) Top-down is bottom-up: does predation in the rhizosphere regulate aboveground dynamics? Ecology 84, 846–857. Moritsuka, N., Yanai, J., Mori, K., et al. (2004) Biotic and abiotic processes of nitrogen immobilization in the soil-residue interface. Soil Biology and Biochemistry 36, 1141–1148. Neilands, J.B. (1984) Siderophores of bacteria and fungi. Microbiological Sciences 1, 9–14.

Neumann, G. (2007) Root exudates and nutrient cycling. In: Nutrient Cycling in Terrestrial Ecosystems (eds. P. Marschner & Z. Rengel), pp. 123–158, Springer, Berlin. Neumann, G. & Roemheld, V. (2000) The release of root exudates as affected by the plant’s physiological status. In: The Rhizosphere—Biochemistry and Organic Substances at the Soil-Plant-Interface (eds. R. Pinton, Z. Varanini & P. Nannipieri), pp. 41–93, Marcel Dekker, New York. Neumann, G., Massonneau, A., Martinoida, E., et al. (1999) Physiological adaptions to phosphorus deficiency during proteoid root development in white lupin. Planta 208, 373–382. Neumann, G., Massonneau, A., Langlade, N., et al. (2000) Physiological aspects of cluster root function and development in phosphorus-deficient white lupin (Lupinus albus L.). Annals of Botany 85, 909–919. Oberson, A., Friesen, D.K., Rao, I.M., et al. (2001) Phosphorus transformations in an oxisol under contrasting land-use systems: the role of the soil microbial biomass. Plant and Soil 237, 197–201. Parkin, T.B., Kaspar, T.C., & Cambardella, C. (2002) Oat plant effects on net nitrogen mineralization. Plant and Soil 243, 187–195. Qian, J.H., Doran, J.W., & Walters, D.T. (1997) Maize plant contributions to the root zone available carbon and microbial transformations of nitrogen. Soil Biology and Biochemistry 29, 1451–1462. Rengel, Z. (1997) Root exudation and microflora populations in rhizosphere of crop genotypes differing in tolerance to micronutrient deficiency. Plant and Soil 196, 255–260. Rengel, Z., Graham, R.D., & Pedler, J.F. (1993) Manganese nutrition and accumulation of phenolics and lignin as related to differential resistance of wheat genotypes to the take-all fungus. Plant and Soil 151, 255–263. Rengel, Z., Gutteridge, R., Hirsch, P., et al. (1996) Plant genotype, micronutrient fertilization and take-all colonization influence bacterial populations in the rhizosphere of wheat. Plant and Soil 183, 269–277. Reyes, I., Bernier, L., Simard, R.R., et al. (1999) Characteristics of phosphate solubilization by an isolate of a tropical Penicillium rugulosum and two UV-induced mutants. FEMS Microbiology Ecology 28, 291–295. Richardson, A.E. (2001) Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Australian Journal of Plant Physiology 28, 897–906. Roemheld, V. (1986) pH changes in the rhizosphere of various crop plants in relation to the supply of plant nutrients. Potash Review 12, 1–12.

THE ROLE OF THE RHIZOSPHERE IN NUTRIENT USE EFFICIENCY IN CROPS

Roemheld, V. (1991) The role of phytosiderophores in acquisition of iron and other micronutrients in graminaceous species: an ecological approach. Plant and Soil 130, 127–134. Roemheld, V. & Marschner, H. (1983) Mechanisms of iron uptake by peanut plants. I. Fe (III) reduction, chelate splitting, and release of phenolics. Plant Physiology 71, 949–954. Rouatt, J.W. & Katznelson, H. (1961) A study of the bacteria on the root surface and in the rhizosphere soil of crop plants. The Journal of Applied Bacteriology 24, 164–171. Schoenwitz, R. & Ziegler, H. (1989) Interaction of maize roots and rhizosphere microorganisms. Zeitschrift fuer Pflanzenernaehrung und Bodenkunde 152, 217–222. Sharma, A., Johri, B.N., Sharma, A.K., et al. (2003) Plant growth-promoting bacterium Pseudomonas sp strain GPR(3) influences iron acquisition in mungbean (Vigna radiata L. Wilzeck). Soil Biology and Biochemistry 35, 887–894. Skou, J.P. (1981) Morphology and cytology in the colonization process. In: Biology and Control of TakeAll (eds. M.J.C. Asher & P.J. Shipton), pp. 175–197, Academic Press, London. Stevenson, F.J. & Cole, M.A. (1999) Cycles of Soil: Carbon, Nitrogen, Phosphorus, Sulfur, Micronutrients. Wiley, New York. Takagi, S. (1976) Naturally occurring iron-chelating compounds in oat- and rice-root washings. Soil Science and Plant Nutrition 22, 423–433. Timonin, M.I. (1946) Microflora of the rhizosphere in relation to the manganese-deficiency disease of oats. Soil Science Society of America Proceedings 11, 284–292. Tisdall, J.M. (1991) Fungal hyphae and structural stability of soil. Australian Journal of Soil Research 29, 729–743. Trinsoutrot, I., Recous, S., Mary, B., et al. (2000) C and N fluxes of decomposing 13C and 15N Brassica napus L.: effects of residue composition and N content. Soil Biology and Biochemistry 32, 1717–1730. Uren, N.C. (1981) Chemical reduction of an insoluble higher oxide of manganese by plant roots. Journal of Plant Nutrition 4, 65–71. Vetterlein, D. & Marschner, H. (1993) Use of a microtensiometer technique to study hydraulic lift in a sandy soil planted with pearl millet (Pennisetum americanum (L.) Leeke. Plant and Soil 149, 275–282. Von Wiren, N., Roemheld, V., Morel, J.L., et al. (1993) Influence of microorganisms on iron acquisition in maize. Soil Biology and Biochemistry 25, 371–376.

63

Von Wiren, N., Roemheld, V., Shiori, T., et al. (1995) Competition between micro-organisms and roots of barley and sorghum for iron accumulated in the root apoplasm. New Phytologist 130, 511–521. Walter, A., Roemheld, V., Marschner, H., et al. (1994) Iron nutrition of cucumber and maize: effect of Pseudomonas putida YC3 and its siderophore. Soil Biology and Biochemistry 26, 1023–1031. Wan, C., Xu, W., Sosebee, R.E., et al. (2000) Hydraulic lift in drought-tolerant and -susceptible maize hybrids. Plant and Soil 219, 117–126. Wang, Y., Brown, H.N., Crowley, D.E., et al. (1993) Evidence for direct utilization of a siderophore, ferrioxamine B, in axenically grown cucumber. Plant, Cell & Environment 16, 579–585. Watt, M. & Evans, J.R. (1999a) Linking development and determinancy with organic acid efflux from proteoid roots of white lupin grown with low phosphorus and ambient or elevated atmospheric CO2 concentration. Plant Physiology 120, 705–716. Watt, M. & Evans, J.R. (1999b) Proteoid roots. Physiology and development. Plant Physiology 121, 317–323. Weisskopf, L., Abou-Mansour, E., Fromin, N., et al. (2006) White lupin has developed a complex strategy to limit microbial degradation of secreted citrate required for phosphate acquisition. Plant, Cell & Environment 29, 919–927. Welch, R.M. (1995) Micronutrient nutrition of plants. Critical Reviews in Plant Sciences 14, 49–82. Whitelaw, M.A., Harden, T.J., & Helyar, K.R. (1999) Phosphate solubilization in solution culture by the soil fungus Penicillium radicum. Soil Biology and Biochemistry 31, 655–665. Wilhelm, N.S., Graham, R.D., & Rovira, A.D. (1990) Control of Mn status and colonization rate by genotype of both host and pathogen in the wheat take-all interaction. Plant and Soil 123, 267–275. Xu, J.M., Tang, C., & Chen, Z.L. (2006) The role of plant residues in pH change of acid soils differing in initial pH. Soil Biology and Biochemistry 38, 709–719. Yehuda, Z., Shenker, M., Roemheld, V., et al. (1996) The role of ligand exchange in the uptake of iron from microbial siderophores by gramineous plants. Plant Physiology 112, 1273–1280. Yehuda, Z., Shenker, M., Hadar, Y., et al. (2000) Remedy of chlorosis induced by iron deficiency in plants with the fungal siderophore rhizoferrin. Journal of Plant Nutrition 23, 1991–2006.

Chapter 4

Optimizing Canopy Physiology Traits to Improve the Nutrient Utilization Efficiency of Crops M. John Foulkes and Erik H. Murchie

Abstract Mineral nutrient fertilizer represents a significant cost of production for the grower and may also have negative environmental impacts through leaching, use of fossil fuels for manufacture and application, and, in the case of nitrogen fertilizers, nitrous oxide (N2O) emissions associated with denitrification. Reducing excessive fertilizer inputs while maintaining yield of crops will be aided by the development of nutrientefficient cultivars. The physiological canopy processes that may contribute to high nutrient use efficiency are reviewed. Canopy traits operating from the cellular to the whole-crop scale are discussed in relation to their optimization, including those affecting radiation interception per unit nutrient uptake and photosynthetic capacity per unit canopy nitrogen, through: (1) maximizing leaf photosynthesis per unit nitrogen, (2) optimizing vertical nitrogen distribution in the canopy, and (3) optimizing nitrogen remobilization and senescence. Potential avenues for optimizing nitrogen distribution in canopies in relation to light attenuation are considered according to quantitative

theories to explain nitrogen distribution in a canopy. Finally, mechanisms for decreasing respiration in leaves are discussed. Rationale for improved nutrient economy of crops Crops with increased nutrient use efficiency will be of economic benefit to farmers and will help to reduce environmental contamination associated with excessive inputs of fertilizers. Crops with low nutrient use efficiency may be associated with (1) leaching of nutrients polluting ground water, (2) eutrophication of rivers and lakes, and (3) in the case of nitrogen fertilizer, global warming, due to emission of N2O derived from denitrification of nitrate by soil bacteria and the use of fossil fuels in the manufacture of nitrogen fertilizers (Goulding, 2004). Therefore, there is increasing emphasis worldwide on breeding cultivars for improved nutrient use efficiency, for example, improved nitrogen use efficiency in wheat (Ortiz-Monasterio et al., 2001; Guarda et al., 2004; LaPerche et al., 2006; Hirel et al., 2007; Li et al., 2008), barley (Sinebo et al., 2004; Kichey et al., 2009),

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 65

66

NUTRIENT USE EFFICIENCY IN CROPS

Table 4.1.

List of physiological variables, their definitions, and units

Physiological Variable (Symbol) Nutrient use efficiency (NtUE) Nutrient uptake efficiency (NtUpE) Nutrient utilization efficiency (NtUtE) Nitrogen use efficiency (NUE) Nitrogen uptake efficiency (NUpE) Nitrogen utilization efficiency (NUtE) Harvest index (HI) Nitrogen harvest index (NHI) Nitrogen remobilization efficiency (NRE) Green area index (GAI) Fractional interception of radiation by canopy (F) Light extinction coefficient (K) Specific leaf nitrogen content (SLN) Radiation use efficiency (RUE) Light-saturated CO2 exchange rate (Amax)

Definition

Units

Kilogram (grain dry mass) at harvest per kilogram available nutrient (from soil plus fertilizer) Kilogram (aboveground nitrogen) at harvest per kilogram available nutrient (from soil plus fertilizer) Kilogram (grain dry mass) per kilogram (aboveground nutrient) at harvest Kilogram (grain dry mass) at harvest per kilogram available nitrogen (from soil plus fertilizer) Kilogram (aboveground nitrogen) at harvest per kilogram available nitrogen (from soil plus fertilizer) Kilogram (grain dry mass) per kilogram (aboveground nitrogen) at harvest Proportion of aboveground biomass dry weight in the grain at harvest Proportion of aboveground nitrogen in the grain at harvest Proportion of nitrogen in the whole plant or organ at anthesis that is not recovered in the straw at harvest Square meter (green area) per square meter (ground)

kg kg−1 (unitless) kg kg−1 (unitless) kg kg−1 (unitless) kg kg−1 (unitless) kg kg−1 (unitless) kg kg−1 (unitless) None None None

Proportion of incident radiation intercepted by canopy

(m2 m−2) unitless None

Slope of linear regression of ln (1 – F) on GAI

None

Gram (leaf nitrogen) per square meter (leaf area)

g m−2

Gram (aboveground biomass dry weight) per megajoule intercepted radiation Micromoles (CO2) per square meter (leaf area) per second in light-saturated conditions

g MJ−1

rice (Peng et al., 2006; Samonte et al., (2006), and maize (Hirel et al., 2001; Boomsma et al., 2009), and improved phosphorus use efficiency in wheat (Manske et al., 2002), barley (Kajer and Jensen, 1995), rice (Fageria and Baligar, 2005), and maize (Chen et al., 2009). Nutrient use efficiency has been defined as grain dry matter (DM) yield per unit of nutrient available (from the soil and/or fertilizer) and may be divided into two components: (1) nutrient uptake efficiency (crop nutrient uptake/ nutrient available) and (2) nutrient utilization efficiency (grain DM yield/crop nutrient

μmol m−2 s−1

uptake) (Moll et al., 1982; Fageria and Baligar, 2005). This definition of nutrient use efficiency has been found useful by crop physiologists, and Table 4.1 summarizes the definition and abbreviations adopted in the present review, in which we principally address the optimization of canopy physiological traits for improving nutrient utilization efficiency. Since approximately 50% to 60% of nitrogen in cereal canopies is associated with chloroplasts, for example, in maize (Hageman, 1986) and wheat (Evans, 1983), a large amount of acquired nitrogen contrib-

OPTIMIZING CANOPY TRAITS TO IMPROVE NUTRIENT EFFICIENCY

67

INCREASE RADIATION INTERCEPTION PER UNIT NUTRIENT UPTAKE:

MAXIMIZE LEAF PHOTOSYNTHETIC CAPACITY PER UNIT N:

• Optimize light extinction coefficient

• Rubisco catalytic properties

• Optimize leaf-specific nitrogen

• Optimize distribution of nitrogen in phosphorus components of chloroplasts

• Shorter phyllochron

OPTIMIZE VERTICAL N DISTRIBUTION: • Optimize K/nL • Reduce light saturation

• Decrease respiration

OPTIMIZE NITROGEN REMOBILIZATION & SENSECENCE: • Leaf and stem nitrogen storage • Optimize nitrogen remobilization efficiency • Optimize grain N%

Fig. 4.1. Strategies to optimize canopy physiology traits for nutrient use efficiency.

utes to the maintenance of photosynthesis. Up to 75% of the reduced nitrogen in cereal leaves is located in the mesophyll cells, mainly as Rubisco, and is involved in photosynthetic processes (Evans, 1983). Canopy responses to nitrogen stress are therefore typically large and often strongly associated with grain yield. Responses in nitrogenlimited crops often include reductions in total leaf area, leaf expansion and duration, leaf nitrogen and chlorophyll content, leaf stomatal conductance, and photosynthesis per unit leaf area (Sylvester-Bradley et al., 1990; Monneveux et al., 2005; Echarte et al., 2008). These responses reduce radiation interception and radiation use efficiency (aboveground biomass per unit radiation interception; RUE) and hence biomass (Echarte et al., 2008; Foulkes et al., 2009a). With photosynthetic capacity diminished in low nitrogen situations, DM partitioning to ears or panicles normally decreases and grain abortion often increases, for example, in maize (Tollenaar et al., 2000; Monneveux et al., 2005) and wheat (Sinclair and Jamieson, 2006). First, in this review, canopy physiology processes that may be manipulated to increase radiation interception per

unit nutrient uptake are discussed. Second, strategies that may be applied to maximize canopy photosynthetic capacity per unit nitrogen, including (1) maximizing leaf photosynthesis per unit nitrogen, (2) optimizing vertical nitrogen distribution in the canopy, and (3) optimizing nitrogen remobilization and senescence are addressed (Fig. 4.1). Strategies to increase nutrient use efficiency in wheat, although examples of strategies in other major grain crops (e.g., rice and maize) are discussed where appropriate, are particularly focused on. Approaches for integrating traits to estimate trade-offs and the translation to yield and to identify the most promising target traits for future breeding are also discussed. Finally, the practical application of traits in breeding programmes is considered. Canopy traits for enhacing radiation capture and RUE Hypothetical framework It has been demonstrated, that for the major worldwide grain crops, genetic progress in yield increases since the mid-1990s has

68

NUTRIENT USE EFFICIENCY IN CROPS

been increasingly associated with gains in biomass rather than harvest index, for example, in wheat (Shearman et al., 2005), rice (Cheng et al., 2007; Yang et al., 2007), and maize (Tollenaar et al., 2000). Therefore, it will be important for future yield progress to increase biomass production. In particular, it will be crucial to boost biomass during stem elongation since assimilate production in the period before the start of the grain filling is crucial for the determination of grain number, for example, during late booting in wheat (Slafer et al., 2009), the 14 days prior to full heading in rice (Takai et al., 2006), and from −230 to 100°C days from silking (the active ear elongation period) in maize (Otegui and Bonhomme, 1998). The following simple model can be broadly applied in grain crops to describe quantitatively the physiological basis of biomass: BM = RAD × F × RUE,

(4.1)

where BM = aboveground dry matter g m−2; RAD = incident radiation in a defined developmental phase of growth (MJ m−2); F = fraction of RAD intercepted; and RUE = radiation use efficiency in a defined developmental phase of growth (g MJ−1). The prospects for manipulating the processes in Equation 4.1 through optimizing canopy physiology to boost biomass production under low nutrient supply as well as the likely interactions affecting their joint optimization are now considered. Increasing radiation interception per unit nutrient uptake Increasing the thermal duration of the period to anthesis accounted for by the stem elongation period is one logical strategy to increase radiation interception during this period. Ways to manipulate patterns of development

to achieve this, for example, manipulating photoperiod responses affected by photoperiod sensitivity genes, were reviewed by Slafer et al. (2009) in wheat and by Foulkes et al. (2009a) across a range of grain crops, and are therefore not discussed further here. Increasing radiation interception during the crucial periods of growth could also be achieved without changing the relative durations of the phases by increasing the average fractional interception during the period. For 95% radiation interception assuming a light extinction coefficient (K) value of 0.5, a green area index (GAI) of 6 is required: K = − ln( I/Io) /L,

(4.2)

where Io is the incident radiation and I is the amount of radiation transmitted below a GAI value of L. At anthesis, modern cultivars of rice (Mitchell et al., 1998), wheat (Whaley et al., 2000; Gaju, 2007), and maize (Ogola et al., 2002) all produce canopies with GAI values in the region of 6, hence achieving full interception at this stage. Therefore, the only realistic way to increase fractional interception in the preanthesis phase is by increasing fractional interception at the start of the stem elongation phase when less than full interception occurs. A mathematical function for crop growth developed by Goudriaan and Monteith (1990) included the concept of the time “lost” for growth while the canopy is closing (i.e., when the crop is in the first exponential phase of growth, when growth rate increases from a very small value on the day of emergence to a maximum rate), which is proportional to the logarithm of the initial fractional radiation interception (Equation 4.3): tb = − ln( F0 /[1 − F0 ]) /Rm

(4.3)

where tb = time “lost” for growth while the canopy is closing; F0 = initial value of frac-

OPTIMIZING CANOPY TRAITS TO IMPROVE NUTRIENT EFFICIENCY

tional radiation interception; Rm = initial maximum relative growth rate. Typically fractional interception at the start of stem elongation is in the region of 0.6–0.7 in wheat (Whaley et al., 2000; Shearman et al., 2005; Gaju, 2007) and 0.4– 0.6 in rice (Mitchell et al., 1998), so only marginal improvements in F0 and time “lost” for growth (hence “lost” biomass) seem possible. Physiological avenues worth investigating for increasing F0 specifically under low nutrient supply may include a larger specific leaf nitrogen area (leaf area per unit leaf nitrogen; SLN) and/or more prostrate nonculm leaves. Phenotypic differences in specific leaf nitrogen in temperate cereals have been associated with embryo size (López-Castañeda et al., 1996) and genetic variation in specific leaf nitrogen could potentially be exploited to achieve earlier canopy closure (Rebetzke & Richards, 1999). The light extinction coefficient is mainly influenced by leaf angle, and for modern cultivars is approximately 0.65 for maize (Varlet-Grancher et al., 1989) and 0.55 for wheat, barley, and rice (Abbate et al., 1998; Mitchell et al., 1998; Bingham et al., 2007), when calculated based on the interception of photosynthetically active radiation (i.e., radiation with wavelengths between 400 and 700 nm; PAR). These values are associated with semi-erect to erect leaf angles, which help to reduce light saturation in the upper canopy leaves, hence boosting RUE, and a shift back to higher values of K seems unlikely to be desirable overall due to the trade-off with RUE. Studies that have collected data on K through the season generally show no change over the stem elongation period, for example, in maize (Flenet et al., 1996) and wheat (Thorne et al., 1988). Therefore, breeding for more prostrate leaves during early vegetative growth and more upright leaves during later vegetative growth, although desirable, may be difficult to achieve in practice. A shorter

69

phyllochron (the thermal duration between successive emerging leaves) would favor earlier canopy closure through more rapid leaf emergence and/or tiller production, although this mechanism may not intrinsically increase nutrient use efficiency. In summary, although gains in biomass may be possible through increasing the average fractional interception during stem elongation, they seem likely to be small. The strategies that may be specifically beneficial under low nitrogen supply would appear to be a lower SLN and more prostrate culm leaves. However, in both cases, the trade-offs must be considered carefully. For example, there are interdependencies since RUE could not be maintained if K was increased; and further work is required to review these interdependencies before prioritizing breeding strategies to optimize canopy architecture and light extinction coefficients for improved nitrogen use efficiency (grain DM/N available from soil nitrogen plus fertilizer nitrogen; NUE). Canopy traits to increase photosynthetic capacity per unit nutrient uptake If radiation interception and harvest index are approaching an upper limit, yield ultimately must be improved mainly by increasing RUE. Current general values of RUE (g MJ−1 PAR) in modern cultivars are in the region of 2.2 for rice, 2.5 for wheat and barley, and 3.3 for maize (Mitchell et al., 1998; Shearman et al., 2005; Bingham et al., 2007). The higher RUE for maize is associated with C4 metabolism, and the lower value for rice than for wheat is thought to be associated largely with higher losses from photorespiration (Mitchell et al., 1998). There are three main potential avenues to manipulate canopy physiology traits to boost photosynthesis per unit nitrogen uptake: (1) increase leaf photosynthetic rate per unit

70

NUTRIENT USE EFFICIENCY IN CROPS

of nitrogen, (2) increase canopy net photosynthetic rate due to a more optimal distribution of incident light across the leaf canopy and/or in relation to nitrogen distribution across the leaf canopy, and (3) reduce respiration. Increase leaf photosynthetic rate per unit nitrogen Increasing the rate of photosynthesis for a given concentration of leaf nitrogen could improve nitrogen utilization efficiency (grain DM/crop nitrogen uptake; NUtE). In C3 cereals, the light-saturated leaf photosynthetic rate (Amax) typically increases to values of the order of 20–30 μmol CO2 m−2 s−1 at leaf nitrogen concentrations of 2 g N m−2 under favorable conditions. Assuming an asymptotic relationship between Amax and leaf nitrogen concentration, for example, in wheat (Evans, 1983; Sinclair and Horie, 1989; Pask, 2009) and rice (Hirasawa et al., (2010), one possible strategy to raise NUtE is to decrease specific leaf nitrogen (nitrogen content per leaf lamina area; SLN) while maintaining Amax. The SLN required for potential photosynthetic function under high light levels has been estimated to be ca. 2.1 g N m−2 in wheat (Pask, 2009). Since leaves of modern wheat genotypes accumulate more nitrogen than this under favorable conditions with no increase in RUE (Critchley, 2001; Pask, 2009), NUtE could be increased by selecting for lower SLN to decrease the transient “storage” nitrogen components of leaves. A sensitivity analysis using the wheat Sirius model (Semenov et al., 2007) predicted that decreasing leaf SLN in the range 1–2 g m−2 increased NUE by 10–15% when nitrogen was limiting. Additionally, Tambussi et al. (2005) in barley demonstrated the potential advantage of a low SLN and its possible translation to yield. An alternative strategy of selecting

for SLN above values of ca. 2 g N m−2 to increase Amax seems unlikely to be advantageous overall since RUE only increases at a low rate as Amax increases above values of about 20–30 μmol CO2 m−2 s−1 in cereals (Monteith, 1977; Sinclair and Horie, 1989). This is because individual leaves may operate well below light saturation in the canopy and because of the need to account for dark respiration (Reynolds et al., 2000). Selection for photosynthetic parameters other than Amax may therefore be more beneficial for closed canopies (Murchie et al., 2009). There may also be pleiotropic tradeoffs with greater SLN being associated with reduced leaf size and light interception (Austin et al., 1982). Genetic variability in SLN ranged from 1.4 to 2.6 g m−2 for 144 durum wheat genotypes (Araus et al., 1997) and from 2.1 to 2.4 g m−2 for 17 durum wheat cultivars (Giunta et al., 2002), whereas in leaf nitrogen concentration the range was from 43.7 to 47.6 mg g−1 for eight bread wheat cultivars (Fischer et al., 1998). The heritability of SLN or leaf nitrogen concentration in wheat is largely unknown. However, it is encouraging that the heritability for straw (leaf lamina, leaf sheath, and stem) nitrogen at anthesis for winter wheat was >0.60 under low nitrogen (LaPerche et al., 2006), indicating that breeding to manipulate the amount of global canopy nitrogen should be possible. Since leaf size and thickness are associated with anatomical structure, there may be merit in investigating the cellular basis of differences in leaf-specific weight including the number of chloroplasts per cell, and mesophyll cell size. Changes in mesophyll cell size and area per leaf induced by ploidy have been linked to photosynthetic rate in wheat (Austin et al., 1982). However, sacrificing leaf size to gain a higher rate of leaf photosynthesis per unit leaf area in future breeding strategies must be considered carefully.

OPTIMIZING CANOPY TRAITS TO IMPROVE NUTRIENT EFFICIENCY

Mooney and Gulmon (1979) introduced the concept of costs and benefits in the study of resource use by plants. The rate of photosynthesis increases with increasing protein content of a leaf. A question then arises: Why don’t all plants have high leaf nitrogen contents and hence high photosynthetic rates? First, there is the issue of intraleaf shading in thick leaves: Many chloroplasts would end up in a light-limited state. Additionally, in a light-limited environment, photosynthesis saturates at a lower enzyme content than in a light-unlimited environment, so plants growing in low-light conditions should have leaves with low enzyme contents (Mooney and Gulmon, 1979; Murchie et al., 2002). Since there are costs in manufacturing enzyme proteins, and the marginal gain decreases with an increase in enzyme investment, plants should invest to the point at which the marginal cost exceeds the marginal gain. This is also complicated by the observation that leaves in cereal canopies are exposed to higher irradiance when first produced and then become progressively shaded as the canopy develops. Therefore, over the full lifetime of the leaf, there has to be an efficient response to both full and limited-light conditions. This means that lifetime analyses will be needed for the improvement of NUE.

Increase canopy photosynthesis via canopy architecture Effects of modifying the light extinction coefficient Altering canopy architecture to raise RUE could be easier than altering single metabolic processes such as leaf photosynthesis. Effects of canopy architecture on RUE have been observed, with RUE being higher for large wheat canopies with more erect leaves,

71

associated with reduced light saturation of the upper leaves (Evans, 1973; Araus et al., 1993). Thus, a decrease in the light extinction coefficient associated with more erect leaves might in theory raise nutrient use efficiency. However, most modern cereals cultivars worldwide already have semi-erect or erect flag leaves. So there is likely limited scope for improvement of RUE via this route under favorable conditions. The control of leaf angle seems to be under simple genetic control so it should be easy to confirm predictions. However, under low nutrient conditions under which canopy size may be reduced to below a GAI of ca. 4, increasing K (more prostrate leaves) could reduce the green canopy area and hence canopy nitrogen required to maximize light interception in wheat as mentioned above (Sylvester-Bradley and Kindred, 2009). Further work is therefore required to review these interdependencies before prioritizing breeding strategies to optimize canopy architecture.

Effects of optimizing DM and nitrogen partitioning Nitrogen use efficiency could theoretically be increased by modifying either DM or nitrogen partitioning. Altering DM partitioning to minimize any sink limitation in the extending stem internodes could offer an avenue for raising RUE during stem elongation. There is a large body of evidence that greater sink strength may support higher photosynthetic rates via feedback regulation. In wheat, greater RUE was observed in response to manipulating the crop by opening rows in the booting/ear emergence phase to increase light interception, ear growth, and grain number (Reynolds et al., 2005). Similarly, enhanced RUE was found in response to a preanthesis shading treatment

72

NUTRIENT USE EFFICIENCY IN CROPS

in winter wheat (i.e., increasing sink relative to source size) compared with the control treatment (Beed et al., 2007). Therefore, strategies to minimize sink limitation and to optimize source/sink balance, particularly during the latter stages of stem elongation, could potentially boost ear biomass and nutrient use efficiency. Such strategies, however, may not lower the nitrogen fertilizer requirement (i.e., the economic fertilizer nitrogen amount) per se. Indeed, there is evidence that genetic progress in yield potential has been associated with higher economic nitrogen optima in winter wheat in the United Kingdom (Foulkes et al., 1998; Sylvester-Bradley and Kindred, 2009). Nevertheless, the absolute yield under low nitrogen supply could be expected to be positively associated with gains in biomass through optimizing source–sink balance (Ortiz-Monasterio et al., 1997; Foulkes et al., 1998; Muurinen et al., 2006). Partitioning of DM among leaves, stems, and roots is also an important consideration because greater aerial biomass without an associated increase in root biomass could affect water and nutrient uptake and could lead to greater incidence of lodging. In addition, the nutrient composition of leaves, stems, and roots differs considerably and so changing the relative abundance of these organs will alter plant nutrient needs. Favoring a greater capacity to store nitrogen in nonphotosynthetic organs such as the stem internodes may enable the translocation of a larger amount of nitrogen to grains without reducing plant photosynthetic capacity (Bertheloot et al., 2008), although the respiratory cost of maintaining a large nonphotosynthetic pool of storage nitrogen is unclear. In wheat, about 60% of the total nitrogen in a canopy at anthesis is in the leaf lamina and leaf sheath (Jamieson and Semenov, 2000; Critchley, 2001). The truestem nitrogen accounts for up 25% of total canopy nitrogen at anthesis (Critchley,

2001). There is relatively little information on the size of the structural and reserve nitrogen pools of the true stem. A recent investigation on UK winter wheat cultivars indicated that aspproximately 60% of the true-stem nitrogen is contained in the “reserve nitrogen” pool, that is, nitrogen that is neither “structural nitrogen” nor “photosynthetic nitrogen” under optimal conditions (Pask, 2009). Genetic variation in stem nitrogen content is reported in winter wheat (Triboï and Ollier, 1991; Critchley, 2001) and in rice (Tirol-Padre et al., 1996). A high capacity to absorb nitrogen in the true stem before flowering could theoretically favor a high maximum rate of nitrogen uptake, hence higher nitrogen uptake efficiency (NUpE; Foulkes et al., 2009b). Some studies in maize report early remobilization of nitrogen from the stem before the leaf lamina (Beauchamp et al., 1976; Friedrich and Schrader, 1979) consistent with the use of stem nitrogen as a buffer for the flow of nitrogen from the leaf lamina to the grain. In this case, a high stem nitrogen absorption capacity coupled with high stem nitrogen remobilization efficiency (proportion of nitrogen in the stem at anthesis which is not recovered in the stem at harvest; stem nitrogen remobilization efficiency [NRE]) would potentially favor high NUtE through delayed senescence of the leaf lamina. The relationship between canopy nitrogen dynamics and senescence patterns is discussed further below. The capacity of a genotype to retain green leaf area for longer than a standard genotype during grain filling has been referred to as the “stay-green” phenotype (Thomas and Smart, 1993). Several investigations have concluded that the genetic control of nitrogen remobilization seems likely to be involved in the regulation of leaf senescence (Sinclair and De Wit, 1975; Masclaux et al., 2001). Although under optimal conditions wheat crops are, in

OPTIMIZING CANOPY TRAITS TO IMPROVE NUTRIENT EFFICIENCY

general, little limited by the assimilate supply during grain filling (Dreccer et al., 1997; Borrás et al., 2004; Calderini et al., 2006), under low to moderate nitrogen fertilizer levels, yields may be more limited by postanthesis assimilate supply. Genetic variation in functional stay-green (delayed senescence associated with extended photosynthesis) lines has been reported in bread wheat (Silva et al., 2000; Verma et al., 2004; Foulkes et al., 2007), although the underlying physiological mechanisms have not been studied extensively. Christopher et al. (2008) found that the stay-green phenotype in the spring wheat, SeriM82, was associated with extraction of deep soil water in Australia. More studies have been carried out on the mechanisms underlying genetic variation in stay-green in sorghum. Nitrogen dynamics are an important factor in the maintenance of green leaf area in sorghum, with stay-green in sorghum hybrids linked to changes in the balance between nitrogen demand and supply during grain filling, resulting in a slower rate of nitrogen translocation from the leaves to the grain compared with senescent genotypes (Borrell and Hammer, 2000; van Oosterom et al., 2010a,b). The latter study showed that the onset and rate of leaf senescence were explained by a supply–demand framework for nitrogen dynamics, in which individual grain nitrogen demand was sink determined and was initially met through nitrogen translocation from the stem and rachis, and then if these nitrogen pools were insufficient, from leaf nitrogen translocation. Stay-green mutants have also been identified in durum wheat (Triticum turgidum spp. durum) (Spano et al., 2003), with delayed senescence being correlated with a higher rate and duration of grain filling. A transcription factor (NAM-B1) accelerates senescence and increases nitrogen remobilization from leaves to grains in emmer wheat (an ancient cultivated tetraploid species, T. turgidum

73

ssp. dicoccoides), whereas modern durum wheat cultivars carry the nonfunctional NAM-B1 allele (Uauy et al., 2006). In summary, a better understanding of the mechanisms underlying the genetic variation in leaf and stem NRE would offer scope for raising NUE in wheat and other grain crops, and further investigations with this objective seem justified. In wheat, for cultivars targeted at the feed, distilling, or biofuel markets (high grain starch to nitrogen ratio requirement), a low nitrogen lamina NRE, potentially associated with the stay-green trait, would be a strategy to boost NutE. For bread-making cultivars (low grain starch to nitrogen to ratio requirement), on the other hand, a high lamina NRE is still required to maintain acceptable grain N%. A further question is whether the nitrogen concentration in the grain at anthesis is important for floral development. There is current debate on this topic, with some physiologists highlighting the close relationship between grain number per ear and ear nitrogen content in field data sets (Demotes-Mainard et al., 1999; Demotes-Mainard and Jeuffroy, 2004; Sinclair and Jamieson, 2006), and others suggesting there is no evidence for effects of nitrogen on grain number apart from those operating via dry matter accumulation (Fischer, 2007). Ears of small grain cereals have a capacity to accumulate large amounts of nitrogen in the preanthesis period in the glumes at a time when there are few alternative sinks around (Lopes et al., 2006). Again, this could be a mechanism of buffering leaf lamina senescence in the postanthesis period similar to that suggested above for true-stem nitrogen, and further work seems justified on the patterns of remobilization on ear nitrogen during early grain filling and associations with staygreen behavior. At the metabolic level, there are several avenues for increasing photosynthetic efficiency. These include (1) relaxing the

74

NUTRIENT USE EFFICIENCY IN CROPS

photoprotected state more rapidly, (2) reducing photorespiration through Rubisco with decreased oxygenase activity, (3) introducing higher catalytic rate forms of Rubisco, and (4) introducing C4 photochemistry into C3 plants (see recent reviews by Reynolds et al., 2000; Parry et al., 2003; Long et al., 2006; Murchie et al., 2009; Parry et al. 2011). Largest potential improvements are available from reduced photorespiration (ca. 30%), with improvement from other mechanisms estimated to be in the range of 15– 22% (Long et al., 2006). There is considerable interest in decreasing the wasteful oxygenase activity of Rubisco. In spite of the lack of any formal enzyme binding site for the gaseous substrates, Rubisco has an inherent ability to discriminate between CO2 and O2 as described by the specificity factor. Attempts to select for specificity and low photorespiration in crop plants have historically met with little success (e.g., Evans, 1983). However, thermophilic red algae, for example, Galderia partita, have Rubisco, which is up to three times more efficient than that in C3 cereals due to greater specificity for CO2 (Uemura et al., 1997). If Rubisco in wheat plants had the specificity factor of that of red alga, the leaf Amax would be increased by about 20% (Austin, 1999). On the other hand, red algal Rubisco has a lower reaction rate than the Rubisco in terrestrial plants, so there could well be a trade-off between specificity and reaction rate. Prospects of boosting RUE of crop plants by introducing Rubisco with greater specificity for CO2 may therefore depend on addressing this apparent trade-off. Recently, species have been identified with properties that deviate from this trend, suggesting that it may be possible to improve both specificity and maximum catalytic activity (Parry et al., 2007). It should be noted that plants selected for superior performance on the basis of superior leaf photosynthetic rate in

a high-radiation environment would not necessarily be superior in more temperate, lower-radiation environments. This would be the case if differences in leaf photosynthesis were not apparent at relatively low light intensities. It has been suggested, following modeling of Rubisco function, that it would benefit canopy photosynthesis if Rubisco properties were tailored to the light profile with a high capacity (low specificity) for leaves in saturating light and a high specificity (low capacity) in light-limited regions (Long et al., 2006). Such a change would require an age-dependent or irradiancedependent shift in the gene expression level to occur during the leaf ’s life cycle. Alternatively, if higher leaf photosynthetic rate reflects higher quantum efficiency as a result of less carbon loss in photorespiration, and these effects were consistent at lower temperatures, then higher leaf photosynthesis should result in benefits in temperate environments as well. For the most rapid gains in RUE, an additional issue is to define leaf photosynthetic traits in terms that are relevant to the canopy, so that their impact on canopy photosynthetic rate may be quantified. Traits that scale to the canopy and promote high rates of canopy photosynthesis should be prioritized in future physiological investigations applied to breeding, and integrative measurements such as leaf cooling exploited in the assessment of photosynthetic traits at the crop level (Foulkes et al., 2009a). Optimizing nitrogen distribution in canopies in relation to light attenuation It has been suggested that to maximize carbon gain by a canopy, nitrogen should be optimally distributed so that leaves receiving the greatest photon flux densities have the largest SLN (Field, 1983; Grindlay, 1997). Theoretically, canopy photosynthesis

OPTIMIZING CANOPY TRAITS TO IMPROVE NUTRIENT EFFICIENCY

is maximized when each leaf in the canopy receives irradiance in proportion to the associated photosynthetic capacity (Farquhar, 1989). A vertical nitrogen distribution that follows the light gradient would allow higher photosynthesis compared with that expected from a uniform nitrogen distribution (Mooney and Gulmon, 1979). The “optimization” theory proposed by Hirose and Werger (1987) suggests that lamina nitrogen distribution within a vegetative canopy optimizes whole canopy photosynthesis. It proposes that, within a dense canopy, leaf lamina nitrogen distribution is driven by the light gradient such that SLN follows an exponential function of the downward cumulative leaf area index with an extinction coefficient for nitrogen (KN) equal to that for light (K). Nitrogen distribution is primarily determined by the structure of the canopy (K), by the total amount of leaf canopy nitrogen, and by the amount of nitrogen invested to structural components of the leaf. Assuming that the photosynthetic capacity is proportional to the amount of nitrogen directly involved in photosynthesis, Anten et al. (1995) derived the optimal nitrogen distribution: nL =

K (nt − nbLAIT ) e − KLAIc + nb, 1 − e − KLAIt

(4.4)

where nL is the nitrogen content per area; LAIc is the leaf area index (LAI) cumulated from the top of the canopy; LAIt is the total LAI; nt is the total amount of leaf canopy nitrogen; nb is the leaf nitrogen per area that is not involved in photosynthesis; and K is the light extinction coefficient. Equation 4.4 indicates that the photosynthetic nitrogen (nL − nb) decreases exponentially as a function of LAIc, where the proportionality constant is K, the light extinction coefficient (see Equation 4.2). An important issue raised from the optimal allocation theory is what controls

75

nitrogen distribution within the canopy. Since young leaves are usually produced at the top of the canopy and are normally high in nitrogen content, either leaf age or light gradient could be proposed as the drivers of nitrogen allocation (Field, 1983; Field and Mooney, 1983). From an investigation of nitrogen distribution in a stand of a Carex acutiformis, a monocotyledenous plant having a meristem at the base of the plant, with the youngest part of a leaf blade subjected to the lowest irradiance within the canopy, it was observed that nitrogen distribution was correlated strongly with the light gradient in the canopy, with only a small effect of leaf age (see a review by Hirose, 2005 for an extended discussion on this area). Alternatively, Chen et al. (1993) proposed the coordination theory to explain nitrogen distribution in a canopy. This theory explicitly takes photosynthetic processes into account at the leaf scale to explain the relationship between light and nitrogen vertical distribution in vegetative canopies. Specific leaf nitrogen is computed to maintain a balance between the Rubisco-limited rate of carboxylation and the electron transport-limited rate of carboxylation, which depends on the amount of intercepted light. This coordination theory may explain nL decreasing with increasing depth in the canopy, but does not explain nL that changes depending on nitrogen availability as well. For this reason, the application of this coordination theory has been very limited. Hikosaka and Terashima (1995) determined the pattern of nitrogen partitioning among photosynthetic components in chloroplasts that maximizes the daily carbon gain for various light environments and leaf nitrogen contents. At high irradiance, nitrogen was allocated more to Calvin cycle enzymes and electron carriers, while at low irradiance, nitrogen was allocated more to chlorophyll–protein complexes. This enabled modelers working on canopy

76

NUTRIENT USE EFFICIENCY IN CROPS

photosynthesis with leaf nitrogen distribution to scale up from chloroplast biochemistry to canopy carbon grain. Zhu et al. (2008) modeled mesophyll cell metabolism, using evolutionary algorithms that imposed variation of allocation of nitrogen between different components (such as the Calvin cycle and photorespiration) and selected for higher photosynthetic rate in each generation. Crucially, the total nitrogen content was kept constant. It was concluded that significantly higher photosynthesis per unit nitrogen could be achieved by diverting nitrogen away from photorespiration and toward Calvin cycle enzymes, raising the hope that leaf photosynthesis increases could be achieved via straightforward means. It was suggested that the current allocation represented a lack of adaptation to rising atmospheric CO2. In wheat, observed nitrogen gradients are generally less steep than predicted with the optimization theory, but do demonstrate that SLN follows an exponential gradient with vertical depth in the canopy (Critchley, 2001; Pask, 2009). In sunflower, Connor et al. (1995) measured the profiles of leaf area and leaf nitrogen from mid-flowering to maturity at contrasting nitrogen supply in Argentina. Compared with a model of estimated daytime net photosynthesis of canopies, observed nitrogen profiles at mid-flowering tended to hold “excessive” nitrogen in their lower canopies. In peanut (Arachis hypogaea L.), Wright and Hammer (1994) reported on leaf nitrogen gradients in canopies, and their impact on RUE in Australia. There was a marked decline in SLN from the top to the base of the canopy, and the gradient appeared to be largely controlled by the light environment within the canopy. RUE was up to 32% higher than the theoretical RUE assuming a uniform SLN distribution in the canopy. Anten et al. (1995) showed that profits from nonuniform

distribution in actual plants were larger in dicots (41%in Glycine max and 35% in Amaranthus cruentus) than in monocots (14% in Oryza sativa and 13% in Sorghum bicolor). However, none of these plants attained the optimal nitrogen distribution. There is relatively little information on genetic diversity in the vertical distribution of nitrogen in the canopy, despite the large amount of work published on light interception and attenuation by crop canopies. In wheat, small differences were observed in the distribution of nitrogen in the top four leaves at anthesis between two UK winter wheat cultivars, Soissons and Spark, but overall the distributions were close to that predicted by the optimization theory (Hirose and Werger, 1987) in both cultivars (Critchley, 2001). Similarly, the vertical distribution of nitrogen at anthesis was close to the optimum, as defined in the optimization theory (Hirose and Werger, 1987), and did not differ significantly for two French winter wheat cultivars, Apache and Isengrain, until almost the end of grain filling (Bertheloot et al., 2008). The role of nitrogen dynamics on canopy photosynthesis and crop productivity will likely become even more important in the future because of the increase in atmospheric CO2 concentration (Kim et al., 2001; Anten et al., 2004). Overall, the reported investigations indicate that actual plants tend to distribute nitrogen more uniformly than the optimal distribution. The difference between actual and optimal distribution implies that optimal nitrogen distribution leads to leaf nitrogen per unit area that is too low at the bottom and too high at the top to be realized. Hirose (2005) suggested that the reason for this may be that some nitrogen may not be capable of translocation, and a certain amount of nitrogen is necessary to utilize sunflecks that leaves receive in lower layers in the canopy (see also Pons et al., 1993).

OPTIMIZING CANOPY TRAITS TO IMPROVE NUTRIENT EFFICIENCY

Decrease respiration Respiration in crops has received less attention than photosynthesis due to difficulties in measurement and the fact that it is rather heterogeneous in the plant depending on tissue type and substrate. Respiration, nonetheless, is critical in determining yield and therefore potentially influences nutrient use efficiency. It is highly responsive to temperature and may become an issue as global temperatures rise (Peng et al., 2004). Respiration is commonly divided into growth and maintenance respiration, with each exerting differing effects. An important early observation was that leaf area index (ratio of leaf lamina area to ground area) does not increase proportionally with respiration rate, meaning that canopy assimilation can continue to respond positively to irradiance. Respiration therefore may be positively but nonlinearly related to photosynthesis. Canopy architecture can influence nutrient use in this way: In rice, leaf erectness can determine the ability of the canopy to retard senescence and retain nitrogen by increasing the amount of light on the lower leaves (Sinclair and Sheehy, 1999). Leaf senescence in this case is assumed to be induced when irradiance falls below a threshold level. The maintenance of erect leaves prevents this from happening in the lowest leaves, retaining nitrogen for photosynthesis and for subsequent remobilization. Is it possible to reduce “wasteful” respiration in crop canopies? The ratio of respiration to photosynthesis is thought to be close to the optimum (Amthor, 2000), although it is suggested that the data for respiration and regulation in different tissue types during development need refinement and that improvements may come from reduction of maintenance respiration (Amthor, 2000). There has been recent work showing that

77

genetic manipulation of respiration in tomato has a knock-on effect on both biomass and yield Nunes-Nesi et al. (2005). Recent work highlighting the importance of increased night time temperature on productivity in rice (Peng et al., 2004) and wheat (Tester and Langridge, 2010) and the high sensitivity of respiration to temperature in general suggests that the environmental responses of crop respiration to temperature is an important area on which to focus. Conclusions This chapter is concerned with identifying strategies to increase the efficiency of nutrient use by crop canopies. Future progress will depend on identifying canopy traits at the biochemical, cellular, and plant levels and integrating toward field performance. In particular, it will be important to increase understanding of the extent to which the genetic control of nutrient accumulation and/or distribution among canopy components and canopy photosynthesis are intrinsically linked. The development of more advanced crop simulation models may be a way to link model parameters with physiological traits and thus facilitate research to identify trade-offs and the joint optimization of the key traits. The preliminary analysis presented in this chapter has highlighted some of the key physiological processes that may underlie nutrient-efficient genotypes. Promising avenues for future improvements in nutrient use efficiency include (1) more efficient radiation capture per unit nutrient uptake, through a shorter phyllochron, decreased leaf-specific nitrogen content and optimized light extinction coefficient, and (2) maximizing photosynthetic capacity per unit nitrogen, through manipulation of Rubisco properties, optimizing distribution of nitrogen in plant organs and in photosynthetic

78

NUTRIENT USE EFFICIENCY IN CROPS

components of chloroplasts and also decreasing respiration. The present analysis has indicated that there is significant scope for improving nutrient use efficiency within adapted germplasm. For some traits, however, it may be necessary to exploit a wider gene pool, utilizing either exotic lines or wide crosses with alien species related to the relevant crop species. Long-term success will depend on a multidisciplinary approach, linking fundamental and strategic research with applied plant breeding to develop new genotypes with high nutrient use efficiency for sustainable agriculture. This review attempts to provide the background for such an integrated approach. References Abbate, P.E., Andrade, D.H., Lazaro, L., et al. (1998) Grain yield in recent argentine wheat cultivars. Crop Science 38, 1203–1209. Amthor, J.S. (2000) The McCree-de Wit-Penning de Vries-Thornley respiration paradigms: 30 years later. Annals of Botany 86, 1–20. Anten, N.P.R., Schieving, F., & Werger, M.J.A. (1995) Patterns of light and nitrogen distribution in relation to whole canopy carbon gain in C3 and C4 mono- and dicotyledonoous species. Oecologia 101, 504–513. Anten, N.P.R., Hirose, T., Onoda, Y., et al. (2004) Elevated CO2 and nitrogen availability have interactive effects on canopy carbon gain in rice. New Phytologist 161, 459–471. Araus, J.L., Reynolds, M.P., & Acedevo, E. (1993) Leaf structure, leaf posture, growth, grain yield and carbon isotope discrimination in wheat. Crop Science 33, 1273–1279. Araus, J.L., Amaro, T., Zuhair, Y., & Nachit, M.M. (1997) Effect of leaf structure and water status on carbon isotope discrimination in field grown durum wheat. Plant, Cell & Environment 20, 1484–1494. Austin, R.B. (1999) Yield of wheat in the United Kingdom: recent advances and prospects. Crop Science 39, 1604–1610. Austin, R.B., Morgan, C.L., Ford, M.A., & Bhagwat, S.G. (1982) Flag leaf photosynthesis of triticum aestivum and related diploid and tetraploid species. Annals of Botany 49, 177–189. Beauchamp, E.G., Kannenberg, L.W., & Hunter, R.B. (1976) Nitrogen accumulation and translocation in

corn genotypes following silking. Agronomy Journal 68, 418–422. Beed, F.D., Paveley, N.D., & Sylvester-Bradley, R. (2007) Predictability of wheat growth and yield in light-limited conditions. Journal of Agricultural Science 145, 63–79. Bertheloot, J., Martre, P., & Andrieu, B. (2008) Dynamics of light and nitrogen distribution during grain filling within wheat canopy. Plant Physiology 148, 1707–1720. Bingham, I.J., Blake, J., Foulkes, M.J., & Spink, J. (2007) Is barley yield in the UK sink limited?: I. Post-anthesis radiation interception, radiation-use efficiency and source–sink balance. Field Crops Research 101, 198–211. Boomsma, C.R., Santini, J.B., Tollenaar, M., & Vyn, T.J. (2009) Maize morphophysiological responses to intense crowding and low nitrogen availability: an analysis and review. Agronomy Journal 101, 1426–1452. Borrás, L., Slafer, G.A., & Otegui, M.E. (2004) Seed dry weight response to source–sink manipulations in wheat, maize and soybean: a quantitative reappraisal. Field Crops Research 86, 131–146. Borrell, A.K. & Hammer, G.L. (2000) Nitrogen dynamics and the physiological basis of stay-green in sorghum. Crop Science 40, 1295–1307. Calderini, D.F., Reynolds, M.P., & Slafer, G.A. (2006) Source-sink effects on grain weight of bread wheat, durum wheat, and triticale at different locations. Australian Journal of Agricultural Research 57, 227–233. Chen, J.L., Reynolds, J.F., Harley, P.C., & Tenhunen, J.D. (1993) Coordination theory of leaf nitrogen distribution in a canopy. Oecologia 93, 63–69. Chen, J., Xu, L., Cai, Y., & Xu, J. (2009) Identification of QTLs for phosphorus utilization efficiency in maize (Zea mays L.) across P levels. Euphytica 167, 245–252. Cheng, S., Cao, L.-Y., Zhuang, J.-Y., et al. (2007) Super hybrid rice breeding in China: achievements and prospects. Journal of Integrative Plant Biology 49, 805–810. Christopher, J.T., Manschadi, A.M., Hammer, G.L., & Borell, A.K. (2008) Developmental and physiological traits associated high yield and stay-green phenotype in wheat. Australian Journal of Agricultural Research 59, 354–364. Connor, D.J., Sadras, V.O., & Hall, A.J. (1995) Canopy nitrogen distribution and the photosynthetic performance of sunflower crops during grain filling—a quantitative analysis. Oecologia 101, 274–281. Critchley, C.S. (2001) A physiological explanation for the canopy nitrogen requirement of wheat. PhD thesis, University of Nottingham.

OPTIMIZING CANOPY TRAITS TO IMPROVE NUTRIENT EFFICIENCY

Demotes-Mainard, S. & Jeuffroy, H.-H. (2004) Effects of nitrogen and radiation on dry matter and nitrogen accumulation in the spike of winter wheat. Field Crops Research 87, 221–233. Demotes-Mainard, S., Jeuffroy, M.-H., & Robin, S. (1999) Spike dry matter and nitrogen accumulation before anthesis in wheat as affected by nitrogen fertilizer: relationship to kernels per spike. Field Crops Research 64, 249–259. Dreccer, M.F., Grashoff, C., & Rabbinge, R. (1997) Source-sink ratio in barley (Hordeum vulgare L.) during grain filling: effects on senescence and grain protein concentration. Field Crops Research 49, 269–277. Echarte, L., Rothstein, S., & Tollenaar, M. (2008) The response of leaf photosynthesis and dry matter accumulation to nitrogen supply in an older and a newer maize hybrid. Crop Science 48, 656–665. Evans, L.T. (1973) The effect of light on plant growth, development and yield. In: Plant Response to Climatic Factors (ed. R.O. Slatyer), pp. 21–35, UNESCO, Paris. Evans, J.R. (1983) Nitrogen and photosynthesis in the flag leaf of wheat (Triticum aestivum L.). Plant Physiology 72, 297–302. Fageria, N.K. & Baligar, V.C. (2005) Enhancing nitrogen use efficiency in crop plants. Advances in Agronomy 88, 97–185. Farquhar, G.D. (1989) Models of integrated photosynthesis of cells and leaves. Philosophical Transactions of the Royal Society of London. Series B 323, 357–367. Field, C. (1983) Allocating leaf nitrogen for the maximization of carbon gain: leaf age as a control on the allocation program. Oecologia 56, 341–347. Field, C. & Mooney, H.A. (1983) Leaf age and seasonal effects on light, water and nitrogen use efficiency in a California shrub. Oecologia 56, 348–355. Fischer, R.A. (2007) The importance of grain or kernel number in wheat: a reply to Sinclair and Jamieson. Field Crops Research 105, 15–21. Fischer, R.A., Rees, D., Sayre, K.D., Lu, Z.M., Condon, A.G., & Saavedra, A.L. (1998) Wheat yield progress associated with higher stomatal conductance and photosynthetic rate and cooler canopies. Crop Science 38, 1467–1475. Flenet, F., Kiniry, J.R., Board, J.E., Westgate, M.E., & Reicosky, D.C. (1996) Row spacing effects on light extinction coefficients of corn, sorghum, soybean and sunflower. Agronomy Journal 88, 185–190. Foulkes, M.J., Sylvester-Bradley, R., & Scott, R.K. (1998) Evidence for differences between winter wheat cultivars in acquisition of soil mineral nitrogen and uptake and utilization of applied fertiliser nitrogen. Journal of Agricultural Science, Cambridge 130, 29–44.

79

Foulkes, M.J., Sylvester-Bradley, R., Weightman, R., & Snape, J.W. (2007) Identifying physiological traits associated with improved drought resistance in winter wheat. Fields Crop Research 103, 11–24. Foulkes, M.J., Reynolds, M.P., & Sylvester-Bradley, R. (2009a) Genetic improvement of grain crops: yield potential. In: Crop Physiology Applications for Genetic Improvement and Agronomy (eds. V.O. Sadras & D.F. Calderini), pp. 355–386, Academic Press, Elsevier, Amsterdam. Foulkes, M.J., Hawkesford, M.J., Barraclough, P.B., et al. (2009b) Identifying traits to improve the nitrogen economy of wheat: recent advances and future prospects. Field Crops Research 114, 329–342. Friedrich, J.W. & Schrader, L.E. (1979) N deprivation in maize during grain-filling. II. Remobilisation of 15 N and 35S and the relationship between N and S accumulation. Agronomy Journal 71, 466–472. Gaju, O. (2007) Identifying physiological processes limiting genetic improvement of ear fertility in wheat. PhD thesis, University of Nottingham, UK, p. 230. Giunta, F., Rosella, M., & Deidda, M. (2002) SPAD readings and associated leaf traits in durum wheat, barley and triticale cultivars. Euphytica 125, 197–205. Goudriaan, J. & Monteith, J.L. (1990) A mathematical function for crop growth based on light interception and leaf area expansion. Annals of Botany 66, 695–701. Goulding, K.W.T. (2004) Minimising losses of nitrogen from UK agriculture. Journal of Royal Agricultural Society, England 165, 1–11. Grindlay, D.J.C. (1997) Towards an explanation of crop nitrogen demand based on the optimisation of leaf nitrogen per unit leaf area. Journal of Agricultural Science, Cambridge 128, 377–396. Guarda, G., Padovan, S., & Delogu, G. (2004) Grain yield, nitrogen-use efficiency and baking quality of old and modern Italian bread-wheat cultivars grown at different nitrogen levels. European Journal of Agronomy 21, 181–192. Hageman, R.H. (1986) Nitrate metabolism in roots and leaves. In: Regulation of Carbon and Nitrogen Reduction and Utilization in Maize (eds. J.C. Shannon, D.P. Knievel, & C.D. Boyer), pp. 105–116, Waverly Press, Baltimore, MD. Hikosaka, K. & Terashima, I. (1995) A model of the acclimation of photosynthesis in the leaves of C3 plants to sun and shade with respect to nitrogen use. Plant, Cell & Environment 18, 605–618. Hirasawa, T., Ozawa, S., Taylaran, R.D., & Ookawa, T. (2010) Varietal differences in photosynthetic rates in rice plants, with special reference to the nitrogen content of leaves. Plant Production Science 13, 53–57.

80

NUTRIENT USE EFFICIENCY IN CROPS

Hirel, B., Bertin, P., Quillere, I., et al. (2001) Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize. Plant Physiology 125, 1258–1270. Hirel, B., Le Gouis, J., Ney, B., & Gallais, A. (2007) The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. Journal of Experimental Botany 58, 2369–2387. Hirose, T. (2005) Development of the Monsi-Saeki theory on canopy structure and function. Annals of Botany 94, 483–495. Hirose, T. & Werger, M.J.A. (1987) Maximizing daily canopy photosynthesis with respect to the leaf nitrogen allocation pattern in the canopy. Oceologia 72, 520–526. Jamieson, P.D. & Semenov, M.A. (2000) Modelling nitrogen uptake and redistribution in wheat. Field Crops Research 68, 21–29. Kajer, B. & Jensen, J. (1995) The inheritance of nitrogen and phosphorous content in barley analyzed by genetic markers. Hereditas 123, 109–119. Kichey, T., Holme, I., Møller, I.S., Jahn, T.P., Holm, P.B., & Schjoerring, J.K. (2009) Improving nitrogen use efficiency in barley (Hordeum vulgare L.) through the cisgenic approach. The Proceedings of the International Plant Nutrition Colloquium XVI, Department of Plant Sciences, UC Davis, UC Davis. Available at: http://escholarship.org/uc/ item/0xn9c24x. Kim, H.Y., Lieffering, M., Miura, S., Kobayashi, K., & Okada, M. (2001) Growth and nitrogen uptake of CO2-enriched rice under field conditions. New Phytologist 150, 223–230. LaPerche, A., Brancourt-Hulmel, M., Heumez, E., Gardet, O., & Le Gouis, J. (2006) Estimation of genetic parameters of a DH wheat population grown at different N stress levels characterized by probe genotypes. Theoretical and Applied Genetics 112, 797–807. Li, Z., Li, B., & Tong, Y. (2008) The contribution of distant hybridization with decaploid Agropyron elongatum to wheat improvement in China. Journal of Genetics and Genomics 35, 451–456. Long, S.P., Zhu, X.-G., Naidu, S.L., & Ort, D.R. (2006) Can improvement in photosynthesis increase crop yields? Plant, Cell & Environment 29, 315–330. Lopes, M.S., Cortadellas, N., Kichey, T., Dubois, F., Habash, D.Z., & Araus, J.L. (2006) Wheat nitrogen metabolism during grain filling: comparative role of glumes and the flag leaf. Planta 225, 165–181. López-Castañeda, C., Richards, R.A., Farquhar, G.D., & Williamson, R.E. (1996) Seed and seedling characteristics contributing to variation in early vigor

among temperate cereals. Crop Science 36, 1257–1266. Manske, G.G.B., Ortiz-Monasterio, J.I., van Ginkel, R.M., et al. (2002) Phosphorus use efficiency in tall, semi-dwarf and dwarf near-isogenic lines of spring wheat. Euphytica 125, 113–119. Masclaux, C., Quillere, I., Gallais, A., & Hirel, B. (2001) The challenge of remobilisation in plant nitrogen economy. A survey of physio-agronomic and molecular approaches. Annals of Applied Biology 138, 69–81. Mitchell, P.L., Sheehy, J.E., & Woodward, F.I. (1998) Potential Yields and Efficiency of Radiation Use in Rice. IRRI Discussion Paper Series No. 32. International Rice Research Institute. Manila, Philippines, p. 62. Moll, R.H., Kamprath, E.J., & Jackson, W.A. (1982) Analysis and interpretation of factors which contribute to efficiency to nitrogen utilization. Agronomy Journal 75, 562–564. Monneveux, P., Zaidi, P.H., & Sanchez, C. (2005) Population density and low nitrogen affects yieldassociated traits in tropical maize. Crop Science 45, 535–545. Monteith, J.L. (1977) Climate and the efficiency of crop production in Britain. Philosophical Transactions of the Royal Society of London. Series B 281, 277–294. Mooney, H.A. & Gulmon, S.L. (1979) Environmental and evolutionary constraints on the photosynthetic characteristics of higher plants. In: Topics in Plant Population Biology (eds. O.T. Solbrig, S. Jain, G.B. Johnson & P.H. Raven), pp. 316–337, Columbia University Press, New York. Murchie, E.H., Hubbart, S., Chen, Y.Z., Peng, S.B., & Horton, P. (2002) Acclimation of rice photosynthesis to irradiance under field conditions. Plant Physiology 130, 1999–2010. Murchie, E.H., Pinto, M., & Horton, P. (2009) Agriculture and the new challenges for photosynthesis research. New Phytologist 181, 532–552. Muurinen, S., Slafer, G.A., & Peltonen-Sainio, P. (2006) Breeding effects on nitrogen use efficiency of spring cereals under northern conditions. Crop Science 46, 561–568. Nunes-Nesi, A., Carrari, F., Lytovchenko, A., et al. (2005) Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants. Plant Physiology 137, 611–622. Ogola, J.B.O., Wheeler, T.R., & Harris, P.M. (2002) Effects of nitrogen and irrigation on water use of maize crops. Field Crops Research 78, 105– 117. Ortiz-Monasterio, J.I., Sayre, K.D., Rajaram, S., & McMahom, M. (1997) Genetic progress in wheat

OPTIMIZING CANOPY TRAITS TO IMPROVE NUTRIENT EFFICIENCY

yield and nitrogen use efficiency under four nitrogen rates. Crop Science 37, 898–904. Ortiz-Monasterio, J.I., Manske, G.G.B., & van Ginkel, M. (2001) Nitrogen and phosphorus use efficiency. In: Application of Physiology in Wheat Breeding (eds. M.P. Reynolds, J.I. Ortiz-Monasterio, & A. McNab), pp. 200–207, CIMMYT, Mexico. Otegui, M.E. & Bonhomme, R. (1998) Grain yield components in maize I. Ear growth and kernel set. Field Crops Research 56, 247–256. Parry, M.A.J., Andralojc, P.J., Mitchell, R.A.C., Madgwick, P.J., & Keys, A.J. (2003) Manipulation of Rubisco: its amount, activity, function and regulation. Journal of Experimental Botany 54, 1321–1333. Parry, M.A.J., Madgwick, P.J., Carvalho, J.F.C., & Andralojc, P.J. (2007) Prospects for increasing photosynthesis by overcoming the limitations of Rubisco. Journal of Agricultural Science 145, 31–43. Parry, M.A.J., Reynolds, M.P., Salvucci, M.E., Raines, C., Andralojc, P.J., Zhu, X-G., Price, G.D., Condon, A.G., & Furbank, R.T. (2011) Raising yield potential of wheat. II. Increasing photosynthetic capacity and efficiency. Journal of Experimental Botany 62, 453–467. Pask, A. (2009) Optimising nitrogen storage in wheat canopies for genetic reduction in fertiliser nitrogen inputs. PhD thesis, University of Nottingham, U.K. Peng, S.B., Huang, J.L., Sheehy, J.E., et al. (2004) Rice yields decline with higher night temperature from global warming. Proceedings of the National Academy of Sciences of the United States of America 101, 9971–9975. Peng, B., Huang, J.L., Zhong, X.Y., et al. (2006) Strategies for overcoming low agronomic nitrogen use efficiency in irrigated rice systems in China. Field Crops Research 96, 37–47. Pons, T.L., Van Rijnberk, H., Scheurwater, I., & Van der Werf, A. (1993) Importance of the gradient in photosynthetically active radiation in a vegetation stand for leaf nitrogen allocation in two monocotyledons. Oecologia 95, 416–424. Rebetzke, G.L. & Richards, R.A. (1999) Genetic improvement of early vigour in wheat. Australian Journal of Agricultural Research 50, 291–301. Rebetzke, G.J., Condon, A.G., Richards, R.A., & Farquhar, G.J. (2002) Selection for reduced carbonisotope discrimination increases aerial biomass and grain yield of rainfed bread wheat. Crop Science 42, 739–745. Reynolds, M.P., Van Ginkel, M., & Ribaut, J.M. (2000) Avenues for genetic modification of radiation use efficiency in wheat. Journal of Experimental Botany 51, 459–473.

81

Reynolds, M.P., Pellegrineschi, A., & Skovmand, B. (2005) Sink-limitation to yield and biomass: a summary of some investigations in spring wheat. Annals of Applied Biology 146, 39–49. Samonte, S., Wilson, L.T., Medley, J.C., Pinson, S.R.M., McClung, A.M., & Lales, J.S. (2006) Nitrogen utilization efficiency: relationships with grain yield, grain protein, and yield-related traits in rice. Agronomy Journal 98, 168–176. Semenov, M.A., Jamieson, P.D., & Martre, P. (2007) Deconvoluting nitrogen use efficiency in wheat: a simulation study. European Journal of Agronomy 26, 283–294. Shearman, V.J., Sylvester-Bradley, R., Scott, R.K., & Foulkes, M.J. (2005) Physiological changes associated with wheat yield progress in the UK. Crop Science 45, 165–175. Silva, S.A., de Carvalho, F.I.F., Caetano, V.R., et al. (2000) Genetic basis of stay-green trait in bread wheat. Journal of New Seeds 2, 55–68. Sinclair, T.R. & De Wit, C.T. (1975) Photosynthate and nitrogen requirements for seed production by various crops. Science 189, 565–567. Sinclair, T.R. & Horie, T. (1989) Leaf nitrogen, photosynthesis and crop radiation use efficiency: a review. Crop Science 29, 90–98. Sinclair, T.R. & Jamieson, P.D. (2006) Grain number, wheat yield, and bottling beer: an analysis. Field Crops Research 98, 60–67. Sinclair, T.R. & Sheehy, J.E. (1999) Erect leaves and photosynthesis in rice. Science 283, 1456–1457. Sinebo, W., Gretzmacher, S., & Edelbauer, A. (2004) Genotypic variation for nitrogen use efficiency in Ethiopian barley. Field Crops Research 85, 43–60. Slafer, G.A., Kantolic, A.G., Appendino, M.L., Miralles, D.J., & Savin, R. (2009) Crop development: genetic control, environmental modulation and relevance for genetic improvement of crop yield. In: Crop Physiology: Applications for Genetic Improvement and Agronomy (eds. V.O. Sadras & D.F. Calderini), pp. 277–308, Elsevier, The Netherlands. Spano, G., Di Fonzo, N., Perrotta, C., et al. (2003) Physiological characterization of “stay green” mutants in durum wheat. Journal of Experimental Botany 54, 1415–1420. Sylvester-Bradley, R. & Kindred, D.R. (2009) Analysing nitrogen responses of cereals to prioritize routes to the improvement of nitrogen use efficiency. Journal of Experimental Botany 60, 1939–1951. Sylvester-Bradley, R., Stokes, D.T., Scott, R.K., & Willington, V.B.A. (1990) A physiological analysis of the diminishing responses of winter wheat to applied nitrogen. 2. Evidence. Aspects of Applied Biology 25, 289–300.

82

NUTRIENT USE EFFICIENCY IN CROPS

Takai, T., Matsuura, S., Nishio, T., Ohsumi, T., Shiraiwa, T., & Horie, T. (2006) Rice yield potential is closely related to crop growth rate during late reproductive period. Field Crops Research 96, 328–335. Tambussi, E.A., Nogués, S., Ferrio, P., Voltas, J., & Araus, J.L. (2005) Does higher yield potential improve barley performance in Mediterranean conditions? A case study. Field Crops Research 91, 149–160. Tester, M. & Langridge, P. (2010) Breeding technologies to increase crop production in a changing world. Science 327, 818–822. Thomas, H. & Smart, C.M. (1993) Crops that stay green. Annals of Applied Biology 123, 193– 219. Thorne, G.N., Pearman, W., Day, W., & Todd, A.D. (1988) Estimation of radiation interception by winter wheat from measurements of leaf area. Journal of Agricultural Science, Cambridge 110, 101–108. Tirol-Padre, A., Ladha, J.K., Singh, U., Laureles, E., Punzalan, G., & Akita, S. (1996) Grain yield performance of rice genotypes at suboptimal levels of soil N as affected by N uptake and utilization efficiency. Field Crops Research 46, 127–143. Tollenaar, M., Ying, J., & Duvick, D.N. (2000) Genetic gain in corn hybrids from the northern and central corn belt. Proc. 55th Corn Sorghum Res. Conf., Chicago, IL. 5–8 Dec. 2000. pp. 53–62. ASTA, Washington, DC. Triboï, E. & Ollier, J. (1991) Kinetic and role of carbon and nitrogen stored in stem on 21 wheat genotypes. Agronomie 11, 239–246. Uauy, C., Distelfeld, A., Fahima, T., Blechl, A., & Dubcovsky, J. (2006) A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314, 1298–1301. Uemura, K., Anwaruzzaman, S., & Yokota, A. (1997) Ribulose-1, 5-bisphosphate carboxylase/oxygenase from thermophilic red algae with strong specificity for CO2 fixation. Biochemical and Biophysical Research Communications 233, 568–571.

van Oosterom, E.J., Borrell, A.K., Chapman, S.C., Broad, I.J., & Hammer, G.L. (2010a) Functional dynamics of the nitrogen balance of sorghum: I. N demand of vegetative plant parts. Field Crops Research 115, 19–28. van Oosterom, E.J., Borrell, A.K., Chapman, S.C., Broad, I.J., & Hammer, G.L. (2010b) Functional dynamics of the nitrogen balance of sorghum. II. Grain filling period. Field Crops Research 115, 29–48. Varlet-Grancher, C., Gosse, G., Chartier, M., Sinoquet, H., Bonhomme, R., & Allirand, J.M. (1989) Mise au point: royonnement solaire absorbe ou intercepte par un covert vegetal. Agronomie 9, 419–439. Verma, V., Foulkes, M.J., Caligari, P., SylvesterBradley, R., & Snape, J. (2004) Mapping quantitative trait loci for flag leaf senescence as a yield determinant in winter wheat under optimal and drought-stressed environments. Euphytica 135, 255–263. Whaley, J.M., Sparkes, D.L., Foulkes, M.J., Spink, J.H., Semere, T., & Scott, R.K. (2000) The physiological response of winter wheat to reductions in plant density. Annals of Applied Biology 137, 167–178. Wright, G.C. & Hammer, G.L. (1994) The allocation pattern of leaf nitrogen throughout a crop canopy can theoretically affect crop photosynthetic performance and radiation use efficiency (RUE). Australian Journal of Agricultural Research 45, 565–574. Yang, W., Peng, S., Laza, R.C., Visperas, R.M., & Dionisio-Sese, M.L. (2007) Grain yield and yield attributes of new plant type and hybrid rice. Crop Science 49, 1393–1400. Zhu, X.-G., Long, S.P., & Ort, R.D. (2008) What is the maximum efficiency with which photosynthesis can convert solar energy into biomass? Current Opinion in Biotechnology 19, 153–159.

Chapter 5

Senescence and Nutrient Remobilization in Crop Plants Per L. Gregersen

Abstract

Introduction

In one respect, nutrient use efficiency in seed crop plants is about bringing as much as possible of the available nutrients in the soil into the harvested seed. However, before most of the nutrients reach the seed, they have to reside temporarily in the vegetative parts of the plant, partially in order to make these parts photosynthetically productive. The senescence process, that is, the dyingoff of vegetative plant parts during seed maturation, is at the core of the nutrient use efficiency issue, as the nutrients need to be remobilized from these parts and translocated into the developing seed. This process is highly controlled, with important regulatory components being plant hormones, sensing the water and nutrient status of the soil and other stress impacts, and gene transcription factors that cause massive changes in gene expression patterns, which eventually control degradation processes in and export of nutrients from the senescing tissues.

The typical agricultural crop plants, such as the cereals, are annual and monocarpic, meaning that seed setting and maturation is the end result of a whole-plant senescence process in which the plant dies and the only surviving living parts are the embryo and specialized tissues of the seed. These living tissues are necessary in order to initiate the germination process as the start of the next round of the life cycle. The transition from the stage of a fully developed, photosynthesizing green plant to a completely senesced plant with mature seeds is a complex process, involving the translocation of photoassimilates and nutrients from the vegetative plant parts to the developing seeds and the regulated dying-off of the vegetative parts. The process can take place stepwise in the way that senescence starts at the organ level, in particular in lower, shaded leaves, whereby nutrients are remobilized, not necessarily to the developing seeds, but also to younger leaves that are still actively photosynthesizing (Fig. 5.1). Since the senescence

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 83

84

NUTRIENT USE EFFICIENCY IN CROPS

N N

N

N

N

Fig. 5.1. The senescence process in cereals. The leaves senesce from the lower parts of the plants while translocat-

ing nutrients, in particular nitrogen (N), to other green tissues or to the developing grain. At maturity, nutrients are concentrated in the grains. The efficiency of this process is genotype-specific.

process involves massive translocations of nutrients within the plant, it is rather obvious to consider this process in relation to nutrient use efficiency of crop plants (Hirel et al., 2007; Foulkes et al., 2009). The general senescence process has been reviewed by a number of authors (e.g., Thomas and Stoddart, 1980; Noodén, 1988; Feller and Fischer, 1994; Thomas, 1994; Buchanan-Wollaston, 1997; BuchananWollaston et al., 2003), and the general understanding of the senescence process as a genuine part of plant developmental processes is well established. This chapter aims to bridge the gap between the many crop physiological studies performed on senescence/nutrient remobilization and the biochemical and molecular studies that over the recent two decades have started to unravel the complex mechanisms of the senescence process and its regulation, which are still only vaguely understood. In particular, the regulation of senescence has been so far a rather black box, with a range of suggestions for important regulatory components, notably hormones (e.g., Schippers

et al., 2007) and transcription factors (e.g., Balazadeh et al., 2008). If future developments in plant productivity and nutrient use efficiency are to involve the regulation of the senescence process, which this chapter intends to show is an important aim, then clear knowledge about the regulatory mechanisms is crucial. There is still more to learn in this context, but a rather large bulk of insights in the processes is already now available from a tremendous amount of crop physiology studies and from emerging studies on the molecular biology and biochemistry of the senescence process. Since considerable investigations have been performed in the cereals on the aspects of nutrient remobilization from senescing leaf tissues, reference to these agronomically important crop species will be predominant. This comprises studies on hormonal regulation of senescence and transport of amino acids that go back several decades, as well as more recent studies on molecular processes at the level of gene regulation. However, the presentation is of a general nature and will include other species where relevant.

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

The senescence process The senescence process of plants is evident to the naked eye as a progressive yellowing of the leaves, followed by a rapid necrotization phase that ends in the wilting of the leaves, and eventually of the whole plant. At the whole-plant level, senescence usually proceeds acropetally, starting from the lower leaves and working its way up the plant (e.g., van Oosterom et al., 2010); at the single leaf level, senescence usually starts at the tip, working its way back to the leaf base, most typically seen in the elongated leaves of grasses (Fig. 5.1). Thus, the visual phenotype is quite clear; however, in order to more precisely quantify the degree of senescence, more precise measurements are required, and in most cases the methods for this reflect the dismantling of the photosynthetic apparatus during yellowing of the leaf, for example, measurements of photosynthetic activity (O2-release or Fv/Fm (maximum quantum efficiency)), chlorophyll content, or abundance of chloroplast proteins (e.g., Humbeck et al., 1996). Stay-green phenotypes The yellowing of leaves during senescence reflects chlorophyll degradation, which, however, is not always correlated with the degradation processes of senescence since a number of stay-green phenotypes have been described where leaves maintain the green color but lose the chloroplastic capacity to conduct photosynthesis (Thomas and Howarth, 2000; Hörtensteiner, 2009). These nonfunctional (“cosmetic”) stay-green phenotypes are caused by defects in the degradation pathway of chlorophyll (Park et al., 2007; Schelbert et al., 2009) but not necessarily in other degradation processes of senescence. As opposed to this, functional stay-green phenotypes imply a delayed senescence with sustained photosynthetic capacity and hence a delay in degradation of the plant tissue. There are a number of crop

85

varieties described with strong phenotypes of this kind showing increased biomass production (e.g., Spano et al., 2003; Gong et al., 2005; Yoo et al., 2007). In addition, there is long tradition for studies on green leaf area duration in cereals, that is, differences in senescence phenotypes, and its importance for the yield potential of cereal crops (e.g., Lupton et al., 1974; Helsel and Frey, 1978; Rawson et al., 1983; Tollenaar and Wu, 1999; Borrell et al., 2000; Kichey et al., 2007). Environmental modulation of senescence The growth and development of a plant is always influenced by the environment in which it resides. Only experimentally is it possible to create near-stress-free environments, which result in extensive vegetative growth and delayed seed maturation. In nature and in agricultural systems, plants usually meet constraints from the environment, in particular related to the annual climate cycle and fluctuating weather conditions, which makes water availability and temperatures essential environmental factors affecting the senescence process (e.g., Wolfe et al., 1988). The environmental modulation of senescence is a finetuning of the genetic predisposition of the plant to age and senesce, supposedly in order to make the plant able to cope with a fluctuating environment. The intrinsic senescence properties of a crop variety result from the genetic adaptation to the regional environment in which the plant grows, in order to make it possible for it to complete its life cycle successfully, even under adverse conditions. A typical example is the traditional durum wheat that is adapted to Mediterranean conditions with dry and hot summers. Old landraces of durum wheat from the region typically have an early and rapid senescence, adapted to the dry and hot environment (Hafsi et al., 2000). The genetic element of senescence in modern crop cultivars is indirectly subject to selec-

86

NUTRIENT USE EFFICIENCY IN CROPS

tion via plant breeding, along with important parameters for flowering control and earliness (Snape et al., 2001), so that the maturation profile of the crop fits the average environmental conditions and agricultural practices of the region where the crop is grown. In addition to water and temperatures, the nutrient status of the soil is an important environmental factor that affects senescence. Limitations in nutrients, in particular nitrogen (N), will cause early senescence and surplus of nitrogen can delay senescence to a degree that is unfavorable to the crop, causing, for example, lodging problems (Yang and Zhang, 2006). The high degree of genetic– environmental interaction for the senescence process implies a considerable overlap between senescence regulation and the response to abiotic stresses (see below on the regulation of senescence). This brings senescence regulation to the heart of the development of environmentally robust plants. Remobilization of nutrients The remobilization of nutrients is at the core of the senescence process when dealing with the issue of nutrient use efficiency. By remobilization, we shall refer here to the process of reallocation of a nutrient from an organ where it has resided for a shorter or longer period, either as part of structural components (e.g., nitrogen in structural proteins) or as part of steady-state levels of compounds in metabolic fluxes. A prevailing general consensus is that the overall physiological role of senescence consists of this remobilization process, where nutrients, via translocation from the senescing organ, are made available to other, developing organs, for example, seeds. Another physiological role of senescence could be a “scorched earth” strategy of the plant where minimal nutrients are left for opportunistic pathogens to grow on, thus preventing harmful pathogen attacks or pathogen buildup (Thomas, 1994).

The remobilization of carbon as a nutrient from the leaves poses a special case, insofar as this remobilization takes place from fully photosynthesizing leaves, in the form of photoassimilates translocated to the seed, for example, during grain filling of cereals. However, the remobilization can also take place from temporary stores of carbohydrates, for example, water-soluble carbohydrates from the stems of cereals (Yang et al., 2001; Ehdaie et al., 2006). For the latter, the mechanisms and regulation of the remobilization are presumably similar to that for minerals (see below) and as such this carbon remobilization may be an important aspect of the senescence process in relation to crop yields (Yang and Zhang, 2006). Nitrogen is the main limiting nutrient for the growth and development of plants, and for many crops a high content in the harvested seed is also an important quality parameter (Frink et al., 1999). Hence, this nutrient element has been a major focus in studies of remobilization of nutrients during senescence. Numerous studies over several decades have shown that senescing leaves in maturing crop plants are depleted of nitrogen (or of nitrogen-containing protein), thus demonstrating that nitrogen is remobilized from the leaves (e.g., Feller et al., 1977; Waters et al., 1980; Simpson et al., 1983; Dreccer et al., 1997; Kichey et al., 2006; Howarth et al., 2008). Furthermore, several studies have shown that a large part of this remobilized nitrogen is translocated to the developing seed, demonstrated either indirectly from mass flows of nitrogen or directly in radiolabeling experiments (Palta and Fillery, 1995; Kichey et al., 2007). Typically, only 10–30% of the nitrogen (protein) in fully developed cereal leaves is retained in the senesced leaves, whereas most of the remaining part is remobilized. However, in stay-green phenotypes, the retained nitrogen level is higher; thus, Heidlebaugh et al.

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

(2008) showed that a slow senescing barley variety retained more than 50% of the nitrogen in the flag leaves at 35 days after anthesis, that is, close to seed maturity. A minor part of the nitrogen content of the green leaf might also be lost to the atmosphere as ammonia and hence not translocated to the seed (Schjoerring et al., 1993). Studies on remobilization during senescence of nutrients other than N are sparser. Nevertheless, a number of studies have shown that most other nutrients, to varying degrees, are remobilized in parallel to nitrogen from senescing leaves (Hill et al., 1979; Mauk and Nooden, 1992; Pearson and Rengel, 1994; Himelblau and Amasino, 2001; Garnett and Graham, 2005; Howarth et al., 2008). Major exceptions seem to be calcium and manganese, which appear to be immobile nutrients in senescing leaves of wheat (Hill et al., 1979; Pearson and Rengel, 1994) and Arabidopsis (Himelblau and Amasino, 2001). Different reports on the remobilization of non-nitrogen elements show inconsistent results for some of the elements; for example, iron is in some cases shown to be efficiently remobilized from wheat flag leaves (Garnett and Graham, 2005) and in others not (Birsin et al., 2010). Species specific mechanisms might be involved here, and additionally the nutritional status of the growth medium might influence the remobilization of the different elements. The dynamics of the remobilization of the different nutrient elements show different patterns, probably reflecting different mechanisms, for example, different transporter systems for export from senescing tissues. Importantly, the two major nonnitrogen elements, potassium and phosphorous, seem to be efficiently remobilized in cereals to the same extent as nitrogen (e.g., Hill et al., 1979; Birsin et al., 2010), although there are conflicting results for potassium, with some reports showing low remobilization.

87

Degradation and transport It is well established that the remobilization of nutrients from vegetative plant parts is a result of degradation processes in which low-molecular-weight breakdown products of cell components are exported from the senescing tissue. The dismantling of chloroplasts and their endomembrane systems are central to these processes in leaves since the chloroplasts contain up to 75% of the nitrogen contained in photosynthetically active cells (Hörtensteiner and Feller, 2002). In the chloroplasts, the fate of Rubisco during senescence has gained special attention since this protein comprise around 50% of the total mass of leaf proteins (Feller et al., 2008). However, in addition, the proteins associated with the light-harvesting complexes constitute a major fraction of the total leaf protein, and, hence, the breakdown of chlorophylls to release this nitrogencontaining protein pool for degradation and remobilization has been of major interest (Matile et al., 1996; Hörtensteiner, 2009).

Proteolysis Proteolytic activities in senescing plant tissues have been studied for decades (Wittenbach, 1979) and are found to be heavily upregulated during the senescence process (reviewed by, e.g., Hörtensteiner and Feller, 2002; Gregersen et al., 2008; Martinez et al., 2008). Over the last decade these results have been supported by a number of studies on gene expression that show transcriptional upregulation of a wide range of genes encoding proteolytic enzymes (e.g., Gepstein et al., 2003; BuchananWollaston et al., 2005; Gregersen and Holm, 2007). In addition to protease activity, there are indications that some of the initial fragmentations of Rubisco can take place via the action of reactive oxygen species (Nakano et al., 2006). This is in accordance with

88

NUTRIENT USE EFFICIENCY IN CROPS

observations that reduced production of reactive oxygen species from the photosystems in chloroplasts can delay the senescence process in tobacco (Zapata et al., 2005, 2007). The focus of studies on the degradation processes has shifted more recently to the mechanisms of the process, that is, the sequence of reactions starting from the first steps of protein degradation in the chloroplasts to complete dismantling of the chloroplast and proteins associated with the chloroplast endomembrane system (Martinez et al., 2008). The picture is emerging that the first steps are governed by chloroplast intrinsic proteases, for example, FtsH (Zelisko et al., 2005) or Clp proteases (Nakashima et al., 1997), which are involved in the turnover of chloroplast proteins during normal, nonsenescing conditions (Kato and Sakamoto, 2009). A chloroplast-located senescence-induced protease activity was also shown for an aspartase protease in tobacco (Kato et al., 2005). Nevertheless, only for a few of the chloroplast-located proteases has the involvement in senescencerelated proteolysis been supported, and the main bulk of senescence-associated genes in this context, in fact, encode extraplastidial proteases (Martinez et al., 2008). Thus, internal chloroplast proteolysis appears to be followed or accompanied by a far more extensive extraplastidial proteolysis, which is also indicated from studies on wholetissue protease activities, where the major activity is caused by vacuolar proteases (e.g., Lin and Wittenbach, 1981; Martinez et al., 2007). The protein content of degraded chloroplasts thus appears to be moved from the plastid to the central vacuole for final degradation; this is supported by several cytological and biochemical studies (see review by Martinez et al., 2008). Different vesicular bodies containing chloroplast-derived proteins have been shown to occur in leaf cells during leaf

senescence, for example, Rubisco-containing bodies (Chiba et al., 2003) and senescenceassociated vacuoles (SAV) (Otegui et al., 2005). How these bodies are derived from the chloroplasts or their fate during the senescence process is not clear. They may fuse with the central vacuole, leaving the final phase of the proteolytic process to the proteases of this compartment, or proteolysis might proceed in the bodies themselves (see discussion in Gregersen et al., 2008). The autophagy (ATG) pathway might play a role in this process, insofar as ATG genes have been shown to be upregulated during senescence (Buchanan-Wollaston et al., 2005; Gregersen and Holm, 2007). This pathway is generally believed to be a mechanism for the bulk turnover of proteins and organelles in eukaryotes under stress and starvation conditions, as opposed to the proteasome pathway that specifically degrades regulatory components of the cell, for example, transcription factors (Kwon and Park, 2008). Recent reports showing impaired movement of proteins from chloroplasts to Rubiscocontaining bodies, and further on to the central vacuole in an atg mutant of Arabidopsis, substantiate a role for the ATG pathway in senescence-associated degradation of chloroplasts (Ishida et al., 2008; Wada et al., 2009). However, other atg mutants show an earlier senescence phenotype than wild-type plants (Doelling et al., 2002), and the role of the autophagy pathway in chloroplast degradation is hence disputable. Fatty acid degradation The breakdown of the chloroplast endomembrane systems and other membranes of the cell evidently also involves fatty acid degradation. The studies on gene expression during senescence clearly show upregulation of genes encoding a range of lipases and enzymes for the β-oxidation of fatty acids (Buchanan-Wollaston et al., 2003, 2005;

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

Gregersen and Holm, 2007). Furthermore, the biochemical evidence for lipid and fatty acid degradation is ample (e.g., Thompson et al., 1998), and there is a clear involvement of enzymes in the classical β-oxidation pathway (Yang and Ohlrogge, 2009). These enzyme systems are located in the peroxisomes, whose role appears to change considerably from being involved in photorespiratory metabolism in green leaves to being involved in fatty acid degradation and in the glyoxylate cycle in senescing leaves (Pracharoenwattana and Smith, 2008). Different studies on the glyoxylate cycle, however, show ambiguous results since the activities of enzymes in this cycle seem to increase in barley leaves during senescence (Gut and Matile, 1988), whereas they do not seem affected in Arabidopsis (Charlton et al., 2005). Evidently, the upregulated fatty acid degradation and glyoxylate cycle remobilize some of the carbon elements captured in the high-molecular-weight fatty acids from membranes; however, at the physiological level, the importance of this remobilization presumably relates to the generation of C-skeletons for carboxylic and amino acid metabolism and is thus primarily important for nitrogen remobilization (Gregersen and Holm, 2007). Hence, via the web of reactions originating in the peroxisomes, the mitochondria, and the linking cytosolic reactions of carboxylic acid metabolism, C-skeletons are channeled into amino acids that are amenable for export from the senescing cell. In this web of reactions, where amino acid transferases play a major role, 2-oxoglutarate (2-OG) comprises a central compound hub, constituting the main C-skeleton source for amino acid synthesis (Hodges, 2002; Foyer et al., 2003). In nonsenescing tissue, the origin of 2-OG is primarily the mitochondrial tricarboxylic acid cycle; however, in senescing tissue, it could as well be derived via a cytosolic route from the citrate gener-

89

ated in peroxisomal fatty acid degradation (Gregersen and Holm, 2007). This would mean a direct integration of degradation of fatty acids and proteins with the pathways catalyzing the efficient remobilization of nitrogen in the senescing tissue (Fig. 5.2). Amino acid metabolism During protein turnover and degradation, there is a release of ammonia in leaf tissues, which is accelerated considerably in senescing leaves. This ammonia can be channeled into amino acid structures again via the action of glutamine synthetases (GS) that appear to play a central role in the remobilization of nitrogen from senescing leaves (Martin et al., 2006; Tabuchi et al., 2007; Bernard and Habash, 2009). A number of studies have shown that during the late stages of leaf senescence the cytosolic form of GS, GS1, is upregulated at the transcriptional level (e.g., Buchanan-Wollaston et al., 2005; Gregersen and Holm, 2007; Bernard et al., 2008). However, the total GS activity of the senescing leaf decreases in senescing leaves (Kichey et al., 2006), evidently because the chloroplast-located GS2 is disappearing along with the breakdown of chloroplasts. There are more isoforms of GS1, presumably reflecting tissue-specific expression. Thus, for maize, it was shown by Martin et al. (2006) that one isoform, GS1-3, was expressed in mesophyll cells, whereas another, GS1-4, was expressed in bundle sheath cells. The central role of the cytosolic GS for nitrogen remobilization has been demonstrated in knockout lines of maize (Martin et al., 2006) and of rice (Tabuchi et al., 2005), which both showed impairments of plant growth. Supporting these results, constitutive overexpression of GS1-3 in maize (Martin et al., 2006) showed increased grain yield; however, this was not shown in a similar approach for rice GS1, which instead

90

NUTRIENT USE EFFICIENCY IN CROPS

Cellular components Chloroplasts, membranes, cell walls

Protein degradation

Fatty acid degradation

Proteases, autophagy, ubiquitin pathway

lipases, β-oxidation

Amino acids, NH4+

Acetyl-CoA oxaloacetate

AAT

glutamate

2-oxoglutarate

GS

glutamine

Amino acids for export

Fig. 5.2. The integration of protein and fatty acid degradation during the senescence process for the efficient remobilization of nitrogen. Cellular components are broken down via specific pathways and C-skeletons for amino acid formation are provided from acetyl-CoA. Amino acid transferases (AAT) play an important role in redistributing amino groups among different C-skeletons, eventually leading to the formation of amino acids that can be exported from the senescing tissue. A central process for the reassimilation of ammonia is the formation of glutamine from glutamate by glutamine synthetase (GS).

showed decreases in grain yield (Cai et al., 2009). These conflicting results may have been due to different levels of constitutive expression in the two situations, emphasizing that fine-tuning of expression should presumably be made in overexpressing strategies for GS-1. Nevertheless, the results from manipulating GS expression illustrate that manipulations of key factors in the amino- and carboxylic acid metabolism look promising with respect to improving nutrient use efficiency (Hirel et al., 2007). Transport As a reflection of the central role of GS1 in generation of amino acids during the late phase of senescence, glutamine appears as a

predominant amino acid in senescing leaves (Simpson and Dalling, 1981; Howarth et al., 2008) and even more in phloem exudates from the leaves (Simpson and Dalling, 1981; Hayashi and Chino, 1990). Hence, the export of amino acids, that is, of nitrogen, from the senescing tissue seems highly controlled. Specific transporters for the exported compounds presumably play a role in this process, and in fact results from gene expression profiling studies of senescing tissues show upregulation of a number of transporters, including not only amino acid transporters but also transporters for elements/ compounds such as potassium and phosphate (Buchanan-Wollaston et al., 2005; Van der Graaff et al., 2006; Gregersen and Holm, 2007; P.L. Gregersen, unpublished data for

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

senescing barley leaves). The transport must take place over compartmental membranes such as the tonoplast (e.g., TIPs transporting water and ammonia [Jahn et al., 2004]) and across plasma membranes, for example, facilitating transport from mesophyll cells to bundle sheath cells; eventually it must involve loading of compounds or elements into the phloem for the long distance transport out of the leaf. The specific role and importance in senescing tissues of most of the putative transporters is not known, but the clear transcriptional upregulation strongly suggests an involvement of these transporters in active transport mechanisms, even though the putative functions are based on gene sequence homologies. In addition to transporters, a number of genes appear upregulated for the generation of chelating factors of minerals, such as deoxymugineic acid and nicotianamine, that chelate zinc and iron (Tauris et al., 2009) and hence may be important for the remobilization of these minerals from senescing leaves. Regulation of senescence The cell death that terminates the senescence process has similarities to programmed cell death (PCD) processes in plants, and many authors thus describe the terminating cell death of senescence as a type of PCD (e.g., van Doorn and Woltering, 2004; Lim et al., 2007). The very last necrotization phase of senescence might thus be regulated in similar ways to PCD processes described in other contexts, for example, for the hypersensitive response in disease resistance responses (Lam et al., 2001). However, PCD is essentially a cell-confined process, whereas the senescence process, from initiation to cell death, involves the interplay between organs at the whole-plant level, that is, nutrient and water status in the soil, environmental stress factors, and the development of reproductive organs. Hence, the senescence process in the individual organs and tissues depends highly

91

on regulation at the whole-plant physiological level. A major component in this wholeplant regulation appears to be the hormonal signaling system of the plant that sets the scene for downstream changes in gene expression patterns. Hormonal regulation of senescence The hormonal regulation of senescence, in particular in relation to drought responses, has been studied for decades, and especially the role of abscisic acid (ABA) has been a focus (reviewed by Wilkinson and Davies, 2002, and Liu et al., 2005). ABA appears to be a primary signal produced during water stress in the roots and also involved in hightemperature induction of senescence (Harding et al., 1990). ABA is initially translocated from the root via the xylem to the shoots, primarily to the leaves. Here, the primary physiological effect of ABA is the closure of stomatal openings to reduce water loss, but in addition a range of stress-related genes are turned on by ABA (Seki et al., 2002; Choudhury and Lahiri, 2010). The composition of these genes varies between experiments (Choudhury and Lahiri, 2010); however, there is a clear overlap with genes induced by drought and during senescence. In senescing leaves, a secondary wave of ABA can be produced that is translocated to other parts of the plant, particularly in an acropetal direction (Wilkinson and Davies, 2002). There has been some controversy about the signal transduction mechanisms for ABA, in particular about the characterization of putative receptors (Pennisi, 2009). However, a promising signaling model for ABA has emerged, in which ABA is perceived by receptor proteins interacting with both ABA and a protein phosphatase 2C, thereby inhibiting the activity of the latter (Ma et al., 2009; Park et al., 2009). This inhibition causes the activation of a kinase that phosphorylates a bZIP transcription

92

NUTRIENT USE EFFICIENCY IN CROPS

factor, which subsequently turns on ABAactivated genes by interacting with ABAresponsive elements (ABRE) in the promoters of these genes (Choi et al., 2000; Fujii and Zhu, 2009). ABA constitutes a putative link between the environmentally regulated senescence processes in vegetative plant parts and the coordinated development of reproductive organs. It has been known for a long time that ABA can be transported from leaves to the developing seed (e.g., Goldbach et al., 1977), thereby contributing to the increasing amounts of ABA that are observed during the first phase of seed development (e.g., Nambara and Marion-Poll, 2003; Yang et al., 2003). ABA plays a major role in the regulatory network that controls accumulation of seed proteins, presumably via the activation of transcription factors that drive the expression of genes for the seed storage proteins (Cao et al., 2007; Verdier and Thompson, 2008). When senescence in vegetative plant parts is promoted, for example, by water deficiency, the production of ABA in and translocation from these plant parts is accelerated, which in turn could give rise to increased storage protein synthesis in reproductive organs. Hence, this model could explain the observed strong coordination of seed development with senescence progression (e.g., Okatan et al., 1981). However, ABA is not the only plant hormone, and, as is well known from physiological studies of hormonal regulation of plant development, the effects of ABA are highly influenced by the effects of other hormones during senescence, particularly cytokinins (CK) and ethylene. ABA appears as the water sensor in roots of plants, and similarly CK appears to be involved in senescence regulation as a sensor of the nitrogen status of the plant (for a review, see Sakakibara et al., 2006). CK is mainly synthesized in the roots in a nitrogen-regulated manner. Following translocation to the

leaves, the hormone has the opposite effect of ABA, that is, impairing the senescence process and the nitrogen remobilization process (Gan and Amasino, 1995; Xie et al., 2004; Criado et al., 2009). This effect has been elegantly shown via the in planta expression of the CK-forming enzyme isopentenyltransferase using senescenceinduced promoters (Gan and Amasino, 1995; Huynh et al., 2005; Rivero et al., 2009). It has been clearly demonstrated that senescing plant tissues produce ethylene, a hormore that is thought to promote senescence in plants (e.g., Zacarias and Reid, 1990). However, the interaction with ABA, the other senescence-promoting hormone, is not clear, in as much as these two hormones in other aspects of plant development act antagonistically (Lim et al., 2007). Ethylene has a senescence-promoting effect only when plants have reached a certain age (Schippers et al., 2007); hence, it seems to accelerate senescence rather than induce it. Nevertheless, treatments with ethylene clearly induce a change in gene expression patterns (Zhong and Burns, 2003), similar to senescence-associated changes, indicating a fundamental regulation of senescence by this hormone. Another way that ethylene might influence the senescence process is via its relation to the synthesis and reoxidation of ascorbic acid, an antioxidant that may have a role in delaying senescence (Garg and Kapoor, 1972; Borraccino et al., 1994). Thus, Gergoff et al. (2010) showed that ethylene treatment decreased the amount of ascorbic acid in spinach leaves. Regulation of senescence by changes in gene expression As already mentioned, a number of studies have shown that immense changes in gene expression patterns take place during the senescence process, supposedly encoding

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

the gene products necessary for the ordered degradation and transport processes taking place (Buchanan-Wollaston et al., 2003, 2005; Gregersen et al., 2008; Jukanti et al., 2008). Since changes in gene expression patterns are governed by the regulatory network of transcription factors and other regulating elements, it is evident that this level of regulation is highly important for the senescence process. Accordingly, there have been a range of reports on different transcription factors that are putatively involved as key factors in regulation. However, since it is a complex network of regulation, it is not easy to pinpoint transcription factors that are essential for senescence regulation. In a targeted experiment to analyze changes for transcription factors at the transcriptome level, Balazadeh et al. (2008) showed more than 40 transcription factors to be upregulated during senescence in Arabidopsis, which is in line with general microarray data from, for example, Buchanan-Wollaston et al. (2005), who showed upregulation of up to 100 transcription factor genes. The involvement of a network of transcription factors in the regulation of senescence emphasizes that there are many ways to modulate this process, presumably reflecting its high plasticity in relation to environmental factors. As noted above, the response to environmental impacts is presumably brought about via hormonal regulation, which, however, has to be channeled into changes of gene expression patterns in order to promote major changes in the development of the plant. Despite the large number of transcription factor genes involved, several specific transcription factors have been shown, for example, in mutant lines, to regulate the senescence process. Thus, several WRKY transcription factors, for example, WRKY6 (Robatzek and Somssich, 2001, 2002) and WRKY53 (Zentgraf et al., 2002, 2010), have specifically been shown to influence the

93

senescence process in Arabidopsis. WRKY53 appears to directly regulate senescence-associated genes by interacting with their promoters (Miao et al., 2004). A large number of genes from the NAC transcription factor family have been shown in transcriptome studies to be associated with the senescence process (e.g., Guo et al., 2004; Buchanan-Wollaston et al., 2005), and several studies suggest a central role of the NAC transcription factors for the regulation of senescence. Guo and Gan (2006) showed that the knockout of AtNAP in Arabidopsis delayed the senescence process considerably and that overexpression of this gene promoted senescence. ORE1/AtNAC2, another NAC gene in Arabidopsis closely related to AtNAP, showed similar phenotypes when mutated or overexpressed (Kim et al., 2009). In addition, the expression level of this gene appeared to be controlled by a microRNA, miRNA164, thus illustrating the complex network of regulation of senescence. A direct link between senescence, nutrient remobilization, and NAC transcription factors in a crop plant appeared from the finding, by map-based cloning, that the GpcB1 gene occurring in some wheat lines in fact encoded a NAC transcription factor (Uauy et al., 2006b). The Gpc-B1 gene increases grain protein content and accelerates senescence when introgressed into wheat lines not carrying the gene (Joppa et al., 1997; Olmos et al., 2003; Uauy et al., 2006a). The NAMB1 gene, as Gpc-B1 was denoted, is phylogenetically closely related to the AtNAP and AtNAC2 genes, all belonging to the subgroup NAC-a of the NAC gene family (Shen et al., 2009). A recent study on wheat lines with silencing of NAM-B1-related genes (Waters et al., 2009) suggests that NAC genes can have considerable effects on the remobilization from the leaves to the grain in wheat of a number of different nutrient elements and not only nitrogen. These effects on remobilization were correlated with the effects on the

94

NUTRIENT USE EFFICIENCY IN CROPS

senescence process. The putative role of NAC transcription factors as important regulators of senescence suggests NAC genes to be possible targets in attempts to manipulate the developmental process of plants with the aim of improving nutrient remobilization during senescence. Regulation by the proteasome pathway One of the important ways that transcription factors are themselves regulated is via turnover by the proteasome pathway (Moon et al., 2004; Vierstra, 2009), and, indeed, transcriptome studies on senescence have shown upregulation of specific genes involved in this pathway (BuchananWollaston et al., 2005; Gregersen and Holm, 2007). In addition, it has become quite evident that many hormonal effects are conveyed via interaction with specific proteins of the proteasome pathway (Santner and Estelle, 2010). Hence, it is conceivable that the interconnection of hormone and transcription factor effects during senescence is partly due to specific regulation by the proteasome pathway (Raab et al., 2009). Indeed, a few studies directly suggest this to be the case. Two mutations of components in the proteasome pathway, ore9, influencing hormonal signaling (Woo et al., 2001), and dsl1, affecting protein degradation (Yoshida et al., 2002), caused delay in senescence, whereas another mutation, saul1, promoted senescence (Raab et al., 2009). SAUL1 was shown to be involved in degradation of an enzymatic component in the biosynthetic pathway of ABA, aldehyde oxidase 3. Mutation in SAUL3 led to increased levels of ABA and hence to premature senescence. These studies clearly emphasize the importance of the proteasome pathway in finetuning the regulation of senescence in relation to hormonal effects and transcription factor levels, reflecting the general

importance of this pathway for regulation of plant developmental processes (Vierstra, 2009). Conclusions: the dilemma of senescence The ideal senescence phenotype of a seed producing crop was described by Thomas and Stoddart (1980) in this way: “The ideal crop may be visualized as striking a balance between, on the one hand, extension of the mature phase of canopy development when it is largely a carbon source and, on the other, achieving rapid recovery of minerals during the self-destruct phase when the leaves are senescent.” However, as this description indicates, there are counteracting forces in play here, in the way that maximal yields seem associated with low nutrient use efficiency, that is, a low nutrient harvest index, whereas high nutrient use efficiency and high percentage grain protein contents are associated with low yields (Fig. 5.3). The problem arising from these conflicting forces could be denoted as the dilemma of senescence, which, in short, states that it is difficult to combine delayed senescence with high nutrient use efficiency. This dilemma is presumably reflected in the overall negative correlation between total grain yield and grain protein content (Monaghan et al., 2001), which seems very difficult to break in breeding programs (Simmonds, 1995). The difficulty in comDelayed senescence High yield

Ineffecient nitrogen remobilization Low harvest index

Accelerated senescence Low yield

Effecient nitrogen remobilization High grain protein content

The dilemma of senescence: Delayed senescence can give rise to high yields, but with low nutrient use efficiency and low harvest index. On the other hand, accelerated senescence can improve the percentage protein content of grains, but is associated with lower grain yields. Fig. 5.3.

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

bining high protein contents/high nutrient use efficiency with high yields is illustrated by the case of the Gpc-B1/NAM-B1 gene in wheat. Thus, in wheat field trials at the International Maize and Wheat Improvement Center (CIMMYT) in Mexico, the presence of the Gpc-B1/NAM-B1 gene, which promotes senescence, appeared to give a yield penalty of around 10% in high-yielding environments, whereas this was not the case in trials with drought or heat stress (R. Singh, CYMMIT, pers. comm.). Hence, in conclusion, the importance of senescence as a process that could potentially be manipulated in order to optimize the nutrient economy, in particular of nitrogen, and yield of crop plants is twofold. (1) An efficient remobilization of nitrogen from senescing tissues is an important part of the nitrogen use efficiency, ensuring that most of the nitrogen in vegetative plant parts is translocated to the harvested seeds (Hirel et al., 2007). This may also lead to a higher percentage protein content of the seed (Uauy et al., 2006a; Jukanti and Fischer, 2008). (2) Delayed senescence, in the form of staygreen phenotypes, is associated with high yields of plants, both of total biomass and of grains, due to a longer period of high photosynthetic activity (Benbella and Paulsen, 1998; Spano et al., 2003; Yang and Zhang, 2006). The question is whether these two essential, positive effects of senescence can be combined. The dilemma of senescence states that this might be rather difficult to achieve. However, it is hoped that this review has shown that insights into the regulatory mechanisms of senescence have increased tremendously during the last decade. This gives hope that new strategies can be opened with the aim of creating crop plants for which the dilemma of senescence can be overcome or at least forced more in the direction of delayed senescence and high yields combined with high nutrient use effi-

95

ciency. The targets to work on, in classical plant breeding or in transgenic strategies, appear primarily to be the transcription factors associated with senescence, such as the AtNAP and NAM-B1 genes, for which the expression levels, temporarily and spatially in the plant, could be manipulated, in order to modulate the progression of senescence, both in terms of kinetics and timing. Ideally, the latter should aim at a delayed, but still rapid senescence process of the crop. In addition, key components of nitrogen metabolism, for example, proteases or glutamine synthetases, could be important targets for manipulation, in order to affect the senescence phenotype and improve nutrient remobilization. In order to create the optimal plant phenotype with respect to nutrient use efficiency, senescence clearly only comprises one part of the picture. Optimal senescence parameters have to be combined with optimization of a range of other factors, for example, root length, nutrient uptake, nutrient transport, and loading into the seed, some of which are discussed in other chapters of this book. Reference Balazadeh, S., Riano-Pachon, D.M., & Mueller-Roeber, B. (2008) Transcription factors regulating leaf senescence in Arabidopsis thaliana. Plant Biology 10, 63–75. Available from: ISI:000258078400007. Benbella, M. & Paulsen, G.M. (1998) Efficacy of treatments for delaying senescence of wheat leaves: II. Senescence and grain yield under field conditions. Agronomy Journal 90(3), 332–338. Available from: ISI:000074765600004. Bernard, S.M. & Habash, D.Z. (2009) The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytologist 182(3), 608–620. Available from: ISI:000265229400006. Bernard, S.M., Moller, A.L.B., Dionisio, G., et al. (2008) Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.). Plant Molecular Biology 67(1-2), 89–105. Available from: ISI:000254965000007.

96

NUTRIENT USE EFFICIENCY IN CROPS

Birsin, M.A., Adak, M.S., Inal, A., Aksu, A., & Gunes, A. (2010) Mineral element distribution and accumulation patterns within two barley cultivars. Journal of Plant Nutrition 33(2), 267–284. Available at: http://www.informaworld.com/10.1080/019041 60903435391. Borraccino, G., Mastropasqua, L., Deleonardis, S., & Dipierro, S. (1994) The role of the ascorbic-acid system in delaying the senescence of oat (Avena sativa L) leaf segments. Journal of Plant Physiology 144(2), 161–166. Available from: ISI:A1994PC76300006. Borrell, A.K., Hammer, G.L., & Henzell, R.G. (2000) Does maintaining green leaf area in sorghum improve yield under drought? II. Dry matter production and yield. Crop Science 40(4), 1037–1048. Available at: https://www.crops.org/publications/cs/ abstracts/40/4/1037. Buchanan-Wollaston, V. (1997) The molecular biology of leaf senescence. Journal of Experimental Botany 48(307), 181–199. Available from: ISI:A1997WL16900001. Buchanan-Wollaston, V., Earl, S., Harrison, E., et al. (2003) The molecular analysis of leaf senescence—a genomics approach. Plant Biotechnology Journal 1(1), 3–22. Available from: ISI:000188993900002. Buchanan-Wollaston, V., Page, T., Harrison, E., et al. (2005) Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/ starvation-induced senescence in Arabidopsis. Plant Journal 42(4), 567–585. Available from: ISI:000228746000011. Cai, H.M., Zhou, Y., Xiao, J.H., Li, X.H., Zhang, Q.F., & Lian, X.M. (2009) Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Reports 28(3), 527–537. Available from: ISI:000263783 200017. Cao, X., Costa, L.M., Biderre-Petit, C., et al. (2007) Abscisic acid and stress signals induce viviparous1 expression in seed and vegetative tissues of maize. Plant Physiology 143(2), 720–731. Available at: http://www.plantphysiol.org/cgi/content/ abstract/143/2/720. Charlton, W.L., Johnson, B., Graham, I.A., & Baker, A. (2005) Non-coordinate expression of peroxisome biogenesis, β-oxidation and glyoxylate cycle genes in mature Arabidopsis plants. Plant Cell Reports 23(9), 647–653. Available at: http:// dx.doi.org/10.1007/s00299-004-0879-7. Chiba, A., Ishida, H., Nishizawa, N.K., Makino, A., & Mae, T. (2003) Exclusion of Ribulose-1,5bisphosphate Carboxylase/oxygenase from chloroplasts by specific bodies in naturally senescing

leaves of wheat. Plant and Cell Physiology 44(9), 914–921. Available at: http://pcp.oxfordjournals.org/ cgi/content/abstract/44/9/914. Choi, H.I., Hong, J.H., Ha, J.O., Kang, J.Y., & Kim, S.Y. (2000) ABFs, a family of ABA-responsive element binding factors. The Journal of Biological Chemistry 275(3), 1723–1730. Available at: http:// www.jbc.org/content/275/3/1723.abstract. Choudhury, A. & Lahiri, A. (2011) Comparative analysis of abscisic acid-regulated transcriptomes in Arabidopsis. Plant Biology 13(1), 28–35. Criado, M.V., Caputo, C., Roberts, I.N., Castro, M.A., & Barneix, A.J. (2009) Cytokinin-induced changes of nitrogen remobilization and chloroplast ultrastructure in wheat (Triticum aestivum). Journal of Plant Physiology 166(16), 1775–1785. Available from: ISI:000271524900008. Doelling, J.H., Walker, J.M., Friedman, E.M., Thompson, A.R., & Vierstra, R.D. (2002) The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. Journal of Biological Chemistry 277(36), 33105–33114. Available at: http://www.jbc.org/content/277/36/33105.abstract. Dreccer, M.F., Grashoff, C., & Rabbinge, R. (1997) Source-sink ratio in barley (Hordeum vulgare L.) during grain filling: effects on senescence and grain protein concentration. Field Crops Research 49(2–3), 269–277. Available at: http:// www.sciencedirect.com/science/article/B6T6M3W3FGNS-H/2/8356558215b1cbf9e2cbcd06 6213e81c. Ehdaie, B., Alloush, G.A., Madore, M.A., & Waines, J.G. (2006) Genotypic variation for stem reserves and mobilization in wheat: II. Postanthesis changes in internode water-soluble carbohydrates. Crop Science 46(5), 2093–2103. Available at: https:// www.crops.org/publications/cs/abstracts/46/5/ 2093. Feller, U. & Fischer, A. (1994) Nitrogen-metabolism in senescing leaves. Critical Reviews in Plant Sciences 13(3), 241–273. Available from: ISI:A1994PB67600003. Feller, U., Anders, I., & Mae, T. (2008) Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated. Journal of Experimental Botany 59(7), 1615–1624. Available at: http:// jxb.oxfordjournals.org/cgi/content/ abstract/59/7/1615. Feller, U.K., Soong, T.S., & Hageman, R.H. (1977) Leaf proteolytic activities and senescence during grain development of field-grown corn (Zea mays L). Plant Physiology 59(2), 290–294. Available at: http://www.plantphysiol.org/cgi/content/ abstract/59/2/290.

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

Foulkes, M.J., Hawkesford, M.J., Barraclough, P.B., et al. (2009) Identifying traits to improve the nitrogen economy of wheat: recent advances and future prospects. Field Crops Research 114(3), 329–342. Available at: http://www.sciencedirect.com/science/ article/B6T6M-4XDKCBJ-2/2/08bfa420 2beb8251819f4efc69336077. Foyer, C.H., Parry, M., & Noctor, G. (2003) Markers and signals associated with nitrogen assimilation in higher plants. Journal of Experimental Botany 54(382), 585– 593. Available from: ISI:000180649800016. Frink, C.R., Waggoner, P.E., & Ausubel, J.H. (1999) Nitrogen fertilizer: retrospect and prospect. Proceedings of the National Academy of Sciences of the United States of America 96(4), 1175–1180. Available at: http://www.pnas.org/content/96/4/ 1175.short. Fujii, H. & Zhu, J.K. (2009) Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proceedings of the National Academy of Sciences 106(20), 8380–8385. Available at: http://www.pnas.org/ content/106/20/8380.abstract. Gan, S. & Amasino, R.M. (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270(5244), 1986–1988. Available at: http://www.sciencemag.org/cgi/content/ abstract/270/5244/1986. Garg, O.P. & Kapoor, V. (1972) Retardation of leaf senescence by ascorbic acid. Journal of Experimental Botany 23(3), 699–703. Available at: http://jxb.oxfordjournals.org/cgi/content/ abstract/23/3/699. Garnett, T.P. & Graham, R.D. (2005) Distribution and remobilization of iron and copper in wheat. Annals of Botany 95(5), 817–826. Available at: http:// aob.oxfordjournals.org/cgi/content/ abstract/95/5/817. Gepstein, S., Sabehi, G., Carp, M.J., et al. (2003) Largescale identification of leaf senescence-associated genes. Plant Journal 36(5), 629–642. Available from: ISI:000186532400005. Gergoff, G., Chaves, A., & Bartoli, C.G. (2010) Ethylene regulates ascorbic acid content during dark-induced leaf senescence. Plant Science 178(2), 207–212. Available from: ISI:0002748777 00018. Goldbach, H., Goldbach, E., & Michael, G. (1977) Transport of abscisic acid from leaves to grains in wheat and barley plants. Naturwissenschaften 64(9), 488–489. Available at: http:// dx.doi.org/10.1007/BF00446269. Gong, Y.H., Zhang, J., Gao, J.F., Lu, J.Y., & Wang, J.R. (2005) Slow export of photoassimilate from stay-

97

green leaves during late grain-filling stage in hybrid winter wheat (Triticum aestivum L.). Journal of Agronomy and Crop Science 191(4), 292–299. Available from: ISI:000230728000007. Gregersen, P.L. & Holm, P.B. (2007) Transcriptome analysis of senescence in the flag leaf of wheat (Triticum aestivum L.). Plant Biotechnology Journal 5(1), 192–206. Available from: ISI:000242963700018. Gregersen, P.L., Holm, P.B., & Krupinska, K. (2008) Leaf senescence and nutrient remobilisation in barley and wheat. Plant Biology 10, 37–49. Available from: ISI:000258078400005. Guo, Y., Cai, Z., & Gan, S. (2004) Transcriptome of Arabidopsis leaf senescence. Plant, Cell & Environment 27(5), 521–549. Available from: ISI:000221231600001. Guo, Y.F. & Gan, S.S. (2006) AtNAP, a NAC family transcription factor, has an important role in leaf senescence. Plant Journal 46(4), 601–612. Available from: ISI:000237098000006. Gut, H. & Matile, P. (1988) Apparent induction of key enzymes of the glyoxylic acid cycle in senescent barley leaves. Planta 176(4), 548–550. Available at: http://dx.doi.org/10.1007/BF00397663. Hafsi, M., Mechmeche, W., Bouamama, L., Djekoune, A., Zaharieva, M., & Monneveux, P. (2000) Flag leaf senescence, as evaluated by numerical image analysis, and its relationship with yield under drought in durum wheat. Journal of Agronomy and Crop Science 185(4), 275–280. Available from: ISI:000165669300009. Harding, S.A., Guikema, J.A., & Paulsen, G.M. (1990) Photosynthetic decline from high temperature stress during maturation of wheat: I. Interaction with senescence processes. Plant Physiology 92(3), 648– 653. Available at: http://www.plantphysiol.org/cgi/ content/abstract/92/3/648. Hayashi, H. & Chino, M. (1990) Chemical composition of phloem sap from the uppermost internode of the rice plant. Plant and Cell Physiology 31(2), 247– 251. Available at: http://pcp.oxfordjournals.org/cgi/ content/abstract/31/2/247. Heidlebaugh, N.M., Trethewey, B.R., Jukanti, A.K., Parrott, D.L., Martin, J.M., & Fischer, A.M. (2008) Effects of a barley (Hordeum vulgare) chromosome 6 grain protein content locus on whole-plant nitrogen reallocation under two different fertilisation regimes. Functional Plant Biology 35(7), 619–632. Available from: ISI:000258592100009. Helsel, D.B. & Frey, K.J. (1978) Grain yield variations in oats associated with differences in leaf area duration among oat lines. Crop Science 18(5), 765–769. Available at: https://www.crops.org/publications/cs/ abstracts/18/5/CS0180050765.

98

NUTRIENT USE EFFICIENCY IN CROPS

Hill, J., Robson, A.D., & Loneragan, J.F. (1979) The effect of copper supply on the senescence and the retranslocation of nutrients of the oldest leaf of wheat. Annals of Botany 44(3), 279–287. Available at: http:// aob.oxfordjournals.org/cgi/content/abstract/44/3/279. Himelblau, E. & Amasino, R.M. (2001) Nutrients mobilized from leaves of Arabidopsis thaliana during leaf senescence. Journal of Plant Physiology 158(10), 1317–1323. Available at: http:// www.sciencedirect.com/science/article/B7GJ74DPXFGH-5R/2/363dfa7c687 66d2d60ed83b76622430e. Hirel, B., Le Gouis, J., Ney, B., & Gallais, A. (2007) The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. Journal of Experimental Botany 58(9), 2369–2387. Available from: ISI:000248846400010. Hodges, M. (2002) Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation. Journal of Experimental Botany 53(370), 905–916. Available from: ISI:000174853500014. Hörtensteiner, S. (2009) Stay-green regulates chlorophyll and chlorophyll-binding protein degradation during senescence. Trends in Plant Science 14(3), 155–162. Available from: ISI:000264681000007. Hörtensteiner, S. & Feller, U. (2002) Nitrogen metabolism and remobilization during senescence. Journal of Experimental Botany 53(370), 927–937. Available from: ISI:000174853500016. Howarth, J.R., Parmar, S., Jones, J., et al. (2008) Coordinated expression of amino acid metabolism in response to N and S deficiency during wheat grain filling. Journal of Experimental Botany 59(13), 3675–3689. Available from: ISI:000259973800016. Humbeck, K., Quast, S., & Krupinska, K. (1996) Functional and molecular changes in the photosynthetic apparatus during senescence of flag leaves from field-grown barley plants. Plant, Cell & Environment 19(3), 337–344. Available at: http://www.blackwell-synergy.com/doi/abs/ 10.1111/j.1365-3040.1996.tb00256.x. Huynh, L.N., VanToai, T., Streeter, J., & Banowetz, G. (2005) Regulation of flooding tolerance of SAG12:ipt Arabidopsis plants by cytokinin. Journal of Experimental Botany 56(415), 1397–1407. Available at: http://jxb.oxfordjournals.org/cgi/ content/abstract/56/415/1397. Ishida, H., Yoshimoto, K., Izumi, M., et al. (2008) Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant Physiology 148(1), 142–155. Available at: http:// www.plantphysiol.org/cgi/content/abstract/148/ 1/142.

Jahn, T.P., Møller, A.L.B., Zeuthen, T., et al. (2004) Aquaporin homologues in plants and mammals transport ammonia. FEBS Letters 574(1–3), 31–36. Available at: http://www.sciencedirect.com/science/ article/B6T36-4D2XB26-1/2/7cd9297f5f4b99b48 2a2722d6a2075a5. Joppa, L.R., Du, C., Hart, G.E., & Hareland, G.A. (1997) Mapping gene(s) for grain protein in tetraploid wheat (Triticum turgidum L.) using a population of recombinant inbred chromosome lines. Crop Science 37(5), 1586–1589. Available at: https://www.crops.org/ publications/cs/abstracts/37/5/CS0370051586. Jukanti, A. & Fischer, A. (2008) A high-grain protein content locus on barley (Hordeum vulgare) chromosome 6 is associated with increased flag leaf proteolysis and nitrogen remobilization. Physiologia Plantarum 132(4), 426–439. Available at: http:// onlinelibrary.wiley.com/doi/10.1111/j.1399-3054. 2007.01044.x/abstract. Jukanti, A.K., Heidlebaugh, N.M., Parrott, D.L., Fischer, I.A., McInnerney, K., & Fischer, A.M. (2008) Comparative transcriptome profiling of near-isogenic barley (Hordeum vulgare) lines differing in the allelic state of a major grain protein content locus identifies genes with possible roles in leaf senescence and nitrogen reallocation. New Phytologist 177(2), 333–349. Available from: ISI:000251766300008. Kato, Y. & Sakamoto, W. (2009) Protein quality control in chloroplasts: a current model of D1 protein degradation in the photosystem II repair cycle. Journal of Biochemistry 146(4), 463–469. Available from: ISI:000270709400003. Kato, Y., Yamamoto, Y., Murakami, S., & Sato, F. (2005) Post-translational regulation of CND41 protease activity in senescent tobacco leaves. Planta 222(4), 643–651. Available at: http://dx.doi. org/10.1007/s00425-005-0011-4. Kichey, T., Heumez, E., Pocholle, D., et al. (2006) Combined agronomic and physiological aspects of nitrogen management in wheat highlight a central role for glutamine synthetase. New Phytologist 169(2), 265–278. Available from: ISI:00023448 2900006. Kichey, T., Hirel, B., Heumez, E., Dubois, F., & Le Gouis, J. (2007) In winter wheat (Triticum aestivum L.), post-anthesis nitrogen uptake and remobilisation to the grain correlates with agronomic traits and nitrogen physiological markers. Field Crops Research 102(1), 22–32. Available from: ISI:000246533800003. Kim, J.H., Woo, H.R., Kim, J., et al. (2009) Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science 323(5917), 1053–1057.Available from: ISI:000263478 400040.

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

Kwon, S. & Park, O. (2008) Autophagy in plants. Journal of Plant Biology 51(5), 313–320. Available at: http://dx.doi.org/10.1007/BF03036132. Lam, E., Kato, N., & Lawton, M. (2001) Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411(6839), 848–853. Available from: ISI:000169246400062. Lim, P.O., Kim, H.J., & Gil Nam, H. (2007) Leaf senescence. Annual Review of Plant Biology 58(1), 115– 136. from: http://dx.doi.org/10.1146/annurev.arplant. 57.032905.105316. Lin, W. & Wittenbach, V.A. (1981) Subcellular localization of proteases in wheat and corn mesophyll protoplasts. Plant Physiology 67(5), 969–972. Available at: http://www.plantphysiol.org/cgi/ content/abstract/67/5/969. Liu, F.L., Jensen, C.R., & Andersen, M.N. (2005) A review of drought adaptation in crop plants: changes in vegetative and reproductive physiology induced by ABA-based chemical signals. Australian Journal of Agricultural Research 56(11), 1245–1252. Available from: ISI:000233570500015. Lupton, F.G.H., Oliver, R.H., & Ruckenbauer, P. (1974) An analysis of the factors determining yields in crosses between semi-dwarf and taller wheat varieties. The Journal of Agricultural Science 82(03), 483–496. Available at: http:// journals.cambridge.org/action/displayAbstract ?fromPage=online&aid=4705320&fulltextType=R A&fileId=S0021859600051388. Ma, Y., Szostkiewicz, I., Korte, A., et al. (2009) Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324(5930), 1064– 1068. Available at: http://www.sciencemag.org/cgi/ content/abstract/324/5930/1064. Martin, A., Lee, J., Kichey, T., et al. (2006) Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell 18(11), 3252–3274. Available from: ISI:000243093700030. Martinez, D.E., Bartoli, C.G., Grbic, V., & Guiamet, J.J. (2007) Vacuolar cysteine proteases of wheat (Triticum aestivum L.) are common to leaf senescence induced by different factors. Journal of Experimental Botany 58(5), 1099–1107. Available at: http://jxb.oxfordjournals.org/cgi/content/abstract/ 58/5/1099. Martinez, D.E., Costa, M.L., & Guiamet, J.J. (2008) Senescence-associated degradation of chloroplast proteins inside and outside the organelle. Plant Biology 10, 15–22. Available from: ISI:000258078400003. Matile, P., Hortensteiner, S., Thomas, H., & Krautler, B. (1996) Chlorophyll breakdown in senescent leaves. Plant Physiology 112(4), 1403–1409. Available at: http://www.plantphysiol.org/content/

99

112/4/1403.full.pdf+html?sid=9b2b77c4 - 8c56 46bd-8633-001686f3c20e. Mauk, C.S. & Nooden, L.D. (1992) Regulation of mineral redistribution in pod-bearing soybean explants. Journal of Experimental Botany 43(256), 1429–1440. Available from: ISI:A1992JZ 53100006. Miao, Y., Laun, T., Zimmermann, P., & Zentgraf, U. (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Molecular Biology 55(6), 853–867. Available from: ISI:000226091100007. Monaghan, J.M., Snape, J.W., Chojecki, A.J.S., & Kettlewell, P.S. (2001) The use of grain protein deviation for identifying wheat cultivars with high grain protein concentration and yield. Euphytica 122, 309–317. Moon, J., Parry, G., & Estelle, M. (2004) The ubiquitinproteasome pathway and plant development. Plant Cell 16(12), 3181–3195. Available from: ISI:000225780700003. Nakano, R., Ishida, H., Makino, A., & Mae, T. (2006) In vivo fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species in an intact leaf of cucumber under chilling-light conditions. Plant and Cell Physiology 47(2), 270–276.Available at: http://pcp.oxfordjournals. org/cgi/content/abstract/47/2/270. Nakashima, K., Kiyosue, T., YamaguchiShinozaki, K., & Shinozaki, K. (1997) A nuclear gene, erd1 encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana. Plant Journal 12(4), 851–861. Available from: ISI:A1997YF 45400011. Nambara, E. & Marion-Poll, A. (2003) ABA action and interactions in seeds. Trends in Plant Science 8(5), 213–217. Available at: http:// www.sciencedirect.com/science/article/B6TD148CFPX8-2/2/01a1510ecd757727 aff3a118c3a7976a. Noodén, L.D. (1988) The phenomena of senescence and aging. In: Senescence and Aging in Plants (eds. L.D. Noodén & A.C. Leopold), pp. 1–50, Academic Press, San Diego, CA. Okatan, Y., Kahanak, G.M., & Nooden, L.D. (1981) Characterization and kinetics of soybean maturation and monocarpic senescence. Physiologia Plantarum 52(2), 330–338. Available from: ISI:A1981ME 32400031. Olmos, S., Distelfeld, A., Chicaiza, O., et al. (2003) Precise mapping of a locus affecting grain protein content in durum wheat. Theoretical and Applied Genetics 107(7), 1243–1251. Available from: ISI:000186401200012.

100

NUTRIENT USE EFFICIENCY IN CROPS

Otegui, M.S., Noh, Y.S., Martinez, D.E., et al. (2005) Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean. Plant Journal 41(6), 831–844. Available from: ISI:000227350700004. Palta, J.A. & Fillery, I.R.P. (1995) N-Application enhances remobilization and reduces losses of preanthesis-N in wheat grown on a duplex soil. Australian Journal of Agricultural Research 46(3), 519–531. Available from: ISI:A1995QV27500005. Park, S.Y., Yu, J.W., Park, J.S., et al. (2007) The senescence-induced staygreen protein regulates chlorophyll degradation. The Plant Cell 19(5), 1649–1664. Available at: http://www.plantcell.org/ content/19/5/1649.abstract. Park, S.Y., Fung, P., Nishimura, N., et al. (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324(5930), 1068–1071. Available at: http://www.sciencemag.org/cgi/content/abstract/ 324/5930/1068. Pearson, J.N. & Rengel, Z. (1994) Distribution and remobilization of Zn and Mn during grain development in wheat. Journal of Experimental Botany 45(12), 1829–1835. Available at: http:// jxb.oxfordjournals.org/cgi/content/ abstract/45/12/1829. Pennisi, E. (2009) Stressed out over a stress hormone. Science 324(5930), 1012–1013. Available at: http:// www.sciencemag.org/content/324/5930/1012. summary?sid=72c7d455-b883-41e7-960e2d411961acd7. Pracharoenwattana, I. & Smith, S.M. (2008) When is a peroxisome not a peroxisome? Trends in Plant Science 13(10), 522–525. Available from: ISI:000260645400004. Raab, S., Drechsel, G., Zarepour, M., et al. (2009) Identification of a novel E3 ubiquitin ligase that is required for suppression of premature senescence in Arabidopsis. Plant Journal 59(1), 39–51. Available from: ISI:000267428400004. Rawson, H.M., Hindmarsh, J.H., Fischer, R.A., & Stockman, Y.M. (1983) Changes in leaf photosynthesis with plant ontogeny and relationships with yield per ear in wheat cultivars and 120-progeny. Australian Journal of Plant Physiology 10(6), 503– 514. Available from: ISI:A1983SC29800003. Rivero, R.M., Shulaev, V., & Blumwald, E. (2009) Cytokinin-dependent photorespiration and the protection of photosynthesis during water deficit. Plant Physiology 150(3), 1530–1540. Available at: http://www.plantphysiol.org/cgi/content/ abstract/150/3/1530. Robatzek, S. & Somssich, I.E. (2001) A new member of the Arabidopsis WRKY transcription factor family,

AtWRKY6, is associated with both senescence- and defence-related processes. Plant Journal 28(2), 123– 133. Available from: ISI:000172099500001. Robatzek, S. & Somssich, I.E. (2002) Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes & Development 16(9), 1139–1149. Available from: ISI:000175556800011. Sakakibara, H., Takei, K., & Hirose, N. (2006) Interactions between nitrogen and cytokinin in the regulation of metabolism and development. Trends in Plant Science 11(9), 440–448. Available at: http://www.sciencedirect.com/science/article/ B6TD1-4KKNNHJ-1/2/665741e23d139642aae073 9c055a3091. Santner, A. & Estelle, M. (2010) The ubiquitinproteasome system regulates plant hormone signaling. Plant Journal 61(6), 1029–1040. Available from: ISI:000275394300012. Schelbert, S., Aubry, S., Burla, B., et al. (2009) Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. The Plant Cell 21(3), 767–785. Available at: http://www.plantcell. org/cgi/content/abstract/21/3/767. Schippers, J.H.M., Jing, H.-C., Hille, J., & Dijkwel, P.P. (2007) Developmental and hormonal control of leaf senescence. In: Senescence Processes in Plants (ed. S. Gan), pp. 145–170, Blackwell Publ., Oxford Schjoerring, J.K., Kyllingsbaek, A., Mortensen, J.V., & Byskovnielsen, S. (1993) Field investigations of ammonia exchange between barley plants and the atmosphere. 1. Concentration profiles and flux densities of ammonia. Plant, Cell & Environment 16(2), 161–167. Available from: ISI:A1993KV93700005. Seki, M., Ishida, J., Narusaka, M., et al. (2002) Monitoring the expression pattern of around 7000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray. Functional & Integrative Genomics 2(6), 282–291. Available at: http://dx.doi.org/10.1007/s10142-002-0070-6. Shen, H., Yin, Y., Chen, F., Xu, Y., & Dixon, R. (2009) A bioinformatic analysis of NAC genes for plant cell wall development in relation to lignocellulosic bioenergy production. BioEnergy Research 2(4), 217–232. Available from: CABI:20093331713. Simmonds, N.W. (1995) The relation between yield and protein in cereal grain. Journal of the Science of Food and Agriculture 67(3), 309–315. Available from: ISI:A1995QM87300005. Simpson, R.J. & Dalling, M.J. (1981) Nitrogen redistribution during grain-growth in wheat (Triticum aestivum L). 3. Enzymology and transport of amino-acids from senescing flag leaves. Planta 151(5), 447–456. Available from: ISI:A1981LP 99900006.

SENESCENCE AND NUTRIENT REMOBILIZATION IN CROP PLANTS

Simpson, R.J., Lambers, H., & Dalling, M.J. (1983) Nitrogen redistribution during grain-growth in wheat (Triticum aestivum L). 4. Development of a quantitative model of the translocation of nitrogen to the grain. Plant Physiology 71(1), 7–14. Available from: ISI:A1983PY81500002. Snape, J.W., Butterworth, K., Whitechurch, E., & Worland, A.J. (2001) Waiting for fine times: genetics of flowering time in wheat. Euphytica 119(1), 185–190. Available at: http://dx.doi.org/10.1023/A: 1017594422176. Spano, G., Di Fonzo, N., Perrotta, C., et al. (2003) Physiological characterization of “stay green” mutants in durum wheat. Journal of Experimental Botany 54(386), 1415–1420. Available from: ISI:000182630300013. Tabuchi, M., Sugiyama, K., Ishiyama, K., et al. (2005) Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase1;1. Plant Journal 42(5), 641–651. Available from: ISI:000229180400003. Tabuchi, M., Abiko, T., & Yamaya, T. (2007) Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L). Journal of Experimental Botany 58(9), 2319–2327. Available at: http://jxb. oxfordjournals.org/cgi/content/abstract/58/9/2319. Tauris, B., Borg, S., Gregersen, P.L., & Holm, P.B. (2009) A roadmap for zinc trafficking in the developing barley grain based on laser capture microdissection and gene expression profiling. Journal of Experimental Botany 60(4), 1333–1347. Available at: http://jxb.oxfordjournals.org/cgi/content/abstract/ 60/4/1333. Thomas, H. (1994) Aging in the plant and animal kingdoms? The role of cell death. Reviews in Clinical Gerontology 4(01), 5–20. Available at: http:// journals.cambridge.org/action/displayAbstract ?fromPage=online&aid=2184512&fulltextType=R V&fileId=S0959259800002227. Thomas, H. & Howarth, C.J. (2000) Five ways to stay green. Journal of Experimental Botany 51, 329– 337. Available from: ISI:000085386600003. Thomas, H. & Stoddart, J.L. (1980) Leaf senescence. Annual Review of Plant Physiology and Plant Molecular Biology 31, 83–111. Available from: ISI:A1980JU72700004. Thompson, J.E., Froese, C.D., Madey, E., Smith, M.D., & Yuwen, H. (1998) Lipid metabolism during plant senescence. Progress in Lipid Research 37(2–3), 119–141. Available at: http://www.sciencedirect. com/science/article/B6TBP-3TXDH372/2/9bad60fe8b61c093334c0d6465677888. Tollenaar, M. & Wu, J. (1999) Yield improvement in temperate maize is attributable to greater stress tolerance. Crop Science 39(6), 1597–1604. Available

101

at: https://www.crops.org/publications/cs/abstracts/ 39/6/1597. Uauy, C., Brevis, J.C., & Dubcovsky, J. (2006a) The high grain protein content gene Gpc-B1 accelerates senescence and has pleiotropic effects on protein content in wheat. Journal of Experimental Botany 57(11), 2785–2794. Available from: ISI:000239 901500030. Uauy, C., Distelfeld, A., Fahima, T., Blechl, A., & Dubcovsky, J. (2006b) A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314(5803), 1298–1301. Available from: ISI:000242215800043. Van der Graaff, E., Schwacke, R., Schneider, A., Desimone, M., Flugge, U.I., & Kunze, R. (2006) Transcription analysis of Arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence. Plant Physiology 141(2), 776–792. Available from: ISI:000238168800051. van Doorn, G. & Woltering, E. (2004) Senescence and programmed cell death: substance or semantics? Journal of Experimental Botany 55, 2147–2153. Available at: http://jxb.oxfordjournals.org/content/ 55/406/2147.abstract. van Oosterom, E.J., Chapman, S.C., Borrell, A.K., Broad, I.J., & Hammer, G.L. (2010) Functional dynamics of the nitrogen balance of sorghum. II. Grain filling period. Field Crops Research 115(1), 29–38. Available at: http://www.sciencedirect.com/ science/article/B6T6M-4XK9J3K-2/2/ b51461b7947f768a531219e9e9cd0fef. Verdier, J. & Thompson, R.D. (2008) Transcriptional regulation of storage protein synthesis during dicotyledon seed filling. Plant and Cell Physiology 49(9), 1263–1271. Available at: http://pcp.oxfordjournals. org/cgi/content/abstract/49/9/1263. Vierstra, R.D. (2009) The ubiquitin-26S proteasome system at the nexus of plant biology. Nature Reviews. Molecular Cell Biology 10(6), 385–397. Available at: http://dx.doi.org/10.1038/nrm2688. Wada, S., Ishida, H., Izumi, M., et al. (2009) Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiology 149(2), 885–893. Available at: http://www.plantphysiol.org/cgi/content/abstract/ 149/2/885. Waters, S.P., Peoples, M.B., Simpson, R.J., & Dalling, M.J. (1980) Nitrogen redistribution during graingrowth in wheat (Triticum-Aestivum L). 1. Peptidehydrolase activity and protein breakdown in the flag leaf, glumes and stem. Planta 148(5), 422–428. Available from: ISI:A1980JW58600002. Waters, B.M., Uauy, C., Dubcovsky, J., & Grusak, M.A. (2009) Wheat (Triticum aestivum) NAM proteins

102

NUTRIENT USE EFFICIENCY IN CROPS

regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain. Journal of Experimental Botany 60(15), 4263–4274. Available at: http://jxb.oxfordjournals.org/cgi/ content/abstract/60/15/4263. Wilkinson, S. & Davies, W.J. (2002) ABA-based chemical signalling: the co-ordination of responses to stress in plants. Plant, Cell & Environment 25(2), 195–210. Available from: ISI:000174061500007. Wittenbach, V.A. (1979) Ribulose bisphosphate carboxylase and proteolytic activity in wheat leaves from anthesis through senescence. Plant Physiology 64(5), 884–887. Available from: ISI:A1979HV 88000043. Wolfe, D.W., Henderson, D.W., Hsiao, T.C., & Alvino, A. (1988) Interactive water and nitrogen effects on senescence of maize. I. Leaf area duration, nitrogen distribution, and yield. Agronomy Journal 80(6), 859–864. Available at: https://www.agronomy.org/ publications/aj/abstracts/80/6/AJ0800060859. Woo, H.R., Chung, K.M., Park, J.H., et al. (2001) ORE9, an F-Box protein that regulates leaf senescence in Arabidopsis. The Plant Cell 13(8), 1779– 1790. Available at: http://www.plantcell.org/cgi/ content/abstract/13/8/1779. Xie, Z., Jiang, D., Dai, T., Jing, Q., & Cao, W. (2004) Effects of exogenous ABA and cytokinin on leaf photosynthesis and grain protein accumulation in wheat ears cultured in vitro. Plant Growth Regulation 44(1), 25–32. Available at: http:// dx.doi.org/10.1007/s10725-004-1880-4. Yang, Z.L. & Ohlrogge, J.B. (2009) Turnover of fatty acids during natural senescence of Arabidopsis, brachypodium, and switchgrass and in Arabidopsis beta-oxidation mutants. Plant Physiology 150(4), 1981–1989. Available from: ISI:000268692200032. Yang, J.C. & Zhang, J.H. (2006) Grain filling of cereals under soil drying. New Phytologist 169(2), 223– 236. Available from: ISI:000234482900002. Yang, J., Zhang, J., Wang, Z., Zhu, Q., & Liu, L. (2001) Water deficit-induced senescence and its relationship to the remobilization of pre-stored carbon in wheat during grain filling. Agronomy Journal 93(1), 196–206. Available at: https://www.agronomy.org/ publications/aj/abstracts/93/1/196. Yang, J.C., Zhang, J.H., Wang, Z.Q., Zhu, Q.S., & Liu, L.J. (2003) Involvement of abscisic acid and cytokinins in the senescence and remobilization of carbon reserves in wheat subjected to water stress during grain filling. Plant, Cell & Environment 26(10), 1621–1631. Available from: ISI:00018553 6000003. Yoo, S.C., Cho, S.H., Zhang, H., et al. (2007) Quantitative trait loci associated with functional

stay-green SNU-SG1 in rice. Molecules and Cells 24(1), 83–94. Available at: http://www.scopus.com/ inward/record.url?eid=2-s2.0-35048855476&par tnerID=40&md5=3638e8d644722a1e2418628237 8a7c6f. Yoshida, S., Ito, M., Callis, J., Nishida, I., & Watanabe, A. (2002) A delayed leaf senescence mutant is defective in arginyl-tRNA: protein arginyltransferase, a component of the N-end rule pathway in Arabidopsis. Plant Journal 32(1), 129–137. Available from: ISI:000178374100011. Zacarias, L. & Reid, M.S. (1990) Role of growthregulators in the senescence of Arabidopsis thaliana leaves. Physiologia Plantarum 80(4), 549–554. Available from: ISI:A1990ER94300009. Zapata, J.M., Guera, A., Esteban-Carrasco, A., Martin, M., & Sabater, B. (2005) Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhFdefective tobacco. Cell Death and Differentiation 12(10), 1277–1284. Available at: http:// dx.doi.org/10.1038/sj.cdd.4401657. Zapata, J.M., Gasulla, F., Esteban-Carrasco, A., Barreno, E., & Guera, A. (2007) Inactivation of a plastid evolutionary conserved gene affects PSII electron transport, life span and fitness of tobacco plants. New Phytologist 174(2), 357–366. Available from: ISI:000245413900018. Zelisko, A., Garcia-Lorenzo, M., Jackowski, G., Jansson, S., & Funk, C. (2005) AtFtsH6 is involved in the degradation of the light-harvesting complex II during high-light acclimation and senescence. Proceedings of the National Academy of Sciences of the United States of America 102(38), 13699– 13704. Available from: ISI:000232115100062. Zentgraf, U., Laun, T., & Miao, Y. (2002) The complex regulation of WRKY53 during leaf senescence of Arabidopsis thaliana. European Journal of Cell Biology 89(2–3), 133–137. Available at: http://www.sciencedirect.com/science/article/ B7GJ2-4XWMGG1-1/2/ d4cd48b1af888fc622e3d56a87bd7774. Zentgraf, U., Laun, T., & Miao, Y. (2010) The complex regulation of WRKY53 during leaf senescence of Arabidopsis thaliana. European Journal of Cell Biology 89(2–3), 133–137. Available from: ISI:000275308300003. Zhong, G.V. & Burns, J.K. (2003) Profiling ethyleneregulated gene expression in Arabidopsis thaliana by microarray analysis. Plant Molecular Biology 53(1), 117–131. Available from: ISI:000187 356900010.

Chapter 6

Effects of Nitrogen and Sulfur Nutrition on Grain Composition and Properties of Wheat and Related Cereals Peter R. Shewry

Abstract

Introduction

Nitrogen is a major determinant of cereal grain quality, with nitrogen supply determining the total protein content and also modulating the protein composition, including the ratio of storage to nonstorage proteins, the proportions of individual storage proteins, and the ratios of storage protein polymers to monomers. Protein composition is also affected by sulfur supply, which is required in adequate amounts to allow the synthesis of sulfur-containing amino acids including cysteine, which is required for the formation of disulphide bonds. The nutritional effects on protein synthesis and composition have significant impacts on grain end-use quality, notably the nutritional quality of all cereals for livestock feed, the processing quality of wheat for making bread and processed foods, and the malting quality of barley. Understanding the molecular basis for the effects of nutrition on grain protein composition should facilitate the combination of protein quality with high grain yield and good nitrogen use efficiency.

Seeds fulfill an important biological role in plant propagation and dispersal following sexual reproduction. Seed germination requires the provision of energy and building blocks to support the growth of the seedling until it is capable of independent growth. These are usually provided by the mobilization of specific groups of storage components, although in some species, such as cereals, the digestion of the storage organ itself also contributes. The accumulation of specific groups of storage compounds in high concentrations means that seeds have become important sources of food, feed, and raw material for humankind, with the added attraction that seeds have low water content and hence are readily transported and stored. Breeding for increased yields of seeds has resulted in massive increases in their contents of storage compounds, which in cultivated crops are vastly in excess of the biological requirements of the plant. Nevertheless, they still need to comply with the biological requirements for synthesis, trafficking, and deposition

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 103

104

NUTRIENT USE EFFICIENCY IN CROPS

within the cell, and mobilization during germination. The three major groups of storage components in seed are lipids, starch, and proteins, which are deposited in specific organelles within the cells of the storage tissues (oil bodies, amyloplasts, and protein bodies, respectively). This location facilitates their packaging into dense deposits with low water contents and eliminates any potential effects of accumulation on cell function. However, different storage compounds occur in different species, with legume seeds being particularly diverse in composition. Thus, while most commercially grown legumes store either protein and starch (e.g., pea and bean) or protein and oil (e.g., peanut and soybean), a number of noncultivated species store nitrogen and carbon in other groups of compounds. For example, the nonprotein amino acids canavanine, hydroxytryptophan, and dihydroxyphenylalanine (L-dopa) are found in seeds of Dioclea, Griffonia, and Mucuna, respectively. Similarly, cell wall galactomannans account for up to 20% of the dry weight in other legume species (Ceratonia, Cyanopsis, Gleditsia, and Medicago) (the reader is referred to Bewley and Black, 1978 for an excellent discussion of the diversity of seed storage components). Because storage components often account for over 50% of the seed dry weight, their amount, composition, and properties have major impacts on the yield and end-use characteristics of the whole seeds. Mineral nutrition may affect storage compound synthesis in two ways. First, the mineral may contribute to the structure of the storage compound; notably both nitrogen and sulfur are usually required for storage protein synthesis due to their role in amino acid structure. In this case, the availability of the mineral is a prerequisite for synthesis, and deficiency may affect the amount and/or composition. However, minerals may also have indirect effects by

affecting the availability of substrates or the uptake, transport, or biosynthesis of other components that compete for substrates. The present review focuses not on mechanisms, but on the impacts of nitrogen, sulfur, and other minerals on composition and end-use quality, whether for foods, feed, or raw material for industry. It is also largely focused on wheat for several reasons. First, because much of the wheat grown in the world is processed for consumption by humans, the composition and quality of the grain is of greater importance for breeders, producers, and processors than for other cereals. Second, the volume of literature on wheat is massive compared with that on other cereals. Third, the conclusions from these studies are largely also applicable to other cereals. However, related studies on barley, rye, and oats are also included, with brief references to maize and rice where relevant. Effect of nitrogen on grain protein content The relationship between available nitrogen and grain nitrogen content has been studied intensively, with Benzian and Lane (1981) summarizing data from 20 years (1954– 1973) of wheat experiments carried out in Southern England. They defined four types of response curve for applications of up to 175 kgN ha−1, with most experiments showing linear or convex responses, without reaching a maximum value. In addition, about a quarter of these experiments (9196 in total) showed a “dilution effect,” in which the addition of nitrogen up to about 50 kg ha−1 resulted in decreased %N. This is illustrated in Figure 6.1A, taken from a more recent study (2004–2007) of the Rothamsted Broadbalk experiment (Godfrey et al., 2010), and indicates that low levels of nitrogen stimulate yield (i.e., starch deposition) before impacting on protein content.

NUTRITION AND GRAIN COMPOSITION

105

35

A

B % Wet gluten content

2.2

Flour %N

2.0 1.8 1.6 1.4

30

25

20

15

10

1.2 0

50

100

150

200

250

300

1.2

1.4

1.6

N Application rate

2.4

2.0

2.2

Flour %N 27.5

C

D

25.0

2.2 2.0

Extensibility

Flour %N

1.8

1.8 1.6 1.4

22.5 20.0 17.5 15.0 12.5

1.2

10.0

1.0 0.90 0.95 1.00 1.05 1.10 1.15 1.20

(F3+F4)/(F1+F2)

0.90

0.95

1.00

1.05

1.10

1.15

1.20

(F3+F4)/(F1+F2)

Relationship between the application rate of nitrogen fertilizer (kgNha−1) and the nitrogen content (%DW) of the wheat cultivar Hereward grown in the Broadbalk experiment in 2005, 2006, and 2007 (mean values). (A) Flour %N and nitrogen application rate. (B) Flour %N and wet gluten content. (C) Flour %N and gliadin : gluten ratio (F3 + F4/F1 + F2). (D) Gliadin : glutenin ratio and dough extensibility. Taken from Godfrey et al. (2010), with permission. Fig. 6.1.

Because most of the nitrogen present in the grain is in the form of proteins (with small amounts of free amino acids and peptides), it is usual to convert grain % to % protein by multiplying by a standard factor. The factor applied to most biological materials is 6.25, whereas 5.7 N is usually used for cereal grain, reflecting their high content of

glutamine, which contains two nitrogen atoms. In fact, the differences in composition between the individual cereals, and the fact that the proportions of storage proteins increase with increased nitrogen fertilization (as discussed below), means that the true conversion factor varies from about 5.1 to 5.4 between different cereals at low grain

106

NUTRIENT USE EFFICIENCY IN CROPS

Lysine (g 16 g N–1)

B

5.8 ze

Mai

Sorghum

5.6 Barley Oats

5.4

Rye 5.2

5.0

Rice

Nitrogen to protein conversion factor

A

0

Wheat

2 Grain N (g 100 g–1 dry wt.)

4

5

Oats

4 Rice

B a rl ey

3

Wh e Mai at ze So rgh um

2

1 4 2 Grain N (g 100 g–1 dry wt.) Fig. 6.2. (A) Variation in the nitrogen to protein conversion factor K as a function of grain nitrogen for different cereal species. Taken from Mossé (1990) with permission. (B) Relationship between total seed nitrogen and lysine content for cereals. Taken from Mossé and Huet (1990), with permission. 0

nitrogen and between about 5.4 and 5.8 at high grain nitrogen (Mossé, 1990) (Fig. 6.2A). Nevertheless, a factor of 5.7 is still routinely used for all cereals. Effect of nitrogen on grain protein nutrition and quality Protein nutritional quality is determined by the content of essential amino acids, which

cannot be synthesized by humans or other animals and hence must be provided in the diet. If only one of these essential amino acids is limiting the protein is not efficiently utilized with other (nonlimiting) amino acids being excreted. Ten of the 20 amino acids that commonly occur in proteins are strictly essential: lysine, isoleucine, leucine, phenylalanine, tyrosine, threonine, tryptophan, valine, histidine, and methionine. However, cysteine may be included as it can only be synthesized from methionine, and combined values for these two amino acids are often presented. Similarly, the aromatic amino acids tyrosine and phenylalanine are also biosynthetically related and therefore also combined. There has been much debate about the precise requirements of essential amino acids for adults and children, who have higher requirements due to rapid growth. Furthermore, in the developed world, the emphasis is on nutrition for livestock, particularly pigs and poultry, with diets being specifically designed to provide the most efficient weight gain at different growth stages. Table 6.1 shows the contents of essential amino acids in “typical” grain and flour fractions from a range of cereals, compared with the levels recommended for human adults and children by the Food and Agriculture Organization (FAO/WHO/ UNU, 1985). In all cases, lysine is the first limiting amino acid for children, with values reaching up to 4.2% (in oats) compared with the FAO recommendation of 6.6%. Deficiencies of other amino acids are less severe, but it should be noted that tryptophan is particularly low in maize. None of the essential amino acids in cereal grain are below the recommended levels for adult humans, who can live adequately on a cereal-based diet. The deficiencies in essential amino acids in cereal grain result from their low proportions in the grain storage proteins, particularly in the prolamins, which are the major

NUTRITION AND GRAIN COMPOSITION

Table 6.1.

107

Essential amino acid contents of cereal grain and flour compared with the FAO recommended levels Wheat

Histidine Isoleucine Leucine Lysine Cysteine Methionine Phenylalanine Tyrosine Threonine Tryptophan Valine

Barley

Oats

Rye

Rice

Maize

FAO Recommendations

Grain

White Flour

Grain

Groat

Grain

Milled

Cornflour

Children

Adults

2.3 3.7 6.8 2.8 2.3 1.2 4.7 1.7 2.9 (1.1) 4.4 *

2.2 3.6 6.7 2.2 2.5 1.3 4.8 1.5 2.6 (1.1) 4.1 *

2.3 3.7 7.0 3.5 2.3 1.7 5.2 2.9 3.6 1.9 4.9 †

2.2 3.9 7.4 4.2 1.6 2.5 5.3 3.1 3,3 ND 5.3 ‡

2.2 3.5 6.2 3.4 1.9 1.4 4.5 1.9 3.3 1.1 4.8 §

2.4 3.8 8.2 3.7 1.6 2.1 4.8 2.5 3.4 1.3 5.8 §

2.7 3.6 12.5 2.7 1.6 1.9 5.0 3.8 3.7 0.6 4.8 §

2.6 4.6 9.3 6.6 4.2

1.6 1.3 1.9 1.6 1.7

7.2

1.9

4.3 1.7 5.5 ¶

0.9 0.5 1.3 ¶

*Means of values for wholemeal and white flour samples of five types of wheat (hard red winter, hard red spring, soft red winter, club, and durum). Calculated from data in Shoup et al. (1966). Values for tryptophan are taken from single analysis reported in Paul and Southgate (1978). †Means of values for eight samples each of six-rowed and two-rowed barleys. Calculated from data in Newman and McGuire (1985). ‡Means of values for 289 samples (Robbins et al., 1971). §Calculated from single analyses reported by Paul and Southgate (1978). ¶FAO/WHO/UNU (1985). Taken from Shewry et al. (1999), with kind permission of Springer Science and Business Media. Values are g 100 g−1 protein or g 16 N−1.

storage proteins in all of the major cereals, except oats and rice. For example, lysine generally accounts for 1% or less of the amino acids in prolamins of all cereals, while tryptophan is absent from maize prolamins. In oats and rice, prolamins are only minor grain components (contributing up to about 10% of the total grain protein), with the major storage proteins being related to the 11S globulins of legume seeds. These proteins are richer in most essential amino acids but are nevertheless still deficient in lysine (about 2.9 mol% in oat globulin, 2.3 mol% in rice proteins, which are termed glutelin on account of their insolubility). Consequently, whole rice and oat grains have higher lysine contents than other cereals (Table 6.1). The reader is referred to Shewry (2007) for a more detailed discussion.

The proportion of storage proteins in the grain is not stable, but increases in response to increased availability of nitrogen (and sulfur) as discussed below. In terms of nutritional quality, this results in a dilution of the “high nutritional quality” nonstorage proteins with the poor quality storage proteins, and hence decreased proportions of essential amino acids. This has been described in detail for a number of cereals and amino acids, particularly by Mossé and colleagues (Mossé et al., 1985, 1988a,b; Landry and Delhaye, 1993), whose data for lysine are summarized in Figure 6.2B. Clear differences in the % lysine content have been observed in all species, with the dilution being less in oats and rice, in which the globulin/glutelin storage proteins are richer in lysine than the prolamin storage proteins in other species.

108

NUTRIENT USE EFFICIENCY IN CROPS

Effect of grain nitrogen on protein composition and functional properties of wheat The disproportional increase in storage proteins that occurs with increased nitrogen availability has been referred to above (and shown in Fig. 6.1). In fact, the impact is more complex than this because of differential effects on individual protein components. In order to understand these effects, it is necessary to first describe the wheat storage proteins in more detail. The classification of seed proteins dates back over 200 years but was formalized by TB Osborne in a series of studies reported between about 1890 and 1920 (see Osborne, 1924). Osborne classified proteins into groups based on their solubility and established a sequential extraction procedure that is still used today in many laboratories. This “Osborne fractionation” procedure results in separate fractions called albumins (watersoluble), globulins (soluble in dilute saline), prolamins (soluble in alcohol-water mixtures), and glutelins, the last comprising insoluble proteins that can only be extracted in dilute acids or alkalis, chaotropic agents (e.g., urea), or detergents. The prolamins and glutelins of wheat are termed gliadins and glutelins, respectively, and together form the gluten protein fraction that forms a viscoelastic network in dough. Although the gliadins and glutenins were long thought to be distinct groups of proteins, we now know that the individual components of these groups are related and that the difference in solubility results from their polymerization behavior rather than from fundamental differences in sequence or structure. Thus, the alcohol-soluble gliadins are monomeric proteins that are classified on the basis of their electrophoretic mobility at low pH and amino acid compositions into three types: the ωgliadins, which lack cysteine residues and thus cannot form disulfide bonds, and the α-

type (α- and β-) and γ-type gliadins, which form only intrachain disulfide bonds. The alcohol-insoluble glutenins comprise polymers stabilized by interchain disulfide bonds which probably have molecular weights of up to about 10 million. On reduction, the component subunits are readily soluble in alcohol– water mixtures (like the gliadins) and can be separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) into high-molecular-weight (HMW) and lowmolecular-weight (LMW) groups of subunits. The latter are further divided into a major group of B-type subunits and minor groups of subunits related to α-type/γ-type gliadins (C-type) and ω-gliadins (D-type). Based on their structural and evolutionary relationships, the gluten proteins (gliadins and glutenin subunits) can be classified into three groups: the HMW subunits; the ωgliadins and D-type LMW subunits (called sulfur-poor); and the α-type/γ-type gliadins and B/C type LMW subunits (called sulfurrich). This classification is summarized in Figure 6.3 (taken from Shewry et al., 2003a). The classification of gluten proteins into gliadins and glutenins has been remarkably durable and is still used by cereal chemists today. This is because the two fractions are associated with different biomechanical properties. The glutenin polymers are considered to form the “backbone” of the gluten network, and to confer elasticity (also referred to as strength) to gluten and dough, while the gliadins interact with the glutenin polymers and other gliadins by strong noncovalent forces (notably hydrogen bonds) and confer viscosity and extensibility. An appropriate balance of these properties is required for processing, with high dough strength required for bread-making and more extensible dough for cakes and biscuits. A range of studies carried out over 30 years has shown that variation in dough strength is associated with allelic variations in the HMW subunits, and hence this group

NUTRITION AND GRAIN COMPOSITION

109

Fig. 6.3. The classification and nomenclature of wheat gluten proteins separated by SDS-PAGE and electrophoresis

at low pH. The D-type LMW subunits are only minor components and are not clearly resolved in the separation shown. Taken from Shewry et al. (1999) with permission.

of proteins has been studied in particular detail (reviewed by Shewry et al., 2003b). Several studies have shown that increasing grain protein by nitrogen fertilization is associated with an increased proportion of gliadins, and hence an increase in the gliadin : glutenin ratio. For example, Kindred et al. (2008) extracted total proteins by sonication with 1% sodium dodecylsulfate and separated the components by size exclusion high performance liquid chromatography (SE-HPLC). They calculated that the proportion of gliadins increased from 42.7% and 41.4% in the cultivars Option and Riband, respectively, grown with 120 kg N ha−1, to 44.8% and 44.0% in the same two cultivars grown with 240 kg N ha−1. Figure 6.1 shows similar data from our own analysis of the wheat cultivar Hereward grown on the longterm Broadbalk experiment at Rothamsted with nitrogen levels ranging from 0 to 288 kg ha−1 (taken from Godfrey et al., 2010). This shows linear increases in both wet gluten content (Fig. 6.1B) and the gliadin : glutenin ratio (the ratio of peaks F3 + F4/F1 + F2 separated by SE-HPLC) with increasing flour nitrogen (i.e., protein)

(Fig. 6.1C). Similar increases in the proportions of gliadins with increasing grain protein content have been reported by Jia et al. (1996b) for the cultivar Soissons, by Daniel and Triboi (2000) for the cultivar Thésée, and by Wieser and Seilmeier (1998) for a collection of 13 cultivars, all grown with and without fertilization. Zhu and Khan (2001) and Panozzo and Eagles (2000) also showed increased proportions of monomeric proteins and gliadins, respectively, with increasing grain protein when comparing samples in which the variation in protein content resulted from cultivation under different environmental conditions rather than different nitrogen fertilization. However, other studies have shown that increased grain protein is not always associated with an increased proportion of gliadins. Thus, Pechanek et al. (1997) reported an increase in gliadin proportion in the cultivar Capo but a decrease in Renan, and no change in Lindos when comparing material grown with and without nitrogen fertilization. Zhu et al. (1999) reported that nitrogen fertilization resulted in an increased proportion of

110

NUTRIENT USE EFFICIENCY IN CROPS

polymers compared with monomers (i.e., glutenins compared with gliadins) when comparing samples of the cultivar Warigal grown with and without nitrogen fertilization. The effects of grain protein content on the composition of the glutenin fraction, and in particular on the proportion of high molecular mass polymers that are associated with dough strength, are less clear. Whereas Zhu and Khan (2001) showed a significant decrease in the ratio of SDS-soluble polymers to SDS-insoluble polymers with increased protein content, and Pechanek et al. (1997) a similar decrease in the ratio of LMW : HMW polymers, other studies indicate that genotype and environment may have greater effects than grain protein content on glutenin polymerization (Jia et al., 1996a; Zhu et al., 1999, b). Wieser and Seilmeier (1998) reported specific increases in the proportions of HMW subunits within the glutenin fraction for 13 cultivars grown with nitrogen levels varying from 0 to 200 kg ha−1, while Pechanek et al. (1997) showed increases in the ratio of HMW : LMW subunits in three cultivars grown with nitrogen fertilization compared to without nitrogen fertilization. Gliadin composition has been studied in less detail in relation to nitrogen nutrition than to sulfur nutrition (see below). However, Wieser and Seilmeier (1998) showed an increased proportion of ω-gliadins and a decreased proportion of γ-gliadins in total protein extracts from flour of 13 cultivars grown with 0–200 kg N ha−1, while Daniel and Triboi (2000) showed increased proportions of ω-gliadins and γ-gliadins and decreased proportions of α-/β-gliadins in total gliadin extracts from the cultivar Thésé grown at three nitrogen levels. The discrepancies between the results reported in the various studies discussed above may relate to several factors. First,

despite a massive volume of research, the gluten proteins remain difficult to analyze and quantify, with no single methods being widely adopted. Hence, some of the differences may relate to the use of different methodologies. Second, the levels of nitrogen applied varied widely, from 0 to 280 kg ha−1 in various experiments, as did the wheat genotypes and the local environmental conditions. Furthermore, in some cases, the differences in grain protein content resulted not from varying the application of nitrogen as fertilizer in replicated field experiments, but from other environmental impacts. In particular, the assembly of glutenin polymers is likely to be highly sensitive to local environmental factors such as temperature and water availability, as well as affected by genotype. When nitrogen fertilization effects alone are considered, the most common response is an increase in the gliadin to glutenin ratio, which may result in increased dough extensibility and viscosity (Pedersen and Jørgensen, 2007; Godfrey et al., 2010). This is illustrated in Figure 6.1D, which shows that dough extensibility is positively correlated with an increased ratio of gliadin : glutenin (the ratio of peaks F3 + F4/F1 + F2 separated by SE-HPLC) in the wheat cultivar Hereward grown with nitrogen applications ranging from 0 to 280 kg ha−1 (Godfrey et al., 2010). However, these effects of grain protein content on functionality are not universal, and several other reports have shown that differences related to the genotype and environment are stronger than the effects of nutrition (Panozzo and Eagles, 2000; Johansson et al., 2001; Ames et al., 2003). Also, any dough-weakening effect resulting from nitrogen fertilization may be outweighed by the increased total protein content, resulting in an overall increase in bread-making performance with higher applications of nitrogen fertilizer.

NUTRITION AND GRAIN COMPOSITION

Effect of sulfur on grain protein composition and functional properties of wheat Sulfur is an essential component of most proteins, being present in the amino acids cysteine and methionine. However, the impact of sulfur nutrition on cereal grain composition, as well as the requirement for sulfur fertilization, has only been appreciated in the past 30 years. Historically, crops in the United Kingdom received sulfur in two forms, as fertilizer (single superphosphate and ammonium sulfate) and from deposition from the atmosphere. However, the change to using fertilizers that do not contain sulfur (triple superphosphate, ammonium nitrate, and urea) and decreased deposition of sulfur from the atmosphere due to reduced emissions from industry have resulted in requirement for sulfur fertilization over much of the arable production area. The situation is highlighted in the United Kingdom by two surveys of the sulfur content in UK wheat grain grown a decade apart, in 1981 and 1982 (Byers et al., 1987) and 1992 and 1993 (Zhao et al., 1995). These showed a significant decrease in grain sulfur (by about a third) over the 10-year period, as shown in Figure 6.4. The N : S ratio also increased over this period, with up to 10% of the samples in 1992 and 1993 exceeding the S : N ratio of 17, which is regarded as the maximum for bread-making quality. The sulfur levels in UK soils have continued to decline over the past two decades, and sulfur is now routinely applied to a large proportion of the UK wheat crop grown for bread-making, at a recommended level of 15–20 kg ha−1. However, these studies in the United Kingdom were preceded by a series of detailed studies of sulfur deficiency in wheat and other crops in Australia which were ini-

111

tiated in the early 1980s (Moss et al., 1983; Wrigley et al., 1984, reviewed by Randall and Wrigley, 1986). These studies showed that sulfur deficiency differentially affected the proportions of the different groups of gluten proteins, in relation to their contents of cysteine and methionine. In particular, the proportion of the sulfur-poor ω-gliadins was significantly increased and the proportions of the sulfur-rich α-type and γ-type gliadins decreased (Wrigley et al., 1984). Similar effects on gliadin composition have since been reported in a number of other studies carried out in Europe and the United States, comparing either samples grown +/− sulfur or samples grown with increasing levels of nitrogen fertilization without the application of corresponding levels of sulfur (Fullington et al., 1987; Wieser et al., 2004; DuPont et al., 2006; Rogers et al., 2006; Zörb et al., 2009). The proportion of sulfur amino acids in the HMW subunits of glutenin (about 1–2 mol%) is intermediate between those of the sulfur-poor ω-gliadins (generally absent) and the LMW subunits and α-type and γtype gliadins (3–5 mol%) (Shewry et al., 2009a). The proportion of HMW subunits therefore generally increases and the proportion of LMW subunits decreases when sulfur is limited relative to nitrogen, although the effects on the proportions of the glutenin subunits tend to be limited compared with the effects on the gliadin fraction (Wrigley et al., 1984; Wieser et al., 2004; Zörb et al., 2009). Because the balance of gluten proteins determines the properties of dough, the changes in gluten protein composition with increasing sulfur fertilization result in reduced resistance to extension and increased extensibility of the dough, reducing the energy input required for mixing and improving the breadmaking performance (Zhao et al., 1999a,b; Wooding et al., 2000a,b; Flæte et al., 2005; Zörb et al., 2009).

112

NUTRIENT USE EFFICIENCY IN CROPS

Frequency (%) F

40 1993

1992

30

1982

20

1981

10 0

<1.0 1.0– 1.1– 1.2– 1.3– 1.4– 1.5– 1.6– 1.7– 1.8– 1.9– >2.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

Sulfur (mg g–1)

Frequency (%)

50 40

1981

1982

1993 1992

30 20 10 0

<10 10–11 11–12 12–13 13–14 14–15 15–16 16–17 17–18 18–19 19–20 >20

N:S ratio Frequency distributions of (A) grain sulfur and (B) grain N : S ratio in 1981, 1982, 1992, and 1993. Taken from Zhao et al. (1995), with permission.

Fig. 6.4.

Similar effects are observed when nitrogen and sulfur fertilizers are applied as foliar sprays rather than to the soil (Kettlewell et al., 1998; Tea et al., 2004, 2007). Effects on gluten protein polymerization may be mediated via effects on the form and content of glutathione, in addition to the direct effects resulting from the changes in protein composition (Tea et al., 2005; Reinbold et al., 2008). However, the extent of such secondary effects is not known. Finally, although most studies of sulfur fertilization have focused on the gluten proteins, early studies also showed effects on nongluten proteins (see, for example,

Wrigley et al., 1984). These effects have recently been studied in more detail by Flæte et al. (2005) and Grove et al. (2009), who used two-dimensional proteomic analysis to study the impact of sulfur fertilization on individual components. Effects of nitrogen and sulfur on protein composition and malting quality of barley The effects of nitrogen and sulfur fertilization on prolamin (hordein) composition in barley are similar to that in wheat. Early analyses of grain of the cultivar Julia grown

NUTRITION AND GRAIN COMPOSITION

in field experiments with 50, 100, 150, and 200 kg N ha−1 showed a decrease in the lysine content from over 4% total amino acids to less than 3%, and an increase in the proportion of hordein from about 37% to 50% of the total grain nitrogen (Kirkman et al., 1982). This was accompanied by a decrease in the ratio of sulfur-rich prolamins (B hordein) to sulfur-poor prolamins (C hordein) from 6.7 to 3.2, indicating that sulfur was limiting under conditions of high nitrogen fertilization. The importance of sulfur availability was confirmed by growing barley under severely restricted sulfur supply. This resulted in greatly decreased accumulation of total hordein (from about 50% to 27% of total grain nitrogen), and a dramatic increase in the proportion of C hordein from 32% to 71% of the total fraction in the cultivar Athos and from 25.5% to 82.5% in the cultivar Sundance (Shewry et al., 1983). In addition, the proportion of D hordein (the HMW prolamin of barley) increased from only traces to 15% and 5% of the total hordein fraction in the two cultivars, respectively. The same study also showed that hydrolysates of the sulfur-deficient grain contained high levels of aspartic acid, which was suggested to result from the hydrolysis of free asparagine, which accumulated as a storage pool due to the restricted synthesis of proteins. Similar relationships between the proportions of B hordein and C hordein and grain nitrogen content have been reported by Griffiths (1987), Molina-Cano et al. (2001), and Savin et al. (2006). Although low protein content is preferred for malting barley, the diastatic power actually increases with higher grain protein as the synthesis of β-amylase, the major diastatic enzyme, responds positively to nitrogen fertilization in the same way as hordeins (Giese and Hejgaard, 1984; Arends et al., 1995; Qi et al., 2006). The application of sulfur under conditions of nitrogen limita-

113

tion may result in substantial improvements in malt quality, with increased diastatic power, friability, α-amylase activity, and homogeneity, and reduced protein content by increasing yield (Zhao et al., 2006). The balance of nitrogen and sulfur is also important in determining the level of sulfur– methylmethionine (the precursor of dimethyl sulphoxide) in malt, with higher levels being present in grain grown with either low nitrogen (Nanamori et al., 2008) or high sulfur (Zhao et al., 2006). It is therefore important to maintain high levels of protein synthesis to ensure that the pools of nonprotein sulfur are low. Effects of nitrogen on β-glucan in oats The content of β-glucan in oats is an important quality criterion because of its accepted benefits for human health in lowering serum cholesterol (FDA, 1997, 2005). The application of nitrogen fertilizer to oats increases both the yield and the grain protein content, as reported for other cereals (Welch and Yong, 1980; Welch and Leggett, 1997; Zhou et al., 1998). Humphreys et al. (1994) and Saastamoinen et al. (1992) showed either no relationship or a negative relationship between nitrogen fertilization, grain protein, and βglucan content. By contrast, Weightman et al. (2004) showed that the β-glucan content increased when nitrogen was applied, in line with increases in yield and protein content. Weightman et al. (2004) also suggested that the application of foliar urea at flowering was preferable to application of nitrogen to soil in the spring as this reduced the risk of lodging. Effect of nitrogen and sulfur nutrition on other cereal grain components Free amino acids and asparagine Shewry et al. (1983) showed high levels of aspartic acid in hydrolyses of barley grain

114

NUTRIENT USE EFFICIENCY IN CROPS

grown under conditions of severe sulfur deficiency and concluded that it may have been derived from the accumulation of free asparagine in the grain. This observation became relevant to grain processing when it was demonstrated that asparagine was a major precursor for the formation of acrylamide, a neurotoxin and carcinogen, during the processing of cereal products (Mottram et al., 2002; Stadler et al., 2002). A direct relationship between sulfur deficiency of wheat, accumulation of grain asparagine, and increased acrylamide formation during processing has since been demonstrated by analysis of wheat grown under conditions of varying sulfur nutrition, in both pot experiments and field trials (Muttucumaru et al., 2006; Granvogel et al., 2007; Elmore et al., 2008; Curtis et al., 2009). The extent of the differences varied with the fertilizer regimes, but the effects observed were clearly relevant to the sulfur status of wheat crops grown commercially in the United Kingdom and other countries. Free amino acids, including asparagine, also increase with nitrogen fertilization and grain protein content in wheat (Claus et al., 2006), barley (Winkler and Schön, 1980) and rye (Dembinski and Bany, 1991). Consequently, high protein grain produced for bread-making will contain a higher content of asparagine than grain of low protein content. Analysis of milling fractions and mill streams demonstrated that asparagine was concentrated in the bran and embryo of the grain of both wheat and rye (Fredriksson et al., 2004; Shewry et al., 2009b), although the asparagine content of white flour (i.e., the starchy endosperm) of wheat was increased disproportionally compared with that of the bran fractions under conditions of sulfur deficiency (Shewry et al., 2009b). This indicated that the asparagine content of white flour could not be greatly reduced by

decreasing the milling yield to exclude a higher proportion of the bran from the white flour fraction. Essential trace elements Selenium is an essential micronutrient for humans, being required for a number of enzymes and also possibly protecting against oxidative damage (Arthur et al., 1997). Wheat is an important source of dietary selenium, but because the amount present in the grain is largely determined by selenium availability in the soil, the contribution of wheat to dietary intake of selenium varies widely with the region of cultivation. The selenium content of wheat is particularly important in the United Kingdom, where the contribution of wheat products to the total dietary intake of selenium fell from about 60 μg day−1 in the 1970s to 29–29 μg day−1 in 1995 (MAFF, 1997), due to the replacement of selenium-rich wheat imported from North America with selenium-poor wheat grown in the United Kingdom. One option to increase selenium density in wheat is “agronomic fortification” with seleniumcontaining fertilizers, as practiced in some other countries (Varo et al., 1988; Eurola et al., 1990). However, selenate and sulfate compete for the same enzymes and transporters in wheat (Terry et al., 2000; Hawkesford and Zhao, 2007; Shinmachi et al., 2010). Consequently, fertilization with sulfur, which may be required to improve the breadmaking quality or reduce the asparagine level for processing, may also result in decreased grain selenium (Stroud et al., 2010a,b). A similar effect of sulfur fertilization was also observed on the content of a second essential trace element, molybdenum (Stroud et al., 2010b), although in this case it is unlikely to affect the requirement for human health, which is adequately met in normal diets in most countries.

NUTRITION AND GRAIN COMPOSITION

Nutritional control of storage protein gene expression Early studies showed that the expression of prolamin genes in developing cereal grain, including wheat and barley, is primarily controlled at the level of gene transcription, with transcript abundance reflecting protein accumulation (Rahman et al., 1984; Bartels and Thompson, 1986; Sørensen et al., 1989). The levels of gene expression also respond positively to the availability of nitrogen and sulfur in the grain (Giese and Hopp, 1984; Duffus and Cochrane, 1992; DuPont et al., 2006). However, studies of barley grown with high and low levels of sulfur indicated that the efficiency of translation may also be moderated to a limited extent by the availability of the amino acids required for protein synthesis (Rahman et al., 1983). Most prolamin genes of barley and wheat have a conserved sequence of about 30 bp located about 300 bp upstream of the ATG translation initiation site (Forde et al., 1985). This “-300 element” or “prolamin box” contains two conserved motifs, called the E or endosperm motif and the N or nitrogen motif, which interact with transcription factors to regulate the transcription of the gene. Müller and Knudsen (1993) showed that these elements had distinct roles in regulating the expression of a barley C hordein gene, with the N motif acting as a negative regulator at low nitrogen levels, and interacting with the E motif to give high expression when sufficient nitrogen was available. Transcription factors that bind to both elements have been identified (HammondKosack et al., 1993; Albani et al., 1997; Vincente-Carbajosa et al., 1997), but the sensing mechanisms that link these transcription factors to nutritional status have not been identified. Whereas full prolamin boxes are present in the genes encoding sulfur-rich and sulfurpoor prolamins, the HMW prolamin genes

115

contain a sequence (the primary prolamin enhancer) that shows only limited similarity to prolamin box (Colot et al., 1987; Halford et al., 1989; Robert et al., 1989). Sequences corresponding to parts of the E and N motifs are also repeated further upstream and called the “partial enhancer repeat” and “N motif” (Norre et al., 2002). Although these motifs have been shown to bind nuclear proteins from maize (Norre et al., 2002), less is known about their regulation than about the prolamin box. The reader is referred to Halford and Shewry (2007) for a more detailed account of prolamin gene structure and expression. Conclusions Nitrogen and sulfur nutrition are immensely important for the production of wheat and other cereals. First, nitrogen is a major determinant of grain yield, as it is necessary to build the crop canopy to fix carbon. However, both minerals also have important effects on crop quality for the major end uses, by determining the amount and composition of the seed storage proteins. In particular, it is important to optimize the balance of the two minerals if optimum quality of wheat for bread-making and barley for malting is to be achieved. Acknowledgments Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. References Albani, D., Hammond-Kosack, M.C.U., Smith, C., et al. (1997) The wheat transcriptional activator SPA: a seed-specific BZIP protein that recognizes the GCN4-like motif in the biofactorial endosperm box of prolamin genes. The Plant Cell 9, 171–184.

116

NUTRIENT USE EFFICIENCY IN CROPS

Ames, N.P., Clarke, J.M., Dexter, J.E., Woods, S.M., Selles, F., & Marchylo, B. (2003) Effects of nitrogen fertilizer on protein quantity and gluten strength parameters in durum wheat (Triticum turgidum L. var. durum) cultivars of variable gluten strength. Cereal Chemistry 80, 203–211. Arends, A.M., Fox, G.P., Henry, R.J., Marschke, R.J., & Symons, M.H. (1995) Genetic and environmental variation in the diastatic power of Australian barley. Journal of Cereal Science 21, 63–70. Arthur, J.R., Brown, K.M., Fairweather-Tait, S.J., & Crews, H.M. (1997) Dietary selenium: why do we need it and how much is enough. Nutrition and Food Science 6, 225–228. Bartels, D. & Thompson, R.D. (1986) Synthesis of messenger-RNAs coding for abundant endosperm proteins during wheat grain development. Plant Science 46, 117–125. Benzian, B. & Lane, P. (1981) Interrelationship between nitrogen concentration in grain, grain yield and added fertiliser nitrogen in wheat experiments of South-east England. Journal of the Science of Food and Agriculture 32, 35–43. Bewley, J.D. & Black, M. (1978) Physiology and Biochemistry of Seeds. Vol. 1 Development Germination and Growth. Springer-Verlag, BerlinHeidelberg, p. 306. Byers, M., McGrath, S.P., & Webster, R. (1987) A survey of the sulphur content of wheat grown in Britain. Journal of the Science of Food and Agriculture 38, 151–160. Claus, A., Schreiter, P., Weber, A., et al. (2006) Influence of agronomic factors and extraction rate on the acrylamide contents in yeast-leavened breads. Journal of Agricultural and Food Chemistry 54, 8968–8976. Colot, V., Robert, L.S., Kavanagh, T.A., Bevan, M.W., & Thompson, R.D. (1987) Localization of sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco. EMBO Journal 6, 3559–3564. Curtis, T.Y., Muttucumaru, N., Shewry, P.R., et al. (2009) Effects of genotype and environment on free amino acid levels in wheat grain: implications for acrylamide formation during processing. Journal of Agricultural and Food Chemistry 57, 1013–1021. Daniel, C. & Triboi, E. (2000) Effects of temperature and nitrogen nutrition on the grain composition of winter wheat: effects on gliadin content and composition. Journal of Cereal Science 32, 45–56. Dembinski, E. & Bany, S. (1991) The amino acid pool of high and low protein rye inbred lines (Secale cereale L.). Journal of Plant Physiology 138, 494–496.

Duffus, C.M. & Cochrane, M.P. (1992) Grain structure and composition. In: Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology (ed. P.R. Shewry), pp. 291–317, CAB International, Wallingford, U.K. DuPont, F.M., Hurkman, W.J., Vensel, W.H., et al. (2006) Differential accumulation of sulfur-rich and sulfur-poor wheat flour proteins is affected by temperature and mineral nutrition during grain development. Journal of Cereal Science 44, 101–112. Elmore, S.J., Parker, J.K., Halford, N.G., Muttucumaru, N., & Mottram, D.S. (2008) Effects of plant sulfur nutrition on acrylamide and aroma compounds in cooked wheat. Journal of Agricultural and Food Chemistry 56, 6173–6179. Eurola, M., Ekholm, P., Ylinin, M., Koivistoinen, P., & Varo, P. (1990) Effects of selenium fertilization on the selenium content of cereal grains, flour and bread produced in Finland. Cereal Chemistry 67, 334–337. FAO/WHO/UNU (1985) necessidades de Energia y Proteinas. World Health Organisation, Geneva. FDA (1997) 21 CFR Part 101. Food labelling, health claims: soluble dietary fiber from certain foods and coronary heart disease. Federal Register 62, 3584–3601. FDA (2005) 21 CFR Part 101. Food labeling, health claims: soluble dietary fiber from certain foods and coronary heart disease. Federal Register 70, 76150–76162. Flæte, N.E.S., Hollung, K., Ruud, L., et al. (2005) Combined nitrogen and sulphur fertilisation and its effect on wheat quality and protein composition measured by SE-HPLC and proteomics. Journal of Cereal Science 41, 357–369. Forde, B.G., Heyworth, A., Pywell, J., & Kreis, M. (1985) Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucleic Acids Research 134, 7327–7339. Fredriksson, H., Tallving, J., Rosén, J., & Åman, P. (2004) Fermentation reduces free asparagine in dough and acrylamide content in bread. Cereal Chemistry 81, 650–653. Fullington, J.G., Miskelly, D.M., Wrigley, C.W., & Kasarda, D.D. (1987) Quality-related endosperm proteins in sulfur-deficient and normal wheat grain. Journal of Cereal Science 5, 233–245. Giese, H. & Hejgaard, J. (1984) Synthesis of saltsoluble proteins in barley. Pulse-labeling study of grain filling in liquid-cultured detached spikes. Planta 161, 172–177. Giese, H. & Hopp, E. (1984) Influence of nitrogen nutrition on the amount of hordein, protein Z and

NUTRITION AND GRAIN COMPOSITION

β-amylase messenger RNA in developing endosperms of barley. Carlsberg Research Communications 49, 365–383. Godfrey, D., Hawkesford, M., Powers, S., Millar, S., & Shewry, P.R. (2010) Nutritional effects on wheat grain composition and end use quality. Journal of Agricultural and Food Chemistry 58, 3012–3021. Granvogel, M., Wiester, H., Koehler, P., von Tucher, S., & Schieberle, P. (2007) Influence of sulfur fertilization on the amounts of free amino acids in wheat. Correlation with baking properties as well as with 3-aminopropionamide and acrylamide generation during baking. Journal of Agricultural and Food Chemistry 55, 4271–4277. Griffiths, D.W. (1987) The ratio of B to C hordeins in barley as estimated by high performance liquid chromatography. Journal of the Science of Food and Agriculture 38, 229–235. Grove, H., Hollung, K., Moldestad, A., Færgestad, E.M., & Uhlen, A.K. (2009) Proteome changes in wheat subjected to different nitrogen and sulfur fertilizations. Journal of Agricultural and Food Chemistry 57, 4250–4258. Halford, N.G. & Shewry, P.R. (2007) The structure and expression of cereal storage protein genes. In: Plant Cell Monographs, Endosperm Developmental and Molecular Biology (ed. O.-A. Olsen), pp. 195–218, Springer-Verlag, Berlin, Germany. Halford, N.G., Forde, J., Shewry, P.R., & Kreis, M. (1989) Functional analysis of the upstream regions of a silent and an expressed member of a family of wheat seed protein genes in transgenic tobacco. Plant Science 62, 207–216. Hammond-Kosack, M.C.U., Holdsworth, M.J., & Bevan, M.W. (1993) In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm. EMBO Journal 12, 545–554. Hawkesford, M.J. & Zhao, F.J. (2007) Strategies for increasing the selenium content of wheat. Journal of Cereal Science 46, 282–292. Humphreys, D.G., Smith, D.L., & Mather, D.E. (1994) Nitrogen fertilizer and seeding date induced changes in protein, oil and β-glucan contents of four oat cultivars. Journal of Cereal Science 20, 283–290. Jia, Y.-Q., Fabre, J.-L., & Aussenac, T. (1996a) Effects of growing location on response of protein polymerization to increased nitrogen fertilization for the common wheat cultivar Soissons: relationship with some aspects of the breadmaking quality. Cereal Chemistry 73, 526–532. Jia, Y.-Q., Masbou, V., Aussenac, T., Fabre, J.-L., & Debaeke, P. (1996b) Effects of nitrogen fertilization and maturation conditions on protein aggregates and on the breakmaking quality of Soissons, a

117

common wheat cultivar. Cereal Chemistry 73, 123–130. Johansson, E., Prieto-Linde, M.L., & Jonsson, J.O. (2001) Effects of wheat cultivar and nitrogen application on storage protein composition and breadmaking quality. Cereal Chemistry 78, 19–25. Kettlewell, P.S., Griffiths, M.W., Hocking, T.J., & Wallington, D.J. (1998) Dependence of wheat dough extensibility on flour sulphur and nitrogen concentrations and the influence of foliar-applied sulphur and nitrogen fertilisers. Journal of Cereal Science 28, 15–23. Kindred, D.R., Verhoeven, T.M.O., Weightman, R.M., et al. (2008) Effects of variety and fertiliser nitrogen on alcohol yield, grain yield, starch and protein content, and protein composition of winter wheat. Journal of Cereal Science 48, 46–57. Kirkman, M.A., Shewry, P.R., & Miflin, B.J. (1982) The effect of nitrogen nutrition on the lysine content and protein composition of barley seeds. Journal of the Science of Food and Agriculture 33, 115– 127. Landry, J. & Delhaye, S. (1993) The tryptophan contents of wheat, maize and barley grains as a function of nitrogen content. Journal of Cereal Science 18, 259–266. MAFF (1997) Dietary intake of selenium. Food Surveillance Info Sheet No. 126. Molina-Cano, J.L., Polo, J.P., Romera, E., Araus, J.L., Zarco, J., & Swanston, J.S. (2001) Relationships between barley hordeins and malting quality in a mutant of cv Triumph 1. Genotype by environment interaction of hordein content. Journal of Cereal Science 34, 285–294. Moss, H.J., Randall, P.J., & Wrigley, C.W. (1983) Alteration to grain, flour and dough quality in three wheat types with variation in soil sulfur supply. Journal of Cereal Science 1, 255–264. Mosse, J. (1990) Nitrogen to protein conversion factor for ten cereals and six legumes or oilseeds. A reappraisal of its definition and determination. Variation according to species and to seed protein content. Journal of Agricultural and Food Chemistry 38, 18–24. Mossé, J. & Huet, J.-C. (1990) Amino acid composition and nutritional score for ten cereals and six legumes or oilseeds: causes and ranges of variations according to species and to seed nitrogen content. Sciences des Alimentations 10, 151–173. Mossé, J., Huet, J.-C., & Baudet, J. (1985) The aminoacid composition of wheat-grain as a function of nitrogen-content. Journal of Cereal Science 3, 115–130. Mossé, J., Huet, J.-C., & Baudet, J. (1988a) The amino acid composition of triticale grain as a function of

118

NUTRIENT USE EFFICIENCY IN CROPS

nitrogen content: comparison with wheat and rye. Journal of Cereal Science 7, 49–60. Mossé, J., Huet, J.-C., & Baudet, J. (1988b) The amino acid composition of rice grain as a function of nitrogen content as compared with other cereals: a reappraisal of rice chemical stores. Journal of Cereal Science 8, 165–175. Mottram, D.S., Wedzicha, B.L., & Dodson, A.T. (2002) Acrylamide is formed in the Maillard reaction. Nature 419, 448–449. Müller, M. & Knudsen, S. (1993) The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box. Plant Journal 4, 343–355. Muttucumaru, N., Halford, N.G., Elmore, S.J., et al. (2006) Formation of high levels of acrylamide during the processing of flour derived from sulphate-deprived wheat. Journal of Agricultural and Food Chemistry 54, 8951–8955. Nanamori, M., Kanatani, R., Kihara, M., et al. (2008) Effects of nitrogen application on malt modification and dimethyl sulphide precursor production in two Japanese barley cultivars. Journal of the Science of Food and Agriculture 88, 1464–1471. Newman, C.W. & McGuire, C.F. (1985) Nutritional quality of barley. In: Barley Agronomy Monograph 26 (ed. D.C. Rasmussen), pp. 403–456, CSSASSSA, Madison, WI. Norre, F., Peyrot, C., Garcia, C., et al. (2002) Powerful effect of an a typical bifactorial endosperm box from wheat HMWG-Dx5 promoter in maize endosperm. Plant Molecular Biology 50, 699–712. Osborne, T.B. (1924) The Vegetable Proteins. Longmans Green & Co, London, p. 154. Panozzo, J.F. & Eagles, H.A. (2000) Cultivar and environmental effects on quality characters in wheat. II Protein. Australian Journal of Agricultural Research 51, 629–636. Paul, A.A. & Southgate, D.A.T. (1978) McCance and Widdowson’s the Composition of Foods, 4th ed. Elsevier, Amsterdam. Pechanek, U., Karger, A., Gröger, S., Charvat, B., Schöggl, G., & Lelley, T. (1997) Effect of nitrogen fertilization on quantity of flour protein components, dough properties and breadmaking quality of wheat. Cereal Chemistry 74, 800–805. Pedersen, L. & Jørgensen, J.R. (2007) Variation in rheological properties of gluten from three biscuit wheat cultivars in relation to nitrogen fertilisation. Journal of Cereal Chemistry 46, 132–138. Qi, J.-C., Zhang, G.-P., & Zhou, M.-X. (2006) Protein and hordein content in barley seeds as affected by nitrogen level and their relationship to beta-amylase activity. Journal of Cereal Science 43, 102–107.

Rahman, S., Shewry, P.R., Forde, B.G., Kreis, M., & Miflin, B.J. (1983) Nutritional control of storageprotein synthesis in developing grain of barley (Hordeum vulgare L.). Planta 159, 366–372. Rahman, S., Kreis, M., Forde, B.G., Shewry, P.R., & Miflin, B.J. (1984) Hordein-gene expression during development of the barley (Hordeum vulgare) endosperm. The Biochemical Journal 223, 315–322. Randall, P.J. & Wrigley, C.W. (1986) Effects of sulfur supply on the yield, composition, and quality of grain from cereals, oilseeds and legumes. In: Advances in Cereal Science and Technology, Vol. VIII (ed. Y. Pomeranz), pp. 171–206, AACC, St Paul, MN. Reinbold, J., Rychlik, M., Asam, S., Wieser, H., & Koehler, P. (2008) Concentrations of total glutathione and cysteine in wheat flour as affected by sulfur deficiency and correlation to quality parameters. Journal of Agricultural and Food Chemistry 56, 6844–6850. Robbins, G.S., Pomeranz, Y., & Briggle, L.W. (1971) Amino acid composition of oat groats. Journal of Agricultural Food Chemistry 19, 536–539. Robert, L.S., Thompson, R.D., & Flavell, R.B. (1989) Tissue-specific expression of a wheat high molecular weight glutenin gene in transgenic tobacco. The Plant Cell 1, 569–578. Rogers, W.J., Cogliatti, M., Lerner, S.E., et al. (2006) Effects of nitrogen and sulfur fertilizers on gliadin composition of several cultivars of durum wheat. Cereal Chemistry 83, 677–683. Saastamoinen, M., Plaami, S., & Kumpulainen, J. (1992) Genetic and environmental variation in βglucan content of oats cultivated or tested in Finland. Journal of Cereal Science 16, 279–290. Savin, R., Prystupa, P., & Araus, J.L. (2006) Hordein composition as affected by post-anthesis sourcesink ratio under different nitrogen availabilities. Journal of Cereal Science 44, 113–116. Shewry, P.R. (2007) Improving the protein content and composition of cereal grain. Journal of Cereal Science 46, 239–250. Shewry, P.R., Franklin, J., Parmar, S., Smith, S.J., & Miflin, B.J. (1983) The effects of sulphur starvation on the amino acid and protein compositions of barley grain. Journal of Cereal Science 1, 21–31. Shewry, P.R., Tatham, A.S., & Halford, N. (1999) The prolamins of the Triticeae. In: Seed Proteins (eds. P.R. Shewry & R. Casey), pp. 35–78, Kluwer Academic Publishers, Dordrecht. Shewry, P.R., Halford, N.G., & Tatham, A.S. (2003a) Genetics of wheat gluten proteins. II Wheat gluten proteins. In: Advances in Genetics, Vol. 49 (eds. J.C. Hall, J.C. Dunlap & T. Friedman), pp. 111–184, Academic Press, San Diego, CA.

NUTRITION AND GRAIN COMPOSITION

Shewry, P.R., Halford, N.G., & Tatham, A.S. (2003b) The high molecular weight subunits of wheat glutenin and their role in determining wheat processing properties. In: Advances in Food and Nutrition Research, Vol. 45 (ed. S.L. Taylor), pp. 219–302, Academic Press, San Diego, CA. Shewry, P.R., D’Ovidio, R., Lafiandra, D., Jenkins, J.A., Mills, E.N.C., & Békés, F. (2009a) Wheat grain proteins. In: Wheat: Chemistry and Technology, 4th ed. (eds. K. Khan & P.R. Shewry), pp. 223–298, AACC, St Paul, MN. Shewry, P.R., Zhao, F.-J., Gowa, G.B., et al. (2009b) Sulphur nutrition differentially affects the distribution of asparagine in wheat grain. Journal of Cereal Science 50, 407–409. Shinmachi, F., Buchner, P., Stroud, J.L., et al. (2010) Influence of sulfur deficiency on the expression of specific sulfate transporters and the distribution of sulfur, selenium and molybdenum in wheat. Plant Physiology 153, 327–336. Shoup, F.K., Pomeranz, Y., & Deyoe, C.W. (1966) Amino acid compositions of wheat varieties and flours varying widely in bread-making potentialities. Journal of Food Science 31, 94–101. Sørensen, M.B., Cameron-Mills, V., & Brandt, A. (1989) Transcriptional and post-transcriptional regulation of gene expression in developing barley endosperm. Molecular & General Genetics 217, 195–201. Stadler, R.H., Blank, I., Varga, N., et al. (2002) Acrylamide from Maillard reaction products. Nature 419, 449–450. Stroud, J.L., Li, H.F., Lopez-Bellido, F.J., et al. (2010a) Impact of sulphur fertilisation on crop response to selenium fertilisation. Plant Soil 332, 31–40. Stroud, J.L., Zhao, F.J., Buchner, P., et al. (2010b) Impacts of sulphur nutrition on selenium and molybdenum concentrations in wheat grain. Journal of Cereal Science 52, 111–113. Tea, I., Genter, T., Naulet, N., Boyer, V., Lummerzheim, M., & Kleiber, D. (2004) Effect of foliar sulfur and nitrogen fertilization on wheat storage protein composition and dough mixing properties. Cereal Chemistry 81, 759–766. Tea, I., Genter, T., Violleau, F., & Kleiber, D. (2005) Changes in the glutathione thiol-disulfide status in wheat grain by foliar sulphur fertilization: consequences for the rheological properties of dough. Journal of Cereal Science 41, 305–315. Tea, I., Genter, T., Naulet, N., Lummerzheim, M., & Kleiber, D. (2007) Interaction between nitrogen and sulfur by foliar application and its effects on flour bread-making quality. Journal of the Science of Food and Agriculture 87, 2853–2859.

119

Terry, N., Zayed, A.M., de Souza, M.P., & Tarun, A.S. (2000) Selenium in higher plants. Annual Review of Plant Physiology 51, 401–432. Varo, P., Alfthan, G., Ekholm, P., Aro, A., & Koivistoinen, P. (1988) Selenium intake and serum selenium in Finland—effects of soil fertilisation with selenium. American Journal of Clinical Nutrition 48, 324–329. Vincente-Carbajosa, J., Moose, S.P., Parsons, R., & Schmidt, R.J. (1997) A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2. Proceedings of the National Academy of Sciences of the United States of America 94, 7685–7690. Weightman, R.M., Heywood, C., Wade, A., & South, J.B. (2004) Relationship between grain (1→3,1 →4)-β-D-glucan concentration and the response of winter-sown oats to contrasting forms of applied nitrogen. Journal of Cereal Science 40, 81–86. Welch, R.W. & Leggett, J.M. (1997) Nitrogen content, oil content and oil composition of oat cultivars (A. sativa) and wild Avena species in relation to nitrogen fertility, yield and partitioning of assimilates. Journal of Cereal Science 26, 105–120. Welch, R.W. & Yong, Y.Y. (1980) The effects of variety and nitrogen fertiliser on protein production in oats. Journal of the Science of Food and Agriculture 31, 541–548. Wieser, H., Gutser, R., & von Tucher, S.W. (2004) Influence of sulphur fertiliser on the quantities and proportions of gluten protein types in wheat flour. Journal of Cereal Science 40, 239–244. Wieser, H. & Seilmeier, W. (1998) The influence of nitrogen fertilisation on quantities and proportions of different protein types in wheat flour. Journal of Science of Food and Agriculture 76, 49–55. Wieser, H., Gutser, R., & von Tucher, S. (2004) Influence of sulphur fertilisation on quantities and proportions of gluten protein types in wheat flour. Journal of Cereal Science 40, 239–244. Winkler, U. & Schön, W.J. (1980) Amino acid composition of the kernel proteins in barley resulting from nitrogen fertilization at different stages of development. Journal of Agronomy and Crop Science 149, 503–512. Wooding, A.R., Kavale, S., MacRitchie, F., Stoddard, F.L., & Wallace, A. (2000a) Effects of nitrogen and sulfur fertilizer on protein composition, mixing requirements, dough strength of four wheat cultivars. Cereal Chemistry 77, 798–807. Wooding, A.R., Kavale, S., Wilson, A.J., & Stoddard, F.L. (2000b) Effects of nitrogen and sulfur fertilization on commercial-scale wheat quality and mixing requirements. Cereal Chemistry 77, 791–797.

120

NUTRIENT USE EFFICIENCY IN CROPS

Wrigley, C.W., Du Cros, D.L., Fullington, J.G., & Kasarda, D.D. (1984) Changes in polypeptide composition and grain quality due to sulfur deficiency in wheat. Journal of Cereal Science 2, 15–24. Zhao, F.-J., McGrath, S.P., & Crosland, A.R. (1995) Changes in the sulphur status of British wheat grain in the last decade, and its geographical distribution. Journal of the Science of Food and Agriculture 68, 507–514. Zhao, F.-J., Salmon, S.E., Withers, P.J.A., et al. (1999a) Responses of breadmaking quality to sulphur in three wheat varieties. Journal of the Science of Food and Agriculture 79, 1865–1874. Zhao, F.-J., Salmon, S.E., Withers, P.J.A., et al. (1999b) Variation in the breadmaking quality and rheological properties of wheat in relation to sulphur nutrition under field conditions. Journal of Cereal Science 30, 19–31. Zhao, F.J., Fortune, S., Barbosa, V.L., et al. (2006) Effects of sulphur on yield and malting quality of barley. Journal of Cereal Science 43, 369–377.

Zhou, M.X., Roberts, G.L., Robards, K., GlennieHolmes, M., & Helliwell, S. (1998) Effects of sowing date, nitrogen application and sowing rate on oat quality. Australian Journal of Agricultural Research 49, 845–852. Zhu, J. & Khan, K. (2001) Separation and quantification of HMW glutenin subunits by capillary electrophoresis. Cereal Chemistry 78, 737–742. Zhu, J., Khan, K., Huang, S., & O’Brien, L. (1999) Allelic variation at Glu-D1 locus for high molecular weight (HMW) glutenin subunits: quantification by multistacking SDS-PAGE of wheat grown under nitrogen fertilization. Cereal Chemistry 76, 915–919. Zörb, C., Steinfurth, D., Seling, S., et al. (2009) quantitative protein composition and baking quality of winter wheat as affected by late sulfur fertilization. Journal of Agricultural and Food Chemistry 57, 3877–3885.

Part II

Nitrogen as a Key Driver of Production

Chapter 7

Genetic Improvement of Nutrient Use Efficiency in Wheat Jacques Le Gouis

Abstract Breeding for an efficient use of nitrogen (N) was not a major breeding objective for wheat while nitrogen fertilizers were relatively cheap and environmental concerns were low. However, more recently, the improvement of nitrogen use efficiency (NUE) has taken a larger part in genetic studies and breeding strategies. In addition, very different situations exist because of the variety of management systems and end-use qualities. Although few studies have reported directly on NUE genetic progress, numerous studies have demonstrated an increase in grain yield with variety date of release. However, generally, genetic progress has been lower when estimated at low nitrogen inputs compared with high inputs. Furthermore, no difference between old and modern cultivars was observed for NUE at the optimum nitrogen level. As grain protein concentration is a major quality trait for wheat, its correlation to NUE was reported. There is a negative correlation between NUE and grain protein concentration, which must be taken into account if high grain protein concentrations are needed. While the majority of wheat cul-

tivars are pure lines, in several studies, hybrid varieties of wheat have been shown to be more efficient in using and taking up nitrogen. With regard to breeding strategies, it has been shown that direct selection will be more efficient than indirect selection at high nitrogen inputs to improve performance at low nitrogen levels. Nitrogen uptake rather than nitrogen utilization efficiency has been reported to be the main trait related to NUE. Finally, several quantitative genetic studies have identified chromosomal regions involved in the control of NUE and the responsiveness to nitrogen rate. The next challenge for breeding improved NUE will be to take into account environmental modifications associated with climate change. Introduction Breeding for an efficient use of nitrogen (N) has not been a major objective for wheat in most of the last century. As mineral nitrogen fertilizers were relatively cheap and readily available, on farm yields were largely not limited by nitrogen nutrition (Legg, 2005). Previously, breeding programs, registration trials, and evaluation performed by produc-

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 123

124

NUTRIENT USE EFFICIENCY IN CROPS

ers have been conducted at high nitrogen levels. One of the main contributions of selection was the introduction in commercial cultivars of semi-dwarfing genes from the Japanese variety Norin10 as part of wheat improvement programs in the United States and at the International Maize and Wheat Improvement Center (CIMMYT) in Mexico in the 1960s (Ellis et al., 2002). A reduction in plant height improved lodging resistance and enabled the crop to sustain larger nitrogen fertilizer applications. Both economic and environmental reasons now put nitrogen use efficiency (NUE) at the center of breeding programs (Hirel et al., 2007). Developments in fertilizer production and application may also be used to improve nutrient efficiency and minimize environmental impacts as discussed by Chien et al. (2008). The simple statement that it is necessary to select and release new varieties showing better NUE hides a more complex situation because different management systems and end-use qualities have to be considered (see also Chapter 1). Huge differences exist in terms of available nitrogen between intensive cropping systems and low-input or organic farming systems. A large amount of readily available nitrogen can be applied in conventional agricultural systems to fulfill requirements depending on crop cycles. In France, the average fertilizer nitrogen applied was 165 kg ha−1 in three to four split applications in 2006. Low-input or organic systems often have limited pools of mineral nitrogen and rely greatly on organic sources and internal cycling of nitrogen, with seasonal nitrogen availability not necessarily matching crop demand (Dawson et al., 2008). Nitrogen is also the constituent of grain proteins, and concentration and composition are major determinants of wheat end-use value. High grain protein concentrations (GPC), up to 12–13%, are required for bread-making depending on the baking process, while lower concentrations would

be adequate for feed wheat. Biofuel or distilling will require low-protein grain as a strong negative correlation has been reported between grain nitrogen content and alcohol yield (Kindred et al., 2008). Harvestable dry matter (DM) and grain nitrogen do not increase in direct proportion to nitrogen supply so efficiencies of nitrogen use are highly dependent on the level of nitrogen supply at which they are measured (Sylvester-Bradley and Kindred, 2009). Different considerations will then fix nitrogen rates. If environmental concerns are the first priority, low nitrogen rates will be considered as both nitrogen uptake (limiting nitrogen leaching, nitrogen volatilization, and energy use to produce nitrogen fertilizer) and nitrogen utilization efficiencies are higher at low nitrogen. If, in the context of the increasing world demand for grain, production per unit of land is the priority, the optimum nitrogen level, defined as the minimal nitrogen rate enabling the maximal yield, will be considered. In this case an optimized nitrogen management during the whole crop cycle will possibly control nitrogen losses through leaching and volatilization. However, an insurance strategy will usually be considered where over-optimal rates will be used to ascertain maximum grain yield. Finally, an intermediate and price-dependent strategy may be used if nitrogen rates are set according to economic principles where the relative prices of fertilizer nitrogen and grain are taken into account (Sylvester-Bradley and Kindred 2009). In any case, the optimum nitrogen level would then have to be adapted if high GPC are also required as maximum grain yield (GY) and maximum GPC are generally not obtained at the same nitrogen rate and with the same application strategy (Lopez-Bellido et al., 2006). Moll et al. (1982) stated that an ideal wheat genotype should capture more N from the soil and the fertilizer, produce a high GY per unit of absorbed nitrogen, and have little

GENETIC IMPROVEMENT OF NUTRIENT USE EFFICIENCY IN WHEAT

nitrogen in the straw at harvest. Given the large genotype × environment × management interaction usually observed for most quantitative traits including NUE (Le Gouis et al., 2000; Barraclough et al., 2010), it is highly probable that different varieties are required that are adapted to specific management systems and utilization. Wheat grown in the United Kingdom has been estimated to have an average NUE of 25 kg DM kg−1 nitrogen available, with an average fertilizer and soil nitrogen recovery of 65% and an average nitrogen utilization of 39 kg DM kg−1 nitrogen captured (Sylvester-Bradley and Kindred, 2009). This leaves some room for genetic improvement as higher values for both nitrogen capture efficiency (NUpE) and nitrogen utilization efficiency (NUtE) have been reported in field experiments. The question remains as to whether nitrogen capture has to tend to 100%, as it may have an impact on long-term soil fertility, and if no residual nitrogen is left in the soil, more would have to be applied for the following crop. Apparent fertilizer recovery has, however, been estimated to be 45% at optimum nitrogen for 27 cultivars tested in the United Kingdom (Foulkes et al., 1998), so that at least more nitrogen coming from fertilizers should be captured. High values have been reported in experimental trials for NUtE, from 27 to 77 kg DM kg−1 in the United Kingdom (Barraclough et al., 2010) and from 32 to 55 in France (calculated from Le Gouis et al., 2000). The existence of genotype × environment interactions, generally meaning that cultivar ranking will depend on the nitrogen level, has been reported for NUE and its components. Interestingly, it seems that these interactions are higher for NUpE than for NUtE. Le Gouis et al. (2000) did not find significant genotype × nitrogen interaction for NUtE, while most of the interaction for GY was explained by the interaction for NUpE. In the same way, Barraclough et al. (2010) showed a stronger correlation between rank-

125

ings at 0 versus 200 kg ha−1 for NUtE than for NUpE. An extensive literature exists on NUE and its improvement (Good et al., 2004; Hirel et al., 2007; Dawson et al., 2008; Foulkes et al., 2009; Spiertz, 2010; Chapters 1, 4, 8–11, this book) that will provide additional information not treated in this chapter. Here principally four topics are addressed: the genetic progress for NUE, the relationship between NUE and GPC, heterosis (hybrid vigor) for NUE, and finally, breeding for NUE. The genetic progress for NUE In a very simple way, NUE can be defined as the ratio of GY to nitrogen available to the plant. As GY is the main wheat breeding trait, data are available for almost any wheat trial. However, as noted by many authors, available nitrogen is much more difficult to assess (see Dawson et al., 2008 for a discussion) and is either not reported or is inconsistently estimated, hindering comparisons. This tends to limit the possibilities to estimate evolution of NUE. However, SylvesterBradley and Kindred (2009) used available data to estimate the average evolution of NUE in the United Kingdom over the last 30 years. They used national survey data for GY, grain nitrogen content, and nitrogen applications, and a fixed value for soil nitrogen (estimated at 80 kg N ha−1) and nitrogen harvest index (NHI = 0.75). They estimated that NUE had increased by +1.2 kg grain DM kg−1 available N decade−1 from 1974 to 2008, reaching a mean NUE of 24.4 kg grain DM kg−1 available N over the last decade. Assuming a constant NHI, NUE may have improved through better nitrogen capture (+3.7% decade−1) rather than through better nitrogen conversion to GY (no significant trend). A similar trend was reported for maize in Iowa (USA) as nitrogen application has stabilized since the late 1970s, while GY increased, inferring

126

NUTRIENT USE EFFICIENCY IN CROPS

that soil and fertilizer use efficiency has increased notably since then (Fischer and Edmeades, 2010). This increase in NUE was due in part to genetic progress, and also to the changes in agricultural practices, in the biotic/abiotic environment (climate and soil changes), and to the adaptation of varieties to these changes (the genotype × environment × management interaction). To partition the different effects, experiments are usually conducted where genotypes bred at different periods are grown in the same experiments. The biotic and abiotic environments may of course change over the time span of the varieties compared (e.g., climate or soil evolution, ozone, or CO2 increase), in which case apparent breeding progress in gain yield could include adaptation to these changes (Fischer and Edmeades, 2010). As already noted, very few studies were conducted on NUE, as available nitrogen is often not estimated. If it is assumed that available N is the same for all the genotypes in a single field trial, then NUE is directly related to GY. This can be considered a first estimate that does not take into account difference in the length of the growth cycle that may cause a bias in the estimation, as early and late genotypes would have access to different amounts of nitrogen due to mineralization of organic matter. Brancourt-Hulmel et al. (2003) reported a GY increase of 6 to 59 kg ha−1 year−1, surveying 16 studies on wheat genetic progress, suggesting a similar genetic progress for NUE. However, a few studies have reported directly on NUE change (Ortiz-Monasterio et al., 1997; Muurinen et al., 2006; SylvesterBradley and Kindred, 2009). An NUE increase of 0.149 kg GY kg−1 N year−1 was observed at high nitrogen levels (200 kg ha−1 N applied) for the last 30 years (SylvesterBradley and Kindred, 2009). Several authors have compared the genetic progress at different nitrogen levels (Table 7.1). All report a higher absolute genetic progress (in kg ha−1),

at high compared with low nitrogen inputs. For example, Ortiz-Monasterio et al. (1997) reported genetic progress nearly three times higher at 300 kg N ha−1 compared to without fertilizer. A common explanation for this situation is that breeding is usually carried out at a high nitrogen input (Brancourt-Hulmel et al., 2005; Muurinen et al., 2006), thus selecting genotypes adapted to these conditions. However, when relative genetic progress (in % ha−1) is considered, the difference between different nitrogen levels is usually less obvious (Table 7.2). Very few studies have compared cultivars at the optimum nitrogen levels because such a study requires large experiments with numerous nitrogen levels for each cultivar. However, SylvesterBradley and Kindred (2009) were able to compute NUE at the optimum nitrogen level, which they termed “on-farm NUE.” They used multisite nitrogen response experiments, where genotypes were compared at several nitrogen levels, enabling the identification of the optimum nitrogen level (defined as the nitrogen level giving the maximum economic margin with a fertilizer nitrogen : grain price ratio = 5). They showed that the optimum nitrogen level increased by 1.24 kg N ha−1 year−1, reaching 174 kg N ha−1 for the cultivars released during the 2001–2007 period. Similar results showing an increased optimum nitrogen level with modern cultivars were previously reported by Foulkes et al. (1998) and Guarda et al. (2004). Sylvester-Bradley and Kindred (2009) were then able to compute the change of NUE at the optimum nitrogen level, and in this case they did not observe any significant change. Old (released between 1977 and 1987) and modern (2001–2007) cultivars showed approximately the same NUE at the nitrogen optimum. Both old and modern cultivars had therefore approximately the same NUpE (0.83 kg kg−1) and NUtE (35 kg kg−1) at the nitrogen optimum. Apparently, breeding has not increased onfarm NUE but mainly has increased the optimum nitrogen level.

Table 7.1. Assessment of genetic gain in wheat yields at different nitrogen levels from direct comparison of old

and modern cultivars grown simultaneously Country

Period

Number of Genotypes

Nitrogen Level (kg N ha−1) Paternoster field + 38 Camp field + 104 0 75 150 300 0 Noptimum No fungicide + 0 Fungicides + 0 No fungicide + 40–100 Fungicides + 40–100 0 80 160 0 40 215–250 0 50–200 0 200 Noptimum

UK

1908–1978

12

Mexico

1950–1985

10

UK

1969–1988

22

France

1946–1992

14

Italy

1900–1994

16

Ireland

1977–1991

10

Mexico

1966–200

24

UK

1977–2007

25

Gain (kg GY ha−1 year−1) ,

18.3 * § 30.3 *,§ 32.1* 42.8* 59.2* 89.4* ns 96† 35.7* 41.6* 54.4* 62.9* 19.2‡,§ 38.5‡,§ 43.0‡,§ 21† 25† 65† NS 14*,§ 21† 53† 55†

Reference Austin et al. 1980 OrtizMonasterio et al., 1997 Foulkes et al., 1998 BrancourtHulmel et al. 2003 Guarda et al., 2004 White and Wilson 2006 Serret et al. 2008 SylvesterBradley and Kindred, 2009

*100% DM. †85% DM. ‡87% DM. §Estimated from publication data, NS: not significant.

Table 7.2. Assessment of yearly percent genetic gain in wheat grain yields, nitrogen uptake, and nitrogen utilization efficiency from direct comparison of old and modern cultivars grown simultaneously

Period 1908–1978

1950–1985

Nitrogen Level (kg N ha−1)

Grain Yield (% year−1)

Nitrogen Uptake (% year−1)

Nitrogen Utilization (% year−1)

Paternoster Field + 38 Camp Field + 104 0 75 150 300

0.49*

0.19*

0.31*

0.50* 1.15 1.00 1.26 1.87

0.20* 0.92 0.58 0.40 0.70

0.30* 0.23 0.42 0.86 1.17

Reference Austin et al. 1980 OrtizMonasterio et al., 1997

*Estimated from publication data.

127

128

NUTRIENT USE EFFICIENCY IN CROPS

NUE relation to GPC As several studies have shown a negative correlation between genetic progress for GY and grain nitrogen concentration (GNC), the relation between GPC and NUE must be discussed (see also Chapter 1). GPC and composition are major determinants of wheat end-use and value. Depending on final use, different protein concentrations may be required. Usually, high concentrations are required for baking quality (e.g., Branlard et al., 2001; Oury et al., 2010). The composition of grain storage protein is also known to determine wheat end-use value as it influences dough cohesiveness and viscoelasticity (Weegels et al., 1996), with highly elastic (strong) dough being suited for breadmaking and more extensible (weak) dough being suited for cakes and biscuit making. Selection has not been so stringent for feed wheat so that breeding has resulted in differences in protein content of about 2% dry weight between high-protein bread-making varieties and low-protein varieties suitable for livestock feed, as illustrated by the two UK cultivars Avalon (feed) and Hobbit (bread) (Snape et al., 1993). In recent years, wheat has been considered a possible energy crop and for grain distilling (Loyce et al., 2002). As the demand for biofuels derived from plants is likely to increase, so is the demand for wheat destined for bioethanol production. A strong negative correlation has been reported between grain nitrogen content and alcohol yield (Kindred et al., 2008), so that low-protein but high-yielding cultivars will be ideal for this use. Numerous studies have shown a negative correlation between GY and GNC in wheat (e.g., Kibite and Evans, 1984; Simmonds, 1995; Triboï et al., 2006) and in other cereals (Feil, 1997). Oury et al. (2003) have shown that on average the coefficient of correlation is about −0.8. The slope of the regression is

such that a genetic GY increase of 1 t ha−1 will mean a 1% decrease in GPC. Barraclough et al. (2010) also reported a near-functional inverse relationship between NUtE and grain %N (r2 = 0.96). The physiological bases of the negative GY–grain protein correlation are still largely unknown, in spite of various hypotheses proposed. The most common are a trade-off between nitrogen remobilization from the leaves and photosynthetic activity (Feil, 1997), a dilution effect of nitrogen by carbon-based compounds (Acreche and Slafer, 2009), and an energetic competition between carbon and nitrogen assimilation (Munier-Jolain and Salon, 2005). For energy, biofuel, distilling, and even feed use, wheat cultivars with low grain nitrogen content and high NUE may be selected. The minimum percentage of nitrogen in the straw and grain at harvest will determine how much biomass or grain can be produced per unit of absorbed nitrogen, provided it is higher than the amount of nitrogen required for optimal radiation interception and photosynthesis by the canopy. Values as low as 7.2% GPC were reported by Martre et al. (2006) and Bogard et al. (2010). Kindred et al. (2008) reported a value of 6.61% for the UK feed cultivar Riband grown without nitrogen fertilizer. Applying the usual nitrogen concentration– protein concentration relationship (N = protein/5.7), this value corresponds to a nitrogen concentration of 1.16%. It is difficult to assess whether lower values may be reached, as no direct conscious selection for low GNC has probably been carried out in wheat. Conversely, a long-term divergent selection experiment for protein concentration was started in 1896 for maize (Dudley, 2007) and two main selected strains were established: Illinois High Protein (IHP) and Illinois Low Protein (ILP). Mass selection, based on analysis of individual ears, was

GENETIC IMPROVEMENT OF NUTRIENT USE EFFICIENCY IN WHEAT

applied with a selection intensity of approximately one ear out of five. No progress in ILP was observed after generation 76, while selection continued to show progress for IHP after 100 generations. ILP values as low as 4.0 were reached, probably corresponding to the lower limit in this background (Dudley and Lambert, 1992). Although maize and wheat kernels and proteins are quite different, this useful information may be considered when assessing the possible lower limit for cereal grain GPC. For baking quality, a value of 10% GPC is generally considered the lower limit accepted for baking bread without significant adaptation of the process. Increasing the proportion of nitrogen that is remobilized from the vegetative part to the grain would be a first solution to maintain GPC. An alternative to high-protein wheat for bread-making is to modify protein composition to increase dough strength and viscoelasticity, allowing for lower protein grain to be suitable for bread-making (see Chapter 6 for an extended description of wheat storage proteins). Guarda et al. (2004) have already observed that the lower GPC of the recent cultivars is matched by a quality improvement in protein composition. Storage proteins are divided into two fractions, gliadins and glutenin, which are the main components of gluten. Glutenins, which are polymeric proteins, mainly determine viscoelastic properties, whereas gliadins, which are monomeric proteins, strongly influence dough extensibility (Hoseney, 1986). Breeding for high NUE wheat with lower GPC would avoid the current situation in which high GPC is generally associated with an increased the gliadin : glutenin ratio, resulting in increased extensibility of the dough (Kindred et al., 2008). Although it may be possible to select for low gliadin content (Kindred et al., 2008), it is unlikely that this will lead to the

129

development of low-protein varieties suitable for bread-making (Foulkes et al., 2009). However, recently, changes in allocation between protein fractions were associated to polymorphism in the sequence of the storage protein activator (SPA) transcription factor (Ravel et al., 2009). The haplotypes for the three homoeologous copies were associated with significant quantitative differences in SPA expression. Significant differences were found in the quantity of total grain nitrogen allocated to the gliadin protein fractions for different haplotypes of the A-genome copy. Transgenesis may be another possibility, by increasing the proportion of the high-molecular-weight subunits of glutenin (Jones et al., 2009) in a low-protein background (Foulkes et al., 2009). Heterosis for NUE Since the mid-1990s, F1 hybrid wheat cultivars have been regularly registered in Europe, although they still represent only a small part of the wheat-growing area. There are also initiatives to grow F1 hybrid wheat cultivars in some developing countries (Matuschke et al., 2007). Commercial hybrids are usually produced thanks to specific chemical hybridizing agents, which induce male sterility when applied at the right stage. One limitation to their development is the cost of the seed due to the difficulty of producing them on a regular basis. However, they may have particular characteristics for NUE. In experiments conducted in plots sown at typical seeding density, limited but consistent best-parent heterosis has been reported for GY under highyielding conditions, for example, +4.3% for 10 hybrids (Borghi et al., 1988), +7.3% for 17 hybrids (Brears et al., 1988), and +3.6% for 430 hybrids (Morgan et al., 1989). Moreover, Perenzin et al. (1992) and Oury

130

NUTRIENT USE EFFICIENCY IN CROPS

et al. (1994, 1995) have reported either a higher grain protein content of the hybrids for the same yield or the same protein content despite a higher GY. These results tend to indicate a higher NUE and NUpE for hybrids compared with pure lines. Some results also showed that best-parent heterosis was higher at low nitrogen levels than at high nitrogen levels (Le Gouis and Pluchard, 1996), even though year effects were high (Le Gouis et al., 2002). This was, however, not confirmed by Kindred and Gooding (2005) using a limited set of commercial hybrids grown at two extreme nitrogen levels (0 and 200 kg N ha−1). In this case, heterosis was only apparent at high nitrogen levels. Components of NUE that may be altered also differed between experiments. While Le Gouis et al. (2002) observed a best-parent heterosis for total nitrogen at anthesis and harvest, meaning a better NUpE, Kindred and Gooding (2004) reported only little heterosis for total nitrogen in the above-ground biomass but the hybrids showed an increased NUtE. Values reported for mid-parent heterosis for NUpE at flowering and maturity could be related to a more efficient root system. Indeed, heterosis was shown for different root characteristics such as root length, root DM, and root area (Kraljevic-Balalic et al., 1988). Genetic effects may be separated into general combining ability (GCA) and specific combining ability (SCA) (Sprague and Tatum, 1942). Simply, GCA is the mean of all hybrids obtained with a given parent and represents primarily additive effects. SCA is basically the difference between the expected performance of a hybrid based on the GCA of its two parents and the observed performance of this hybrid. It was shown that GCA effects were higher than SCA effects and that a significant interaction existed with the nitrogen level (Le Gouis et al., 2002). The implication is that the parents used in creating hybrids should be chosen as a function of the

nitrogen level at which the hybrids will be grown. Selection for increased NUE As already stated, direct selection for NUE has not been a major trait for breeding in the past. However, several studies have reported genetic variation for NUE and for its components. Field experiments have shown that genetic variability for nitrogen uptake exists in different cereals (e.g., Löffler et al., 1985; Van Sanford and MacKown, 1986; Fossati et al., 1993; Le Gouis et al., 2000). In an investigation of 39 cultivars at five nitrogen rates in the United Kingdom, Barraclough et al. (2010), however, only found cultivar differences in NUpE at the highest three nitrogen rates, while no significant differences were observed when 0 or 50 kg N ha−1 was applied. Genetic variation has been reported for post-flowering nitrogen absorption (e.g., Löffler et al., 1985; Van Sanford and MacKown 1986; Monaghan et al., 2001; Bogard et al., 2010), in spite of this trait being difficult to measure due to large experimental errors (Kichey et al., 2007). Similarly, genotypic variation was reported for NUtE calculated as the ratio of GY to total plant nitrogen (e.g., Cox et al., 1985; Van Sanford and MacKown, 1987; Dhugga and Waines, 1989; May et al., 1991; Le Gouis et al., 2000; Barraclough et al., 2010). Variation for the ability to remobilize nitrogen absorbed before anthesis to the grain has been reported in wheat grown in the field (Cox et al., 1985; Van Sanford and MacKown, 1987), although Barbottin et al. (2005) showed that when there are no environmental factors limiting grain filling, the differences in the amount of remobilized nitrogen taken up before anthesis were mainly due to the capacity of the plant to store nitrogen in sink organs until this period. Kichey et al. (2007), using both usual sampling and 15N labeling, showed

GENETIC IMPROVEMENT OF NUTRIENT USE EFFICIENCY IN WHEAT

that the range for remobilization efficiency was between 70% and 89% with an average of 78.5%. Several authors have reported on the relation between NUE and its two main components, NUpE and NUtE. It is often reported in both maize and wheat that contributions of NUpE and NUtE to NUE are dependent on the nitrogen level (Moll et al., 1982; Dhugga and Waines, 1989; OrtizMonasterio et al., 1997; Le Gouis et al., 2000). Differences between species and between studies, however, do exist. In maize, it seems that the NUtE contribution to NUE is higher at low nitrogen, but higher for NUpE at high nitrogen (Moll et al., 1982; Bertin and Gallais, 2000). In wheat, nitrogen uptake generally explains variation in GY more than nitrogen utilization (Van Sanford and MacKown, 1987; Dhugga and Waines, 1989; May et al., 1991; Muurinen et al., 2006). Moreover, Ortiz-Monasterio et al. (1997) and Le Gouis et al. (2000) showed that at low nitrogen inputs, NUpE accounted for more of the genetic variation in NUE compared with high nitrogen inputs. For example, in an investigation of 20 winter wheat cultivars, NUpE accounted for 64% of the variation of NUE at low nitrogen (0 kg N ha−1) and only 31% at high nitrogen (170 kg N ha−1) (Le Gouis et al., 2000). More important, 63% of the genotype × nitrogen level interaction for NUE was explained by the interaction for NUpE. The importance of nitrogen uptake in determining NUE may be related to different traits. The first one is the ability to explore the soil profile and to capture nitrogen (see Foulkes et al., 2009 and Chapter 2 for a review of roots traits to be considered). The second one is the ability to acquire nitrogen late in the season, while nitrogen mineralization may be active due to high temperatures, provided that soil water availability is still sufficient. Both Ortiz-Monasterio et al. (1997) and Laperche et al. (2006a) reported a positive correlation between nitrogen uptake and flowering date

131

at low nitrogen levels, indicating that late genotypes were able to acquire more nitrogen. Studies conducted at high nitrogen levels generally have shown genetic progress for NUE and its components (Table 7.1). Although the genetic progress is slower, it is usually observed at low nitrogen inputs, indicating that some genetic variability is still present in breeders’ gene pool. Despite some significant genotype × nitrogen level interactions, modern cultivars are usually better than older cultivars at acquiring and utilizing nitrogen. However, the lack of necessity to access a larger genetic base was challenged by the results from Foulkes et al. (1998) which showed that old cultivars were more able to capture soil nitrogen, while modern cultivars were more able to acquire fertilizer nitrogen. Other traits may be difficult to find in modern wheat cultivars either because they were lost during domestication or subsequent breeding or because they do not exist in the species. A promising alternative for increasing available genetic variability would be to use crosses with wild relatives of wheat (Calderini and Ortiz-Monasterio, 2003). In relation to nitrogen, one of the best known examples is the increased GPC achieved in bread wheats by using the GPC-B1 quantitative trait locus (QTL) from Triticum turgidum var. dicoccoides (Kade et al., 2005; DePauw et al., 2007). Uauy et al. (2006) cloned a NAC transcription factor, NAMB1, which is responsible for the GPC-B1 QTL on chromosome 6BS, increasing GPC without reducing GY in the environment it was tested. The NAM-B1 gene was shown to accelerate canopy senescence during grain filling and to be responsible for a higher nitrogen remobilization and a better partitioning of nitrogen to the grain (Waters et al., 2009). It was hypothesized that a nonfunctional allele of this gene has been fixed during domestication (Uauy et al., 2006).

132

NUTRIENT USE EFFICIENCY IN CROPS

However, it was shown recently, by genotyping 63 historical seed samples originating from the 1862 International Exhibition in London, that the ancestral allele was present in two spelt wheat and two bread wheat cultivars widely cultivated at that time (Asplund et al., 2010). In addition, several T. turgidum var. dicoccoides genotypes of high grain protein percentage showed a higher capacity for nitrogen uptake than modern wheat cultivars (Kushnir and Halloran, 1984). During the last decades, CIMMYT has developed an important program of wide crosses and hybridization between tetraploids (e.g., Triticum durum) and wild diploid relatives (e.g., Aegilops tauschii) to create synthetic wheats that may provide a wide range of variability for biotic and abiotic stresses (Calderini and Ortiz-Monasterio, 2003). Wheat, rice, and maize do not possess the ability to inhibit biological nitrification that would result in less flux of N2O and nitric oxide (NO) and retention of nitrogen fertilizer for longer time in the soil (Subbarao et al 2007a,b). Leymus racemosus, a wild relative of wheat, was found to be a source for inhibiting or reducing soil nitrification by releasing inhibitory compounds from roots and suppressing Nitrosomonas bacteria (Subbarao et al 2007a,b). A Leymus chromosome containing the relevant trait was introduced into a wheat line that thereafter showed the ability for biological nitrification inhibition. Research is underway to characterize and quantify this ability in field trials (Ortiz et al., 2008). The ability to efficiently select for a given trait will directly depend on its heritability, the part of the total variance that is due to genetic variance, the rest being the variance due to the environment. The lower genetic progress observed at low nitrogen may be due to the fact that heritabilities are generally lower under low-input or in stressed environments compared with high-input (Ud-din et al., 1992; Calhoun et al., 1994;

Bänziger et al., 1997; Bertin and Gallais, 2000; Sinebo et al., 2002; Laperche et al., 2006a). In maize, this has been explained either by the fact that at low nitrogen inputs, genetic variance decreased more than environmental variance (Bänziger et al., 1997), or by the fact that environmental variance increased more than genetic variance (Bertin and Gallais, 2000; Sinebo et al., 2002). In wheat, this decrease of heritability has been explained both by an increase of environmental variance and a decrease of genetic variance under low nitrogen inputs (Brancourt-Hulmel et al., 2005; Laperche et al., 2006a). If the heritability is low for a trait, for example, NUE at low nitrogen levels, indirect selection may be considered. In this case, genetic progress will be expected at low nitrogen levels while selecting at high nitrogen levels. The relative gain of indirect versus direct selection, considering equal selection intensities, depends on heritabilities in both environments and genetic correlation between the environments (Falconer, 1974). It has been shown both in maize and in wheat that the correlation between the performances observed in two environments decreased when the difference in nitrogen stress intensity increased between the two environments (Bänziger et al., 1997; Presterl et al., 2003; Laperche et al., 2006a). This confirms that cultivars adapted to low-input practices are different from cultivars adapted to high-yielding environments. Bänziger et al. (1997) showed that selection under high nitrogen for performance under low nitrogen was significantly less efficient than selection under low nitrogen when relative yield reduction due to nitrogen stress exceeded 43%. Brancourt-Hulmel et al. (2005) compared direct and indirect selection for GY to increase GY at low nitrogen levels. The relative efficiency of indirect selection to direct selection for each pair of environments ranged from 0.15 to 0.99, indicating that indirect selection was never more efficient

GENETIC IMPROVEMENT OF NUTRIENT USE EFFICIENCY IN WHEAT

than direct selection. Therefore, it was concluded that breeding programs targeting low-input environments should include lowinput selection environments to maximize selection gains. At CIMMYT, the Bread Wheat Breeding program has selected breeding lines under intermediate nitrogen levels for many years (Van Ginkel et al., 2001). The resulting lines have shown improved levels of nitrogen uptake efficiency (UPE), nitrogen utilization efficiency (UTE), and nitrogen use efficiency (NUE) (Ortiz-Monasterio et al., 1997). To verify whether this method of managing segregating populations is effective in identifying the most nitrogen-useefficient wheat, different selection schemes were compared. Selection was either conducted at low, medium, high, or alternated between low and high nitrogen levels. The selection regime where alternate selection between high nitrogen in the F2 generation, low nitrogen in the F3, and so on was used (shuttling) gave the highest yields at intermediate and high nitrogen levels. No difference was observed between the selection schemes at low nitrogen levels for yield, biomass, NUE, or straw nitrogen content. Even the regime where selection was always carried out at low nitrogen failed to show superior performance when tested at low nitrogen in yield trials. If shuttling was commenced with nitrogen stress at the F2, and then alternated between high and low nitrogen levels, the population was severely truncated in responsiveness to nitrogen, yielding even less than populations always selected at medium nitrogen levels. Sylvester-Bradley and Kindred (2009) reported that no genetic progress was observed for NUE at nitrogen optimum (Table 7.1). If NUE at nitrogen optimum is to be improved by breeding, the design and analysis of the experiments need to be adapted. Sylvester-Bradley et al. clearly stated that it is prohibitive, both logistically

133

and financially, to undertake analysis of most traits relevant to NUE and for the estimation of the optimum, which requires many nitrogen levels, on multiple genotypes. Experiments comparing canopy and grain traits for numerous genotypes will more likely use a single nitrogen amount, which inevitably will differ from the optimum nitrogen amounts that would be used if those genotypes were being managed individually on-farm. The alternative would be to conduct large experiments on multiple lines with multiple nitrogen amounts but with the measurement of crop parameters restricted to those that are easy to measure, enabling the statistical fitting of a simple physiological model as proposed. Molecular approaches to improving NUE NUE is a complex trait that is likely to be dependent on many genes. Identifying key chromosomal regions with molecular markers linked to them would be a means to facilitate selection. Therefore, quantitative genetic studies have been undertaken to identify the basis of nitrogen uptake and utilization in several crops including barley (Kjaer and Jensen, 1995), maize (Agrama et al., 1999; Bertin and Gallais, 2001; Hirel et al., 2001; Gallais and Hirel, 2004; Coque et al., 2006), rice (Obara et al., 2001; Lian et al., 2005), and wheat (An et al., 2006; Habash et al., 2007; Laperche et al., 2006b, 2007; Fontaine et al., 2009). Regardless of the crop examined, these studies allowed the identification of chromosomal regions controlling plant root or shoot vegetative growth or yield through the efficiency of nitrogen uptake, assimilation, and recycling. Several studies were also conducted at different nitrogen levels to access the response to nitrogen (An et al., 2006; Laperche et al., 2007, 2008). It is usually observed that less QTL are detected at low nitrogen levels,

134

NUTRIENT USE EFFICIENCY IN CROPS

probably because of the lower heritability already mentioned. Both nitrogen-level-specific QTL and nonspecific QTL are detected, confirming the significant genotype x environment interactions reported. None of these QTL has been yet cloned, but progress in establishment of physical maps of wheat chromosomes to prepare for high quality sequencing (Paux et al., 2008) will render this task feasible if not rapid and simple. Conclusions and perspectives Breeding for higher NUE has become an important trait in several breeding programs. Conventional breeding has enabled an increase in GY, resulting in a higher NUE. Although molecular markers linked to chromosomal regions are available, their application is still limited in breeding. The development of cheap and high-throughput genotyping techniques will probably alter this situation and facilitate more comprehensive genetic studies. Particularly promising is the use of association genetics to identify relevant chromosomal regions for complex traits such as NUE. These new statistical methods are based on linkage disequilibrium and take the structure of the population used into account (Kang et al., 2008). In wheat, association genetics has already proved successful for the study of the influence of candidate genes on traits such as grain composition and earliness with a population based on worldwide genetic resources (Ravel et al., 2006; Bonnin et al., 2008; Charmet et al., 2009). The first wholegenome-scale studies were devoted to yield and disease resistance (Crossa et al., 2007) but will soon be conducted on NUE and its components. A new challenge will be to improve NUE in the context of climate change. There may be favorable effects linked to CO2 concentrations, which were about 270 ppm before the industrial revolution, have now reached

380 ppm, and are predicted to exceed 550 ppm by 2050. On average, across several species and under unstressed conditions, compared with current atmospheric CO2 concentrations, crop yield increases in the range of 10–20% are observed for C3 plants at 550 ppm CO2 (Tubiello et al., 2007). This increase in GY may, however, accentuate the decline in GPC already observed, with a large impact on grain quality (Piikki et al., 2008). Moreover, climate change is associated with other factors that may have a detrimental influence on NUE; this would be the case particularly for high temperatures during grain set and early grain formation, or with low water availability that will lower GY either directly or by limiting nitrogen uptake. In summary, much further selection is required to improve NUE, and its components, and to anticipate climate change. References Acreche, M.M. & Slafer, G.A. (2009) Variation of grain nitrogen content in relation with grain yield in old and modern Spanish wheats grown under a wide range of agronomic conditions in a mediterranean region. Journal of Agricultural Science, Cambridge 147, 657–667. Agrama, H.A.S., Zakaria, A.G., Said, F.B., & Tuinstra, M. (1999) Identification of quantitative trait loci for nitrogen use efficiency in maize. Molecular Breeding 5, 187–195. An, D.G., Su, J.Y., Liu, Q.Y., et al. (2006) Mapping QTLs for nitrogen uptake in relation to the early growth of wheat (Triticum aestivum L.). Plant and Soil 284, 73–84. Asplund, L., Hagenblad, J., & Leino, M.W. (2010) Reevaluating the history of the wheat domestication gene NAM-B1 using historical plant material. Journal of Archaeological Science 37, 2303–2307. Austin, R.B., Bingham, J., Blackwell, R.D., Evans, L.T., Ford, M.A., Morgan, C.L., & Taylor, M. (1980) Genetic improvements in winter wheat yields since 1900 and associated physiological changes. Journal of Agricultural Science, Cambridge 94, 675–689. Bänziger, M., Betran, F.J., & Lafite, H.R. (1997) Efficiency of high-nitrogen selection environments for improving maize for low-nitrogen target environments. Crop Science 37, 1103–1109.

GENETIC IMPROVEMENT OF NUTRIENT USE EFFICIENCY IN WHEAT

Barbottin, A., Lecomte, C., Bouchard, C., & Jeuffroy, M.-H. (2005) Nitrogen remobilization during grain filling in wheat: genotypic and environmental effects. Crop Science 45, 1141–1150. Barraclough, P.B., Howarth, J.R., Jones, J., et al. (2010) Nitrogen efficiency of wheat: genotypic and environmental variation and prospects for improvement. European Journal of Agronomy 33, 1–11. Bertin, P. & Gallais, A. (2000) Genetic variation for nitrogen use efficiency in a set of recombinant maize inbred lines. I. Agrophysiological results. Maydica 45, 53–66. Bertin, P. & Gallais, A. (2001) Genetic variation for nitrogen use efficiency in a set of recombinant maize inbred lines. II. QTL detection and coincidences. Maydica 46, 53–68. Bogard, M., Allard, V., Brancourt-Hulmel, M., et al. (2010) Deviation from the grain protein concentration—grain yield negative relationship is highly correlated to post-anthesis N uptake in winter wheat. Journal of Experimental Botany 61, 4303–4312. Bonnin, I., Rousset, M., Madur, D., et al. (2008) FT genome A and D polymorphisms are associated with the variation of earliness components in hexaploid wheat. Theoretical and Applied Genetics 116, 383–394. Borghi, B., Perenzin, M., & Nash, R.J. (1988) Agronomic and qualitative characteristics of ten bread wheat hybrids produced using a chemical hybridizing agent. Euphytica 39, 185–194. Brancourt-Hulmel, M., Doussinault, G., Lecomte, C., Bérard, P., Le Buanec, B., & Trottet, M. (2003) Genetic improvement of agronomic traits of winter wheat cultivars released in France from 1946 to 1992. Crop Science 43, 37–45. Brancourt-Hulmel, M., Heumez, E., Pluchard, P., et al. (2005) Indirect versus direct selection of winter wheat for low input or high input levels. Crop Science 45, 1427–1431. Branlard, G., Dardevet, M., Saccomano, R., Lagoutte, F., & Gourdon, J. (2001) 2001. Genetic diversity of wheat storage proteins and bread wheat quality. Euphytica 119, 59–67. Brears, T., Hydon, A.G., & Bingham, J. (1988) An assessment of the feasibility of producing F1 and F2 hybrids for the UK. Proc 7th Int Wheat Genet Symp, 1057–1062. Calderini, D.F. & Ortiz-Monasterio, I. (2003) Are synthetic hexaploids a means of increasing grain element concentrations in wheat? Euphytica 134, 169–178. Calhoun, D.S., Gebeyehu, G., Miranda, A., et al. (1994) Choosing evaluation environments to increase wheat-grain yield under drought conditions. Crop Science 34, 673–678.

135

Charmet, G., Masood-Quraishi, U., Ravel, C., et al. (2009) Genetics of dietary fibre in bread wheat. Euphytica 170, 155–168. Chien, S.H., Prochnow, L.I., & Cantarella, H. (2008) Recent developments of fertilizer production and use to improve nutrient efficiency and minimize environmental impacts. Advances in Agronomy 102, 267–322. Coque, M., Bertin, P., Hirel, B., & Gallais, A. (2006) Genetic variation and QTLs for 15N natural abundance in a set of maize recombinant lines. Field Crop Research 97, 310–321. Cox, M.C., Qualset, C.O., & Rains, D.W. (1985) Genetic variation for nitrogen assimilation and translocation in wheat. I. Dry matter and nitrogen accumulation. Crop Science 25, 430–435. Crossa, J., Burgueno, J., Dreisickacker, S., et al. (2007) Association analysis of historical bread wheat germplasm using additive genetic covariance of relatives and population structure. Genetics 177, 1889–1913. Dawson, J.C., Huggins, D.R., & Jones, S.S. (2008) Characterizing nitrogen use efficiency in natural and agricultural ecosystems to improve the performance of cereal crops in low-input and organic agricultural systems. Field Crops Research 107, 89–101. DePauw, R.M., Knox, R.E., Clarke, F.R., et al. (2007) Shifting undesirable correlations. Euphytica 157, 409–415. Dhugga, K.S. & Waines, J.G. (1989) Analysis of nitrogen accumulation and use in bread and durum wheat. Crop Science 29, 1232–1239. Dudley, J.W. (2007) From means to QTL: the Illinois long-term selection experiment as a case study in quantitative genetics. Crop Science 47, S20–S31. Dudley, J.W. & Lambert, R.J. (1992) 90-generations of selection for oil and protein in maize. Maydica 37, 81–87. Ellis, M.H., Spielmeyer, W., Gale, M.D., Rebetzke, G.J., & Richards, R.A. (2002) “Perfect” markers for the Rht-B1b and Rht-D1b dwarfing genes in wheat. Theoretical and Applied Genetics 105, 1038–1042. Falconer, D. (1974) Introduction à la génétique quantitative. Masson, Paris. Feil, B. (1997) The inverse yield-protein relationships in cereals: possibilities and limitations for genetically improving the grain protein yield. Trends in Agronomy 1, 103–119. Fischer, R.A. & Edmeades, G. (2010) Breeding and cereal yield progress. Crop Science 50, S85–S98. Fontaine, J.-X., Ravel, C., Pageau, K., et al. (2009) A quantitative genetic study for elucidating the contribution of glutamine synthetase, glutamate dehy-

136

NUTRIENT USE EFFICIENCY IN CROPS

drogenase and other nitrogen-related physiological traits to common wheat agronomic performance. Theoretical and Applied Genetics 119, 645–662. Fossati, D., Fossati, A., & Feil, B. (1993) Relationship between grain yield and grain nitrogen concentration in winter triticale. Euphytica 71, 115–123. Foulkes, M., Sylvester-Bradley, R., & Scott, R. (1998) Evidence for differences between winter wheat cultivars in acquisition of soil mineral nitrogen and uptake and utilization of applied fertilizer nitrogen. Journal of Agricultural Science, Cambridge 130, 29–44. Foulkes, M., Hawkesford, M., Barraclough, P., et al. (2009) Identifying traits to improve the nitrogen economy of wheat: recent advances and future prospects. Field Crop Research 114, 329–342. Gallais, A. & Hirel, B. (2004) An approach to the genetics of nitrogen use efficiency in maize. Journal of Experimental Botany 55, 295–306. Good, A.G., Shrawat, A.K., & Muench, D.G. (2004) Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production. Trends in Plant Science 9, 597–605. Guarda, G., Padovan, S., & Delogu, G. (2004) Grain yield, nitrogen-use efficiency and baking quality of old and modern Italian brad-wheat cultivars grown at different nitrogen levels. European Journal of Agronomy 21, 181–192. Habash, D.Z., Bernard, S., Schondelmaier, J., Weyen, J., & Quarrie, S.A. (2007) The genetics of nitrogen use in hexaploid wheat: N utilisation, development and yield. Theoretical and Applied Genetics 114, 403–419. Hirel, B., Bertin, P., Quilleré, I., et al. (2001) Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize. Plant Physiology 125, 1258–1270. Hirel, B., Le Gouis, J., Ney, B., & Gallais, A. (2007) The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. Journal of Experimental Botany 58, 2369–2387. Hoseney, R.C. (1986) Principles of Cereal Sciences and Technology. Am. Assoc. Cereal Chem., St Paul, MN. Jones, H.D., Sparks, C.A., & Shewry, P.R. (2009) Transgenic manipulation of wheat quality. In: Wheat Chemistry and Technology (eds. K. Khan & P.R. Shewry), AACC, St Paul, MN. Kade, M., Barneix, A.J., Olmos, S., et al. (2005) Nitrogen uptake and remobilization in tetraploid “Langdon” durum wheat and a recombinant substitution line with the high grain protein gene Gpc-B1. Plant Breeding 124, 343–349.

Kang, H.M., Zaitlen, N.A., Wade, C.M., et al. (2008) Efficient control of population structure in model organism association mapping. Genetics 178, 1709–1723. Kibite, S. & Evans, L.E. (1984) Causes of negative correlations between grain yield and grain protein concentration in common wheat. Euphytica 33, 801–810. Kichey, T., Hirel, B., Heumez, E., Dubois, F., & Le Gouis, J. (2007) In wheat (Triticum aestivum L.), post-anthesis nitrogen uptake and remobilisation to the grain correlate with agronomic traits and nitrogen physiological markers. Field Crops Research 102, 22–32. Kindred, D.R. & Gooding, M.J. (2004) Heterotic and seed rate effects on nitrogen efficiencies in wheat. Journal of Agricultural Science, Cambridge 142, 639–657. Kindred, D.R. & Gooding, M.J. (2005) Heterosis for yield and its physiological determinants in wheat. Euphytica 142, 149–159. Kindred, D.R., Verhoeven, T.M.O., Weightman, R.M., et al. (2008) Effects of variety and fertiliser nitrogen on alcohol yield, grain yield, starch and protein content, and protein composition of winter wheat. Journal of Cereal Science 48, 46–57. Kjaer, B. & Jensen, J. (1995) The inheritance of nitrogen and phosphorus content in barley analysed by genetic markers. Hereditas 123, 109–119. Kraljevic-Balalic, M., Kastori, R., & Vojvodic, M. (1988) Inheritance of total root area, length and dry weight in F1 wheat crosses. Genetika 20, 229–234. Kushnir, U. & Halloran, G.M. (1984) Plant nitrogen distribution in wild tetraploid (Triticum turgidum dicoccoides) and hexaploid wheat (Triticum aestivum). Euphytica 33, 641–649. Laperche, A., Brancourt-Hulmel, M., Heumez, E., Gardet, O., & Le Gouis, J. (2006a) Estimation of genetic parameters of a DH wheat population grown at different N stress levels characterized with probe genotypes. Theoretical and Applied Genetics 112, 797–807. Laperche, A., Devienne-Barret, F., Maury, O., Le Gouis, J., & Ney, B. (2006b) A simplified conceptual model of carbon and nitrogen functionning for QTL analysis of winter wheat adaptation to nitrogen deficiency. Theoretical and Applied Genetics 113, 1131–1146. Laperche, A., Brancourt-Hulmel, M., Heumez, E., et al. (2007) Using genotype x nitrogen interaction variables to evaluate the QTL involved in wheat tolerance to nitrogen constraints. Theoretical and Applied Genetics 115, 399–415. Laperche, A., Le Gouis, J., Hanocq, E., & BrancourtHulmel, M. (2008) Modelling nitrogen stress with

GENETIC IMPROVEMENT OF NUTRIENT USE EFFICIENCY IN WHEAT

probe genotypes to assess genetic parameters and genetic determinism of winter wheat tolerance to nitrogen constraint. Euphytica 161, 259–271. Le Gouis, J. & Pluchard, P. (1996) Genetic variation for nitrogen use efficiency in winter wheat (Triticum aestivum L.). Euphytica 92, 221–224. Le Gouis, J., Béghin, D., Heumez, E., & Pluchard, P. (2000) Genetic differences for nitrogen uptake and nitrogen utilisation efficiencies in winter wheat. European Journal of Agronomy 12, 163–173. Le Gouis, J., Béghin, D., Heumez, E., & Pluchard, P. (2002) Diallel analysis of winter wheat at two nitrogen levels. Crop Science 42, 1129–1134. Legg, B.J. (2005) Crop improvement technologies for the 21st century. In: Yields of Farmed Species: Constraints and Opportunities in the 21st Century (eds. R. Sylvester-Bradley & J. Wiseman), pp. 31– 50, Nottingham University Press, Nottingham. Lian, X., Xing, Y., Yan, H., Xu, C., Li, X., & Zhang, Q. (2005) QTLs for low nitrogen tolerance at seedling stage identified using a recombinant inbred line population derived from an elite rice hybrid. Theoretical and Applied Genetics 112, 85–96. Löffler, C.M., Rauch, T.L., & Busch, R.H. (1985) Grain and plant protein relationships in hard red spring wheat. Crop Science 25, 521–524. Lopez-Bellido, L., Lopez-Bellido, R., & Lopez-Bellido, F. (2006) Fertilizer nitrogen efficiency in durum wheat under rainfed Mediterranean conditions: effect of split application. Agronomy Journal 98, 55–62. Loyce, C., Rellier, J.P., & Meynard, J.M. (2002) Management planning for winter wheat with multiple objectives (2): ethanol-wheat production. Agricultural Systems 72, 33–57. Martre, P., Jamieson, P.D., Semenov, M.A., Zyskowski, R.F., Porter, J.R., & Triboi, E. (2006) Modelling protein content and composition in relation to crop nitrogen dynamics for wheat. European Journal of Agronomy 25, 138–154. Matuschke, I., Mishra, R.R., & Qaim, M. (2007) Adoption and impact of hybrid wheat in India. World Development 35, 1422–1435. May, L., Van Sanford, D.A., MacKown, C.T., & Cornelius, P.L. (1991) Genetic variation for nitrogen use in soft red x hard red winter wheat populations. Crop Science 31, 626–630. Moll, R.H., Kamprath, E.J., & Jackson, W.A. (1982) Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agronomy Journal 74, 562–564. Monaghan, J.M., Snape, J.W., Chojecki, A.J.S., & Kettlewell, P.S. (2001) The use of grain protein deviation for identifying wheat cultivars with high

137

protein concentration and yield. Euphytica 122, 309–317. Morgan, C.L., Austin, R.B., Ford, M.A., Bingham, J., Angus, W.J., & Chowdhury, S. (1989) An evaluation of F1 hybrid winter wheat genotypes produced a chemical hybridizing agent. Journal of Agricultural Science, Cambridge 112, 143–149. Munier-Jolain, N. & Salon, C. (2005) Are the carbon costs of seed production related to the quantitative and qualitative performance ? An appraisal for legumes and other crops. Plant, Cell & Environment 28, 1388–1395. Muurinen, S., Slafer, G.A., & Peltonen-Sainio, P. (2006) Breeding effects on nitrogen use efficiency of spring cereals under Northern conditions. Crop Science 46, 561–568. Obara, M., Kajiura, M., Fukuta, Y., et al. (2001) Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice (Oryza sativa L.). Journal of Experimental Botany 52, 1209–1217. Ortiz, R., Braun, H.-J., Crossa, J., et al. (2008) Wheat genetic resources enhancement by the international maize and wheat improvement center (CIMMYT). Genetic Resources and Crop Evolution 55, 1095–1140. Ortiz-Monasterio, J.I., Sayre, K.D., Rajaram, S., & McMahon, M. (1997) Genetic progress in wheat and nitrogen use efficiency under four nitrogen rates. Crop Science 37, 898–904. Oury, F.X., Brabant, P., Pluchard, P., Bérard, P., & Rousset, M. (1994) Une étude de la qualité des blé hybrides à travers différents tests technologiques. Agronomie 14, 377–385. Oury, F.X., Triboï, E., Bérard, P., Ollier, J.L., & Rousset, M. (1995) Etude des flux de carbone et d’azote chez des blés hybrides et leurs parents, pendant la période de remplissage du grain. Agronomie 15, 193– 204. Oury, F.-X., Bérard, P., Brancourt-Hulmel, M., et al. (2003) Yield and grain protein concentration in bread wheat: a review and a study of multi-annual data from a French breeding program. Journal of Genetics and Breeding 57, 59–68. Oury, F.-X., Chiron, H., Faye, A., et al. (2010) The prediction of bread wheat quality: joint use of the phenotypic information brought by technological tests and the genetic information brought by HMW and LMW glutenin subunits. Euphytica 171, 87–109. Paux, E., Sourdille, P., Salse, J., et al. (2008) A physical map of the 1-Gigabase bread wheat chromosome 3B. Science 322, 101–104. Perenzin, M., Pogna, N.E., & Borghi, B. (1992) Combining ability for breadmaking in wheat. Canadian Journal of Plant Science 72, 743–754.

138

NUTRIENT USE EFFICIENCY IN CROPS

Piikki, K., De Temmerman, L., Ojanperä, K., Danielsson, H., & Pleijel, H. (2008) The grain quality of spring wheat (Triticum aestivum L.) in relation to elevated ozone uptake and carbon dioxide exposure. European Journal of Agronomy 28, 245–254. Presterl, T., Seitz, G., Landbeck, M., et al. (2003) Improving nitrogen-use efficiency in European maize: estimation of quantitative genetic parameters. Crop Science 43, 1259–1265. Ravel, C., Praud, S., Murigneux, A., et al. (2006) Identification of Glu-B1-1 as a candidate gene for the quantity of high-molecular-weight glutenin in bread wheat (Triticum aestivum L.) by means of an association study. Theoretical and Applied Genetics 112, 738–743. Ravel, C., Martre, P., Romeuf, I., et al. (2009) Nucleotide polymorphism in the wheat transcriptional activator Spa influences its pattern of expression and has pleiotropic effects on grain protein composition, dough viscoelasticity, and grain hardness. Plant Physiology 151, 2133–2144. Serret, M.D., Ortiz-Monasterio, I., Pardo, A., & Araus, J.L. (2008) The effects of urea fertilisation and genotype on yield, nitrogen use efficiency, d15N and d13C in wheat. Annals of Applied Biology 153, 243–257. Simmonds, N.W. (1995) The relation between yield and protein in cereal grain. Journal of Science Food and Agriculture 67, 309–315. Sinebo, W., Gretzmacher, R., & Edelbauer, A. (2002) Environment of selection for grain yield in low fertilizer input in barley. Field Crops Research 74, 151–162. Snape, J.W., Hyne, V., & Aitken, K. (1993) Targeting genes in wheat using marker mediated approaches. Proceedings of Eighth International Wheat Genetics Symposium, Beijing, p 749–759. Spiertz, J.H.J. (2010) Nitrogen, sustainable agriculture and food security. A review. Agronomy for sustainable. Development 30, 43–55. Sprague, G.F. & Tatum, L.A. (1942) General versus specific combining ability in single crosses of corn. Journal of the American Society of Agronomy 34, 923–932. Subbarao, G., Ban, T., Kishii, M., et al. (2007a) Can biological nitrification inhibition (BNI) genes from perennial Leymus racemosus (Triticeae) combat nitrification in wheat farming? Plant and Soil 299, 55–64. Subbarao, G., Rondon, M., Ito, O., et al. (2007b) Biological nitrification inhibition (BNI)—is it a

widespread phenomenon. Plant and Soil 294, 5–18. Sylvester-Bradley, R. & Kindred, D.R. (2009) Analysing nitrogen responses of cereals to prioritize routes to the improvement of nitrogen use efficiency. Journal of Experimental Botany 60, 1939–1951. Triboï, E., Martre, P., Girousse, C., Ravel, C., & TriboïBlondel, A.-M. (2006) Unravelling environmental and genetic relationships between grain yield and nitrogen concentration for wheat. European Journal of Agronomy 25, 108–118. Tubiello, F.N., Soussana, J.-F., & Howden, M.S. (2007) Crop and pasture response to climate change. Proceedings of the National Academy of Sciences of the United States of America 104, 19686–19690. Uauy, C., Distelfeld, A., Fahima, T., Blechl, A., & Dubcovsky, J. (2006) A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314, 1298–1301. Ud-din, N., Carver, B.F., & Clutter, A. (1992) Genetic analysis and selection for wheat yield in droughtstressed and irrigated environments. Euphytica 62, 89–96. Van Ginkel, M., Ortiz-Monasterio, I., Trethowan, R., & Hernandez, E. (2001) Methodology for selecting segregating populations for improved N-use efficiency in bread wheat. Euphytica 119, 223–230. Van Sanford, D.A. & MacKown, C.T. (1986) Variation in nitrogen use efficiency among soft red winter wheat genotypes. Theoretical and Applied Genetics 72, 158–163. Van Sanford, D.A. & MacKown, C.T. (1987) Cultivar differences in nitrogen remobilization during grain fill in soft red winter wheat. Crop Science 27, 295–300. Waters, B.M., Uauy, C., Dubcovsky, J., & Grusak, M.A. (2009) Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain. Journal of Experimental Botany 60, 4263–4274. Weegels, P.L., Hamer, R.J., & Schofield, J.D. (1996) Critical review: functional properties of wheat glutenin. Journal of Cereal Science 23, 1–18. White, E.M. & Wilson, F.E.A. (2006) Responses of grain yield, biomass and harvest index and their rates of genetic progress to nitrogen availability in ten winter wheat varieties. Irish Journal of Agricultural and Food Research 45, 85–101.

Chapter 8

The Molecular Genetics of Nitrogen Use Efficiency in Crops Bertrand Hirel and Peter J. Lea

Abstract In this chapter, recent findings and future developments for improving nitrogen use efficiency in crop species are presented, taking into account the knowledge gained from model species. For crops, the information available concerning the regulatory mechanisms controlling plant nitrogen economy has significantly increased over the last decade, with the aim of improving nitrogen use efficiency and reducing the input of fertilizers, while still maintaining an acceptable yield. This was achieved mainly through the development of whole-plant physiological studies combined with a variety of “omics”-based and genetic approaches. An overview is provided of how the understanding of the physiological and molecular controls of nitrogen assimilation under varying environmental conditions has been improved in crops. This has been achieved through the use of combined approaches, based on whole-plant physiology, quantitative genetics, and forward and reverse genetics. Current knowledge and prospects for future agronomic development and breeding of crops adapted to various

levels of fertilizer input are explored, taking into account the world economic and environmental constraints in the 21st century.

Introduction The main driver for crop improvement over the 20th century has been yield. During this period, the rate of yield improvement has accelerated due primarily to the introduction of an increasingly scientific approach to plant breeding, but also through the extensive use of mineral fertilizers (Miflin, 2000; Tilman et al., 2002). Among these fertilizers, nitrogen (N) is the major limiting factor in agricultural production (Vitousek et al., 1997) and the largest input cost for nonleguminous crops, which can amount to up to 60% of the production costs (Kachanoski, 2009; Robertson and Vitousek, 2009). However, this extensive use of nitrogen fertilizers has caused major detrimental impacts on the diversity and functioning of the nonagricultural bacterial, animal, and plant ecosystems, including the capacity of the biosphere to absorb carbon from the atmosphere (Gruber and Galloway, 2008).

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 139

140

NUTRIENT USE EFFICIENCY IN CROPS

In particular, there are some strong lines of evidence that the intensive use of synthetic fertilizers depletes soil organic matter by promoting microbial carbon utilization and nitrogen mineralization, which, in turn, will increase the need for synthetic fertilization (Mulvaney et al., 2009). In addition, nitrogen fertilizers can be the origin of gaseous emissions of nitrogen oxides that react with stratospheric ozone and the emission of toxic ammonia into the atmosphere (Galloway et al., 2003; Robertson and Vitousek, 2009). At present, a combination of global factors is converging to put unprecedented pressure on agricultural productivity. These factors include increasing demand for food in developing nations with large populations; diminishing supplies and rising costs of fossil fuel energy that is required for food and fertilizer production; and global climate change (Galloway et al., 2008). The consequences of these trends are clear: current agricultural practices are not sustainable. Cereals such as maize, rice, wheat, and to a lesser extent, barley, along with root crops, are the basis for human food and animal feed production in the world. They require large inputs of nitrogen fertilizers (nitrates in particular) to achieve optimum performance. Nitrogen use efficiency (NUE) can be defined as the grain or biomass yield per unit of available mineral nutrient in the soil, which includes the residual mineral nutrients present in the soil and those provided by fertilization. Improving NUE in most cultivated crops is a major challenge, considering (1) the growing demand for agricultural products (Edgerton, 2009), (2) the rising cost of fertilizer, and (3) water pollution caused by excess nitrogen fertilization (Hirel et al., 2007a). It is necessary to select and release new crop varieties requiring less nitrogen fertilizer, but maintaining high yield and good grain quality (protein content, in particular). The research conducted in

many public institutions and in the private sector is currently at the interface between biology, genetics, and agronomy. The aims are to optimize yield and grain quality in model and crop plants, while deepening our fundamental knowledge of key metabolic functions, which are tightly linked to both plant development and plant nutrition (Andrews et al., 2004; Lea and Azevedo, 2006, 2007). Between 2010 and 2025, the human population is expected to increase from 6 to 10 billion people, and therefore the challenge for the next decades will be to accommodate the needs of the expanding world population by developing a highly productive agriculture, while at the same time preserving the quality of the environment (Dyson, 1999). Furthermore, farmers are facing increasing economic pressure with the rising cost of the fossil fuels required for producing nitrogen fertilizers (Pimentel et al., 1973, 2008; Robertson and Vitousek, 2009). There is a consensus recognizing that fossil fuel “won’t last forever” and that we should be prepared in the next decades first for higher prices and then shortage in their supply. Moreover, the increased price of fossil fuels has rekindled interest in biofuel production, the value of which is still under discussion (Walker, 2010), but which would certainly require yield increases (Sims et al., 2006). Lastly, climate change is likely to bring unpredictable weather and seasons, thus increasing the impact of biotic and abiotic stresses including those directly or indirectly linked to the availability and efficient use of nitrogen in various types of soil (Parry et al., 2004). Cereal grains provide 60% of the world’s nutrition either directly in the human diet or indirectly as animal feed (Hirel et al., 2007b). For example, improvements in maize yield of about 1% each year from 1955 have been estimated to be 40% due to improvements in cultural practices and 60% due to genetic

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

gains (Edgerton, 2009). All major maize seed breeding companies such as Monsanto, DuPont-Pioneer, and Syngenta are investing in genomic research and applying molecular marker and transgenic techniques to identify regions, genes, and alleles that can be combined in order to further improve NUE in cereals (Edgerton, 2009). For abiotic stress improvement in crops, NUE has become the second priority after drought. Maize is also recognized not only as a major crop but also as a model species that is well adapted to fundamental research, especially for understanding the genetic basis of yield performance. Many tools are available for most cereal crops, such as mutant collections, wide genetic diversity, recombinant inbred lines (RILs) or Doubled Haploid Line populations (DHLs), straightforward transformation protocols, and physiological, biochemical, and genomic data (Hirel et al., 2007a,b). Thus, the commercial maize research effort is paralleled by research in the public sector, notably with the release of the genome sequences for rice (Goff et al., 2002) and maize (Schnable et al., 2009), and the current development of sequencing projects for wheat (http://www.wheatgenome.org/ and http://cerealsdb.uk.net), barley (http:// barleygenome.org/), and a number of other crops. Therefore, improving NUE is particularly relevant to the majority of crops currently cultivated, for which large amounts of nitrogen fertilizers are required to reach maximum yield and for which global NUE is estimated, on average, to be far less than 50% (Raun and Johnson, 1999; Zhang, 2007). It is therefore crucial to identify the critical steps controlling NUE, in particular the key genes involved. NUE is composed of two components: uptake efficiency (NUpE, that is, the ability of plants to take up a given mineral nutrient from the soil such as nitrate or ammonium), and utilization efficiency (NUtE, that is, the ability of plants to use the

141

mineral nutrient such as nitrogen to produce biomass, grain in particular). Recent studies have demonstrated that there are large variations between different crop species in their ability to grow and yield well on soils with low nitrogen availability, which depends on both NUpE and NUtE for nitrogen (Hirel et al., 2007a). Agronomic and genetic studies were thus undertaken in order to determine if it was possible to improve the capacity of different crops to take up and utilize nitrogen at low and high input, taking into account both species and environmental impacts (Hirel et al., 2007a). Another way to improve crop NUE is to ensure that fertilizers applied to the field are readily available to the plants, thus being beneficial to both crop production and the environment. The various aspects of fertilizer production and application to improve NUE and thus minimize environmental impact have recently been reviewed by Chien et al. (2009) and will not be covered in this chapter. Intensive research has also been conducted with the aim of enhancing the rhizobium–legume symbiosis and nitrogen fixation to improve crop productivity (Andrews et al., 2009; Zahran, 2009). However, the molecular genetics of the symbiosis in terms of NUE on the side of the host plant is far less advanced when compared to non-nitrogen-fixing crops. Nitrogen Metabolism and Its Management For most crop species, the plant life cycle can be roughly divided into two main phases: (1) the vegetative growth phase, when young developing roots and leaves behave as sink organs that efficiently assimilate inorganic nitrogen (mainly in the form of nitrate and to a lesser extent ammonium) for amino acid and protein synthesis, and (2) the remobilization phase when senescing tissues start to behave as source organs

142

NUTRIENT USE EFFICIENCY IN CROPS

translocating organic molecules such as amino acids and carbohydrates to ensure the formation of new developing and/or storage organs. Storage organs are involved in plant survival and reproduction and are represented by grains in cereals (Hirel et al., 2007a). Therefore, during these two phases of plant growth and development, a better understanding is required of the metabolic and genetic control of nitrate uptake, nitrate partitioning between roots and shoots, and nitrate reduction and its subsequent assimilation and transfer into organic molecules. A better knowledge of the regulatory mechanisms controlling these different metabolic processes and their relationships will allow the selection of targeted plant physiological traits, which can then be used as markers, both for breeding new genotypes or new cultivars exhibiting a better NUE and for the rationalization of crop fertilization (Hirel and Lemaire, 2005). Nitrate is the principal nitrogen source for most wild and crop species. Following its uptake by means of specific transporters located in the root cell membrane, the assimilation of nitrate is a two-step process. First, the enzyme nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite. Subsequently, the enzyme nitrite reductase mediates the reduction of nitrite to ammo-

Photorespiration

NO3– NR

Uptake

NO2– NiR

NH4+ Amino acid Nase and protein degradation N2

nium. Root-specific transporters also allow the direct uptake of ammonium when available in the soil, or in particular environments (Ludewig et al., 2007). In addition to nitrate reduction, ammonium can be generated inside the plant by a variety of metabolic pathways such as photorespiration, phenylpropanoid metabolism, utilization of nitrogen transport compounds, and amino acid interconversion during the process of nitrogen recycling, following protein hydrolysis. Whatever the metabolic origin of ammonia, it is the ultimate form of inorganic nitrogen available to the plant, which is then incorporated into the organic form by the enzyme glutamine synthetase (GS) to synthesize glutamine. The reaction catalyzed by the enzyme GS is now considered the major route facilitating the incorporation of inorganic nitrogen into organic molecules in conjunction with the ferredoxin- and NADHdependent forms of glutamate synthase (FdGOGAT/NADH-GOGAT), which recycle glutamate and incorporate carbon skeletons to form the glutamate synthase cycle (Fig. 8.1). Glutamine and glutamate represent two key nitrogen metabolites that act as amino group donors to form other amino acids used for transport, structural, and storage protein synthesis and the synthesis of nucleotides for the formation of RNA and

Glutamate

GS

a-ketoglutarate

GO GAT Glutamate Glutamine

TCA cycle

Amino acids

Fig. 8.1. Main reactions involved in nitrogen assimilation in higher plants. NR, nitrate reductase; NiR, nitrite

reductase; Nase, nitrogenase; GS, glutamine synthetase; GOGAT, glutamate synthase; TCA, tricarboxylic acid.

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

DNA (Hirel and Lea, 2001; Forde and Lea, 2007). The inorganic nitrogen assimilation pathway is therefore one of the major limiting steps controlling crop growth and productivity, with regard to biomass, grain production, and grain protein content (Hirel et al., 2007a). In spite of the significant progress that has been made during the last few years in increasing our knowledge of the regulation of inorganic nitrogen metabolism and the relationships with carbon metabolism, many uncertainties concerning the regulation of nitrogen assimilation and nitrogen recycling both at the cellular and at the organ levels remain to be resolved (Gifford et al., 2008). In addition, this knowledge needs to be integrated at the whole-plant level, not only under controlled growth conditions but also under the constantly changing environmental constraints usually occurring in field situations (Stitt et al., 2002; Hirel et al., 2007a). This integration is required because in addition to regulating a range of cellular processes including nitrogen assimilation itself, through the coordination of nitrate or ammonium uptake and use, nitrate and nitrogen metabolite levels in the cell can regulate directly or indirectly a number of closely related metabolic and developmental processes. These processes, which may also be regulated through the action of hormones, encompass the synthesis and accumulation of amino acids and organic acids and the modification of plant development, including the extent and form of root growth and the timing of flower induction (Vidal and Gutiérrez, 2008). All these processes, acting either individually or synergistically, condition nitrogen allocation in newly developing tissues or in storage organs, to finally ensure plant vegetative or sexual reproduction (Lea et al., 2007; Hirel et al., 2007a; Gregersen et al., 2008; Masclaux-Daubresse et al., 2008).

143

To better understand the regulation of these various processes, several approaches have been undertaken in parallel. They include (1) the study of the transcriptional and posttranslational regulation of the proteins and enzymes involved in nitrogen uptake and assimilation and the subsequent incorporation of reduced nitrogen into organic molecules; (2) the identification of nitrogen compounds that are responsible for the regulation of each specific process under normal or demanding environmental conditions such as high CO2 concentrations, drought or salt stress, nitrogen availability, and variable light intensities and other environmental factors; (3) the development of strategies to alter the content of amino acids via the use of mutants, transgenic plant technology, or conventional breeding, to increase NUE; (4) the discovery of how variations in the relative concentrations of nitrogen compounds are sensed and how the information concerning these metabolic changes are channeled through signal transduction pathways to further develop strategies to modify these transduction pathways in a changing environment; and (5) the development of integrated research both at the whole-plant and at the crop levels in order to evaluate the impact of any genetic or molecular modification of plant NUE and the associated environmental risks (O’Brien and Mullins, 2009). Identification of Key Genes Using Reverse and Forward Genetics Currently, one of the most common ways to establish the function of a structural or regulatory gene in the control of a physiological trait such as NUE, or an agronomic trait such as yield, is to reduce its expression through reverse genetic approaches to further examine the impact of this change on plant phenotype. This can be achieved by using a

144

NUTRIENT USE EFFICIENCY IN CROPS

variety of molecular tools such as antisense RNA, RNA interference (RNAi) (Auer and Frederick, 2009), and screening of mutants in targeting induced local lesions in genomes (TILLING) (Parry et al., 2009), or insertion of mutagenesis (Mutator transposons) populations in the case of maize (Yi et al., 2009). In the early experiments, it was found that the effect of completely reducing the expression of a gene commonly had such a detrimental effect on the phenotype of the mutant plants that it was difficult to carry out an analytical study of NUE. Such problems occurred following the isolation of the original barley mutants that were deficient in GS, GOGAT, and the aminotransferases involved in photorespiratory metabolism, which were only able to grow in elevated CO2 (Blackwell et al., 1988; Leegood et al., 1995), and also those using Arabidopsis thaliana (Igarashi et al., 2006; Potel et al., 2009). It is now possible to construct mutants in which the transcription of specific genes encoding cytoplasmic GS is prevented. Characterization of the individual lines has clearly demonstrated that cytoplasmic GS is of major importance in grain production in rice (Tabuchi et al., 2005) and maize (Martin et al., 2006), and is discussed later. The production of transgenic plants with an improved NUE through the overexpression of a protein or enzyme has been seen as an important goal. The research has been the subject of detailed reviews in the past (Harrison et al., 2000; Yamaya et al., 2002; Andrews et al., 2004; Good et al., 2004), and therefore work published before 2004 is not considered here. Perhaps the most startling and surprising attempts to improve NUE have been carried out by Allen Good and his colleagues using a barley alanine aminotransferase gene (AlaAT). Brassica napus (canola) (Good et al., 2007) and Oryza sativa (rice) (Shrawat et al., 2008; Beatty et al., 2009) containing the overexpressed gene and increased AlaAT enzyme activity exhib-

ited improved NUE, and increased biomass and seed yield (see Chapter 9 for more detail). Attempts to increase the activity of the chloroplast, cytoplasmic, and mitochondrial aspartate aminotransferase isoenzymes in rice were less successful. Although small increases in the protein content of the rice seeds were detected, no major changes in the phenotytpes of the transgenic plants were observed (Zhou et al., 2009). Initial attempts to construct transgenic rice overexpressing the rice cytoplasmic GS gene OsGS1;1 under the control of its own promoter produced plants with no obvious difference in growth or phenotype (Yamaya et al., 2002; Tabuchi et al., 2007). In agreement, when the rice GS genes GS1;1, GS1;2, and Escherichia coli glnA were overexpressed in rice transgenic plants using the CaMV35S promoter, no significant differences were observed in the dry matter production of the vegetative parts of the shoot grown under low nitrogen field conditions (Cai et al., 2009). In addition, somewhat surprisingly, significant decreases in the grain yield were observed when compared with wild-type plants, 25–33% for GS1;1, 7–25% for GS1;2, and 19–39% for glnA overexpressed plants. Higher sensitivity to salt, drought, and cold stresses were observed in the GS1;2-overexpressed rice plants, whereas GS1;1- and glnA-overexpressed plants showed no significant differences in phenotype compared with wild-type plants (Cai et al., 2009). Fei et al. (2006) constructed a range of transgenic pea plants using the soybean cytoplasmic GS gene GS15 and different promoters. Some of the transformants, (in particular, those using the root promoter rolD) exhibited increases in GS activity, but increases in biomass and plant nitrogen content were limited to growth on high concentrations of ammonium. Using the mesophyll-specific promoter of the small subunit of Rubisco and the general CaMV35S

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

promoter, Seger et al. (2009) obtained expression of a soybean cytoplasmic GS1 gene (Gmglnβ1) in both alfalfa and tobacco. There was evidence of small increases in GS activity, leaf protein content, and rates of photosynthesis in the transformed plants. In pine, there are only two GS genes, both of which encode cytoplasmic located proteins GS1a and GS1b, while there is no evidence of a chloroplast-located GS isoenzyme (Canovas et al., 2007). Using the CaMV35S promoter, the cytosolic GS1a gene was overexpressed in poplar. Ectopic expression of the pine GS1a in poplar leaves lead not only to increased GS activity, but also to enhanced levels of chlorophyll, protein, and vegetative growth. In a 3-year field trial, expression of the pine GS led to average heights of the poplar trees that were 21%, 36%, and 41% greater than control plants after the first, second, and third year of growth, respectively. When the poplar plants were grown under 0.3 and 10 mM nitrate, the percentage increases in growth for the GS transgenic lines were greater under low than under high nitrate availability. Taken together with 15 N-enrichment experiments, the data indicated that poplar trees that were overexpressing GS had a higher NUE. In addition, the transgenic GS poplar lines showed enhanced resistance to drought and the herbicide phosphinothricin (Man et al., 2005; Kirby et al., 2006; Pascual et al., 2008). Maize Dof1 is a member of the Dof (DNA-binding with One Finger) family of transcription factors unique to plants and is an activator of the expression of a range of genes associated with organic acid metabolism. Transgenic A. thaliana expressing Dof1 under the control of a maize pyruvate phosphate dikinase (PPDK) promoter exhibited a remarkable elevation of amino acid concentration, especially glutamine, and increased growth under low nitrogen conditions (Yanagisawa et al., 2004). The Dof5

145

transcription factor from maritime pine (Pinus pinaster) has been shown to be a regulator of the expression of GS genes in photosynthetic and nonphotosynthetic tissues. In pine, Dof5-activated transcription of the GS1b promoter, in contrast, repressed transcription of the GS1a promoter. These results suggest a regulatory mechanism for the transcriptional control of the spatial distribution of cytosolic GS isoforms in pine (Rueda-Lopez et al., 2008). Interestingly, a GS gene was upregulated in an A. thaliana callus overexpressing AtDof5.4, which is a close relative of PpDof5 (Tsujimoto-Inui et al., 2009). Additionally, there is now evidence that transcription factors are involved in the control of nitrogen remobilization through the process of leaf senescence and are therefore important in determining the protein content of the wheat grain (Uauy et al., 2006). In contrast, the Dof1 transcription factor does not seem to play a major role in the control of nitrogen or carbon metabolism in maize (Cavalar et al., 2007). Twenty-two different lines of rice overexpressing a rice chimeric OsNADHGOGAT1 gene under the control of its own promoter were shown to contain wideranging enzyme protein and activity. Those with the highest NADH-GOGAT activity exhibited an 80% increase in spikelet weight with no change in number (Yamaya et al., 2002; Tabuchi et al., 2007). Bi et al. (2009) established that the early nodulin gene OsENOD93-1 was upregulated over sevenfold in rice, in response to a change from 1 to 10 mM nitrate and to a slightly lesser extent in the change from 10 to 1 mM nitrate. Although the gene is expressed in both legume and nonleguminous plants, the function of the protein is unknown. Transgenic rice plants in which the OsENOD93-1 gene was overexpressed using the ubiquitin promoter had higher amino acid concentrations in the xylem at lower applied nitrate, which gave rise to

146

NUTRIENT USE EFFICIENCY IN CROPS

higher seed yield and shoot biomass. This work demonstrates that transcriptional profiling, coupled with a transgenic validation approach, is an effective strategy for gene discovery. Exploitation of genetic variability One of the main goals of functional genomics is to identify a portfolio of new structural and regulatory genes and to understand their involvement both in plant physiology and in plant development. This type of inventory will be able to provide an analysis of the biochemical and molecular mechanisms interactively regulating the simultaneous expression of several genes from the transcriptional level up to metabolic fluxes controlled by proteins and enzymes. It is therefore essential to link plant physiology to whole genome expression studies in order to have an integrated view on how the expression of genes can affect overall plant functioning. Modifying gene expression either by genetic engineering or by isolating mutants allows the identification of their function in a targeted manner. By studying the impact of the genetic manipulation or the mutation on the phenotype or the physiology of the plant, it is then possible to determine whether the expression of the gene in question is a limiting step in the development of a particular organ or a given metabolic pathway. In general, this targeted approach, which allows the identification of a single limiting reaction, or a colimiting/ nonlimiting reaction, does not adequately take into account the variation in complex traits such as those controlling agronomic productivity. However, it will be essential to propose genetic targets that could meet the objectives of improving both crop quality and yield. In the last two decades, transgenic and mutagenic approaches have increased our understanding of the role of a number of enzymes or proteins in the control of NUE

(Good et al., 2004; Hirel et al., 2007a) and have allowed the identification of regulatory signals potentially involved in the expression of the corresponding genes (Coruzzi and Bush, 2001; Coruzzi and Zhou, 2001). In particular, these studies have established how the level of expression of genes encoding enzymes involved in both primary nitrogen and carbon metabolism can be modulated by carbon and nitrogen metabolites themselves, thus reflecting the response of the plant to the environment (Scheible et al., 1997; Miller et al., 2007). It now appears that it is necessary to integrate the information gained from such fragmentary studies at the whole-plant level by taking into account environmental effects such as nitrogen limitation. Over the last 10 years, quantitative genetics, through the detection of quantitative trait loci (QTLs), has become a suitable approach for identifying, in an integrated manner, key regulatory or structural genes involved in the expression of complex physiological and agronomic traits and for studying plant responses to environmental constraints (Xu, 1997). When QTLs for agronomic and phenotypic traits are located on a genetic map, it is possible to look for their genetic significance by establishing the colocation of QTLs for physiological or biochemical traits with genes putatively involved in the control of the trait of interest (candidate genes). Validation of candidate genes can then be undertaken using transgenic technologies (forward genetics) or mutagenesis (reverse genetics) or by studying the relationship between allelic polymorphism and the trait of interest (association genetics) either at the single-gene or at the genome-wide level (Yu and Buckler, 2006). Positional cloning is another alternative strategy that can be used to focus on the chromosomal region controlling the trait of interest and that ultimately allows access to a single gene (Salvi and Tuberosa, 2007).

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

A quantitative genetic approach also allows a study of the reciprocal interactions between different regulatory elements controlling a QTL, in order to determine their fitness as a function of environmental constraints (Juenger et al., 2005) and thus of agronomic and economic needs. Eventually, a study of these interactions will include the results obtained from transgenic and mutagenesis experiments when they are placed within the framework of a genetic network controlling the expression of the trait. It thus becomes possible to maximize in a more targeted manner the genetic or the created genetic variability and thus optimize plant productivity (Yano and Tuberosa, 2009). Therefore, in addition to the physiological and agronomic aspects of NUE, quantitative genetic approaches were originally developed in maize for which RIL populations were available, both to construct genetic maps and then to perform QTL studies. The aim of such studies was to identify chromosomal regions involved in the control of yield and its components, and to determine the relative importance of high or low fertilization. Bertin and Gallais (2001) found that most of the chromosomal regions for yield, grain composition, and traits related to NUE detected at low nitrogen input corresponded to QTLs detected at high nitrogen input, whereas Agrama et al. (1999) detected a limited number of QTLs at low nitrogen input. These contrasting results suggest that depending on the RIL population, the response of yield to various levels of nitrogen fertilization could be different and thus controlled by a set of genes specific for a given population. In addition, both of these quantitative genetic studies confirmed that in maize lines, variation in the utilization of nitrogen, including remobilization at low nitrogen input, was greater than the variation of nitrogen uptake before or after flowering, whereas it was the opposite at high nitrogen input (Rajcan and Tollenaar, 1999).

147

Exploiting the genetic variability of these two traits under nitrogen-limiting or nonlimiting conditions appears to be a key target for improving NUE. In a number of other studies performed mostly on rice and maize, QTLs for grain or stover nutritional quality (amino acids, protein, oil, and starch content) as a function of nitrogen fertilization were identified (Wang et al., 2008; Liu et al., 2008a; Xie et al., 2009). However, as for the study performed on root traits (Coque et al., 2008; Liu et al., 2008b) in relation to nitrogen nutrition, colocalization of QTLs with candidate genes of the nitrogen assimilatory pathway was not thoroughly investigated. The work of Bertin and Gallais (2001) was followed by a more detailed investigation in which, in addition to agronomic traits, metabolic functions were associated with DNA markers (Hirel et al., 2001). As for the agronomic traits, a significant genotypic variation was observed for various physiological traits measured in young developing leaves related to nitrogen metabolism. For example, a positive correlation was observed between leaf NO3− content, GS activity, and yield and its components. NR activity, on the other hand, was negatively correlated. It was therefore suggested that increased productivity in maize genotypes could be due to the ability to accumulate NO3− in the leaves during vegetative growth and to efficiently remobilize this stored nitrogen during grain filling. Moreover, coincidences of QTLs for yield and its components with genes encoding cytosolic GS and the corresponding enzyme activity were detected, which could, at least partially, explain the variations in yield. Since coincidences of QTLs for grain yield, GS, NR activity, and nitrate content were also observed, it was also proposed that leaf NO3− accumulation and the reactions catalyzed by NR and GS are coregulated and represent key elements controlling NUE for grain filling in maize (Hirel et al., 2001) (Fig. 8.2).

bnlg109

0

388 Gln1.1 dupssr2

13

umc11 sc309_c_Apx csu59b phi001 pslg204 bnlg2204 gsy366 bc SOD3 1 gsy366_bc_SOD3_1 gsy515_ab_AS2 gsy297a_EMB gsy271_P pslg25 396 bnlg2238 gsy59c_SH2 gsy304_SOD 216 dupssr26 bnlg2295 241

25

NUE traits

32 38 42

% remobilization from leaf Remobilization from stem Remobilization from leaf Remobilization whole plant Postanthesis nitrogen uptake

50

64 72

88

131

umc1035

153

umc1590 bnl559 csu61 gsy473_a_HHU523 195 umc67 gsy351_CS 346 psl18 psl2 psl6 bnlg1057 umc1335 umc58 bnlg615 umc1278 gsy61_BTL2 gsy60b_BT2 umc128 umc83a mmc0041 32 229 bnlg1643 13

162 166 173 179

PHYSIOLOGICAL traits Leaf GS activity N + (young plants) Leaf GS activity N + and N – (adult plants)

185 192 204 209 216

230 239 245 250

gsy282a_CAB_LHCP

pslg210 gsy52_ROOT gsy296_EmbSp Gln1.2 gsy291

272 280 287 292

gsy56_TUA1

300

gsy19_KN1 19 KN1 adh1_iso umc39c gsy177b_MADS pslg208 umc161 bnl829

308 313

YIELD traits

325

+

Grain yield N Kernel number N + TKW N +

335

401 - Gdh1 355

psl44

364 369

umc84 bnlg131 phi064 pslc13 bnl632 pslc33

375 379 386

bnlg1006

405

1

(405 cM)

GENE expression pattern Upregulated in young leaves in N+ Upregulated in young leaves no nitrogen effect Upregulated in old leaves in N+ Upregulated in old leaves in N–

148

399 392 204

307

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

Since a QTL for a thousand kernel weight was coincident with a cytosolic GS (Gln1-4) locus and QTLs for a thousand kernel weight and yield were coincident with another cytosolic GS (Gln1-3) locus (Hirel et al., 2001), further work was undertaken to validate the function of these two putative candidate genes. The impact of the knockout mutations gln1-3 and gln1-4 on kernel yield and its components were examined in plants grown under suboptimal nitrogen feeding conditions (Martin et al., 2006). The phenotype of the two mutant lines was characterized by a reduction of kernel size in the gln1-4 mutant and by a reduction of kernel number in the gln1-3 mutant. In the gln13/1-4 double mutant, a cumulative effect of the two mutations was observed. In transgenic plants overexpressing Gln1-3 constitutively in the leaves, an increase in kernel number was observed, thus providing further evidence that the GS1-3 isoenzyme plays a major role in controlling kernel yield under high (Martin et al., 2006) or low nitrogen fertilization conditions (Hirel et al., 2007a). The hypothesis that GS is one of the key steps in the control of cereal productivity was strengthened by a study performed on rice in which a colocalization of a QTL for the GS1.1 locus and a QTL for one-spikelet weight was identified (Obara et al., 2001). As a confirmation, a strong reduction in growth rate and grain yield was observed in

149

rice GS1.1-deficient mutants (Tabuchi et al., 2005). In contrast, the role of the GS enzyme and other nitrogen-related physiological traits in the control of agronomic performance in wheat still remains to be clearly established. Using a quantitative genetics approach, Fontaine et al. (2009) found only a colocalization between a QTL for GS activity and GSe, a structural gene encoding cytosolic GS, but no obvious colocalization with a QTL for yield, in agreement with previous work published by Habash et al. (2006). Presumably, both the complexity of the hexaploid wheat genome and of the cellular compartmentation of the GS enzyme (Bernard et al., 2008) may explain why such an approach was not as successful as those conducted on maize and rice. The occurrence of a strong genotype and environmental interactions, together with the major influence of vernalization and developmental genes, may also explain why the variation observed for agronomic traits including yield and its components were not so evident in wheat, although there are a number of key traits that could possibly be improved in this species (Foulkes et al., 2009). Interestingly, in a woody species such as maritime pine that is far away from cereals on an evolutionary point of view, a protein QTL for GS colocalized with a GS gene and a QTL for biomass (Plomion et al., 2004). Functional validation of the pine GS gene in

Fig. 8.2. Coincidences between QTLs for physiological traits, traits related to NUE, traits related to grain yield, and newly identified candidate genes on maize chromosome 1. Location of the QTLs for physiological traits (leaf GS activities of young plants at high nitrogen input and leaf GS activities of adult plants at both low and high nitrogen input) for NUE traits (nitrogen remobilization, and postanthesis nitrogen uptake at both high (N+) and low input (N−) and QTLs for grain yield (GY), kernel number (KN), and thousand kernel weight (TKW). The genetic map and the position of the QTL correspond to those originally published by Gallais and Hirel (2004) with additional markers from the IBM (Intermated B73xMo17; http://www.maizemap.org/iMapDB/iMap.html) genetic map. The position of the loci for the most interesting genes is indicated on the genetic map. Each gene is identified by its ID number (indicated in the squares at the right side of the chromosome); the shade of the background corresponds to that used to visualize the four different classes of genes for which transcript abundance is influenced by nitrogen, leaf aging, or both. The position of some known genes involved in nitrogen metabolism encoding cytosolic GS (Gln1.1, Gln1.2) GDH (Gdh1) is indicated and used as a reference to previously published work (Hirel et al., 2001).

150

NUTRIENT USE EFFICIENCY IN CROPS

transgenic poplars (see above), which can be considered a crop for wood production, shows once again that quantitative genetics represent one of the most powerful approaches for identifying NUE candidate genes involved in the control of plant productivity. There is a broad base of knowledge about the model plant A. thaliana, particularly concerning the biology of nitrogen metabolism and the genetic basis of NUE (Loudet and Daniel-Vedele, 2007). However, much work is required before the knowledge acquired on the model plant can be transferred to closely related dicotyledonous plants such as oilseed rape or monocotyledonous cereals, in terms of plant productivity (Gonzalez et al., 2009). In particular, it remains to be determined whether the adaptive mechanisms developed by a weed to take up and utilize nitrogen when it is scarce in its growing environment (Thompson, 1994) are the same as those found in modern crop varieties that have been selected at high fertilizer input (Ceccarelli, 1996). Quantitative genetic studies on NUE have focused, to a large part, on GS because of its central role in nitrogen assimilation and recycling, in relation to yield and biomass production (Bernard and Habash, 2009). Further work is necessary to identify whether other root and shoot enzymes or regulatory proteins could play a specific role under low nitrogen availability, as demonstrated in some cases through the use of mutants and genetically modified plants. Such proteins include those directly involved in nitrogen metabolism or those positioned at the interface between carbon and nitrogen metabolism during plant growth and development (Krapp and Truong, 2005; Gutiérrez et al., 2007b). It will be necessary therefore to identify new nitrogen-responsive genes through detailed analyses of transcriptomic data sets, including using systems biology approaches (see below). The analyses will

be targeted specifically to nitrogen uptake, assimilation, and recycling in vegetative (Wang et al., 2009) and reproductive organs (Cañas et al., 2009) at various stages of plant development, using plants grown under different levels of nitrogen fertilization. It will then be possible to map the newly identified genes, taking advantage of the recent progress in crop genomics through the availability of both physical and genetic high-density maps and QTL or meta-QTL genetic map positions generated by the plant science community (Coque et al., 2008; Geiger, 2009). Comparative genomics and synteny approaches can complete such analyses by linking the genetic maps of maize, rice, barley, and wheat harboring nitrogen-related QTLs, thus allowing the reinforcement of the weight of selected putative candidate genes. In a report by Coque and Gallais (2006), strategies to achieve these goals have been proposed, although it appears that most of the genes expressed under stress conditions (including nitrogen stress) are constitutive but may be differentially regulated under adverse conditions. Altogether, the studies performed on maize suggest that some of the genes involved in the control of yield and its components may be different from those related to the adaptation to nitrogen deficiency. It will therefore be necessary to identify genomic regions responding specifically to nitrogen stress and isolate, via positional cloning, the gene(s) involved in the expression of the trait. Such a strategy has been successfully employed to isolate genes involved in tolerance to a number of abiotic stresses, including drought, salinity, and aluminum (Tuberosa and Salvi, 2006; Collins et al., 2008). However, this has not been achieved in relation to NUE, as developing a reliable phenotypic screen under nitrogen-deficient conditions is not an easy task. Although such screens are generally based on yield performance, they first require the identification of a QTL that is

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

specific for nitrogen-deficient conditions. Moreover, the occurrence of epistatic interactions between genes (Li et al., 1997) under low or high nitrogen input and possibly the presence of nonshared genes within the genome of different genotypes (Brunner et al., 2005) will complicate gene identification and cloning. However, the recent progress made in large genome sequencing and mapping will probably help to decipher part of this complexity. Therefore, to circumvent this problem, it may be necessary to develop, on top of conventional QTL studies, specific breeding programs including nested association mapping (NAM), combining the advantages of linkage analysis and association mapping (Yu et al., 2008). For such studies, the selection of founder parental lines originating from different areas of the world and landraces that are tolerant to nitrogendeficient conditions and a wide range of environments (for example, climate, photoperiod, water availability, flowering precocity, and soil properties) will be essential. This will allow the identification of new QTLs and candidate genes involved in the ability of a plant to grow under lower nitrogen fertilization input. During crop domestication (for maize in particular) a number of genes have been the targets for artificial selection through the process of plant breeding (Yamasaki et al., 2007). However, nothing is known about the nature of the genes that had a strong impact on the selection for improved NUE, which was mostly carried out at high nitrogen input (Raun and Johnson, 1999). It will therefore be interesting to determine whether the selection of NUE candidate genes occurred primarily during crop improvement rather than domestication, which will be a guide for the choice of parental lines, landraces, and crop ancestors before further genetic studies can be developed. Ultimately, following functional validation of candidate genes using all the avail-

151

able approaches offered by mutagenesis and genetic engineering, association genetic marker-assisted selection (MAS) can be then undertaken. MAS consists of introducing the target trait using molecular markers that are closely linked to underlying genes, or that have been developed from the actual candidate gene sequence involved in controlling the agronomic trait of interest. However, there are still a number of technical and scientific challenges that remain to be resolved before MAS can be routinely used in breeding for complex traits such as NUE. This is mainly due to the number of interactions that govern the expression of such traits both at the genetic and at the environmental levels (Xu and Crouch, 2008). In parallel to quantitative genetics, the emerging “systems biology” approach may be a way to unravel such a network of complex interactions when the knowledge gained from model prokaryotic and eukaryotic organisms will be transferred to crops. New developments in systems biology An emerging research field called systems biology consists of taking advantage of various “omics” data sets that can be further analyzed in an integrated manner through the utilization of various mathematical, bioinformatic, and computational tools (Gutiérrez et al., 2007b). Ultimately, such an integrated analysis may allow the identification of the key individual or common regulatory elements involved in the control of a given biological process (Coruzzi et al., 2009). To identify some of the regulatory and structural elements representing the physiological changes associated with NUE, several studies have first evaluated modifications in gene expression. Both in model and in crop species, transcriptome studies have highlighted the complexity of the regu-

152

NUTRIENT USE EFFICIENCY IN CROPS

latory mechanisms involved in the control of leaf gene expression under nitrogen-limiting and nonlimiting conditions, using mutants deficient in key reactions of primary nitrogen and carbon metabolism, as well as during leaf aging in relation to carbon availability (Wang et al., 2003, 2009; Bläsing et al., 2005; Pourtau et al., 2006; Castaings et al., 2009). Map based-cloning studies of mutant plants exhibiting a perturbed adaptation to low-nitrogen nutrition conditions is also another attractive approach that has allowed the identification of key regulatory elements involved in the response to nitrogen limitation (NLA) in A. thaliana (Peng et al., 2007). Whether these elements, including transcription factors such as Dof1 (Yanagisawa et al., 2004), play a similar role in crop species remains to be tested. Further on, tools such as MapMan have been developed to visualize the expression of large gene expression data sets in A. thaliana in order to search for similar global responses across large numbers of microarrays (Usadel et al., 2005). Such a tool, which has now been adapted for maize (Usadel et al., 2009), may provide useful information about the response of whole genome expression to nitrogen nutrition in relation to other cellular and metabolic processes both in model and in crop species and thus identify key regulatory elements involved in their control. Both whole genome expression studies, together with the recent development of network models and genome-wide expression data, confirm the existence of a complex carbon/nitrogen-responsive gene network in plants (Gutiérrez et al., 2007a,b). It is probable that there is a relationship between this network and other cellular processes linked to either external signals such as light (Krouk et al., 2009), or internal signals such as hormones (Nero et al., 2009). Although much more informative than conventional transcriptome studies, such whole genome

expression approaches have remained confined to deciphering regulatory circuits at the transcriptional level since only the steadystate level of transcripts have been considered. Such an approach, originally developed for A. thaliana by virtue of the wealth of information at the transcriptome level, when transferred to crops may help in identifying key master genes involved in the control of NUE. More recently, translatomic studies have been developed to provide another level of information about translation rates both globally and for individual enzymes, taking into account at the same time their turnover (Piques et al., 2009). Now, it is even possible to refine such a translatome approach at the cellular level by isolating cell-specific populations of polysomes (Mustroph et al., 2009). Therefore, it will be theoretically possible in the near future to further expand our knowledge on the regulation of NUE in different organs and cells at both the transcriptional and the translational levels using a combination of molecular and metabolic approaches (Gifford et al., 2008). In comparison to the numerous transcriptomic studies, there is still a paucity of data on proteomics concerning NUE both in model and in crop species. Proteomic approaches are increasingly being used to address biological questions, particularly in the medical domain for examining the effect of environmental factors on diseases and aging (Banks et al., 2000; George and Shukla, 2008). In plants, proteomic studies have not been extensively developed because they involve time-consuming and difficult techniques. Moreover, at best, less than a thousand proteins can usually be separated by two-dimensional gel electrophoresis, and identified using either the available databases (Jorrin et al., 2007), or mass spectrometry techniques. However, proteomic studies can provide additional information on the quantity of expressed protein and their post-

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

translational modifications such as phosphorylation and glycosylation that cannot be identified by only examining transcriptional regulation of the whole genome. The first proteomic studies in relation to nitrogen deficiency were performed on wheat, and the data indicated that it was mostly the concentration of enzymes and proteins involved in carbon metabolism that were modified following nitrogen stress (Bahrman et al., 2004). Changes in the protein profile were also examined in maize (Prinsi et al., 2009) and rice (Kim et al., 2009) under low and high nitrogen supply. Results from these studies showed that the amounts of protein of enzymes having a pivotal role in nitrogen assimilation such as GS, and in carbon metabolism such as phosphoenolpyruvate (PEP) carboxylase, were higher when plants were fed with nitrate. Many other proteins involved in a number of photosynthetic reactions and in maintaining the energy and redox status of the cell were also shown to be nitrogen-responsive, thus confirming the close relationship existing between nitrogen and carbon metabolism and the related energetic processes found at the transcriptional level (Gutiérrez et al., 2007a,b). The finding that posttranslational modifications of PEP carboxylase are occurring demonstrates that such modifications may be important in the overall plant nitrogen economy and thus should be investigated in more detail using both proteomic and biochemical approaches (Feria et al., 2008). However, since in all the proteomic studies there was no direct comparison with transcriptomic studies, it was difficult to tell if the regulation of protein synthesis occurred solely at the translational or posttranslational level or if the amount of protein was correlated with the amount of corresponding transcripts. Nevertheless, there were some indications that correlations between protein and nitrogen metabolite contents could be a way of identifying new structural or regulatory proteins involved in

153

the response to the amount of nitrogen fertilization in different wheat varieties. Such findings also suggest that posttranscriptional modifications may be positively or negatively regulated by plant nitrogen metabolite concentration (Bahrman et al., 2005). There are even fewer metabolomic studies in crops aimed at identifying changes in metabolite levels under various nitrogen treatments. Until now, the vast majority of metabolomic studies have been carried out using A. thaliana. They allowed plant phenotyping by exhaustive metabolic profiling using generally gas chromatography (GC)–mass spectrometry (MS)-based separation techniques (Stitt and Fernie, 2003; OksmanCaldentey and Saito, 2005). More recently, 1 H-NMR spectroscopy approaches have been developed, which are less sensitive but noninvasive compared with those requiring the extraction of plant material (Allwood et al., 2008). 1H-NMR metabolomics appears to be an attractive technique for the development of mapping approaches (Graham et al., 2009) that could support breeding for improved NUE through the establishment of metabolite databases. 1H-NMR was also used successfully to improve the characterization of GS-deficient mutants in maize, indicating that in addition to the glutaminederived amino acid biosynthetic pathways, lignin biosynthesis was also altered (Mesnard and Ratcliffe, 2005; Broyard et al., 2009). Whether these pathways are coordinately regulated through the control of Dof transcription factors, known to be important in the control of NUE, is an interesting hypothesis that remains to be tested (Tsujimoto-Inui et al., 2009). Metabolomic studies are becoming more and more extensively used for the highthroughput phenotyping necessary for largescale molecular and quantitative genetic studies aimed at identifying new candidate genes involved in the control of plant productivity (Meyer et al., 2007; Lisec et al.,

154

NUTRIENT USE EFFICIENCY IN CROPS

2008). However, these analyses only provide a narrow and static picture of the physiological status of a given organ, at a particular stage of plant development. Noninvasive in vivo NMR spectroscopy techniques based on the use of 15N-labeled compounds may represent an interesting complementary approach to metabolomics since the technique can provide additional information on the changes in metabolic fluxes occurring in mutants, genetically modified crops, or genotypes exhibiting contrasting NUE (Labboun et al., 2009). Unlike gene transcription and the translational regulation of proteins, metabolism is probably the best characterized of all molecular interaction networks in biology. This has prompted a number of groups, irrespective of the organism studied, to focus their research effort on developing data integration tools for metabolic reactions, rather than gene expression studies. Large amounts of data relating to metabolic reactions are currently available, but despite this wealth of information, metabolic phenotypes still cannot be accurately predicted (Sweetlove et al., 2008). Knowledge of plant metabolism in particular remains fractional, and efforts to engineer plant metabolism in general, and nitrogen metabolism in particular, have been unsuccessful on most occasions (Good et al., 2007; Hirel et al., 2007b). Thus, a comprehensive understanding of plant metabolism is as yet missing, but it has the potential to bring valuable advances in the improvement of yield, NUE, and nutritional quality of crops. Encouragingly, on the modeling side, an increasing number of genome-scale metabolic models of microorganisms and multicellular organisms have recently been developed with a broad range of applications from metabolic engineering to biological discovery (Shlomi et al., 2008; Koide et al., 2009) (Fig. 8.3). In contrast to these remarkable achievements, metabolic modeling in plants is still in its infancy.

Partial models of A. thaliana have been released (de Oliveira Dal’Molin et al., 2010; Radrich et al., 2010), but they lack the complete stoichiometric model required for metabolic engineering purposes. However, the achievements in microorganisms have resulted in a collection of modeling tools and methods that have now set the stage for the rapid and successful development of metabolic models in plants, if used judiciously. Such metabolic models should help to unravel key reactions and thus limit the steps required for the control of NUE, taking into account both tissue specificities and environmental constraints. Metabolic, biochemical, and molecular markers for diagnostic and breeding Plant adaptation to various nitrogen levels in soils is a quantitative process that proceeds through growth and development. This adaptive biological process integrates many traits and hence is polygenic. Therefore, tools to diagnose differential responses, either for varied inputs or for genotype differences, have to combine many elements that are defined as indicative of function and nitrogen economy at a particular stage of plant development (Fig. 8.4). For example, these diagnostic tools can be developed from the knowledge gained from transcriptomic studies. This will ultimately result in the production of diagnostic microarrays containing marker genes whose expression are regulated by the plant nitrogen status, independent of genotype and environmental conditions. These microarrays will be directly utilizable by breeders to screen for the bestperforming genotypes. From a longer term perspective, they could also be used by farmers to evaluate in real time the nitrogen status of a crop, in order to control precisely the application of fertilizer in an optimal manner. This could be achieved by the pro-

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

A

B

UNCONSTRAINED NETWORK Chloroplast Starch

Mitochondria Phosphate PEPc Trioses

TCA

Glucose

Rubisco CO2

Gly(¥2)

Ser + CO2 + NH4+

Glu GOGAT

aKG

NH4+

GDH Glu

aKG

Gln GS2 AA

NiR

NO2–

Starch Calvin

NR

Oxaloacetate TCA Gly(¥2)

Sucrose

Ser + CO2 + NH4+

GOGAT

aKG

aKG

GDH Glu

Gln

Glu

GS2 AA NiR

NO3–

Mitochondria

Peroxisome

NH4+ NO2–

PEPc

Glucose

AA Glu

Phosphate Trioses

O2

CO2

Peroxisome

Glu

Chloroplast

Rubisco

Sucrose

AA

MULTILEVEL REGULATORY EFFECTS

Oxaloacetate

O2

Calvin

155

Vacuole

NO2–

Plasma membrane

NR NO2–

NO3–

Vacuole

Plasma membrane

Uptake

Uptake

C Biomas /yield

N+ N–

Plant growth rate Fig. 8.3. Concept for a plant NUE predictive model. (A) A simplified drawing of central nitrogen metabolism in

relation to carbon metabolism, where the network is unconstrained. (B) Application of various transcriptional or posttranscriptional regulatory effects indicated by the cross in a gray background symbol. This restricts the number of pathways that compounds can be metabolized through in the network. (C) The model can then be used to predict the growth and productivity of a plant as a function of metabolic product formation and additional nitrogen uptake rates, under low (N−) and high nitrogen (N+) fertilization conditions. PEPc, PEP carboxylase; Rubisco, RuBP carboxylase/oxygenase; TCA, tricarboxylic acid cycle.

duction of microfluidic chips (Senapati et al., 2009) to detect nitrogen use deficiency or efficiency. Cancer research has already demonstrated significant practical and commercial potential for such diagnostic chips (Heath and Davis, 2008). In the near future, farmers and breeders should be able to obtain a diagnosis from a small leaf sample within a few minutes. These quick

test results should not only gain precious time for plant selection and adjustment of fertilization, but could also offer significant savings, as testing can be done at a fraction of the cost of current methods. Based on affordable colorimetric methods, physiological markers of plant nitrogen status could be developed as easyto-use detection kits. They represent an

156

NUTRIENT USE EFFICIENCY IN CROPS

PLANT DEVELOPMENT

MOLECULAR BIOCHEMICAL MARKERS FOR NUE

G

F

N-

L3 L2

PROTEIN`OR METABOLITE COLORIMETRIC DETECTION

L1

VS

FL

M

N+

MICROCHIPS

Fig. 8.4. A theoretical concept for producing diagnostic tools to evaluate or monitor crop NUE during growth and

development. Plant samples from different organs (L1, L2, L3, different leaf stages; S, stems; F, flowers; G, grain) and at different stages of plant development (VS, vegetative stage; FL, flowering; M, maturity) can be used to identify marker genes, protein, or metabolites representative of the plant physiological status with respect to NUE under low (N−) or (N+) fertilization conditions. The level of expression of marker genes (dark gray = low, gray = medium, white = high) can be determined by using microchips. Protein or metabolite content (dark gray = low, white = high) could be measured by using immunochemical or colorimetric detection.

alternative to the DNA chips discussed above, for both breeding and cultivation purposes. A first step toward developing these tools was to perform molecular and physiological studies at the whole-plant level. These studies performed under field growth conditions have allowed the representation, in a dynamic and integrated manner, of the changes in various physiological and biochemical markers representative of nitrogen uptake, nitrogen assimilation, and nitrogen recycling in crops (Hirel et al., 2005; Kichey et al., 2006). The finding that in the model plant, A. thaliana, the metabolic signature of the plant is a good predictive marker of plant biomass production further strengthens the idea that it is possible to use metabolites as biomarkers of plant productivity (Meyer et al., 2007). More recently, 15N-labeling techniques have been used in the field, along with the measurement of physiological traits, to estimate the genetic variability of nitrogen uptake, nitrogen assimilation, and nitrogen recycling in different wheat cultivars. The investigations revealed that GS and NR

activities are potential markers for the estimation of the proportion of nitrogen taken up or nitrogen remobilized. The nitrogen taken up or remobilized is further invested in grain yield or grain nitrogen content (Kichey et al., 2007). Therefore, 15N-labeling techniques combined with the use of simple physiological markers may be a way to assist breeders to estimate crop performance under different levels of nitrogen nutrition, since both methods allow scoring to be carried out relatively easily and cheaply, when using a large number of genotypes. The next step in the development of diagnostic tools will be to fit them into a precision agriculture farming system, combining soil testing, fertilizer application, and projected fertilizer requirement, to determine the application rates of fertilizers under different environmental conditions. They could be used to complement the currently available sensing tools based on leaf and canopy chlorophyll determinations (Foulkes et al., 2009), or polyphenol concentrations (Samborski et al., 2009). In parallel, the development of crop models will be required

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

to simulate the morphogenetic responses of plants to contrasting nitrogen nutrition and thus to understand in a more integrated way the constraints linked to increasing crop NUE on the side of both the plant and the environment. The development of these models will enable breeders to identify crop ideotypes and agronomists to optimize nitrogen management practices under various environmental scenarios (Baret et al., 2007; Semenov and Halford, 2009). Developing a framework based on crop simulation models to improve our ability to analyze complex traits such as NUE will not only become a viable tool for genetics and subsequent genomics research, but will also provide a physiological interpretation of the variation and interactions of key traits representative of NUE. These models may also help to link plant model parameters with simple physiological and biochemical traits and thus facilitate genetic and genomic research to identify the key regulatory or structural genes involved, and how they are controlled. Conclusions and Perspectives Although extensive studies have been carried out over the last two decades to identify the rate-limiting steps of nitrogen uptake, assimilation, and recycling in an increasing number of crops, the identification of key structural and regulatory genes involved in their control remains incomplete. There are a number of possible reasons for the limited success of the various investigations that were developed using either transgenic or quantitative genetic approaches. One of them is that nitrogen uptake and assimilation efficiencies are species-specific (Hirel et al., 2007a) and are constantly changing during plant growth and development. These changes occur not only in an organ-specific and time-dependent manner (including diurnal fluctuations and the

157

various stages of plant development from seed to seed), but also as a function of a constantly changing environment in the soil (nitrogen mineralization, nitrogen and water availability, symbiotic associations, competition with microorganisms, and soil properties) and above the soil (light and climatic conditions). At the whole-plant and canopy level, it is evident that only an agronomic predictive model, taking into account the different parameters listed above, will allow an integrated view of the various inputs or outputs influencing crop NUE (McCown et al., 1996). Although this aspect was beyond the scope of this review, one of the main challenges in the future will be to develop reliable decision support systems with the help of sensors (Samborski et al., 2009) and biological diagnostic tools in precision agriculture, in order to optimize the application of nitrogen in a more sustainable manner. From a physiological and molecular genetic point of view, it can be considered that NUE is mostly confined to the plant itself, being controlled by a complex array of physiological and developmental interactions that are organ- and tissue-specific. Moreover, it appears that such an array of interactions is subject to genetic variability, thus modifying the expression of a single gene or even a group of genes in a given genetic background, may not be as efficient as expected, simply because the expression of the gene or group of genes is not limiting in this particular genetic background (Cañas et al., 2011). This means that a much more extensive survey of a wide range of genotypes covering the genetic diversity of the crop should be performed, using the various available “omics” techniques, in order to identify common and specific elements controlling NUE and plant productivity. Data integration can be then performed using the appropriate bioinformatics tools to link crop phenotype to the various “omics” data sets.

158

NUTRIENT USE EFFICIENCY IN CROPS

Obviously, such a high-throughput type of analysis will require, in addition to largescale field experiments, the appropriate devices for grinding (Stitt et al., 2009) and analyzing the samples (Gibon et al., 2004), as well as tools to support the design and analysis of experiments (Hannemann et al., 2009). Hopefully, the combination of genetic studies (Takeda and Matsuoka, 2009) and system biology approaches (Coruzzi et al., 2009) using the appropriate crop genotypes, will allow the identification of target genes and metabolic pathways involved in the control of NUE, which hitherto have not been exploited.

References Agrama, H.A.S., Zacharia, A.G., Said, F.B., et al. (1999) Identification of quantitative trait loci for nitrogen use efficiency in maize. Molecular Breeding 5, 187–195. Allwood, J.W., Ellis, D.I., & Goodrace, R. (2008) Metabolomic technologies and their application to the study of plants and plant-host interactions. Physiologia Plantarum 132, 117–135. Andrews, M., Lea, P.J., Raven, J.A., et al. (2004) Can genetic manipulation of plant nitrogen assimilation enzymes result in increased crop yield and greater N-use efficiency? An assessment. Annals of Applied Biology 145, 25–40. Andrews, M., Lea, P.J., Raven, J.A., et al. (2009) Nitrogen use efficiency. 3. Nitrogen fixation: genes and costs. Annals of Applied Biology 155, 1–13. Auer, C. & Frederick, R. (2009) Crop improvement using small RNAs: applications and predictive ecological risk assessments. Trends in Biotechnology 27, 644–651. Bahrman, N., Le Gouis, J., Negroni, L., et al. (2004) Differential protein expression assessed by twodimensional gel electrophoresis for two wheat varieties grown at four nitrogen regimes. Proteomics 4, 709–719. Bahrman, N., Gouy, A., Devienne-Barret, F., et al. (2005) Differential change in root protein pattern of two wheat varieties under high and low nitrogen nutrition levels. Plant Science 168, 81–87. Banks, R.E., Dunn, M.J., Hochstrasser, D.F., et al. (2000) Proteomics: new perspectives, new biochemical opportunities. The Lancet 356, 1749–1756.

Baret, F., Houlès, V., & Guérif, M. (2007) Quantification of plant stress using remote sensing observations and crop models: the case of nitrogen. Journal of Experimental Botany 58, 869–880. Beatty, P.H., Shrawat, A.K., Carroll, R.T., et al. (2009) Transcriptome analysis of nitrogen-efficient rice over-expressing alanine aminotransferase. Plant Biotechnology Journal 7, 562–576. Bernard, S.M. & Habash, D.Z. (2009) The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytologist 182, 608–620. Bernard, S.M., Møller, A.L.B., Dionisio, G., et al. (2008) Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat (Triticum aestivum L.). Plant Molecular Biology 67, 89–105. Bertin, P. & Gallais, A. (2001) Physiological and genetic basis of nitrogen use efficiency in maize. II. QTL detection and coincidences. Maydica 46, 53–68. Bi, Y.M., Kant, S., Clark, J., et al. (2009) Increased nitrogen-use efficiency in transgenic rice plants over-expressing a nitrogen-responsive early nodulin gene identified from rice expression profiling. Plant, Cell & Environment 32, 1749–1760. Blackwell, R.D., Murray, A.J.S., Lea, P.J., et al. (1988) The value of mutants unable to carry out photorespiration. Photosynthesis Research 16, 155–176. Bläsing, O.E., Gibon, Y., Günter, M., et al. (2005) Sugars and circadian regulation make major contributions to the global regulation of diurnal gene expression in Arabidopsis. The Plant Cell 17, 3257–3281. Broyard, C., Fontaine, J.X., Molinie, R., et al. (2009) Metabolomic profiling of two cytosolic glutamine synthetase maize mutants (Zea mays L.) using 1 H-nuclear magnetic resonance. Phytochemical Analysis 21, 102–109. Brunner, S., Fengler, K., Morgante, M., et al. (2005) Evolution of DNA sequence non homologies among maize inbreds. The Plant Cell 17, 342–360. Cai, H.M., Zhou, Y., Xiao, J.H., et al. (2009) Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Reports 28, 527–537. Cañas, R.A., Quilleré, I., Christ, A., et al. (2009) Nitrogen metabolism in the developing ear of maize (Zea mays L.): analysis of two lines contrasting in their mode of nitrogen management. The New Phytologist 184, 340–352. Cañas, R.A., Amiour, N., Quilleré, I., et al. (2011) An integrated statistical analysis of the genetic variability of nitrogen metabolism in the ear of three maize inbred lines (Zea mays L). Journal of Experimental Botany 62, 2309–2318.

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

Canovas, F.M., Avila, C., Canton, F.R., et al. (2007) Ammonium assimilation and amino acid metabolism in conifers. Journal of Experimental Botany 58, 2307–2318. Castaings, L., Camargo, A., Pocholle, D., et al. (2009) The nodule interception-like protein7 modulates nitrate sensing and metabolism in Arabidopsis. The Plant Journal 57, 426–435. Cavalar, M., Phlippen, Y., Kreuzaler, F., & Peterhaensel, C. (2007) A drastic reduction in DOF1 transcript levels does not effect C4 specific gene expression in maize. Journal of Plant Physiology 164, 1665–1674. Ceccarelli, S. (1996) Adaptation to low/high input cultivation. Euphytica 92, 203–214. Chien, S.H., Prochnow, L.I., & Cantarella, H. (2009) Recent developments of fertilizer production and use to improve nutrient efficiency and minimize environmental impacts. Advances in Agronomy 102, 267–323. Collins, N.C., Tardieu, F., & Tuberosa, R. (2008) Quantitative trait loci and crop performance under abiotic stress; where do we stand. Plant Physiology 147, 469–486. Coque, M. & Gallais, A. (2006) Genomic regions involved in response to grain yield selection at high and low nitrogen fertilization in maize. Theoretical and Applied Genetics 112, 1205–1230. Coque, M., Martin, A., Veyrieras, J.B., et al. (2008) Genetic variation for N-remobilization and postsilking N-uptake in a set of maize recombinant inbred lines. 3. QTL detection and coincidences. Theoretical and Applied Genetics 117, 729–747. Coruzzi, G.M. & Bush, D.R. (2001) Nitrogen and carbon nutrient and metabolite signalling in plants. Plant Physiology 125, 61–64. Coruzzi, G.M. & Zhou, L. (2001) Carbon and nitrogen sensing and signaling in plants: emerging “matrix effects”. Current Opinion in Plant Biology 4, 247–253. Coruzzi, G.M., Burga, A.R., Katari, M.S., et al. (2009) Systems biology: principles and applications in plant research. In: Annual Plant Reviews, Vol. 35 (eds. G.M. Coruzzi & R.A. Guttiérez), pp. 3–40, Wiley-Blackwell, Oxford, U.K. de Oliveira Dal’Molin, C.G., Quek, L.-E., Palfreyman, W., et al. (2010) AraGEM, a genome-scale reconstruction of the primary metabolic network in Arabidospis thaliana. Plant Physiology 152, 579–589. Dyson, T. (1999) World food trends and prospects to 2025. Proceeding of the National Academy of Science of the United States of America 96, 5929–5936. Edgerton, M.D. (2009) Increasing crop productivity to meet global needs for feed, food and fuel. Plant Physiology 149, 7–13.

159

Fei, H., Chaillou, S., Hirel, B., et al. (2006) Effects of the overexpression of a soybean cytosolic glutamine synthetase gene (GS15) linked to organspecific promoters on growth and nitrogen accumulation of pea plants supplied with ammonium. Plant Physiology and Biochemistry 44, 543–550. Feria, A.B., Alvarez, R., Cochereau, L., et al. (2008) Regulation of phosphoenolpyruvate carboxylase phosphorylation by metabolites and ABA during the development and germination of barley seeds. Plant Physiology 148, 761–774. Fontaine, J.X., Ravel, C., Pageau, K., et al. (2009) A quantitative genetic study for elucidating the contribution of glutamine synthetase, glutamate dehydrogenase and other nitrogen-related physiological traits to the agronomic performance of common wheat. Theoretical and Applied Genetics 119, 645–662. Forde, B.G. & Lea, P.J. (2007) Glutamate in plants: metabolism, regulation and signaling. Journal of Experimental Botany 58, 2339–2358. Foulkes, M.J., Hawkesford, M.J., Barraclough, P.B., et al. (2009) Identifying traits to improve the nitrogen economy of wheat. Field Crops Research 114, 329–342. Gallais, A. & Hirel, B. (2004) An approach to the genetics of nitrogen use efficiency in maize. Journal of Experimental Botany 55, 295–306. Galloway, J.N., Aber, J.D., Ersiman, J.W., et al. (2003) The nitrogen cascade. BioScience 53, 341–356. Galloway, J.N., Townsend, A.R., Erisman, J.W., et al. (2008) Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320, 889–892. Geiger, H.H. (2009) Agronomic traits and maize modifications: nitrogen use efficiency. In: Handbook of Maize: Its Biology (eds. J.L. Bennetzen & S.C. Hake), pp. 405–417, Springer Science + Business Media, New York. George, J. & Shukla, Y. (2008) Prognostic factors of male breast cancer: proteomic approaches for early detection and treatment. Journal of Proteomics & Bioinformatics 1, 112–127. Gibon, Y., Blaesing, O.E., Henneman, J.H., et al. (2004) A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. The Plant Cell 16, 3304–3325. Gifford, M.L., Dean, A., Gutiérrez, R.A., et al. (2008) Cell-specific nitrogen responses mediate developmental plasticity. Proceedings of the National Academy of Sciences of the United States of America 105, 803–808.

160

NUTRIENT USE EFFICIENCY IN CROPS

Goff, S.A., Ricke, D., Lan, T.H., et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296, 92–100. Gonzalez, N., Beemster, G.T.S., & Inzé, D. (2009) David and Goliath: what can the tiny weed Arabidopsis teach us to improve biomass production in crops? Current Opinion in Plant Biology 12, 157–164. Good, A.G., Shrawat, A.K., & Muench, D.G. (2004) Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production? Trends in Plant Science 9, 597–605. Good, A.G., Johnson, S.J., DePauw, M.D., et al. (2007) Engineering nitrogen use efficiency with alanine aminotransferase. Canadian Journal of Botany 85, 252–262. Graham, S.F., Amigues, E., Migaud, M., et al. (2009) Application of NMR based metabolomics for mapping metabolite variation in European wheat. Metabolomics 5, 302–306. Gregersen, P.L., Holm, P.B., & Krupinska, K. (2008) Leaf senescence and nutrient remobilization in barley and wheat. Plant Biology 10, 37–49. Gruber, N. & Galloway, J.N. (2008) An Earth-system perspective of the global nitrogen cycle. Nature 451, 293–296. Gutiérrez, R.A., Gifford, M.L., Poultney, C., et al. (2007a) Insights into the genomic nitrate response using genetics and the sungear software system. Journal of Experimental Botany 58, 2359–2367. Gutiérrez, R.A., Lejay, L.V., Dean, A., et al. (2007b) Qualitative network models and genome-wide expression data define carbon/nitrogen-responsive molecular machines in Arabidopsis. Genome Biology 8, R7. Habash, D.Z., Bernard, S., Shondelmaier, J., et al. (2006) The genetics of nitrogen use on hexaploid wheat: N utilization, development and yield. Theoretical and Applied Genetics 114, 403–419. Hannemann, J., Poorter, H., Usadel, B., et al. (2009) Xeml lab: a tool that supports the design of experiments at a graphical interface and generates computer-readable metadata files, which capture information about genotypes, growth condition, environmental perturbations and sampling strategy. Plant, Cell & Environment 32, 1185–1200. Harrison, J., Brugiere, N., Phillipson, B., et al. (2000) Manipulating the pathway of ammonia assimilation through genetic engineering and breeding: consequences to plant physiology and plant development. Plant and Soil 221, 81–93. Heath, J.R. & Davis, M.E. (2008) Nanotechnology and cancer. Annual Review of Medicine 59, 251– 265.

Hirel, B. & Lea, P.J. (2001) Ammonia assimilation. In: Plant Nitrogen (eds. P.J. Lea & J.F. Morot-Gaudry), pp. 79–99, INRA Paris and Springer-Verlag, Berlin, Heidelberg, New York. Hirel, B. & Lemaire, G. (2005) From agronomy and ecophysiology to molecular genetics for improving nitrogen use efficiency in crops. In: Enhancing the Efficiency of Nitrogen Utilisation in Plants (eds. S.S. Goyal, R. Tischner & A.S. Basra), pp. 213– 257, Haworth Press, Binghampton. Hirel, B., Bertin, P., Quillere, I., et al. (2001) Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize. Plant Physiology 125, 1258–1270. Hirel, B., Andrieu, B., Valadier, M.H., et al. (2005) Physiology of maize II: identification of physiological markers representative of the nitrogen status of maize (Zea mays L.) leaves, during grain filling. Physiologia Plantarum 124, 178–188. Hirel, B., Le Gouis, J., Ney, B., et al. (2007a) The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. Journal of Experimental Botany 58, 2369–2387. Hirel, B., Le Gouis, J., Bernard, M., et al. (2007b) Genomics and plant breeding: maize and wheat. In: Functional Plant Genomics (eds. J.F. MorotGaudry, P.J. Lea & J.F. Briat), pp. 614–635, Science Publishers, Enfield, Jersey, Plymouth. Igarashi, D., Tsuchida, H., Miyao, M., et al. (2006) Glutamate: glyoxylate aminotransferase modulates amino acid content during photorespiration. Plant Physiology 142, 901–910. Jorrin, J.V., Maldonado, A.M., & Castijjero, M.A. (2007) Plant proteomics analysis: a 2006 update. Proteomics 7, 2947–2962. Juenger, T.E., Sen, S., Stowe, K.A., et al. (2005) Epistasis and genotype-environment interaction for quantitative trait loci affecting flowering time in Arabidopsis thaliana. Genetica 123, 87– 105. Kachanoski, R.G. (2009) Crop response to nitrogen fertilizer: the delta yield concept. Canadian Journal of Soil Science 89, 543–554. Kichey, T., Heumez, E., Pocholle, D., et al. (2006) Combined agronomic and physiological aspects of nitrogen management in wheat (Triticum aestivum L.). Dynamic and integrated views highlighting the central role for the enzyme glutamine synthetase. New Phytologist 169, 265–278. Kichey, T., Hirel, B., Heumez, E., et al. (2007) In winter wheat (Triticum aestivum L.), post-anthesis nitrogen uptake and remobilisation to the grain correlates with agronomic traits and nitrogen

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

physiological markers. Field Crops Research 102, 22–32. Kim, D.H., Shibato, J., Kim, D.W., et al. (2009) Gelbased proteomics approach for detecting lownitrogen responsive proteins in cultivated rice species. Physiology and Molecular Biology of Plants 15, 31–41. Kirby, E.G., Gallardo, F., Man, H., et al. (2006) The overexpression of glutamine synthetase in transgenic poplar: a review. Silvae Genetica 55, 278–284. Koide, T., Pang, W.L., & Baliga, N.S. (2009) The role of predictive modelling in rationally re-engineering biological systems. Nature Reviews. Microbiology 7, 297–305. Krapp, A. & Truong, H.N. (2005) C/N interaction in model plant species. In: Enhancing the Efficiency of Nitrogen Utilisation in Plants (eds. S.S. Goyal, R. Tischner & A.S. Basra), pp. 127–173, Haworth Press, Binghampton. Krouk, G., Tranchina, D., Lejay, L., et al. (2009) A systems approach uncovers restrictions for signal interactions regulating genome-wide responses to nutritional cues in Arabidopsis. PLoS Computational Biology 5, 1–12. Labboun, S., Tercé-Laforgue, T., Roscher, A., et al. (2009) Resolving the role of plant glutamate dehydrogenase: I. In vivo real time nuclear magnetic resonance spectroscopy experiments. Plant & Cell Physiology 50, 1761–1773. Lea, P.J. & Azevedo, R.A. (2006) Nitrogen use efficiency. 1. Uptake of nitrogen from the soil. Annals of Applied Biology 149, 243–247. Lea, P.J. & Azevedo, R.A. (2007) Nitrogen use efficiency. 2. Amino acid metabolism. Annals of Applied Biology 151, 269–275. Lea, P.J., Sodek, L., Parry, M.A.J., et al. (2007) Asparagine in plants. Annals of Applied Biology 150, 1–26. Leegood, R.C., Lea, P.J., Adcock, M.D., et al. (1995) The regulation and control of photorespiration. Journal of Experimental Botany 46, 1397–1414. Li, Z., Pinson, S.R.M., Park, W.D., et al. (1997) Epistasis for three grain yield components in rice (Oryza sativa L.). Genetics 145, 453–465. Lisec, J., Meyer, R.C., Steinfath, M., et al. (2008) Identification of metabolic and biomass QTL in Arabidopsis thaliana in a parallel analysis of RIL and Il populations. The Plant Journal 53, 960–972. Liu, J., LI, J., Chen, F., et al. (2008b) Mapping QTLs for root traits under different nitrate levels at the seedling stage in maize (Zea mays L.). Plant and Soil 305, 253–265. Liu, Z.H., Xie, H.L., Tian, G.W., et al. (2008a) QTL mapping of nutrient components in maize kernels

161

under low nitrogen conditions. Plant Breeding 127, 279–285. Loudet, O. & Daniel-Vedele, F. (2007) Analysis of complex characters into elementary components through the use of molecular quantitative genetics in Arabidopsis thaliana. In: Functional Plant Genomics (eds. J.F. Morot-Gaudry, P.J. Lea & J.F. Briat), pp. 559–570, Science Publishers, Enfield, Jersey, Plymouth. Ludewig, U., Neuhäuser, B., & Dynowski, M. (2007) Molecular mechanisms of ammonium transport and accumulation in plants. FEBS Letters 581, 2301–2308. Man, H.M., Boriel, R., El-Khatib, R., et al. (2005) Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. The New Phytologist 167, 31–39. Martin, A., Lee, J., Kichey, T., et al. (2006) Two cytosolic glutamine synthetase isoforms of maize (Zea mays L.) are specifically involved in the control of grain production. The Plant Cell 18, 3252–3274. Masclaux-Daubresse, C., Reisdorf-Cren, M., & Orsel, M. (2008) Leaf nitrogen remobilization for plant development and grain filling. Plant Biology 10, 23–36. McCown, R.L., Hammer, G.L., Hargreaves, J.N.G., et al. (1996) APSIM: a novel software system for model development, model testing and simulation in agricultural systems research. Agricultural Systems 50, 255–271. Mesnard, F. & Ratcliffe, R.G. (2005) NMR analysis of plant nitrogen metabolism. Photosynthesis Research 83, 163–180. Meyer, R.C., Steinfath, M., Lisec, J., et al. (2007) The metabolic signature related to high plant growth rate in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 104, 4759–4664. Miflin, B.J. (2000) Crop improvement in the 21st century. Journal of Experimental Botany 51, 1–8. Miller, A.J., Fan, X., Shen, D., et al. (2007) Amino acids and nitrate as signals for the regulation of nitrogen. Journal of Experimental Botany 59, 111–119. Mulvaney, R.L., Khan, S.A., & Ellsworth, T.R. (2009) Synthetic nitrogen fertilizers deplete soil nitrogen: a global dilemma for sustainable cereal production. Journal of Environmental Quality 38, 2295–2314. Mustroph, A., Zanetti, M.E., Hang, C.J.H., et al. (2009) Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proceedings of the National Academy of Science of the United States of America. 106, 18843–18848.

162

NUTRIENT USE EFFICIENCY IN CROPS

Nero, D., Krouk, G., Tranchina, D., et al. (2009) A system biology approach highlights a hormonal enhancer effect on regulation of genes in a nitrate responsive “biomodule”. BMC Systems Biology 3, 59. O’Brien, M. & Mullins, E. (2009) Relevance of genetically modified crops in light of future environmental and legislative challenges to the agri-environment. Annals of Applied Biology 154, 323–340. Obara, M., Kajiura, M., Fukuta, Y., et al. (2001) Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate in rice (Oryza sativa L.). Journal of Experimental Botany 52, 1209–1217. Oksman-Caldentey, K.M. & Saito, K. (2005) Integrating genomics and metabolomics for engineering plant metabolic pathways. Current Opinion in Biotechnology 16, 174–179. Parry, M.A.J., Madgwick, P.J., Bayon, C., et al. (2009) Mutation discovery for crop improvement. Journal of Experimental Botany 60, 2817–2825. Parry, M.L., Rosenzweig, C., Iglesias, A.I., et al. (2004) Effects of climate change on global food production under SRES emissions and socio-economic scenarios. Global Environmental Change 14, 53–67. Pascual, M.B., Jing, Z.P., Kirby, E.G., et al. (2008) Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin. Phytochemistry 69, 382–389. Peng, M., Hanamm, C., Gu, H., et al. (2007) A mutation in NLA, which encodes a RING-type ubiquitin ligase, disrupts the adaptability of Arabidospis to nitrogen limitation. The Plant Journal 50, 320–337. Pimentel, D., Hurd, L.E., Belloti, A.C., et al. (1973) Food production and the energy crisis. Science 182, 443–449. Pimentel, D., Williamson, S., Alexander, C.E., et al. (2008) Reducing energy inputs in the US food system. Human Ecology 36, 459–471. Piques, M., Shulze, W.X., Höhne, M., et al. (2009) Ribosome and transcript copy numbers, polysomes occupancy and enzyme dynamics in Arabidopsis. Molecular Systems Biology 5, 314. Plomion, C., Bahrman, N., Costa, P., et al. (2004) Proteomics for genetic and physiological studies in forest trees: application in maritime pine. In: Molecular Genetics and Breeding of Forest Trees (eds. S. Kumar & M. Fladung), pp. 53–79, Food Products Press, New York. Potel, F., Valadier, M.H., Ferrario-Mery, S., et al. (2009) Assimilation of excess ammonium into amino acids and nitrogen translocation in Arabidopsis thaliana—roles of glutamate synthases and car-

bamoylphosphate synthetase in leaves. The FEBS Journal 276, 4061–4076. Pourtau, N., Jennings, R., Pelzer, E., et al. (2006) Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta 224, 556–568. Prinsi, B., Negri, A.S., Pesaresi, P., et al. (2009) Evaluation of protein pattern changes in roots and leaves of Zea mays plants in response to nitrate availability by two-dimensional gel electrophoresis analysis. BMC Plant Biology 9, 113. Radrich, K., Tsuruoka, Y., Dobson, P., et al. (2010) Reconstruction of an in silico metabolic model of Arabidopsis thaliana through database integration. BMC Systems Biology 4, 114. Rajcan, I. & Tollenaar, M. (1999) Source: sink ratio and leaf senescence in maize. I. Dry matter accumulation and partitioning during grain filling. Field Crop Research 60, 245–253. Raun, W.R. & Johnson, G.V. (1999) Improving nitrogen use efficiency for cereal production. Agronomy Journal 91, 357–363. Robertson, G.P. & Vitousek, P.M. (2009) Nitrogen in agriculture: balancing the cost of an essential resource. Annual Review of Environment and Resources 34, 97–125. Rueda-Lopez, M., Crespillo, R., Canovas, F.M., et al. (2008) Differential regulation of two glutamine synthetase genes by a single Dof transcription factor. The Plant Journal 56, 73–85. Salvi, S. & Tuberosa, R. (2007) Cloning QTLs. In: Plants Genomics-Assisted Crop Improvement. Vol. 1: Genomics Approaches and Platforms (eds. R.K. Varshney & R. Tuberosa), pp. 207–225, Springer, Netherlands. Samborski, S.M., Tremblay, N., & Fallon, E. (2009) Strategies to make use of plant sensors-based diagnostic information for nitrogen recommendations. Agronomy Journal 101, 800–816. Scheible, W.R., Gonzalez-Fontes, A., Lauerer, M., et al. (1997) Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. The Plant Cell 9, 783–798. Schnable, P.S., Ware, D., Fulton, R.S., et al. (2009) The B73 maize genome: complexity, diversity and dynamics. Science 326, 1112–1115. Seger, M., Ortega, J.L., Bagga, S., et al. (2009) Repercussion of mesophyll-specific overexpression of a soybean cytosolic glutamine synthetase gene in alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.). Plant Science 176, 119–129. Semenov, M. & Halford, N.G. (2009) Identifying target traits and molecular mechanisms for wheat breeding under changing climate. Journal of Experimental Botany 60, 2791–2804.

THE MOLECULAR GENETICS OF NITROGEN USE EFFICIENCY IN CROPS

Senapati, S., Mahon, A.H., Gordon, J., et al. (2009) Rapid on-chip genetic detection microfluidic platform for real world applications. Biomicrofluidics 3, 022407. doi: 1063/1.3127142. Shlomi, T., Cabili, M.N., Herrgard, M.J., et al. (2008) Network based prediction of human tissue specific metabolism. Nature Biotechnology 26, 2003– 2010. Shrawat, A.K., Carroll, R.T., DePauw, M., et al. (2008) Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase. Plant Biotechnology Journal 6, 722–732. Sims, R.E.H., Hastings, A., Schlamindler, B., et al. (2006) Energy crops: current status and future prospects. Global Change Biology 12, 2054–2076. Stitt, M. & Fernie, A.R. (2003) From measurements of metabolites to metabolomics: an “on the fly” perspective illustrated by recent studies of carbon-nitrogen interactions. Current Opinion in Biotechnology 14, 136–144. Stitt, M., Müller, C., Matt, P., et al. (2002) Steps towards an integrated view of nitrogen metabolism. Journal of Experimental Botany 53, 959–970. Stitt, M., Sulpice, R., & Gibon, Y. (2009) System and method for producing weighed portions of powder from at least one biological material at cryotemperatures. Patent application 07118640. Sweetlove, L.J., Fell, D.A., & Fernie, A.R. (2008) Getting to grips with the plant metabolic network. The Biochemical Journal 409, 27–41. Tabuchi, M., Sugiyama, T., Ishiyama, K., et al. (2005) Severe reduction in growth and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase 1;1. The Plant Journal 42, 641–655. Tabuchi, M., Abiko, T., & Yamaya, T. (2007) Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.). Journal of Experimental Botany 58, 2319–2327. Takeda, S. & Matsuoka, M. (2009) Genetic approaches to crop improvement: responding to environmental and population changes. Nature Reviews. Genetics 9, 444–457. Thompson, L. (1994) The spatiotemporal effect of nitrogen and litter on the population dynamics of Arabidopsis thaliana. Journal of Ecology 82, 63–68. Tilman, D., Cassman, K.G., Matson, P.A., et al. (2002) Agricultural sustainability and intensive production practices. Nature 418, 671–677. Tsujimoto-Inui, Y., Naito, Y., Sakurai, N., et al. (2009) Functional genomics of the Dof transcription factor family genes in suspension-cultured cells of Arabidopsis thaliana. Plant Biotechnology 26, 15–28.

163

Tuberosa, R. & Salvi, S. (2006) Genomic based approaches to improve drought tolerance of crops. Trends in Plant Science 11, 405–412. Uauy, C., Distelfeld, A., Fahima, T., et al. (2006) A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 24, 1298–1301. Usadel, B., Nagel, A., Thimm, O., et al. (2005) Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes, and comparison with known responses. Plant Physiology 138, 1195–1204. Usadel, B., Poree, F., Nagel, A., et al. (2009) A guide to using MapMan to visualize and compare Omics data in plants: a case study in the crop species, Maize. Plant, Cell & Environment 32, 1211–1229. Vidal, E.A. & Gutiérrez, R.A. (2008) A systems view of nitrogen nutrient and metabolite responses in Arabidopsis. Current Opinion in Plant Biology 11, 521–529. Vitousek, P.M., Aber, J., Howarth, R.W., et al. (1997) Human alteration of the global nitrogen cycle: sources and consequences. Ecological Applications 7, 737–750. Walker, D.A. (2010) Biofuels-for better or worse? The Annals of Applied Biology 156, 319–327. Wang, R., Okamoto, M., Xin, X., et al. (2003) Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiology 132, 556–567. Wang, L., Zhong, M., Li, X., et al. (2008) The QTL controlling amino acid content in grains of rice (Oryza sativa) are co-localized with the regions involved in the amino acid metabolism pathway. Molecular Breeding 21, 127–137. Wang, R., Xing, X.X., Wang, Y., et al. (2009) A genetic screen for nitrate regulatory mutants captures the nitrate transporter gene NRT1.1. Plant Physiology 151, 472–478. Xie, H.L., JI, H.Q., Liu, Z.H., et al. (2009) Genetic basis for nutritional content of stover in maize under low nitrogen conditions. Euphytica 165, 485–493. Xu, Y. (1997) Quantitative trait loci: separating, pyramiding, and cloning. Plant Breeding Reviews 15, 85–139. Xu, Y. & Crouch, J.H. (2008) Marker-assisted selection in plant breeding: from publication to practice. Crop Science 48, 391–407. Yamasaki, M., Wright, S.I., & McMullen, M.D. (2007) Genomic screening for artificial selection during domestication and improvement in maize. Annals of Botany 100, 967–973.

164

NUTRIENT USE EFFICIENCY IN CROPS

Yamaya, T., Obara, M., Nakajima, H., et al. (2002) Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice. Journal of Experimental Botany 53, 917–925. Yanagisawa, S., Akiyama, A., Kisaka, H., et al. (2004) Metabolic engineering with Dof1 transcription factor in plants: improved nitrogen assimilation and growth under low-nitrogen conditions. Proceeding of the National Academy of Science of the United States of America 101, 7833–7838. Yano, M. & Tuberosa, R. (2009) Genome studies and molecular genetics-from sequence to crops: genomics comes of age. Current Opinion in Plant Biology 12, 103–106. Yi, G., Luth, D., Goodman, T.D., et al. (2009) Highthroughput linkage analysis of Mutator insertion sites in maize. The Plant Journal 58, 883–892. Yu, J. & Buckler, E.S. (2006) Genetic association mapping and genome organization of maize. Current Opinion in Biotechnology 17, 155–160.

Yu, J., Holand, J.B., McMullen, M.D., et al. (2008) Genetic design and statistical power of nested } association mapping in maize. Genetics 178, 539–551. Zahran, H.M. (2009) Enhancement of Rhizobialegumes symbioses and nitrogen fixation for crop productivity improvement. In: Microbial Strategies for Crop Improvement (eds. M. SaghirKhan, A. Zaidi & J. Musarrat), pp. 227–254, Springer, Berlin, Heidelberg. Zhang, Q.F. (2007) Strategies for developing green super rice. Proceeding of the National Academy of Science of the United States of America 104, 16402–16409. Zhou, Y., Cai, H.M., Xiao, J.H., et al. (2009) Overexpression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds. Theoretical and Applied Genetics 118, 1381–1390.

Chapter 9

Biotechnological Approaches to Improving Nitrogen Use Efficiency in Plants: Alanine Aminotransferase as a Case Study Allen G. Good and Perrin H. Beatty

Abstract The application of nitrogen-based fertilizers is essential to maintaining high crop yields, but it comes at a significant cost to both producers and the environment. Global use of nitrogen in 2007 amounted to 110 million metric tons has been projected to increase to between 125 and 236 million metric tons by the year 2050. While the ability of plants to capture nitrogen from the soil is dependent on a number of variables including crop, soil type, and the environment, in many cases up to 50–75% of nitrogen applied to agricultural lands is not used by the crop plants. As a result, there is considerable interest in decreasing fertilizer nitrogen inputs by improving plant nitrogen use efficiency (NUE) in crop plants, using both traditional breeding approaches and biotechnological approaches. This chapter reviews the recent biotechnological approaches that have been used to improving NUE in crop plants, focusing primarily on alanine aminotransferase (AlaAT) as a case study. Additionally, the experience with AlaAT overexpression is

used to highlight some of the key issues that future research in this area should take into account in designing different strategies to improve NUE.

Introduction It is becoming clear that the challenge of meeting the increased demand for food, feed, and fuel stocks (e.g., bioethanol) while reducing the environmental impact of agriculture will require the development of crops and cropping systems where the nutrient supply and the crops’ demand for that nutrient are in better synchrony (Good et al., 2004; Vitousek et al., 2009). There is no greater need for this than with nitrogen (N)based fertilizers. While the economics of improving nitrogen use efficiency (NUE) in crop plants has been highlighted by the sharp increase in nitrogen fertilizer costs in recent years, the environmental importance of improving NUE in crop plants cannot be understated. For example, in the United States, 89% of total nitrogen inputs into the

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 165

166

NUTRIENT USE EFFICIENCY IN CROPS

Mississippi come from agricultural runoff. Therefore, due to leaching, it has been estimated that the Mississippi River transports about 1.6 million metric tons of nitrogen each year from an estimated 21 million tons of nitrogen applied in the US cornbelt (7.6% for a current direct cost of $1.27 billion; US-EPA 2007). Environmentally, as a result of the massive increase in the amount of anthropogenic nitrogen introduced into the environment, there have been a number of significant negative consequences (Galloway et al., 2008). These effects include the impact on drinking-water supplies and the eutrophication of fresh water and marine ecosystems, including the proliferation of harmful algal blooms and “dead zones” in coastal marine ecosystems (Vitousek et al., 2009). Other consequences, such as the health impacts of nitrate in drinking water are less well understood (Powlson et al., 2006). In addition to these aquatic environmental costs, agriculture plays a substantial role in the imbalance of three of the most significant anthropogenic greenhouse gases (GHGs): CO2, N2O (nitrous oxide), and CH4 (methane). The global warming potential (GWP) of each of these gases can be expressed in CO2 equivalents: GWPs of N2O and of CH4 are 296 and 23 times greater, respectively, than a unit of CO2 (Denman et al., 2007). Among these gases, N2O may be the most environmentally damaging gas emitted by fertilizer use because of its large CO2 equivalent influence on global warming. Good et al. (2004) highlighted the need to recognize the different definitions of NUE and provided a starting point by discussing the definitions that have developed over the years (Steenbjerg and Jakobsen, 1963; Moll et al., 1982; Craswell and Godwin, 1984). These definitions differ in a few basic ways. First, the measurement of NUE can be based on either total biomass (Good et al., 2004; equations 1 and 2) or grain weight (Good

et al., 2004; equations 3, 5, 6, and 8). Second, the definitions often differ based on whether synthetic applied nitrogen only, synthetic and organic applied nitrogen, or total available soil nitrogen are being taken into account. Clearly, the appropriate way to estimate NUE depends on the crop and the respective harvested product. The definitions and equations that define NUE can also be partitioned, based on the physiological processes that are affected. For example, Uptake Efficiency (NUpE) looks at the efficiency of extracting nitrogen from the soil, whereas Utilization Efficiency (NUtE) looks at the ability to convert plant nitrogen into harvested biomass or grain yield (Fig. 9.1). Therefore, these equations reflect the efficiency with which applied nitrogen is used to produce either biomass or grain yield and can also be expanded to include additional factors including physiological ones (Masclaux et al., 2001; Rathke et al., 2006). From a researcher ’s perspective, the dissection of NUE into its components may bring focus to improving specific physiological processes such as uptake, assimilation, photorespiration, proteolysis, and remobilization of nitrogen storage proteins. For example, total plant nitrogen could be partitioned into acquired nitrogen and nitrogen loss due to processes such as photorespiration (Fig. 9.1). This would then allow researchers to focus on specific questions such as “What are the N losses that occur in crop plants as a result of photorespiration and other processes?” Whether finetuning the specific components that may affect NUE is realistic in terms of whole crop improvement remains to be seen; however, traditional engineering has shown this strategy to be fruitful. Moreover, in areas such as photosynthesis, the manipulation of a number of key steps may be required to achieve significant improvements in CO2 fixation (Zhu et al., 2007), suggesting that for complex metabolic traits, such as photosynthesis or NUE, there

BIOTECHNOLOGICAL APPROACHES

Available soil nitrogen (Ns)

Grain yield (Gw)

Plant nitrogen (Nt)

Photorespiration

NUE= Gw/Ns

Key shoot traits to modify: Nitrogen storage Nitrogen assimilation Nitrogen translocation Nitrogen remobilization Nitrogen utilization efficiency NUtE=Gw/Nt

Nitrogen uptake efficiency NUpE=Nt/Ns

Nitrogen uptake: NO3-, NH4+, organic nitrogen

167

Key root traits to modify: Nitrogen transporters Primary nitrogen assimilation Architecture

Nitrogen remobilization Nitrogen translocation Nitrogen assimilation

Fig. 9.1. The nitrogen cycle from soil to plant product. Nitrogen use efficiency (NUE) is determined by uptake efficiency (NUpE) and utilization efficiency (NUtE), which correspond to the amount of nitrogen taken up by the plant NUpE, Nt (total plant nitrogen)/Ns (total available soil nitrogen). Gw is grain yield or weight. NUtE, Gw/Nt key components (traits) that have been modified and should be evaluated in more detail are shown.

may well be a need to manipulate several steps to ultimately show significant improvements in the field. Improving crop NUE: Trying the simple solutions first The most rapid way in which nitrogen losses in agriculture can be reduced is by developing better nutrient management practices (NMP) (Keeney, 1982; Goulding et al., 2008). These practices include coordinating fertilizer requirements to particular crops and soils, and where possible, the coordination of nitrogen applications with soil water status. While researchers may debate the merits of any one approach, they all need to encompass the different forms of nitrogen

(organic and synthetic fertilizers) and take into account the four Rs of nutrient stewardship: right source, right rate, right time, and right place (International Plant Nutrition Institute, 2009). Perhaps one of the most widely recognized tools for measuring nutrient inputs and losses is to use a farm gate nutrient budget, although a number of other alternative systems exist. Nutrient budgets quantify inputs and outputs over a time period for a particular farm and are often developed by government agencies to support producers in managing their nutrient inputs. As examples, the United Kingdom (PLANET; http://www.planet4farmers.co.uk) and the European Union (EU) have producer support systems in response to the EU Nitrates Directive (91/676/EEC). This

168

NUTRIENT USE EFFICIENCY IN CROPS

approach has the benefit of using relatively simple accounting procedures and userfriendly spread sheets to evaluate farm-level nutrient inputs and losses (Oenema et al., 2003). However, there is no doubt that these types of accounting procedures place a significant burden on the producer, who has to become knowledgeable about the process and implement all of the documentation required. What is clear from a number of studies is that, provided nitrogen application rates are kept in balance, nitrogen losses can be reduced to a minimum. There is also a clear need to increase the amount of agronomy research focusing on these questions, due to the huge economic implications of these decisions; however, it is beyond the scope of this chapter to address these issues. Parallel to using the most appropriate NMPs, the development of better systems to measure crop nutrient levels, improved slow release fertilizers, and nutrient-efficient crop varieties through either traditional or transgenic approaches are also important components to reducing nitrogen losses (Shrawat and Good, 2009). Traditional approaches: genetic variation for NUE While the purpose of this chapter is to review novel transgenic approaches to improving crop NUE, it would be remiss to not mention the possible improvements that could be made using more traditional plant breeding approaches. A number of groups have attempted to increase NUE via plant breeding in maize, wheat, rice and other crops using two basic approaches. The traditional breeding approach to crop NUE research has used low- and high-nitrogen input systems to select for genotypes that can produce high yield under low nitrogen inputs (OrtizMonasterio et al., 1997; Acreche and Slafer, 2009; Anbessa et al., 2009, 2010). The second approach requires the use of molecu-

lar markers, to identify and track different chromosomal regions that enhance NUE. This technique can also be used to map chromosomal regions associated with NUE in order to identify candidate genes for manipulation using transgenic approaches. These types of approaches in maize and rice have strengthened the case for focusing on candidate genes such as GS1 (Obara et al., 2001; Gallais and Hirel, 2004; Hirel et al., 2007; Chapter 8); however, when a gene affecting NUE is novel, it would be unlikely to be identified using this approach since the number of genes within the quantitative trait locus (QTL) regions that have been mapped is very large. While these breeding approaches have met with some success in developing germplasm that can produce higher yields under low nitrogen inputs, there is still the need for greater acceptance by producers of reducing nitrogen inputs, due to the perceived benefits of maintaining high fertilizer inputs to try and ensure high yields. What has been observed in maize, wheat, and barley has been an increase in NUE in the modern varieties compared with the older varieties. This is presumably because by the 1980s virtually all hybrid development companies used a standard set of conditions to evaluate their germplasm; thus, provided that nitrogen application rates were maintained at a constant level, any yield increase would also, by definition, increase crop NUE. NUE may be calculated as kg yield/ applied kg N. The difficulty with this definition is that it uses applied nitrogen, rather than soil available nitrogen (NUEs = kg yield/soil available nitrogen), and therefore it is often difficult to compare the relative improvements in NUE in crops plants, both over time and between one crop species and another. Another measure, partial factor productivity (PFPN), which is the same as NUE, has increased for maize

BIOTECHNOLOGICAL APPROACHES

from 42 kg yield/applied kg N in 1980 to 57 kg yield/applied kg N in 2000 (an increase of 36%). A number of the chapters in Mosier et al. (2004) provide examples of similar increases in PFPN in barley, wheat, and rice, and for different cropping systems. A similar increase in NUE from 31 kg yield/ applied kg N to 49 kg yield/applied kg N (an increase of 58%) was observed for rice in experimental research plots versus producer trials. Similarly, Abeledo et al. (2008) reported a 29% increase in NUE between old barley varieties (1944; NUE of 38.8 kg yield/applied kg N) and new varieties (1998; NUE of 50 kg yield/applied kg N). Therefore, it seems reasonable that increases in NUE of 30–40% should be achievable, using traditional genetics and breeding. In comparison, improvements in NUE of 20–60% have been reported using targeted transgenic approaches; however, whether these increases will hold up in the field remains to be determined (Brauer and Shelp, 2010). The observation that modern varieties are more nitrogen efficient regardless of nitrogen input suggests that they are intrinsically better at many physiological processes (Moose and Below, 2009). It should be noted that the “Green Revolution” was in a large part based on improving NUE through interaction between “dwarfing genes” and the increased use of nitrogen fertilizers (the dwarfing genes allowed wheat and rice to have much higher harvest indexes without lodging, due to reduced height and stronger straw strength). Thus, the Green Revolution allowed for higher yielding varieties, which were able to benefit from high nitrogen fertilizer regimes. Biofuel crops For certain crops, the value of the crop is based on certain seed characteristics such as storage proteins or enzyme profile, as is the case for wheat (bread-making flour) or

169

barley (grown for malting). However, for biofuel crops or livestock feed crops such as barley, where the key factor is simply available energy, the key yield product is available carbon. Therefore, one way to increase NUE significantly would be to modify the C : N ratio. Plants and seeds that have a high C : N ratio should be able to produce more biomass per unit of applied nitrogen. In these cases, breeders and geneticists may be able to improve crop NUE by focusing on a high C : N ratio crop, much in the same way that plant biotech companies are now focusing on specific maize varieties where the amount of ethanol produced (yield) is determined in part by the ability to mobilize other available sugars more readily (Urbanchuk et al., 2009). Therefore, if one could develop a variety with double the C : N ratio, then for each kilogram of applied nitrogen, there would be a corresponding increase in total yield, if that yield was based on total available carbon. Novel transgenic approaches: targets of genetic improvements Although it is known that cereal varieties differ in their NUE and that this has a genetic component, little is known about the specific genes that control a crop’s response to nitrogen. The variety of transgenic approaches that have been attempted by researchers can be divided into a number of distinct physiological processes for the purpose of genetically dissecting NUE (Fig. 9.1). These include the transport of nitrogen into the plant, the primary assimilation of nitrogen via GS/GOGAT, secondary assimilation of nitrogen via aminotransferases, dehydrogenases, and other enzymes, nitrogen signaling, translocation of nitrogen throughout the plant, and nitrogen storage and remobilization from source to sink organs. Physical processes that affect plant architecture may also be involved in NUE. We will not discuss

170

NUTRIENT USE EFFICIENCY IN CROPS

improvements or research conducted on nitrogen transport into the plant or primary assimilation by GS/GOGAT since they are well covered in other chapters in this book (See Figs. 9.1 and 9.2).

Secondary assimilation of nitrogen (past GS/GOGAT) There are many examples in the literature of manipulation of nitrogen assimilation genes that have resulted in changes in biomass, or increased levels of key nitrogen metabolites like glutamic acid (Glu), glutamine (Gln), and other amino acids. However, most of these studies have not specifically measured NUE, nitrogen uptake, or nitrogen utilization efficiency. Many amino acid synthesis enzymes have been overexpressed in a variety of plants including aspartate aminotransferase (AspAT) in tobacco, Brassica napus, rice, and Arabidopsis; asparagine synthetase (AS) in tobacco, Arabidopsis, Lactuca sativa, and B. napus; and alanine aminotransferase (AlaAT) in B. napus, rice, and Arabidopsis (Table 9.1). Overexpression of AspAT using a CaMV35S promoter resulted in increased phosphoenolpyruvate carboxylase (PEPC) activity in tobacco (Sentoku et al., 2000), an increase in Glu, asparagine (Asn), alanine (Ala), and glycine (Gly) concentrations in Arabidopsis (Murooka et al., 2002), and increased leaf AspAT activity plus greater seed amino acid concentrations and protein content in rice (Zhou et al., 2009). Although none of these studies analyzed NUE, these results do suggest that carbon and nitrogen metabolism, respectively, have been modified. However, when we overexpressed AspAT in B. napus, using both a constitutive (CaMV35S) and tissue-specific promoter (btg26), there was no effect on plant phenotype, biomass, or seed yield (Wolansky, 2005).

The basic biochemistry related to C : N ratios and the fact that asparagine is one of the major nitrogen transport and storage molecules in many plants make AS a logical target for NUE improvement. Overexpression of a bacterial AS in L. sativa with a chimeric promoter resulted in transgenic plants with higher leaf numbers, larger surface area, higher mass, and increased chlorophyll, Asn, aspartic acid (Asp), and Gln concentrations, when provided with sufficient N (Giannino et al., 2008). This bacterial AS gene was constitutively overexpressed in B napus: When the transgenic canola was grown in low nitrogen conditions, the plants had an increased nitrogen content but lower seed yield, while in high nitrogen conditions the transgenic plants had a higher nitrogen harvest index (Seiffert et al., 2004). Transgenic AS overexpressing tobacco plants showed a 10- to 100-fold increase in free Asn in the leaves (Brears et al., 1993). The seeds from Arabidopsis constitutively overexpressing asn1 were found to have double the soluble seed protein content and an increase in total seed protein content of up to 21%, and,seedlings were more tolerant to low nitrogen availability. In addition, these AS overexpressing transgenic Arabidopsis plants showed an increase in Asn transport from source to sink tissues resulting in higher free Asn concentration in the flowers and silique (Lam et al., 2003). The AS gene asn2 was also overexpressed in Arabidopsis, increasing Asn levels under normal nutrient conditions (Igarashi et al., 2009). Since all of these studies used the CaMV35S promoter, with the exception of Giannino et al. (2008), it would be interesting to place the expression of AS under the control of a different promoter and analyze the resulting phenotype for NUE. One of the other nitrogen assimilation genes whose expression has been modified is Lycopersicon esculentum, tobacco, and maize is glutamate dehydrogenase (GDH).

Fig. 9.2. The role of AlaAT in carbon and nitrogen metabolism in plant cells. The lower box represents a root cell,

where nitrogen must be taken up, assimilated, and then transported to the shoots for metabolism and growth. The upper box illustrates a basic C3 leaf cell, where nitrogen is being used for a variety of growth processes and is then remobilized and transported to the developing grain.

171

172 Gene Product

Asparagine synthetase

Asparagine synthetase

Asparagine synthetase

ASN1

asnA

AsnA

E. coli

E. coli

A. thaliana

Pisum sativum

H. vulgare

Alanine aminotransferase Asparagine synthetase AS1 minus gln binding domain.

alaAT

AS1 AS1Δgln

H. vulgare

Alanine aminotransferase

alaAT

Hordeum vulgare

Gene Source

CaMV35S

pMAC

CaMV35S

CaMV35S

CaMV35S

OsAnt1

btg26

Promoter

B. napus

Lactuca sativa

A. thaliana

Arabidopsis thaliana Nicotiana tabacum

Oryza sativa

Brassica napus

Target Plant

No significant increase in growth, 10- to 100-fold higher levels of free asparagine Enhanced seeds protein, nitrogen limitation tolerance in seedlings Improved vegetative growth and enhanced nitrogen status. Increased nitrogen content and reduced seed yield at limited nitrogen, higher seed nitrogen yield, and improved nitrogen harvest index at high nitrogen.

Increased biomass and seed yield both in laboratory and field under low nitrogen Increased biomass and seed yield in laboratory conditions No visible phenotype observed

Phenotype Observed

Seiffert et al., 2004

Giannino et al., 2008

Lam et al., 2003

Miyashita et al., 2007 Brears et al., 1993

Shrawat et al., 2008

Good et al., 2007

Reference

Transgenic approaches to improving nitrogen use efficiency in plants. References within a single box indicate the same gene construct was being evaluated

Aminotransferases and dehydrogenases Alanine alaAT aminotransferase

Gene

Table 9.1.

173

Aspergillus nidulans

NADP-dependent glutamate dehydrogenase NADP-dependent glutamate dehydrogenase Glutamate dehydrogenase

gdhA

gdhA

GDH

gdh1

NADP–Glutamate dehydrogenase

Glycine max

Aspartate aminotransferase

aspAT

aspAT

E. coli

E. coli

L. esculentum

Panicum miliaceum Medicago sativa 3 rice genes, 1 E. coli gene

Aspartate aminotransferase Aspartate aminotransferase Aspartate aminotransferase

aspAT

aspAT

A. thaliana

Asparagine synthetase

ASN2

Gene Source

Gene Product

Gene

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

btg26

CaMV35S

CaMV35S

Promoter

Zea mays

N. tabacum

L. esculentum

Lycopersicon esculentum

A. thaliana

O. sativa

B. napus

N. tabacum

A. thaliana

Target Plant

Asn content increased under normal nutrient conditions. Increased AspAT activity, PEPC activity. Increased AspAT activity, no visible phenotype. Increased AspAT activity in leaves and greater seed amino acid and protein content. Increased AspAT activity in leaves and greater seed amino acid and protein content. Two- to threefold higher levels of free amino acids including Glu. 2.1- to 2.3-fold higher levels of free amino acids including glu Increased biomass and dry weight, increased yield in the field. Increased ammonium assimilation. Higher water potential during water deficit. Increased germination and grain biomass production in the field under water deficit

Phenotype Observed

(Continued)

Lightfoot et al., 2007

Ameziane et al., 2000 Mungur et al., 2005, 2006

Kisaka et al., 2007

Kisaka and Kida, 2003

Murooka et al., 2002

Igarashi et al., 2009 Sentoku et al., 2000 Wolansky, 2005 Zhou et al., 2009

Reference

174 Cytokinin biosynthesis

Cytokinin biosynthesis Cytokinin biosynthesis Ferredoxin NADP + reductase

IPT

IPT

Mitochondrial membrane protein

Hexose transporter

Amino acid permease

OsENOD93-1

STP-13

VfAAP1

FdNADP + reductase

IPT

Cytokinin biosynthesis

IPT

Vicia faba

A. thaliana

O. sativa

Maize

Agrobacterium

Agrobacterium

Agrobacterium

Agrobacterium

Agrobacterium

Cytokinin biosynthesis

Gene Source

IPT*

Gene Product O. sativa

(Continued)

Translocation and senescence-related proteins CKX2 mutation Cytokinin oxidase

Gene

Table 9.1

LeB4

CaMV35S

Ubi1

Ubiquitin

AtSAG12

AtSAG12

AtSAG12

Vicilin

PSEE1

Unknown

Promoter

Vicia narbonensis and pea

A. thaliana

O. sativa

Maize, soybean, rice

Arabidopsis

L. sativa

N. tabacum

N. tabacum

Zea mays

O. sativa

Target Plant

Higher concentration of total amino acids and total nitrogen in roots, increased dry biomass and seed yield. Improved growth, higher biomass and nitrogen use when provided with exogenous sugar. Seed size increased by 20–30%, increase in relative abundance of Asn, Asp, Glu, and Gln in the seed, higher seed storage protein content.

Delayed bolting and flowering, delayed leaf senescence. More biomass and seed yield, higher flood tolerance. Enhanced root growth, ear size, seed weight.

More panicles and a 23–34% increase in grain numbers Delayed senescence (stay-green) when grown in low soil nitrogen Larger embryo and seed, higher seed protein content, increased seedling growth. Delayed leaf senescence, increase in biomass.

Phenotype Observed

Rolletschek et al., 2005

Schofield et al., 2009

Gan and Amasino, 1995; Jordi et al., 2000 McCabe et al., 2001 Huynh et al., 2005 US 7,589,257 Hershey et al., 2009 Bi et al., 2009

Ma et al., 2002

Ashikari et al., 2005 Robson et al., 2004

Reference

175

MADS box gene-rat glucocorticoid receptor Transcription factor PII regulatory protein

ANR1-rGR

Dof1

GLB1

N. tabacum

Rubisco

CaMV35S

CaMV35SS

CaMV35S

C4PPDK35S

N. tabacum

Solanum tuberosum

A. thaliana

A. thaliana

A. thaliana

A. thaliana

A. thaliana

Target Plant

Larger concentrations of malate, Glu, Gln, Asp, Thr, Ala, Gly, and Val. Slower growth rate and transgenic plants showed relief from nitrogen limitation Total nitrogen (total nitrogen/ total mass) increased. Increase in vacuolar nitrate.

Significantly more lateral root growth after plants were treated with synthetic steroid dexamethasone. Enhanced growth rate under nitrogen-limiting conditions. Increased anthocyanin production under low nitrogen conditions

Reduced growth rate, impairs calcium utilization and sensitivity to ionic stress in transgenic plants. Lateral root induction and elongation.

Phenotype Observed

Masle et al., 1993

Rademacher et al., 2002

Yanagisawa et al., 2004 Hsieh et al., 1998

Zhang and Forde, 1998 Filleur et al., 2005

Kim et al., 2001

Reference

*A detailed discussion of genetic modification of cytokinin genes is presented in Ma (2008). Only those genetic modifications in either Arabidopsis or crop plants are listed in this table. AtSAG12, Arabidopsis thaliana promoter specific to senescing leaves; btg26, canola root specific promoter; C4PPDK 35S, derivative of the 35S promoter; CaMV35S, cauliflower mosaic virus 35S promoter; LeB4, Vicia faba seed storage protein legumin B4 promoter; OsAnt1, Oryza sativa antiquitin 1 promoter; pMAC, prokaryotic chimeric 35S/MAS promoter; PSEE1, senescence enhanced promoter from maize; Ubi1, maize ubiquitin 1 promoter; Vicilin, Pisum sativum 7S seed storage protein vicilin.

Rubisco small subunit antisense gene

Solanum tuberosum and Flaveria trinervia

C/N metabolism related proteins ppc modified C3 potato PEPC with a C4 F. trinervia PEPC domain cannot be phosphorylated

A. thaliana

Zea mays

A. thaliana Rat

CaMV35S

CaMV35S

A. thaliana

MADS transcription factor

Promoter

ANR1

Gene Source CaMV35S

Gene Product

Signaling proteins involved in the regulation of nitrogen metabolism AtGluR2 Glutamate receptor A. thaliana

Gene

176

NUTRIENT USE EFFICIENCY IN CROPS

The proposed role of GDH in amino acid metabolism also indicates the potential involvement of GDH in nitrogen remobilization during senescence (Miyashita and Good, 2008). In fact, induction of GDH in senescing leaves has been documented in plants. During the physiological process of senescence, deamination catalyzed by GDH may be an important source of ammonia for reassimilation into nitrogen transport compounds such as Glu and Asn. This potential role of GDH during senescence is particularly interesting since cytosolic GS1, which is induced during senescence, has been shown to be a major component of grain production in maize. The coexpression of cytosolic GS1 and GDH during senescence could provide an efficient pathway for the synthesis of nitrogen transport compounds from the free amino acids derived from proteolysis. Maize plants constitutively overexpressing an Escherichia coli GDH (Lightfoot et al., 2007) showed grain biomass increases in field trials, even under stressful water deficit conditions. Similarly, constitutive expression of this bacterial GDH gene slightly increased tobacco biomass and total leaf amino acid concentration (20–30%) under both field and controlled conditions (Ameziane et al., 2000), as well as increasing ammonium assimilation, metabolic ion abundance in roots and shoots, respectively, and water potential during water deficit conditions (Mungur et al., 2005, 2006). An NADP-dependent gdh gene from either Aspergillus nidulans or tomato has been constitutively overexpressed in tomato plants. The fruit from the transgenic plants showed higher levels of free amino acids, and especially Glu (Kisaka and Kida, 2003; Kisaka et al., 2007). However, in most of these studies, detailed physiological analysis of the impact of the introduced transgene on both nitrogen and carbon assimilation was not thoroughly investigated.

Another gene that has been manipulated resulting in altered secondary nitrogen assimilation is the hexose transporter gene (STP-13) in Arabidopsis (Schofield et al., 2009). Hexose levels in Arabidopsis are regulated by the large monosaccharide transporter gene family (MST), of which STP13 is a member. Constitutively overexpressed STP13 seedlings grown with sufficient available nitrogen showed higher rates of glucose uptake and higher endogenous sucrose levels, accumulated more total carbon, and had more biomass per plant. As well, one of the high-affinity nitrate transporters, NRT2.2, was highly expressed in STP13 overexpressing seedlings, in both roots and shoots. The transgenic seedlings grown with limited nitrogen also showed higher biomass compared with control plants. By increasing the carbon availability in these transgenic Arabidopsis, the NUE was also improved. PEPC has also been constitutively overexpressed in a number of plants, resulting in increased concentrations of specific tricarboxylic cycle acids (TCA) such as malate, and key nitrogen amino acids such as Glu, Gln, and Asp, as well as threonine (Thr), Ala, Gly, and valine (Val). The transgenic potato plants had diminished plant growth with carbon flow being redirected from soluble sugars to organic and amino acids (Rademacher et al., 2002). Translocation of nitrogen throughout the plant Translocation of nitrogen throughout the plant during the vegetative and reproductive stages, and senescence is closely linked to the other physiological processes such as nitrogen uptake, nitrogen assimilation, and nitrogen storage, and remobilization (Lam et al., 1996). Once nitrogen is taken up by the plant by the roots, it is either translocated as inorganic or organic nitrogen to other

BIOTECHNOLOGICAL APPROACHES

tissues for assimilation, stored in vacuoles for future assimilation and translocation, or assimilated immediately and translocated as free amino acids, proteins, or nitrogencontaining compounds. Nitrogen in one form or another is translocated from the roots to the shoots, from older leaves to younger leaves, and from leaves and shoots to seeds. All of these steps would involve a number of membrane-associated proteins involved in translocation, enzymes involved in primary metabolism, and regulatory proteins involved throughout these processes. Recently, a rice mitochondrial membrane protein called OsENOD93-1, thought to play a role in the transport of compounds from root to shoot, was constitutively overexpressed in rice, resulting in higher seed yield and an accumulation of amino acids in roots and xylem sap (Bi et al., 2009). The function of this protein is still unknown; however, it may be speculated that it is involved in nitrogen translocation from roots to shoots. Cytokinin hormones play a part in many aspects of plant growth and development including the signaling involved in the translocation of nitrogen from source to sink organs during senescence (see, for example, Ashikari et al., 2005). Modification of cytokinin biosynthesis has been attempted in many different plants including corn, rice, lettuce, tobacco, and Arabidopsis. Corn plants expressing the Agrobacterium gene isopentenyl transferase (IPT) driven by a novel maize senescence-induced promoter lead to transgenic plants with increased levels of cytokinin within senescing tissues. This produced a “stay green” phenotype which equated to a delay in senescence, and hence, a delay in loss of photosynthetic capability, especially when grown in lownitrogen soil (Robson et al., 2004). The IPT gene driven by a senescence-associated promoter, SAG12, was also introduced into tobacco, lettuce, and Arabidopsis. IPT trans-

177

genic tobacco showed delayed leaf senescence as well as an increase in biomass, while the transgenic Arabidopsis showed higher biomass, higher seed yield, and an increase in flooding tolerance (Gan and Amasino, 1995; Jordi et al., 2000; Huynh et al., 2005). Transgenic lettuce also showed delayed leaf senescence coupled with delayed bolting and flowering (McCabe et al., 2001). The cytokinin-altered rice was engineered with a mutation in the rice gene ckx2 encoding a cytokinin oxidase/ dehydrogenase that functions in cytokinin degradation. By reducing or eliminating CKX2, cytokinin levels remain high in shoot tissues. In rice, this led to an increase in panicle number and grain. This phenotype has been seen in natural variation of high- to low-yielding rice varieties used in plant breeding studies (Takeda and Matsuoka, 2008), where the high-yielding variety has a null allele of ckx2, the next high-yielding variety has a low expressing ckx2 allele, and a standard yielding variety has a gain-offunction ckx2 allele. Ma (2008) provides a more detailed review of cytokinin transgenes; however, in the cases outlined in this chapter, none of them specifically reported on improved NUE. An amino acid transporter gene from Vicia faba, VfAAP1, was overexpressed with a seed specific promoter in both Vicia narbonenesis and pea plants resulting in an increased seed size by 20% to 30% and an increase in relative abundance of Asp, Asn, Glu, and Gln in the seed (Rolletschek et al., 2005). VfAAP1 over-expression increased the amino acid sink seed strength resulting in stimulation of storage protein synthesis and higher storage protein content within the seed. Nitrogen sensing, signaling, and physiological regulation Signaling molecules that are believed to be involved in N signal transduction include

178

NUTRIENT USE EFFICIENCY IN CROPS

inorganic nitrogen (e.g., nitrate) and organic nitrogen (e.g., Glu, Gln, and Asn). Nitrate levels have been found to regulate the expression of energy production, metabolism, glycolysis, and gluconeogenesis genes. Recently, a signal transduction protein called PII, known to be nearly ubiquitous within prokaryotes (Moorhead and Smith, 2003), has been identified in plastids of many different plant species. In photosynthetic bacteria, PII is bound with adenosine triphosphate (ATP) and 2-oxoglutarate (2-OG) and functions as a key regulatory step for nitrate/ nitrite uptake. In plants, PII may also play a key role in regulating nitrogen uptake and the C : N status in plants. Studies on PII from Arabidopsis show that it has a high affinity for binding ATP and then 2-OG. This binding may allow PII to be a sensitive sensor of carbon status within the chloroplast since the binding constant for 2-OG is very close to the estimated concentration of plastidlocated 2-OG (Moorhead and Smith, 2003). A PII protein from Arabidopsis was isolated and constitutively overexpressed in Arabidopsis, resulting in an increased growth rate and higher anthocyanin production under low nitrogen conditions (Hsieh et al., 1998). The importance of glutamate in nitrogen metabolism suggests that glutamate receptors may be of importance in nitrogen signal transduction. Sequencing of the Arabidopsis genome revealed 20 glutamate receptor homolog (AtGLR) genes, divided into three phylogenetically distinct subfamilies, AtGLR1, AtGLR2, and AtGLR3 (Lacombe et al., 2001; Chiu et al., 2002; Davenport, 2002). A study utilizing antisense suppression suggested that AtGLR1.1 plays a role in carbon/nitrogen metabolism, abscisic acid (ABA) biosynthesis, and the control of seed germination (Kang and Turano, 2003). Overexpression of AtGLR3.2 in Arabidopsis resulted in Ca2+-deficiency symptoms, and sensitivity to other ions. This phenotype

could be rescued by applying external Ca2+, suggesting that AtGLR3.2 may function in calcium translocation in plants (Kim et al., 2001). T-DNA insertions of the different glutamate receptors genes in Arabidopsis have been analyzed, but no novel phenotypes have been reported to date (Liu et al., 2007). A number of the transgenes that are currently being studied to increase abiotic stress tolerance are transcription factors, which often regulate the coordinated expression of enzymes involved in a metabolic pathway (Century et al., 2008). The concept is that if multiple genes provide the greatest improvement, then enhanced expression of key transcription factors would be capable of activating the particular set of required genes. Transgenic Arabidopsis lines overexpressing Dof1, a maize protein that belongs to the Dof family of plant-specific transcription factors known to activate the expression of several C-metabolizing genes associated with organic acid metabolism, showed up to 30% higher nitrogen content, higher levels of amino acids, and better growth under low nitrogen conditions (Yanagisawa et al., 2004). In spite of the large alteration in amounts of various metabolites, the C : N ratio was not changed in transgenic plants. Although this report suggests that it is possible to modify nitrogen assimilation in plants using a transcription factor, it will be necessary to investigate whether expressing the Dof1 transcription factor is really effective in improving nitrogen assimilation of crops in the field. One of the difficulties with the research on NUE to date has been that it has largely focused on single gene manipulations. This is only logical, given the number of different candidate genes; however, it may well be that a number of different genes/enzymes need to be manipulated to increase nitrogen uptake or efficiency. Currently, models of carbon metabolism using an “evolutionary”

BIOTECHNOLOGICAL APPROACHES

algorithm have been used to search for multiple alterations in carbon partitioning to increase photosynthetic rate (Zhu et al., 2007). Similarly, we need to develop better models of nitrogen metabolism that might allow us to identify key regulatory steps that may affect nitrogen uptake. Nitrogen storage and nitrogen remobilization from source to sink organs Depending on the developmental state, plants will store nitrogen under conditions of high nitrogen availability as nitrate or amino acids in vacuoles, or as storage proteins, to await future assimilation and transport to other tissues. Nitrogen is also stored in chlorophyll or abundant plant enzymes such as Rubisco, PEPC, and GS, to be released upon senescence. Still other nitrogen storage compounds, in the form of nonenzymatic proteins, may also be present. There have been few studies on the effect of nitrogen storage forms on NUpE and the consequences for NUE of plants. As nitrogen is a limiting factor for plant growth, the efficient reassimilation of metabolically generated ammonia is particularly important for plant productivity. After anthesis, crops generally experience a rapid exhaustion of nitrogen availability in soil and, therefore, grain filling tends to be directly supported by nitrogen remobilization/ recycling as shown for maize and wheat (Plénet and Lemaire, 1999). The ability of plants to accumulate “luxury” nitrogen during periods of large nitrogen supply and nitrogen remobilization, or the recycling of organic nitrogen to young development and storage organs, is closely linked to leaf senescence (Masclaux et al., 2001). Sink to source transition is usually characterized by drastic changes in the concentration of carbon and nitrogen metabolites and the induction of both cytosolic GS1 and NADH-

179

dependent GDH activity (Hirel and Lemaire, 2005). Cytosolic GS1 is induced during leaf senescence; therefore, it has been proposed as a key component of NUtE in plants (Miflin and Habash, 2001), and its metabolism is particularly important for nitrogen remobilization and recycling in plants. However, we will not be discussing the role of GS further, as it is addressed in other chapters of this book. Physical processes that affect plant and specifically root architecture As both organic and inorganic forms of nitrogen are initially sensed and taken up by roots, root architecture has been a key trait to analyze and modify in an attempt to improve NUE (Garnett et al., 2009; see also Chapter 2). There are two general aspects of root architecture that have been seen to play a role in nitrogen sensing and uptake: the exploratory root system (primary and lateral roots) and the root hair system (Leblanc et al., 2008). The various genetic and QTL studies that have been done on roots have provided us with some interesting genetic targets that could be modified for potential NUE improvement (for a review of roots and NUE, see Garnett et al., 2009). Many researchers have suggested that NUE could be improved by the development of a root system that is more efficient at nitrogen uptake. One method to assess the ability of the plant to take up nitrogen is to measure nitrogen uptake efficiency (NUpE = Nt/Ns). Two small-scale NUE studies, one on maize and the other barley, determined that at high applied nitrogen, the most nitrogenefficient varieties also displayed the highest NUpE, while at low applied nitrogen, NUtE appeared to be more responsible for the high NUE varieties (Moll et al., 1982; Beatty et al., 2010). Conversely, the opposite was found in wheat, where at low applied nitrogen, the high NUE varieties showed

180

NUTRIENT USE EFFICIENCY IN CROPS

high NUpE but not NUtE (Le Gouis et al., 2000). Greater root density (or root volume per unit area) can increase the root surface area and hence improve nitrogen uptake by the plant. High NUE wheat lines have shown faster root growth and higher root length densities than wheat lines with lower NUE (Liao et al., 2004, 2006). Maize plants with higher root length densities have shown greater NO3− uptake and, concomitantly, less leaching from the soil (Wiesler and Horst, 1993, 1994). External L-glutamate sensed at the primary root tip by an unknown receptor has been shown to slow primary root growth and stimulate lateral root branching in Arabidopsis, resulting in a greater root length density. In addition, external NO3− sensed at the Arabidopsis primary root tip by the phosphorylated dual affinity nitrate transporter NRT1.1 (operating in highaffinity mode) can antagonize this Glu inhibitory effect, resulting in stimulation of primary root growth (Walch-Liu and Forde, 2008). In gene knockout studies, Walch-Liu and Forde (2008) determined that the mutant NRT1.1, where the nitrate transporter lacked the ability to be phosphorylated, was still able to sense and transport NO3−, but was not able to counteract the Glu inhibition of primary root growth. In Arabidopsis, external NO3− sensed at the lateral root tip results in a localized stimulatory effect where meristematic activity is induced and the lateral root is elongated (Zhang and Forde, 1998). This elongation is induced via a signaling pathway that involves ANR1, a MADS box-related transcription factor. ANR1 has been both overexpressed and knocked out in Arabidopsis. These experiments suggest that the ANR1 gene is activated in lateral roots by NO3− and in turn stimulates lateral root growth until the nitrogen status of the plant is high, then a feedback mechanism kicks in to downregulate ANR1 and slow or stop further lateral root

growth (Zhang and Forde, 2000). ANR1 may be another genetic target to be modified to increase root density and ultimately NUE. Since root hairs have been estimated to make up 70–80% of the root surface, and increase in numbers under nutrient stress, presumably an increase in root hair numbers would enhance nitrogen uptake. The third way in which a plant could be modified to enhance NUE would be to increase the length of time that the root is actively taking up nitrogen, thereby allowing the plant to take up more total nitrogen over its total lifespan and not limited to the vegetative stage. Recently, a nitrogen uptake and nitrogen utilization study was carried out on subtropical maize hybrids growing in Zimbabwe and Kenya. The hybrids that showed an NUE phenotype while grown on low nitrogen soils were determined to be able to take up N during the flowering and grain filling developmental stages (Worku et al., 2007). The importance of understanding nitrogen and carbon metabolism It is well known that C and N metabolism are closely integrated, and while some key steps in the coordination of C : N metabolism are known (such as nitrate reductase [NR], PEPC, and the PII protein), many of the nutrient/metabolic signaling, receptor, and regulatory pathways still need to be determined (Coruzzi and Bush, 2001; Foyer et al., 2003). For example, Oaks (1994) demonstrated that when plants are grown under high levels of CO2, NUE increases (from 22% to 56%) with an increase in available nitrogen. Following the primary assimilation of nitrogen into glutamate, nitrogen is quickly distributed to other amino acids/ proteins, nucleotides, chlorophyll, polyamines, and alkaloids (Lam et al., 2003). The assimilation of nitrogen requires carbon skeletons such as 2-OG or oxaloacetate that are both TCA cycle intermediates, with the

BIOTECHNOLOGICAL APPROACHES

potential products being specific amino acids such as alanine or aspartate. Carbon and nitrogen metabolism is also crossregulated via signal transduction proteins such as the PII protein that senses the level of 2-OG. The activity of carbon metabolism enzymes such as PEPC and sucrose phosphate synthase is regulated by the nitrateinduced phosphorylation of these enzymes, leading to reallocation of the carbon skeletons to amino acid synthesis instead of sucrose synthesis (Schofield et al., 2009). At a whole-plant level, the uptake of nitrogen and the process of nitrate reduction is a very energy-intensive process, consuming ∼20% of the electrons produced by photosynthetic electron transport. For example, in the single cell alga Selenastrum minutum, the addition of NH4+ to anaerobic cells resulted in an approximately twofold increase in the rate of starch breakdown (Vanlerberghe and Turpin, 1990), indicating that this is an energetically expensive step. NH4+ addition also resulted in a large drop in Glu and an increase in Gln. Once Glu and Gln pools have reached a steady state level, the only amino acid accumulated is alanine (Vanlerberghe and Turpin, 1990). More specifically, Vanlerberghe et al. (1991) found that when S. minutum was nitrogen starved and under hypoxic conditions the addition of 15NH4+ resulted in 93% of the 15N taken up being incorporated into alanine. In contrast, under aerobic conditions, only 4% of the 15N was incorporated into alanine. Thus, the availability of carbon skeletons and the effect that it may have on the levels of specific amino acids may be a key factor affecting NUE. Second, the production of specific amino acids is often determined less by a “rational choice” based on the C : N ratio of the plant (e.g., asparagine) than by the availability of carbon skeletons. This is clearly the case for alanine and other amino acids, where changes in the availability of specific carbon skeletons (as influenced by imposing stresses

181

such as hypoxia) will cause a rapid change in the concentration of specific amino acids (Streeter and Thompson, 1972; Vanlerberghe and Turpin, 1990). Yanagisawa et al. (2004) showed the tight interaction between carbon and nitrogen metabolism when the Dof1 transcription factor was overexpressed in Arabidopsis, resulting in an increase in amino acid levels, especially Glu, along with the upregulation of the carbon metabolism enzymes PEPC, PK, citrate synthase, and isocitrate dehydrogenase. By overexpressing a transcription factor that induces or positively affects both nitrogen and carbon metabolism, the transgenic Dof1-OX Arabidopsis also exhibited improved growth under low-nitrogen growth conditions (Yanagisawa et al., 2004). At a whole-plant level, the interaction between nitrogen assimilation and the availability of carbon skeletons has also been demonstrated by manipulations of the C3 carbon fixation cycle (Raines, 2006). For example, nitrogen allocation or nitrogen use has been studied in plants that have been engineered to have modified levels of Rubisco (Masle et al., 1993; Makino et al., 2000). Masle et al. (1993) found that plants with reduced levels of Rubisco grew as fast at high levels of CO2 as the wild-type plants but at a lower organic nitrogen cost. However, in only a few cases have the researchers evaluated any of the components if NUE. The importance of carbon and nitrogen metabolism is also covered in other Chapters in this book. The role of alanine in plants In agricultural systems, plants take up nitrogen primarily in an inorganic form (NO3− or NH4+); however, in some ecosystems, organic forms of nitrogen, primarily in the form of amino acids, can be major sources of nitrogen (Näsholm et al., 1998). In fact, it is known that it is relatively easy for Arabidopsis

182

NUTRIENT USE EFFICIENCY IN CROPS

to grow using certain amino acids as their sole nitrogen source, such as alanine (Miyashita et al., 2007). While seldom considered to be an important amino acid for nitrogen transport or signaling, alanine is of particular interest for several reasons. First, as noted, alanine can be used as the sole source of nitrogen for plants, reinforcing that it is relatively easy to convert alanine to other nitrogen-containing compounds for growth and development. Second, Waters et al. (1998) have shown that alanine is excreted by nitrogen-fixing bacteria, followed by its assimilation into the plant root, suggesting it may have an important role in organic nitrogen metabolism within the nodule and roots of nitrogen-fixing plants. Both of these observations indicate that plants can take alanine up directly from the soil, or from soil bacteria, and use it as a nitrogen source. Third, alanine is produced in relatively large amounts in most plant roots exposed to hypoxic or anoxic conditions, although its role in flood tolerance has not yet been elucidated (Effer and Ranson, 1967; Streeter and Thompson, 1972; Good and Crosby, 1989). However, alanine can be used as the major storage amino acid under certain stresses, (e.g., flooding), perhaps because pyruvate is the only readily available carbon skeleton under anaerobic conditions (Vanlerberghe and Turpin, 1990). Finally, alanine is an endproduct amino acid so that any nitrogen stored as the amino group of alanine can be catalyzed to produce glutamate and pyruvate. In fact, one of the roles of alanine may be to act as a nitrogen storage compound under hypoxic conditions, with the nitrogen being easily remobilized upon the return to normoxic conditions (Miyashita et al., 2007). Thus, alanine can be used in transport and nitrogen storage, but its relative importance presumably varies between species and by the availability of specific carbon skeletons in the form of keto acids.

AlaAT as a gene and enzyme Recent research on alanine aminotransferase arises from an interest in AlaAT as an enzyme induced under anaerobic conditions (Good and Crosby, 1989). AlaAT is ubiquitous in prokaryotic and eukaryotic cells, and in plants alanine appears to be produced solely by AlaAT. This aminotransferase is involved in nitrogen uptake and transport, protein synthesis, and as a transport compound for the movement of fixed carbon between the mesophyll and bundle sheath cells in C4 plants (Hatch, 1987). In addition to the synthesis of alanine and 2-OG, the catalysis of alanine results in the production of glutamate and pyruvate (Fig. 9.2). The early work on AlaAT was performed by researchers interested in its possible role in the malate shuttle in C4 plants. Hatch completed some of the early characterization of the activity, location, and possible role of AlaAT in plants (Hatch, 1987) which is recounted in a personal history (Hatch, 1992). He and others demonstrated that different AlaAT isozymes were associated with the mesophyll and bundle sheath cells in several C4 pathway plants. Panicum miliaceum (broomcorn millet), an NAD-malic enzyme-type C4 plant, has three isoforms of AlaAT in leaf tissue of which AlaAT-2 is the major isoform, and the AlaAT-2 mRNA and enzyme activity levels both increase in leaf tissue during greening (Son et al., 1992). It has also been shown that AlaAT activity, protein and mRNA levels increase in leaf tissue during the recovery from nitrogen stress in a number of species (Son et al., 1992; Son and Sugiyama, 1992). In barley (a C3 plant), AlaAT-2 is hypoxically induced in root tissue for up to 5 days, with the increase in AlaAT activity correlated to an increase in steady-state mRNA and protein levels (Good and Muench, 1993; Muench and Good, 1994). While some of the amino-

BIOTECHNOLOGICAL APPROACHES

transferases can utilize a variety of amino or keto acids as substrates, the extent to which AlaAT can do so is not clear (Good and Crosby, 1989). Although barley and millet have different types of photosynthesis (a C3 plant [barley] versus an NAD-malic enzymetype C4 plant [millet]), the major isoform of AlaAT is very similar at the DNA and protein level, and appears to be expressed the same in way in both roots and leaves and may have similar physiological roles in these species. From a molecular perspective, the hypoxically inducible barley AlaAT was cloned after purifying the AlaAT protein to homogeneity (Good and Muench, 1992) and using the peptide sequence data to design polymerase chain reaction (PCR) primers, which were then used as probes to clone the cDNA from barley (Hordeum vulgare) (Muench and Good, 1994). At the same time, a Japanese group cloned an AlaAT cDNA from Broomcorn millet (P. miliaceum) (Son and Sugiyama, 1992). Subsequently, a wide variety of AlaATs have been reported by different EST sequencing projects. The differences between a number of AlaAT genes across a wide range of Kingdoms have been evaluated by comparing DNA and protein sequences of the different AlaAT genes. Based on sequence analysis, 10 AlaATs were selected for cloning into an expression vector and were shown to have kinetic properties that differed significantly across Kingdoms (McAllister, unpublished). Whether these differences in enzyme kinetics have any functional significance, or whether they can be utilized to attempt novel approaches to modifying nitrogen metabolism remains to be seen. The development of NUE plants While initial interest in AlaAT was based on its inducibility, both enzymatically and tran-

183

scriptionally, the interest in AlaAT as a target for improving NUE was triggered by two serendipitous events and the observations that these triggered. Initially, the cytoplasmic barley AlaAT cDNA (Muench and Good, 1994) was introduced into B. napus (canola) under the control of the btg26 promoter and observed increases in biomass and seed yield when the transgenic plants were grown under low nitrogen conditions. As noted (Good et al., 2007), “In the course of experiments to test the ability of alanine to function as a compatible solute, we noted that transgenic plants expressing AlaAT under the control of a stress inducible promoter displayed a nitrogen efficient phenotype.” The two serendipitous events that allowed the observation of the phenotype were, first, that the transgenic and control plants were inadvertently grown under a low nitrogen regime, and second, that the selection of the promoter (btg26) turned out to be of importance, despite the fact that little was known about the promoter prior to the original observation of NUE (Good et al., 2007). The use of a btg26/GUS reporter construct indicated that AlaAT appeared to be under the control of a root-specific promoter (Stroeher et al., 1995; Good et al., 2007). These results were particularly interesting due to the requirement for a tissue-specific, nonconstitutive promoter to express the phenotype and because the phenotype is only displayed under reduced nitrogen conditions. Comparison of three different promoter/AlaAT fusion genes indicated that it was only with the btg26 promoter, that the phenotype was observed. The NUE phenotype was observed under hydroponic and growth chamber conditions and in the field. Based on an analysis of the concentrations of metabolites in the different genotypes, it was suggested that the increased biomass and seed yield in canola overexpressing barley AlaAT could be attributed to increased

184

NUTRIENT USE EFFICIENCY IN CROPS

Ala accumulation and mobilization (Good et al., 2007). The yield improvements were observed under low (1 mM N), but not high nitrogen (10 mM N) conditions in the lab, and were attributed to increased nitrate influx, which may have been induced by the decreased Gln and Glu concentrations in the shoot. Under field conditions, yield was maintained in the transgenic lines even with a 40% decrease in nitrogen application. The same enzyme, overexpressed under a different tissue-specific promoter in rice, increased shoot biomass, shoot-nitrogen concentration (by 12%), and spikelet yield under adequate concentrations of nitrate and ammonia (Shrawat et al., 2008). In contrast to the canola study, Gln, Glu, and Asn levels in both roots and shoots were increased. While overexpression of AlaAT significantly increased yield and NUpE of both canola and rice, the mechanisms involved appear to be somewhat different, with canola displaying a higher NUE due to changes in shoot dry mass (SDM) while rice displaying both increased SDM and shoot-nitrogen concentrations (Good et al., 2007; Shrawat et al., 2008; Beatty et al., 2009). The intellectual property associated with this invention, the “Brassica patent” as it became known (US Patent #6,084,153), was licensed to Arcadia Bioscience, which conducted field trials over a number of years. The initial field trials were conducted in 2003– 2004 at Brawley, Califonia, in irrigated, sandy tiled soils, where significant levels of nitrogen fertilizer was applied (up to 280 lbs/ acre), and were reported in Good et al. (2007). Arcadia Bioscience has also published data on their field trials conducted in North Dakota and Minnesota (Strange et al., 2008). Subsequent to that, Arcadia and Monsanto also conducted confined field trials on this material, or material based on this genetic construct, in 2004, 2005, 2008, and 2009; however to date there is no indication that there will be a commercial release

of a canola variety with this trait incorporated, in the immediate future. AlaAT as an example of genetic modification The questions that are frequently asked in relation to this research are: “Why choose AlaAT as the gene target?” and “Why does this gene work to increase NUE, while (what appear to be more logical gene choices) nitrate transporters or NR have failed to alter NUE in any plant system studied to date?” The serendipitous events that lead to the discovery of our NUE phenotype have been described, and there are three possible reasons why overexpression of AlaAT has proven to be a successful candidate gene to overexpress. First, alanine synthesis is directly linked both to the major pathway of nitrogen assimilation (the GS/GOGAT cycle) and to carbon sinks in the plant. Given the fact that pyruvate is a readily available source of carbon, it is relatively easy to see how an increase in available nitrogen could be immediately used to produce higher levels of alanine. Second, AlaAT itself is a relatively simple enzyme, and there do not appear to be any protein modifications that regulate the activity of the enzyme. This is in contrast to enzymes such as nitrate reductase, which is regulated posttranslationally by phosphorylation/ dephosphorylation (in part affected by carbohydrate supply) and reversible binding of a 14-3-3 protein inhibitor (Foyer et al., 2003). Therefore, it may be that choosing gene targets should be based, in part, on the complexity of the regulation of the enzyme. Third, based on the unexpected observation of Waters et al. (1998), alanine may have an important, but as of yet unknown role, in organic nitrogen metabolism. The question that is almost always asked is: “What is the basis for the NUE phenotype?” For both Brassica and rice, the AlaAT

BIOTECHNOLOGICAL APPROACHES

overexpressing transgenic lines were analyzed during the vegetative growth phase, and it was clearly shown that these lines had the ability to take up larger amounts of nitrogen (higher NUpE). Vegetative growth for rice is an important phase for efficient nitrogen uptake and assimilation, leading to both the production of biomass and grain filling. The nitrogen that is in the grain is mainly remobilized from nitrogen accumulated in the leaves prior to flowering, with approximately 70–to 90% of the total panicle nitrogen being remobilized from the vegetative organs (Hirel et al., 2007). The NUE phenotype of the AlaAT overexpressing rice was marked by an increase in plant biomass, resulting from more tillers, an increase in grain yield, a denser, bushier root bundle, and an increase in total plant nitrogen (Shrawat et al., 2008). Based on the growth pattern and metabolite data, the following model to explain the transgenic NUE phenotype displayed during vegetative growth is proposed. Overexpression of the AlaAT gene in the roots leads to high levels of AlaAT enzyme at the point of nitrogen uptake and initial assimilation. Overexpressed AlaAT enzyme promotes nitrogen uptake by pulling glutamate from the GS/GOGAT cycle, and thereby allowing the primary assimilation enzymes, GS and GOGAT, to increase the rate of nitrogen assimilation into Gln and Glu, resulting in an increase in the storage of nitrogen in Ala and the transport and translocation of alanine to the shoots (Fig. 9.2). Following amino acid translocation to the shoots and leaves, production of Glu, Gln, and, ultimately, Asn, Asp, and Gly will also be increased in the shoots, allowing the production of a nitrogen pool that is available for remobilization to sink organs. AlaAT is a reversible aminotransferase producing either alanine and 2-OG or pyruvate and glutamate. While the kinetics of the enzyme should favor the production of alanine in the cytoplasm, it remains to be

185

seen whether the kinetics of the specific enzyme is important. Therefore, it is a working model that in the AlaAT overexpressing Brassica plants there was an increase in the total plant nitrogen due to a “de-repression” of nitrogen uptake. Nitrogen uptake is tightly regulated by internal and external levels of nitrogen; however, internal nitrogen status in the form of alanine may not be monitored. Therefore, although the transgenic plants were taking up, assimilating, and storing nitrogen as alanine efficiently, the regulatory network did not register this increasing internal nitrogen excess and so did not shut off or slow nitrogen uptake, which would normally happen in the case of a well-fertilized crop plant (Good et al., 2007). What lessons can we learn from the AlaAT transgene example? A review of the literature makes it clear that the majority of the biotechnological attempts at improving NUE have involved genes known to be involved in primary nitrogen metabolism and have utilized constitutive promoters; however, most of these have been unsuccessful at improving NUE (Table 9.1). While AlaAT may not have initially appeared to be a logical target, it has several features that may be of importance in designing strategies around improving NUE. First, downstream metabolites can often act as storage or transport molecules that may not be recognized by the plant’s nitrogensensing mechanisms; more specifically, it may be possible to increase the concentration of downstream metabolites relatively easily, whereas metabolites in the primary steps of assimilation are more closely monitored and regulated and therefore more difficult to manipulate successfully. Second, as in the case of alanine, there may be features to a specific metabolite that make it a logical target for manipulation. As an example,

186

NUTRIENT USE EFFICIENCY IN CROPS

alanine is a neutral end-product amino acid, which is also nontoxic and is known to be used in both the storage and transport of nitrogen. Thus, the first lesson is to think carefully about your gene target, how it might work, and whether it is regulated in the appropriate fashion or tissue. Relative importance of promoters A major limitation to progress has been the little thought given to the choice of promoters used to express genes of interest, given the number of examples where this has been shown to be critical to the success of the transformation events. While the CaMV35S promoter has been valuable in basic functional genetic studies of specific genes, it has proven to be less valuable for the development of specific commercial traits in crop plants. For example, for the modification of oils, a number of studies have shown that a seed-specific promoter is most useful for the production of modified oils since the developing seed is where oil synthesis occurs. In the case of stress-induced genes, using a stress-induced promoter provides better protection than one that is constitutively expressed (Pino et al., 2007). Therefore, successful transgenic approaches will require controlled overexpression of the specific transgene (by using developmental or tissuespecific promoters) in order to make gains in improving the efficiency of nitrogen use. What is clearly needed is an understanding of the specific expression characteristics that are required for each candidate gene, to properly evaluate whether the particular transgene has any ability to improve NUE. Challenges While the section below discusses the challenges of improving the efficiency with which crop plants utilize nitrogen, it should be noted that there are two other approaches

that would solve the problems associated with excessive nitrogen applications, but are often considered unattainable. These would be to either convince cereal crops to form nitrogen-fixing root nodules (see Chapter 20), or alternatively to introduce an endosymbiotic blue-green algae, which could fix nitrogen within the plant itself—in fact, there are already patents in this area of research (Triplett et al., 2008). What are the specific challenges that need to be addressed in order to bring nutrientefficient crops to the market? First, the selection of the gene of interest is neither trivial nor obvious. AlaAT was not initially a candidate gene, whereas nitrate reductase and nitrate transporters, which were believed to be logical candidate genes, have not provided any gains in NUE, despite significant research, although this may be in part due to inappropriate promoters. Therefore, we need to expand our thinking about possible candidate genes. The ferredoxin reductase gene is another example of a gene that would not logically have been a candidate for improved NUE (Hershey et al., 2009). One approach to this may be to model the flow of nitrogen within the plant, with a view to identifying key rate-limiting steps. This approach is now being used to model carbon assimilation, and additional research in this area in nitrogen metabolism is needed. Second, we have highlighted the need to identify promoters that would be more effective in targeting gene expression to the correct tissue, the correct organelle, and the correct developmental point. Clearly, greater work needs to be done in this area. Third, we need to be able to test any putative NUE transgenes in crops, including cereal crops, to determine whether they truly “work in the field.” This should also be done in a manner that will allow the direct comparison of different transgenic constructs; in other words, we need to use standard genetic material that is relatively well char-

BIOTECHNOLOGICAL APPROACHES

acterized. While this is now the norm for Arabidopsis, in model crop systems such as rice, a variety of different genotypes that differ significantly in growth characteristics and yield parameters are often used for transformation. Fourth, in contrast to what has been the case with herbicide tolerant crops, it may be that for more complex physiological traits, a transgene will work differently, (within a crop species) depending on the genotype that is transformed. Therefore, although the first generation of genetically modified organism (GMO) crops have had traits that worked in virtually any genotype, this will probably not be the case for complex traits such as NUE. Fifth, there will be a need to determine the effect of these transgenes in hybrid crops. For example, in maize, much of the NUE research is performed using inbred lines, which often differ dramatically from the elite hybrid lines. It is naïve to assume that a gene that enhances NUE in a maize inbred, which often produces less than 50% of the biomass of the hybrid, will perform equally well in a hybrid system. Finally, there may be a need to stack a number of subcomponents of NUE, such as carbon fixation, or root architecture, and enhanced NUpE. Clearly, trait and gene stacking will also have to occur with the other agronomics traits that are now ubiquitous, such as herbicide tolerance or insect resistance. In summary, although there have been a number of attempts to improve NUE in crops, to date the successes have been limited. Many challenges need to be addressed to make nutrient use-efficient crops a reality.

References Abeledo, L.G., Calderini, D.F., & Slafer, G.A. (2008) Nitrogen economy in old and modern malting barleys. Field Crops Research 106, 171–178.

187

Acreche, M.M. & Slafer, G.A. (2009) Variation of grain nitrogen content in relation with grain yield in old and modern Spanish wheats grown under a wide range of agronomic conditions in a Mediterranean region. Journal of Agricultural Science 147, 657–667. Ameziane, R., Bernhard, K., & Lightfoot, D. (2000) Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development. Plant and Soil 221, 47–57. Anbessa, Y., Juskiw, P., Good, A., et al. (2009) Genetic variability in nitrogen use efficiency of spring barley. Crop Science 49, 1259–1269. Anbessa, Y., Juskiw, P., Good, A.G., et al. (2010) Selection efficiency across environments for improving barley yield for moderately low N environments. Crop Science 50, 451–457. Ashikari, M., Sakakibari, H., Lin, S., et al. (2005) Cytokinin oxidase regulates rice grain production. Science 309, 741–745. Beatty, P.H., Shrawat, A.K., Carroll, R.T., et al. (2009) Transcriptome analysis of nitrogen-efficient rice over-expressing alanine aminotransferase. Plant Biotechnology Journal 7, 562–576. Beatty, P.H., Anbessa, Y., Juskiw, P., et al. (2010) Nitrogen use efficiencies of spring barley grown under varying nitrogen conditions in the field and growth chamber. Annals of Botany, Plant Nutrition Special Issue. Bi, Y.M., Kant, S., Clark, J., et al. (2009) Increased nitrogen use efficiency in transgenic rice plants over-expressing a nitrogen responsive early nodulin gene identified from rice expression profiling. Plant, Cell & Environment 32, 1749–1760. Brauer, E.K. & Shelp, B.J. (2010) Nitrogen use efficiency: re-consideration of the bioengineering approach. Botany 88, 103–190. Brears, T., Liu, C., Knight, T.J., et al. (1993) Ectopic overexpression of asparagine synthetase in transgenic tobacco. Plant Physiology 103, 1285–1290. Century, K., Reuber, T.L., & Ratcliffe, O.J. (2008) Regulating the regulators: the future prospects for transcription-factor-based agricultural biotechnology products. Plant Physiology 147, 20–29. Chiu, J.C., Brenner, E.D., DeSalle, R., et al. (2002) Phylogenetic and expression analysis of the glutamate-receptor–like gene family in Arabidopsis thaliana. Molecular Biology and Evolution 19, 1066–1082. Coruzzi, G. & Bush, D. (2001) Nitrogen and carbon nutrient and metabolite signaling in plants. Plant Physiology 125, 61–64. Craswell, E.T. & Godwin, D.C. (1984) The efficiency of nitrogen fertilizers applied to cereals grown in

188

NUTRIENT USE EFFICIENCY IN CROPS

different climates. In: Advances in Plant Nutrition, Vol. 1 (eds. P.B. Tinker & A. Lauchli), Praeger Publishers, New York. Davenport, R. (2002) Glutamate receptors in plants. Annals of Botany 90, 549–557. Denman, K.L., Brasseur, G., Chidthaisong, A., et al. (2007) Couplings between changes in the climate system and biogeochemistry. In: IPCC, Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change (eds. S. Solomon, D. Qin, M. Manning, Z. Chen, M. Marquis, et al.), Cambridge University Press, Cambridge, UK. Effer, W.R. & Ranson, S.L. (1967) Some effects of oxygen concentration on levels of respiratory intermediates in buckwheat seedlings. Plant Physiology 42, 1053–1058. Filleur, S., Walch-Liu, P., Gan, Y., & Forde, B.G. (2005) Nitrate and glutamate sensing by plant roots. Biochemical Society Transactions 33 (1), 283–286. Foyer, C.H., Parry, M., & Noctor, G. (2003) Markers and signals associated with nitrogen assimilation in higher plants. Journal of Experimental Botany 54, 585–593. Gallais, A. & Hirel, B. (2004) An approach to the genetics of nitrogen use efficiency in Maize. Journal of Experimental Botany 55, 295–306. Galloway, J.N., Townsend, A.R., Erisman, J.W., et al. (2008) Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320, 889–892. Gan, S. & Amasino, R.M. (1995) Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270, 1986–1988. Garnett, T., Conn, V., & Kaiser, B.N. (2009) Root based approaches to improving nitrogen use efficiency in plants. Plant, Cell & Environment 32, 1272–1283. Giannino, D., Nicolodi, C., Testone, G., et al. (2008) The overexpression of asparagine synthetase A from E. Coli affects the nitrogen status in leaves of lettuce (Lactuca sativa L.) and enhances vegetative growth. Euphytica 162, 11–22. Good, A.G. & Crosby, W.L. (1989) Anaerobic induction of alanine aminotransferase in barley root tissue. Plant Physiology 90, 1305–1309. Good, A.G. & Muench, D.G. (1992) Purification and characterization of an anaerobically induced alanine aminotransferase from barley. Plant Physiology 99, 1520–1525. Good, A.G. & Muench, D.G. (1993) Long-term anaerobic metabolism in root tissue. Metabolic products of pyruvate metabolism. Plant Physiology 101, 1163–1168.

Good, A.G., Shrawat, A.K., & Muench, D.G. (2004) Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production? Trends in Plant Science 9, 597–605. Good, A.G., Johnson, S.J., DePauw, M., et al. (2007) Engineering nitrogen use efficiency with alanine aminotransferase. Canadian Journal of Botany 85, 252–262. Goulding, K., Jarvis, S., & Whitmore, A. (2008) Optimizing nutrient management for farm systems. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 363, 667–680. Hatch, M.D. (1987) C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochimica et Biophysica Acta 895, 6048–6053. Hatch, M.D. (1992) I can’t believe my luck*. Photosynthesis Research 33, 1–14. Hershey, H.P., Simmons, C.R., Loussaert, D., et al. (2009) Genes for enhancing nitrogen utilization efficiency in crop plants. US Patent 7,589,257 B2. Assignee (Pioneer HiBred). Hirel, B. & Lemaire, G. (2005) From agronomy and ecophysiology to molecular genetics for improving nitrogen use efficiency in crops. In: Efficient Nitrogen Use in Crop Production (eds. A. Basra & S. Goyal), pp. 213–257, Haworth’s Food Product Press, Binghamton, NY. Hirel, B., Le Gouis, J., Ney, B., et al. (2007) The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. Journal of Experimental Botany 58, 2369–2387. Hsieh, M.-H., Lam, H.-M., Van De Loo, F.J., et al. (1998) A PII-like protein in Arabidopsis: putative role in nitrogen sensing. Proceedings of the National Academy of Sciences of the United States of America 95, 13965–13970. Huynh, L.N., Van Toai, T., Streeter, J., et al. (2005) Regulation of flooding tolerance of SAG12: ipt Arabidopsis plants by cytokinin. Journal of Experimental Botany 56, 1397–1407. Igarashi, D., Ishizaki, T., Totsuku, K., et al. (2009) ASN2 is a key enzyme in asparagine biosynthesis under ammonium sufficient conditions. Plant Biotechnology Journal 26, 153–159. International Plant Nutrition Institute (2009) The global “4R” nutrient stewardship framework developing fertilizer best management practices for delivering economic, social and environmental benefits. IFA task force on fertilizer best management practices. Available at: http://www.ipni.net/4r (accessed January 2010).

BIOTECHNOLOGICAL APPROACHES

Jordi, W., Schapendonk, A., Davelaar, E., et al. (2000) Increased cytokinin levels in transgenic PSAG12— IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning. Plant, Cell & Environment 23, 279–289. Kang, J. & Turano, F.J. (2003) The putative glutamate receptor1.1 (AtGLR1.1) functions as a regulator of carbon and nitrogen metabolism in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 100, 6872–6877. Keeney, D.R. (1982) Nitrogen management for maximum efficiency and minimum pollution. In: Nitrogen in Agricultural Soils, Agronomy Monograph 22 (ed. F.J. Stevenson), pp. 605–649, American Society of Agronomy, Madison, WI. Kim, S.A., Kwak, J.M., Jae, S.K., et al. (2001) Overexpression of the AtGluR2 gene encoding an Arabidopsis homolog of mammalian glutamate receptors impairs calcium utilization and sensitivity to ionic stress in transgenic plants. Plant and Cell Physiology 42, 74–84. Kisaka, H. & Kida, T. (2003) Transgenic tomato plant carrying a gene for NADP-dependent glutamate dehydrogenase (gdhA) from Aspergillus nidulans. Plant Science 164, 35–42. Kisaka, H., Kida, T., & Miwa, T. (2007) Transgenic tomato plants that overexpress a gene for NADHdependent glutamate dehydrogenase (legdh1). Breeding Science 57, 101–106. Lacombe, B., Becker, D., Hedrich, R., et al. (2001) The identity of plant glutamate receptors. Science 292, 1486–1487. Lam, H.M., Coschigano, K.T., Oliveira, I.C., et al. (1996) The molecular-genetics of nitrogen assimilation into amino acids in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 569–593. Lam, H.M., Wong, P., Chan, H.K., et al. (2003) Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis. Plant Physiology 132, 926–935. Le Gouis, J., Beghin, D., Heumez, E., et al. (2000) Genetic differences for nitrogen uptake and nitrogen utilisation efficiencies in winter wheat. European Journal of Agronomy 12, 163–173. Leblanc, A., Renault, H., Lecourt, J., et al. (2008) Elongation changes of exploratory and root hair systems induced by aminocyclopropane carboxylic acid and aminoethoxyvinylglycine affect nitrate uptake and BnNRT2.1 and BnNRT1.1 transporter gene expression in oilseed rape. Plant Physiology 146, 1928–1940. Liao, M., Fillery, I.R.P., & Palta, J.A. (2004) Early vigorous growth is a major factor influencing nitro-

189

gen uptake in wheat. Functional Plant Biology 31, 121–129. Liao, M.T., Palta, J.A., & Fillery, I.R.P. (2006) Root characteristics of vigorous wheat improve early nitrogen uptake. Australian Journal of Agricultural Research 57, 1097–1107. Lightfoot, D.A., Mungur, R., Ameziane, R., et al. (2007) Improved drought tolerance of transgenic Zea mays plants that express the glutamate dehydrogenase gene (gdhA) of E. coli. Euphytica 156, 103–116. Liu, L.-H., Walch-Liu, P., Qu, X.-Q., et al. (2007) Identification and phenotyping of T-DNA insertion lines of AtGLRs in Arabidopsis. 18th International Conference on Arabidopsis Research. Beijing, China, 20–23 June 2007. Publication: 501722133. Ma, Q.-H. (2008) Genetic engineering of cytokinins and their application to agriculture. Critical Reviews in Biotechnology 28, 213–232. Ma, Q.H., Lin, Z.B., & Fu, D.Z. (2002) Increased seed cytokinin levels in transgenic tobacco influence embryo and seedling development. Functional Plant Biology 29, 1107–1113. Makino, A., Nakano, H., Mae, T., et al. (2000) Photosynthesis, plant growth and N allocation in transgenic rice plants with decreased Rubisco under CO2 enrichment. Journal of Experimental Botany 51, 383–389. Masclaux, C., Quillere, I., Gallais, A., et al. (2001) The challenge of remobilisation in plant nitrogen economy. A survey of physio-agronomic and molecular approaches. Annals of Applied Biology 138, 69–81. Masle, J., Hudson, G.S., & Badger, M.R. (1993) Effects of ambient CO2 concentration on growth and nitrogen use in tobacco (Nicotiana tabacum) plants transformed with an antisense gene to the small subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase. Plant Physiology 103, 1075–1088. McCabe, M.S., Garratt, L.C., Schepers, F., et al. (2001) Effects of PSAG12-IPT gene expression on development and senescence in transgenic lettuce. Plant Physiology 127, 505–516. Miflin, B.J. & Habash, D.Z. (2001) The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. Journal of Experimental Botany 53, 979–987. Miyashita, Y. & Good, A.G. (2008) NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation. Journal of Experimental Botany 59, 667–680. Miyashita, Y., Dolferus, R., Ismond, P., et al. (2007) Alanine aminotransferase catalyses the breakdown

190

NUTRIENT USE EFFICIENCY IN CROPS

of alanine after hypoxia in Arabidopsis thaliana. The Plant Journal 49, 1108–1121. Moll, R.H., Kamprath, E.J., & Jackson, W.A. (1982) Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agronomy Journal 74, 562–564. Moorhead, G.B.G. & Smith, C.S. (2003) Interpreting the plastid carbon, nitrogen and energy status. A role for PII? Plant Physiology 133, 492–498. Moose, S. & Below, F.E. (2009) Biotechnology approaches to improving maize nitrogen use efficiency. In: Molecular Genetic Approaches to Maize Improvement, Biotechnology in Agriculture and Forestry (eds. A.L. Kriz & B.A. Larkins), pp. 65– 77, Springer-Verlag, Berlin. Mosier, A.R., Syers, J.K., & Freney, J.R. (2004) Agriculture and the Nitrogen Cycle. Assessing the Impacts of Fertilizer Use on Food Production and the Environment. SCOPE 65. Island Press, Washington, DC. Muench, D.G. & Good, A.G. (1994) Molecular cloning and expression of an anaerobically induced alanine aminotransferase from barley roots. Plant Molecular Biology 24, 417–427. Mungur, R., Glass, A.D., Goodenow, D., et al. (2005) Metabolite fingerprint changes in transgenic Nicotiana tabacum altered by the Escherichia coli glutamate dehydrogenase gene. Journal of Biomedicine and Biotechnology 25, 198–214. Mungur, R., Wood, A.J., & Lightfoot, D.A. (2006) Water potential is maintained during water deficit in Nicotiana tabacum expressing the Escherichia coli glutamate dehydrogenase gene. Journal of Plant Growth Regulation 50, 231–238. Murooka, Y., Mori, Y., & Hayashi, M. (2002) Variation of the amino acid content of Arabidopsis seeds by expressing soybean aspartate aminotransferase gene. Journal of Bioscience and Bioengineering 94, 225–230. Näsholm, T., Ekblad, A., Nordin, A., et al. (1998) Boreal forest plants take up organic nitrogen. Nature 392, 914–916. Oaks, A. (1994) Efficiency of nitrogen utilization in C3 and C4 cereal. Plant Physiology 106, 407–414. Obara, M., Kajiura, M., Fukuta, Y., et al. (2001) Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice (Oryza sativa L.). Journal of Experimental Botany 52, 1209–1217. Oenema, O., Kros, K., & de Vries, W. (2003) Approaches and uncertainties in nutrient budgets: implications for nutrient management and environmental policies. European Journal of Agronomy 20, 3–16.

Ortiz-Monasterio, R.J.I., Sayre, K.D., Rajaram, S., et al. (1997) Genetic progress in wheat yield and nitrogen use efficiency under four N rates. Crop Science 37, 898–904. Pino, M.-T., Skinner, J.S., Park, E.-J., et al. (2007) Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield. Plant Biotechnology Journal 5, 591–604. Plénet, D. & Lemaire, G. (1999) Relationships between dynamics of nitrogen uptake and dry matter accumulation in maize crops. Plant and Soil 216, 65–82. Powlson, D.S., Addiscott, T.M., Benjamin, N., et al. (2006) When does nitrate become a risk for humans? Journal of Environmental Quality 37, 291–295. Rademacher, T., Rainer, E., Hausler, R.E., et al. (2002) An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants. The Plant Journal 32, 25–39. Raines, C.A. (2006) Transgenic approaches to manipulate the environmental responses of the C3 carbon fixation cycle. Plant, Cell & Environment 29, 331–339. Rathke, G.-W., Behrens, T., & Diepenbrock, W. (2006) Integrated nitrogen management strategies to improve seed yield, oil content and nitrogen efficiency of winter oilseed rape (Brassica napus L.): a review. Agriculture, Ecosystems & Environment 117, 80–108. Robson, P.R.H., Donnison, I.S., Wang, K., et al. (2004) Leaf senescence is delayed in maize expressing the Agrobacterium IPT gene under the control of a novel maize senescence-enhanced promoter. Plant Biotechnology Journal 2, 101–112. Rolletschek, H., Hosein, F., Miranda, M., et al. (2005) Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and Pisum sativum increases storage proteins. Plant Physiology 137, 1236–1249. Schofield, R.A., Bi, Y.M., Kant, S., et al. (2009) Overexpression of STP13, a hexose transporter, improves plant growth and nitrogen use in Arabidopsis thaliana seedlings. Plant, Cell & Environment 32, 271–285. Seiffert, B., Zhou, Z., Wallbraun, M., et al. (2004) Expression of a bacterial asparagine synthetase gene in oilseed rape (Brassica napus) and its effect on traits related to nitrogen efficiency. Physiologia Plantarum 121, 656–665. Sentoku, N., Taniguchi, M., Sugiyama, T., et al. (2000) Analysis of the transgenic tobacco plants expressing Panicum miliaceum aspartate aminotransferase genes. Plant Cell Reports 19, 598–603.

BIOTECHNOLOGICAL APPROACHES

Shrawat, A.K. & Good, A.G. (2009) Genetic engineering approaches for improving nitrogen use efficiency (NUE) in plants. Information Systems for Biotechnology News Report, May 2008, pp 1–5. Shrawat, A.K., Carroll, R.T., DePauw, M., et al. (2008) Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase. Plant Biotechnology Journal 6, 722–732. Son, D. & Sugiyama, T. (1992) Molecular cloning of alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum. Plant Molecular Biology 20, 705–713. Son, D., Kobe, A., & Sugiyama, T. (1992) Nitrogendependent regulation of the gene for alanine aminotransferase which is involved in the C4 pathway of Panicum miliaceum. Plant and Cell Physiology 33, 507–509. Steenbjerg, F. & Jakobsen, S.T. (1963) Plant nutrition and yield curves. Soil Science 95, 69–90. Strange, A., Park, J., Bennett, R., et al. (2008) The use of life-cycle assessment to evaluate the environmental impacts of growing genetically modified, nitrogen use-efficient canola. Plant Biotechnology Journal 6, 337–345. Streeter, J.G. & Thompson, J.F. (1972) Anaerobic accumulation of β-aminobutyrate and alanine in radish leaves (Raphinus sativus). Plant Physiology 49, 572–578. Stroeher, V.L., Boothe, J.G., & Good, A.G. (1995) Molecular cloning and expression of a turgorresponsive gene in Brassica napus. Plant Molecular Biology 27, 541–551. Takeda, S. & Matsuoka, M. (2008) Genetic approaches to crop improvement: responding to environmental and population changes. Nature 9, 444–456. Triplett, E.W., Kaeppler, S.M., & Chelius, M.K. (2008) Klebsiella pneumoniae inoculants for enhancing plant growth. US Patent 7393678-B1. Assignee; Wisconsin Alumni Research Foundation. U.S. Environmental Protection Agency (EPA) (2007) Hypoxia in the Northern Gulf of Mexico: An Update by the EPA Science Advisory Board. (EPA-SAB-08003). EPA Science Advisory Board, Washington, DC. Urbanchuk, J.M., Kowalski, D.J., Dale, B., et al. (2009) Corn amylase: improving the efficiency and environmental footprint of corn to ethanol through plant biotechnology. AgBioForum 12, 149–154. Vanlerberghe, G.C. & Turpin, D.H. (1990) Anaerobic metabolism in the N-limited green alga Selenastrum minutum. II. Assimilation of ammonium by anaerobic cells. Plant Physiology 94, 1124–1130. Vanlerberghe, G.C., Joy, K.W., & Turpin, D.H. (1991) Anaerobic metabolism in the N-limited alga

191

(Selenastrum minutum). III. Alanine is the product of anaerobic ammonium assimilation. Plant Physiology 95, 655–658. Vitousek, P.M., Naylor, R., Crews, T., et al. (2009) Nutrient imbalances in agricultural development. Science 324, 1519–1520. Walch-Liu, P. & Forde, B.G. (2008) Nitrate signalling mediated by the NRT1.1 nitrate transporter antagonises L-glutamate-induced changes in root architecture. The Plant Journal 54, 820–828. Waters, J.K., Hughes, B.L., Purcell, L.C., et al. (1998) Alanine, not ammonia, is excreted from N-2-fixing soybean nodule bacteroids. Proceedings of the National Academy of Sciences of the United States of America 95, 12038–12042. Wiesler, F. & Horst, W.J. (1993) Differences among maize cultivars in the utilization of soil nitrate and the related losses of nitrate through leaching. Plant and Soil 151, 193–203. Wiesler, F. & Horst, W.J. (1994) Root growth and nitrate utilization of maize cultivars under field conditions. Plant and Soil 163, 267–277. Wolansky, M.A. (2005) Genetic manipulation of aspartate aminotransferase levels in Brassica napus: effects on nitrogen use efficiency. M.Sc. thesis, University of Alberta. Worku, M., Banziger, M., Erley, G.S.A., et al. (2007) Nitrogen uptake and utilization in contrasting nitrogen efficient tropical maize hybrids. Crop Science 47, 519–528. Yanagisawa, S., Akiyama, A., Kisaka, H., et al. (2004) Metabolic engineering with Dof1 transcription factor in plants: improved nitrogen assimilation and growth under low-nitrogen conditions. Proceedings of the National Academy of Sciences of the United States of America 101, 7833–7838. Zhang, H. & Forde, B.G. (1998) An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279, 407–409. Zhang, H. & Forde, B.G. (2000) Regulation of Arabidopsis root development by nitrate availability. Journal of Experimental Botany 51, 51–59. Zhou, Y., Cai, H., Xiao, J., et al. (2009) Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds. Theoretical and Applied Genetics 118, 1381–1390. Zhu, X.-G., de Sturler, E., & Long, S.P. (2007) Optimizing the distribution of resources between enzymes of carbon metabolism can dramatically increase photosynthetic rate: a numerical simulation using an evolutionary algorithm. Plant Physiology 145, 513–526.

Chapter 10

Transporters Involved in Nitrogen Uptake and Movement Anthony J. Miller and Nick Chapman

Abstract Families of nitrogen (N) transporters have been identified in the model plant Arabidopsis, and this template is useful for other species. For crops, uptake efficiency is an important component of nitrogen use efficiency (NUE), which has been shown to vary widely between cultivars and species. The root is the most important organ for acquiring soil nitrogen, which is chiefly available as NO3−, NH4+ or amino acids. Soil nitrogen form and availability to roots is transient, and the concentrations can rapidly change in response to climatic factors. Although root architecture is undoubtedly a key trait for nitrogen acquisition efficiency (see Chapter 2) and the rhizosphere influences availability (Chapter 3), the activity of root transporters is important for uptake. Roots have transporter systems to acquire the different nitrogen forms from the soil. Ammonium uptake is achieved by ammonium transporters (AMTs) and amino acids by several different families of transporters. Within the root, NO3− uptake and transport are realized by NO3− transporters (NRTs),

while vacuolar storage is mediated by chloride channels (CLCs). Uniquely, NRT1.1 is capable of functioning in both high- and low-affinity uptake, and has a NO3− sensing and signaling capability, regulating other key players in NO3− uptake, transport, and signaling. NRT expression and function is regulated by plant nitrogen status and can directly influence the root system architecture. Once inside the plant the redistribution of nitrogen is achieved through the activity of more transporters, and each of these steps may be rate limiting. Attempts to increase nitrogen uptake by overexpression of transporters has been unsuccessful because of secondary regulation through posttranslational control mechanisms.

Introduction This chapter covers nitrogen transport and focuses on the nitrogen (N) transporter families, particularly in relation to acquisition by the roots. For this topic, the best characterized plant is Arabidopsis, which provides a model that is useful to all crops. Nitrogen is

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 193

194

NUTRIENT USE EFFICIENCY IN CROPS

an essential element for life and of all the plant nutrients it is required in the largest amounts. In the soil, nitrogen is available in various forms, chiefly as nitrate (NO3−), ammonium (NH4+), and amino acids. In agriculture, the inorganic forms, mostly NO3− and some NH4+, are the main sources, and after uptake these are combined with carbon to produce amino acids (assimilation) before being redistributed. Roots can take up all of these nitrogen forms, which can be directly transported around the plant. Families of membrane carrier proteins that mediate these steps have been identified, and although the transport function of many of these membrane proteins are known, the underlying regulation is much less well understood. The emphasis here is on the transport systems for the inorganic nitrogen forms. High- and low-affinity NO3− transport systems Measurements of the influence of NO3− supply on uptake have led to the conclusion that plants have developed different transport systems to cope with the variable soil supply (Crawford and Glass, 1998). At low NO3− concentrations (below 1 mM), uptake is achieved via two saturable high-affinity transport systems (HATS). The constitutive system (cHATS) is available when plants have been previously starved of NO3−, and the inducible system (iHATS) is stimulated by supplying NO3−. The low-affinity transport system (LATS) mediates NO3− uptake at external NO3− concentrations above 1 mM (Crawford and Glass, 1998). Even at these higher concentrations of NO3− both types of HATS still contribute for uptake. The HATS and LATS are encoded by at least two different gene families of nitrate transporters (NRTs) that are numbered according to the chronology of their discovery, NRT1 and NRT2 (Forde, 2000; Williams and Miller,

2001). In Arabidopsis, the characterization of the transporter protein expressed in Xenopus oocytes and phenotype analysis of gene knockout mutants have both proved to be powerful tools for understanding the function of the transporters. NRT1s The NRT1 gene families are relatively large; for example, there are 53 and 80 in the genomes of Arabidopsis and rice, respectively, but they include members of amino acid and di/tripeptide transporters (PTR) and should more correctly be termed the NRT1/ PTR family (Zhou et al., 1998; Forde, 2000; Orsel et al., 2002). The NRT1 proteins comprise 12 putative transmembrane spanning domains, and a defining feature of the higher plant members is a large hydrophilic loop between the sixth and seventh transmembrane regions. Closely related members of the NRT1/PTR family have evolved distinct functions in planta, which are often defined by a very specific and localized tissue pattern of expression (see Fig. 10.1) such as NO3− transport in the leaf petiole (AtNRT1.4; Chiu et al., 2004), the phloem (AtNRT1.7; Fan et al., 2009), or early embryo (AtNRT1.8; Almagro et al., 2008). In addition to this tissue-specific expression, NRT1/PTR family members can transport a surprising range of unrelated molecules, including NO3− and di/tripeptides. One member of the family, found in the nodule of nitrogenfixing plants, has been shown to transport dicarboxylic acids (Jeong et al., 2004). Recently, AtNRT1.1 was shown to mediate NO3− regulated transport of the plant hormone auxin, which may explain the transporters’ role in sensing available nitrate and lateral root (LR) development (Krouk et al., 2010). The first NRT1/PTR gene to be identified in plants was CHL1 (now known as AtNRT1.1), and it was shown to encode a

TRANSPORTERS INVOLVED IN NITROGEN UPTAKE AND MOVEMENT

195

NRT2.7 NRT1.6

NRT1.4 NRT1.7 NRT2.1 NRT2.2 NAR2.1

NRT1.2 NRT1.5

AMT1;2 AMT1;1 AMT1;3 AMT1;5

NRT1.1

The distribution of some NRT1, NRT2, and AMT genes in Arabidopsis (redrawn from Masclaux-Daubresse et al., 2010).

Fig. 10.1.

proton-coupled NO3− transporter (Tsay et al., 1993). Later, AtNRT1.1 was found to show both HATS and LATS activity indicating that NRT1.1 is a dual-affinity range transporter (Liu et al., 1999). The NO3− affinity of AtNRT1.1 is regulated by the phosphorylation of a threonine residue (Liu and Tsay, 2003; Tsay et al., 2007). The phosphorylated AtNRT1.1 functions as a high-affinity NO3− transporter, while the dephosphorylated AtNRT1.1 is a low-affinity transporter. Plants with defective AtNRT1.1 expression have been shown to exhibit a decreased lateral root response to NO3− patches on agar petri dishes (Remans et al., 2006a). In addition to NO3− uptake and a potential signaling role for NRT1.1, promoter-tagged fluorescent protein lines and immunolocalization studies have been used to demonstrate functional expression of AtNRT1.1 in guard cells (Guo et al., 2003). Mutant lines grown in the presence of NO3− exhibit decreased stomatal opening and therefore are more drought tolerant when compared to wild type. AtNRT1.1 is required for stomatal activity, implying a key role for NO3− transport in guard cell function (Guo et al., 2003). The AtNRT1.1

transporter has an important role in root sensing of external NO3− availability through interaction with kinases (Ho et al., 2009). NRT1.1 demonstrates high-affinity NO3− transport when expressed in yeast (Martin et al., 2008). Brassica napus and rice orthologs have been identified (Zhou et al., 1998; Chen et al., 2008); further research into their function is needed to show if these orthologs show dual-affinity NO3− uptake and nitrate sensing in these crop species. All other AtNRT1s that were studied exhibited lowaffinity NO3− transport activity (Huang et al., 1999; Chiu et al., 2004; Almagro et al., 2008; Lin et al., 2008; Fan et al., 2009). The constitutively expressed AtNRT1.2 is located in the epidermis and functions in the cLATS (Huang et al., 1999). The rice equivalent to AtNRT1.2, OsNRT1, is also a rootepidermal, low-affinity NO3− transporter (Lin et al., 2000). In the leaf petiole, AtNRT1.4 provides low-affinity NO3− uptake (Chiu et al., 2004). A study using Xenopus oocytes demonstrated that NRT1.5 is a low-affinity, pH-dependent, bidirectional NO3− transporter. Localized to the plasma membrane of root pericycle cells in

196

NUTRIENT USE EFFICIENCY IN CROPS

proximity to xylem vessels, NRT1.5 has been postulated to function in the xylem loading of NO3− (Lin et al., 2008); root-toshoot transport of NO3− was not completely lost in the knockout mutant, indicating that there are other proteins involved in the xylem loading of NO3−. A similar approach showed that NRT1.6 is a low-affinity transporter that lacks the capacity to transport dipeptides and functions in the delivery of NO3− from maternal tissues to the developing embryo (Almagro et al., 2008). In aerial tissues, the NO3− transporter NRT1.7 is located in the phloem of the leaf minor veins and functions to transport NO3− from older leaves to younger ones. This tissue localization pattern supports the idea that NO3− can be remobilized via the phloem, and this transport activity can sustain growth (Fan et al., 2009). Peptide transport Peptide transporter activity has been demonstrated in the family by complementation of yeast transporter and plant knockout mutants supplied with peptides as a sole nitrogen source; for example, AtPTR1 was shown to function in di- and tripeptide uptake within the root (Dietrich et al., 2004). Using a similar approach, AtPTR5 was shown to have peptide transport activity in germinating pollen and during seed development (Komarova et al., 2008). Interestingly, NRT1.1 has been implicated in the germination of dormant seeds in response to nitrogen supply (Alboresi et al., 2005), suggesting that some family NRT1/PTR members could have multiple and overlapping functions. Another family of plant PTR has been identified, the oligopeptide transporters (OPTs), which primarily function in the seed and embryo (Stacey et al., 2006). They differ from the unrelated PTRs in transporting tetra- and pentapeptides, although both families use a proton–cotransport mechanism.

NRT2s There are seven NRT2 genes in Arabidopsis. Of these, AtNRT2.1 and AtNRT2.2 are located adjacently on chromosome 1 and both are involved in the HATS (Cerezo et al., 2001; Remans et al., 2006b; Chen et al., 2008). AtNRT2.1 was shown to have a more important role in iHATS due to decreased iHATS activity in nrt2.1 and nrt2.2 knockout lines of 50–72% and 19%, respectively (Li et al., 2007). Both AtNRT2.1 and AtNRT2.2 are NO3− inducible and their activity can influence root architecture through NO3− uptake and sensing, although the effect of AtNRT2.1 can be modified by exogenous sucrose application and light exposure (Lejay et al., 1999; Vidal and Gutiérrez, 2008). AtNRT2.1 is located at the plasma membrane of root cortical and epidermal cells (Krapp et al., 1998; Chopin et al., 2007b). Although the full length protein is involved in NO3− transport and is the most abundant form, other truncated forms can coexist at the cell membrane and their function is unknown (Wirth et al., 2007). To be successfully targeted to the plasma membrane, AtNRT2.1 requires a second protein, AtNAR2.1 (Orsel et al., 2006, 2007; Wirth et al., 2007). An AtNRT2.1 ortholog is known to function in the HATS of wheat and the mRNA accumulates in the root (Yin et al., 2007). The NO3− induced TaNRT2 is located in the root and induced in response to both low and high concentrations of NO3−. Transcripts of one wheat NRT2 were missing in plants grown under nitrogen-limiting conditions or where NH4+ was the sole nitrogen source (Zhao et al., 2004). Some evidence exists for the implication of AtNRT2.1 in a NO3− transport-independent sensing role in LR initiation (Crawford and Glass, 1998; Forde, 2000; Cerezo et al., 2001; Orsel et al., 2002; Little et al., 2005; Remans et al., 2006b; Wirth et al., 2007).

TRANSPORTERS INVOLVED IN NITROGEN UPTAKE AND MOVEMENT

The study of lin1, an AtNRT2.1 mutant line, suggested that AtNRT2.1 acts as a NO3− sensor or signal transducer. In the wild type, a high sucrose : NO3− ratio represses LR initiation, but the repression is removed in lin1. Indeed, this response of the lin1 mutant is observed in media without NO3−, illustrating that this phenotype is independent of NO3− and indicative of a NO3− sensor or signal transducer function for AtNRT2.1 (Little et al., 2005). The lin1 mutant was selected from ethyl methanesulfonate-mutagenized seed and had a single glycine mutation that was likely to compromise NO3− transport (Little et al., 2005). Conversely, a different study demonstrated that the atnrt2.1 gene knockout mutant exhibited the opposite phenotype to lin1: a reduced LR initiation (Remans et al., 2006b). This difference could be partly explained by variations in the growth conditions between studies, but the results suggest that the role of AtNRT2.1 in sensing NO3− still requires clarification. AtNRT2.7 is a vacuolar membrane transporter that plays a role in seed NO3− accumulation, and high expression levels have been detected in reproductive organs and seeds (Chopin et al., 2007a). The vacuolar location is interesting because a totally unrelated transporter family, the CLC transporters, are also implicated in vacuolar accumulation of NO3− (De Angeli et al., 2006). Furthermore, AtNRT1.1 has been implicated in the germination of dormant seeds and is known to regulate AtNRT2.1 expression and may regulate other NRT2 family members. A regulatory interaction between AtNRT1.1 and AtNRT2.7 is possible, perhaps in response to the seed accumulation of NO3− to a critical threshold level at which germination is initiated. The Arabidopsis thaliana mutant atnrt2.1-1 has deletions of both AtNRT2.1 and AtNRT2.2 genes and exhibits suppression in upregulation of the NO3− HATS in response to nitrogen starvation. This mutant

197

was used to investigate root growth morphology in response to low NO3− availability. An increase in the number and growth of visible LRs was reported when wild-type plants were transferred from 10 mM NO3− to lower concentrations. The response of atnrt2.1-1 to moderate NO3− limitation produced root morphology similar to the wild type under severe NO3− stress. The root morphology response could reflect the decreased NO3− uptake measured in the mutant line, suggesting that uptake rate of NO3− could be more important than external NO3− concentration in influencing root architecture. However, the nrt2.1 mutant exhibited inhibited LR initiation in response to nitrogen limitation, independent of the NO3− uptake, and the inhibition persisted even when NO3− was added to the external medium. This result indicates a direct stimulatory role for NRT2.1 in LR initiation and suggests that uptake alone is not responsible for the root morphology responses to nitrogen limitation (Orsel et al., 2004; Little et al., 2005; Remans et al., 2006b). The expression of AtNRT2.1 rapidly increases during early vegetative growth, peaking just before floral stem emergence and decreases to minimal levels in flowering and silique-bearing plants. A series of experiments with altered nitrogen supply and source found that NO3− induced AtNRT2.1 expression and amino acids (specifically glutamine) repressed expression. This provides evidence for a signaling role for glutamine in the regulation of NO3− uptake (Nazoa et al., 2003). In the same study, young roots did not demonstrate NRT2.1 expression despite exhibiting a similar rate of NO3− influx to older roots, suggesting that another high-affinity transporter functions in root tips (Nazoa et al., 2003). Indeed, NRT1.1 has been implicated in glutamine signaling at the root tip and is known to function in both HATS and LATS (Guo et al., 2001; Walch-Liu and Forde, 2008).

198

NUTRIENT USE EFFICIENCY IN CROPS

Two component nitrate transport by NAR2 (NRT3) The NAR2 (NRT3) proteins are required for NRT2.1 function (Tong et al., 2005; Okamoto et al., 2006; Orsel et al., 2006). The NAR2 proteins have a single putative transmembrane spanning domain and they are required for targeting of some NRT2 proteins to the plasma membrane (Orsel et al., 2007; Wirth et al., 2007). Indeed, NO3− elicited currents are only observed in Xenopus oocytes injected with mRNA encoding both components (Zhou et al., 2000; Tong et al., 2005). The yeast splitubiquitin system was used to confirm a direct interaction between the two proteins (Orsel et al., 2006). However, not all NAR2 proteins can form functional interactions with NRT2 proteins. For example, in barley, only HvNAR2.3 can generate an operational unit with HvNRT2.1 (Tong et al., 2005). Arabidopsis was orignally identified as having two NAR2 genes: AtNAR2.1 (AtNRT3.1) and AtNAR2.2. The former has been identified as important in the HATS (Okamoto et al., 2006; Orsel et al., 2006; Wirth et al., 2007). The latter, AtNAR2.2, has recently been annotated as a member of calcineurin-like phosphoesterase family with three splice variants. The nar2.1 null mutant shows extensively decreased HATS activity. Interestingly, the expression of cHATS in the nrt2.1 nrt2.2 double mutant was only decreased by approximately a third of the reduction observed in the nar2.1 null, suggesting that a further unidentified NRT2 is involved in the cHATS (Orsel et al., 2006; Li et al., 2007). Comparison of plasma membrane protein fractions between mutants and wild-type plants suggests that the functional unit for high-affinity nitrate influx in Arabidopsis was a tetramer consisting of two subunits each of AtNRT2.1 and AtNAR2.1 (Yong et al., 2010).

Molecular regulation of nitrate transporters When considering the whole plant, net NO3− uptake is regulated by demand, and the various uptake systems are induced by the presence of NO3− (Crawford and Glass, 1998). However, the uptake systems can be negatively regulated by feedback from assimilatory products, thereby directly linking NO3− influx to plant nitrogen status (Muller and Touraine, 1992; Miller et al., 2009); these levels of regulation will now be briefly described.

Gene expression The regulatory mechanisms of long-distance NO3− transport within the plant remain largely unknown. The inducible HATS is feedback-regulated relative to the plant demand for NO3− , and transcription of the NRT genes is feedback-repressed by the secondary products of NO3− metabolism (Vidal and Gutiérrez, 2008). Regulation of AtNRT2.1 expression has been comprehensively studied and upregulation, from transport activity to promoter activation, is induced by NO3− itself and repressed by downstream nitrogen metabolites (Loqué et al., 2003). AtNRT2.1 transcript accumulates at the epidermis and cortex of mature roots (Nazoa et al., 2003), and is greatly affected by several environmental factors. The expression of AtNRT2.1 is induced by NO3−, downregulated by high nitrogen status via downstream nitrogen metabolites such as NH4+ and amino acids, and positively regulated by sugars and light (Lejay et al., 1999; Nazoa et al., 2003). The positive regulation of AtNRT2.1 by light is achieved indirectly via the reduced repression of AtNRT2.1 by AtNRT1.1, which is mediated by LONG HYPOCOTYL5 (HY5) and HY5 HOMOLOGUE (HYH; Jonassen et al.,

TRANSPORTERS INVOLVED IN NITROGEN UPTAKE AND MOVEMENT

2008). AtNRT2.1 transcript levels are positively correlated with NO3− HATS activity, suggesting that high-affinity NO3− uptake is affected by the transcriptional regulation of AtNRT2.1. As already stated, AtNAR2.1 expression closely parallels that of AtNRT2.1, and it is also repressed by negative feedback from nitrogen metabolites (Krouk et al., 2006). NRT2.1 expression is upregulated by NO3− starvation in wild-type plants and by nitrogen limitation in a NO3− reductase-deficient mutant when grown on NO3− as the sole nitrogen source. Thus, AtNRT2.1 is feedback repressed by the downstream nitrogen metabolites of NO3− reduction. This is not the case for AtNRT1.1, which is not likely to be regulated by the presence of NO3− reductase, but by plant nitrogen status (Lejay et al., 1999). For AtNRT2.1, gene expression is regulated by a cis-acting 150 bp element upstream of the promoter TATA box, which is able to confer regulation to a minimal promoter (Girin et al., 2007). Split-root experiments demonstrate that NO3− activation occurs locally while metabolite-mediated repression is a function of whole-plant nitrogen status. Even sucrose regulation of NRT2.1 is mediated by this element, implying a potential interaction between nitrogen and carbon signaling, and indeed several motifs have been identified within the region that correspond to the regulation of nitrogen and carbon status (Girin et al., 2007). Using a novel systems biology approach, a subnetwork of genes regulated by the downstream metabolites of nitrogen was identified, with the master clock control gene CCA1 involved in the regulation of central nitrogen metabolism enzymes (Gutiérrez et al., 2007). This suggests that nitrogen signaling could influence the endogenous clock of the plant, representing a complex coordination of gene expression.

199

Posttranslational regulation of NO3− transporters The phosphorylation of AtNRT1.1 is controlled by the plant in response to changes in external NO3− concentrations encountered by the root (Liu and Tsay, 2003). The ability of AtNRT1.1 to switch from a high-affinity to a low-affinity transporter is due to the dephosphorylation of the T101 residue (see Fig. 10.2). By using an uptake- and sensingdecoupled mutant, AtNRT1.1 has been shown to function as a NO3− sensor. Two nitrate-inducible calcineurin B-like interacting protein kinases (CIPK8 and CIPK23) have been identified from transcriptome analysis of the AtNRT1.1 mutant (Ho et al., 2009; Hu et al., 2009). A low-level primary response to NO3− is maintained in AtNRT1.1 via the phosphorylation of T101 by a CIPK23 (Kolukisaoglu et al., 2004), enabling NRT1.1 to sense a wide range of NO3− concentrations (Ho et al., 2009). Several NRT and NO3− regulated genes require the protein kinase CIPK8 and a nodule-inception (NIN)-like protein, NLP7, for complete induction by NO3−. The CIPKs are likely to partake in a range of molecular responses to NO3− and clearly implicate calcium signaling in the sensing response. Indeed, CIPK8 has been shown to transduce the NO3− signal in the LATS (Hu et al., 2009). Both kinases are involved in early responses to nitrate, but CIPK23 is a negative controller for the highaffinity response while CIPK8 is a positive regulator of the low-affinity response. Phosphorylation events are known to regulate activity of the dual-affinity transporter AtNRT1.1 in response to environmental cues, and a similar mechanism has been suggested for the regulation of NRT2.1 (Liu and Tsay, 2003). Indeed, a number of conserved protein kinase C recognition motifs are observed in the N- and C-terminal domains of HvNRT2.1 (Forde, 2000). The

200

NUTRIENT USE EFFICIENCY IN CROPS

NRT1.1 high [NO3-]

NRT1.1 low [NO3-] NO3− NO3− HA

CIPK23

LA

NO3−

NO3−

NO3−

NO3−

NRT1.1T101A mutant low [NO3-] high [NO3-]

NO3−

NO3− HA

T101

LA

T101

NO3− HA

LA HA

T101A

HA

LA

T101A

P

P

Cytoplasm −

Schematic representation of NRT1.1 NO3 sensing mediated by CIPK23 (redrawn from Ho et al., 2009). NRT1.1 (oval) is located at the plasma membrane. Small circles indicate the high-affinity (HA) binding site, filled gray when NO3− is bound. Large circles represent the low-affinity (LA) binding site, filled dark gray when NO3− is bound. P denotes phosphorylation of the T101 residue, where the size of P reflects the level of NRT1.1T101 phosphorylation. Fig. 10.2.

presence of several different forms of the AtNRT2.1 protein in the plasma membrane, all of which are likely to rely on posttranslational modification in response to environmental cues, suggests that each could have a specific function (Wirth et al., 2007). Interestingly, no rapid changes in abundance of AtNRT2.1 are detected when the plant is subjected to light, sucrose, or nitrogen treatments, which are known to strongly affect NRT2.1 transcript levels and HATS activity (Wirth et al., 2007). Thus, it is likely that posttranslational modification generates the different forms of NRT2.1 observed at the plasma membrane. Sequence analysis of the NRT2s has identified some possible 143-3 regulatory sites; this is particularly interesting because of the role of these proteins in the regulation of key nitrogen assimilatory enzymes. For example, the C-terminus of the tobacco NRT2.1 gene has a perfect 14-3-3-binding consensus (Miller et al., 2007).

Vacuolar nitrate transport Nitrate storage in the vacuole is important for osmotic balance and as a nitrogen reserve (Van der Leij et al., 1998). Tissue levels are indicators of nitrogen status and this is the basis of tissue testing for crops. Land plants accumulate NO3− in the vacuole, but aquatic plants may have either lost this ability or never developed it (Miller et al., 2007). Giant algal cells did not accumulate NO3− in the vacuole above passive transport levels even after growing in high NO3− concentrations for many months (Miller and Zhen, 1991). Seven members of the CLC gene family have been identified in the Arabidopsis genome and disruption of one of these genes was shown to alter the accumulation of NO3− in leaf tissues (reviewed in De Angeli et al., 2009a). Green fluorescent protein (GFP)tagging of this CLC protein has localized it to the tonoplast, where it is implicated in the storage and remobilization of NO3− and

TRANSPORTERS INVOLVED IN NITROGEN UPTAKE AND MOVEMENT

chloride in the vacuole. The vacuolar NO3− transport activity of this CLC depends on cytoplasmic adenosine triphosphate (ATP) concentrations, and a specific binding site has been identified in the C-terminus of the protein (De Angeli et al., 2009b). Vacuolar transporters are important for understanding and manipulating NUE in crops. Nitrate storage pools in the leaves and stems of crop plants are important for several quite different reasons. First, increased storage in tissue enables overwintered crops to intercept soil NO3− that would otherwise cause environmental damage through leaching in rainwater. Second, when leafy vegetables are eaten there is medical evidence to suggest that high tissue NO3− concentrations are damaging, although low concentrations may actually be beneficial to human health (Leifert and Golden, 2000). The family may have a role in salt tolerance as these genes can also transport chloride. Although the Arabidopsis mutant was deficient in tissue NO3− accumulation, the vacuolar concentrations of NO3− relative to wild type suggest that there are other tonoplast transporters that contribute to loading NO3− into the vacuole. One member of the NRT2 family was found localized to the vacuolar membrane, and this transporter was specifically expressed in the developing seed. Mutant plants deficient in this transporter, AtNRT2.7, did not accumulate NO3− in the seed and showed delayed germination (Chopin et al., 2007a). Nitrate efflux Although plants invest considerable energy in the uptake of NO3−, there can be considerable loss or efflux from cells (Aslam et al., 1996). Despite being an obvious target for improving nitrogen uptake by plants, efflux systems for NO3− have been much less studied than influx systems, although a gene family was identified with activity linked to pH acidification (Segonzac et al., 2007).

201

Aquaporins and anion channels may also be important for efflux, and xylem loading of NO3− for long-distance transport is an example of this process. Concentrations of NO3− in the xylem sap can be quite high (10–30 mM), especially in plants that transport most of the NO3− taken up by roots to the shoot for reduction. The entry of NO3− into the xylem can be mediated by anion channels, assuming cytosolic NO3− concentrations and membrane potentials in xylem parenchyma cells similar to those measured in root epidermal and cortical cells. Genome analysis has identified several different anion channel families that may fulfill this function (De Angeli et al., 2009a). There is a clear diurnal change in xylem sap concentrations, related to changes in the transpiration rate. Nitrate efflux is a component of the signaling pathway leading to pathogen defense responses and hypersensitive cell death in tobacco (Wendehenne et al., 2002). Chemicals that inhibit NO3− efflux inhibitors reduced and delayed hypersensitive cell death and decreased or completely suppressed the induction of several defenserelated genes in tobacco. These results indicated that anion channels are involved intimately in NO3− release from cells and in mediating cell defense responses. Ammonium transporters The high-affinity uptake of ammonium by plants is mediated by membrane proteins of the AMT1 and AMT2 subfamilies (Loqué and von Wirén, 2004). These proteins were first characterized by expression in yeast cells, but later their function has been confirmed by the characterization of Arabidopsis mutants deficient in the proteins. The membrane proteins can function as homo- or hetero-oligmeric complexes, and they mediate the uniport of NH4+ (Ludewig et al., 2003). The accumulation of NH4+ in tissues can be toxic, and to avoid this problem a

202

NUTRIENT USE EFFICIENCY IN CROPS

rapid shut-off mechanism is required. Allosteric regulation of the function of an Arabidopsis AMT was shown to be mediated by the cytosolically located carboxy terminus (Loqué et al., 2007). Mutations in this C-terminal domain, which is conserved between bacteria, fungi, and plants, lead to loss of transport activity. In plants, the AMTs mediate uptake at the cell plasma membrane, where they function not only in nitrogen acquisition from the soil, but also in the recovery of leaked NH4+, which can efflux from the cell as ammonia under certain conditions. Ammonium uptake by roots Like nitrate, the uptake of NH4+ at the plasma membrane by plant root cells can be decreased by increasing the external supply of amino acids and NO3− (e.g., Lee et al., 1992; Rawat et al., 1999). In most agricultural soils, plant roots are usually exposed to more NO3− than NH4+ (Miller et al., 2007). Ammonium uptake is mainly mediated by the AMT family of transporters and the regulation of this transport can occur by several different mechanisms. As this topic has been carefully reviewed in detail recently (Loqué and von Wirén, 2004), only a brief overview of some points that are relevant to transport and nitrogen status is presented here. In Arabidopsis, the AMT family is largely responsible for root uptake of ammonium. By using T-DNA insertion Arabidopsis plants, it was shown that AtAMT1;1 mediates 30% of the total ammonium uptake capacity (Kaiser et al., 2002). Similarly, a gene knockout line AtAMT1;3 also showed a 30% decrease in ammonium uptake and when double lines, deficient in both AMTs, were compared with wild-type plants, the changes were additive (Loqué et al., 2006). Feeding roots with glutamine and the use of a glutamine synthetase inhibitor has led to a model proposing that glutamine in the

cell altered transcription while cytosolic concentrations of NH4+ may posttranslationally regulate one AMT gene (Rawat et al., 1999). More generally, regulation of uptake occurs at the mRNA level and AMT transcripts are strongly dependent on the nitrogen status of the plant, but in Arabidopsis the pattern is different for individual family members. Some AMTs increase expression earlier during nitrogen deficiency while others increase after more prolonged starvation (Loqué and von Wirén, 2004). Split-root experiments have suggested that the local root, rather than whole-plant, nitrogen status regulates the expression of an NH4+ transporter (Gansel et al., 2001). This result was different for a NO3− transporter where the nitrogen status of the whole plant was important (Gansel et al., 2001). The expression of transporters for both NH4+ and NO3− can be stimulated by photosynthesis, and this occurs by changes in the availability of sugars (Lejay et al., 2003). Tobacco plants with 35S promoter-driven expression of an Arabidopsis AMT showed a 30% increase in root uptake of NH4+ relative to wild-type plants when grown hydroponically (Yuan et al., 2007b). However, on soil supplemented with NH4+ as a nitrogen source, these plants showed neither growth nor nitrogen acquisition differences from wild-type plants (Yuan et al., 2007b). Despite expression being driven by the 35S promoter in these tobacco plants, the steady-state transcripts for AtAMT1;1 were not constitutive, changing with nitrogen status of the plants and decreasing after NO3− or NH4+ addition to nitrogen-deficient roots. This result suggests that the nitrogen status of a plant may directly influence mRNA turnover in plants, and this may provide another regulatory mechanism for NH4+ uptake. This result seems to contrast with that for NO3− transporter transcripts. In tobacco, the expression of NpNRT2.1 driven by rolD or the 35S promoters was high, even after treatment with

TRANSPORTERS INVOLVED IN NITROGEN UPTAKE AND MOVEMENT

10 mM NO3−, when the wild-type endogenous gene was repressed (Fraisier et al., 2000). In Arabidopsis, the posttranslational regulation of AtAMT1;1 provides a rapid way of regulating NH4+ transport activity that can be linked to nitrogen supply by the phosphorylation of the C-terminus (Loqué et al., 2007). Furthermore, the differing affinities for NH4+, together with the spatial arrangement of AMT family members, provide a coordinated system for uptake in the root (Yuan et al., 2007a). AtAMT1;1 and AtAMT1;3 are located in the plasma membrane of the rhizodermal cell layer. AtAMT1;2 was expressed in the plasma membrane of endodermis and root cortex cells and therefore mediates the uptake of ammonium entering the root via an apoplasmic transport route. While AtAMT1;5 was expressed in nitrogen-deficient rhizodermal and root hair cells, it also provides a secondary route for entry. Using gene knockout mutants, the in planta NH4+ influx substrate affinities of AMT1;1, AMT1;2, AMT1;3, and AMT1;5 were measured as 50, 234, 61, and 4.5 μM, respectively (Yuan et al., 2007a). The higher affinity for NH4+ of AtAMT1;5 fits with the idea that it is expressed under nitrogen-deficient conditions. Together this arrangement of the Arabidopsis AMTs provides an efficient uptake system for NH4+ in roots. Vacuolar ammonia transport Aquaporins are involved in ammonia transport (Niemietz and Tyerman, 2000; Jahn et al., 2004), and yeast cell expression of two Arabidopsis aquaporins was found to give tolerance to methylammonium, a toxic analog of NH4+ (Loqué et al., 2005). These aquaporins belong to the tonoplast intrinsic protein (TIP) subfamily, and when expressed in Xenopus oocytes, they increased 14 C-methylammonium accumulation with increasing external pH relative to controls.

203

The Arabidopsis TIP-mediated methylammonium detoxification in yeast depended on a functional vacuole, and the subcellular localization of the GFP-fusion proteins on the tonoplast in planta. Taken together these results suggest that these tonoplast aquaporins can mediate the vacuolar transport of ammonia. The transcript levels of both AtTIPs were influenced by nitrogen supply but did not follow those of the nitrogenderepressed ammonium transporter gene AtAMT1;1. However, transgenic Arabidopsis plants overexpressing one of these tonoplast aquaporins, AtTIP2;1, did not show altered NH4+ accumulation in roots after NH4+ supply (Loqué et al., 2005). As discussed above, the equilibrium between ammonia and NH4+ results in the passive accumulation within more acid cellular compartments, such as the vacuole. Decreasing vacuolar pH to give increased acid trapping has even been proposed as a route for genetically engineering increased nitrogen storage in crops to improve NUE (Wood et al., 2006). Transport of NH4+ in the peribacteroid membrane Soil bacteria of the various species of Rhizobium infect roots and induce the formation of nodules. Within certain cells of the nodules, the N2-fixing bacteria differentiate into bacteroids that are surrounded by a plant-derived peribacteroid membrane (PBM) forming an organelle-like structure, the symbiosome. Like the tonoplast surrounding the cellular vacuole, the PBM surrounds an acidic compartment. The PBM controls nutrient exchange between the bacteroids and the host cell that is mainly fixed nitrogen in the form of NH3 or NH4+ from the bacteroids, and reduced carbon from the plant. In these leguminous plants, the membrane that surrounds the acidic peribacteroid compartment has a high NH4+/NH3 transport

204

NUTRIENT USE EFFICIENCY IN CROPS

activity. The transport of NH3 and NH4+ across the PBM has been shown to be mediated by both aquaporins (Niemietz and Tyerman, 2000) and specific channels that were blocked by Mg2+ on the symbiosome lumen side (Obermeyer and Tyerman, 2005). Plastid transport Transport across the plastid envelope is important because it provides the linking point between carbon and nitrogen assimilation. There must be good coordination between the import of nitrite and carbon skeletons into the chloroplast, and these steps provide a possible key site for regulation of the processes (see Fig. 10.3). For example, mutants of the PII protein appear to be altered in chloroplast nitrite transport uptake (Ferrario-Méry et al., 2008). PII protein is a nuclear-encoded plastid protein that regulates the activity of a key enzyme for the biosynthesis of the amino acid argi-

nine. The NH4+ assimilating enzyme glutamine synthetase is found in the cytoplasm, chloroplast, and even the mitochondria, and so transport of NH4+ into the chloroplast may be less important as a site for regulation of carbon and nitrogen assimilation. The genes encoding the route for entry of NH4+ into the plastid have not been demonstrated, but they could belong to either the AMT or TIP families already identified (see above). Several candidate genes for the plastid nitrite transporter have been identified in different species. For example, in algae a formate/ nitrite transporter (Mariscal et al., 2006) and in Arabidopsis a member of the NRT1/PTR family (Sugiura et al., 2007) have been identified, but still a clear demonstration of function is needed. Plants that are defective in plastidic nitrite transport are likely to have a strong nitrogen-deficient phenotype when grown with only NO3− as the nitrogen source. Amino acid selective channels have been identified in the plastid envelope and are important for exchange between the cyto-

NO3– = 0.1–10 mM

Cell

NO3– = 3–5 mM

pH 7.2

Vacuole pH 5.5

H+ Proton pump

ATP

NO3– = 40–120 mM

ADP + Pi Cytosol

Storage

Plastid

Influx NO3– (HATS/LATS)

NO3– Efflux

NR

NO2–

Reduction

NO2–

NiR

NO4+

GS

Amino GOGAT Acids

Xylem Loading Fig. 10.3. A schematic representation of NO3− uptake, transport, and efflux in the root. ADP, adenosine diphosphate; GOGAT, glutamate synthase; NR, nitrate reductase; NiR, nitrite reductase; GS, glutamine synthetase.

TRANSPORTERS INVOLVED IN NITROGEN UPTAKE AND MOVEMENT

plasm and plastid lumen. Intracellular membrane transporters systems have recently been reviewed (see Linka and Weber, 2010). Amino acid transporters Organic nitrogen molecules can be important soil sources for some plants (Miller and Cramer, 2004; Nasholm et al., 2009), and within the plant the long-distance phloemand xylem-transported nitrogen form is usually amino acids or peptides. Excluding the mitochondrial and plastidic transporters, the Arabidopsis genome contains more than 50 genes potentially involved in amino acid transport. These transporters have been classified into two major groups: the amino acid transporter family (ATF) superfamily and the amino acid polyamine choline (APC) transporters (Wipf et al., 2002; Lalonde et al., 2004). The best characterized group of transporters belong to the amino acid permease (AAP) subfamily within the ATF superfamily. In Arabidopsis, AtAAP1 to AtAAP6 and AtAAP8 have been heterologously expressed in yeast and Xenopus oocytes, and they have been shown to transport neutral amino acids and glutamate (Fischer et al., 2002; Okumoto et al., 2002). In root cells, AtAAP1 has a role in acquiring nitrogen from the rhizosphere (Lee et al., 2007), and mutants have been used to demonstrate that amino acids can be an important nitrogen source (Svennerstam et al., 2007). Although the beneficial formation of mycorrhizal associations for phosphorus acquisition by roots is clear for some plants (see Chapter 2), it is less clear for nitrogen (Reynolds et al., 2005), although fungal amino acid transporters that mediate uptake (Wipf et al., 2002), but not transfer to the host plant, have been identified. The importance of soil amino acids as a nitrogen source in natural habitats is well established, and for agricultural crops the topic is worthy of further investigation. Amino acid transport

205

is also important during seed storage and senescence as this organic form is transported around the plant, but the evidence that transport can limit these commercially important processes is limited. Delayed senescence by limiting remobilization may be one way of modifying crop NUE (see Masclaux-Daubresse et al., 2010). Conclusions and future prospects The transport systems for nitrate and ammonium in Arabidopsis are well characterized and the use of gene knockout has been particularly useful for identifying the physiological function of specific genes. This information now enables the rapid identification of equivalent genes in other species. For example, the design of PCR primers designed for conserved parts of the genes can be used to find homologs in other species. The key question is how to use and apply this information to improve NUE. Cultivars with different nitrogen uptake efficiencies can be compared to identify important transporters; these targets may change at different developmental stages. For example, early during vegetative growth the root uptake systems are important for acquisition, but they may also be important during seed filling when late root uptake can influence seed quality, a parameter that is particularly important for seed protein crops. This type of comparison is likely to provide molecular information that can be used to identify management strategies and breeding markers to improve this trait in crops. Another approach may be to learn from the nitrogen ecology and physiology of native species. An understanding of the pattern of nitrogen uptake in plants that grow in nutrient-poor soils can give important insights into strategies for improving nitrogen uptake efficiency by crops. Another area where there is a large gap in our understand-

206

NUTRIENT USE EFFICIENCY IN CROPS

ing is the linking of steps between changes in nitrogen supply and altered levels of growth effectors such as hormones. For example, although the role of auxins and cytokinins in nitrogen root responses are known, and transporter proteins such as AtNRT1.1 and AtNRT2.1, together with CIPKs are the sensors for changes in nitrate availability at the root surface, the way in which the signaling cascade links these “receptors” and “effectors” is unknown. It may be possible to influence this sensing process to directly enhance nitrogen acquisition. Acknowledgments Rothamsted Research is grant-aided by the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. References Alboresi, A., Gestin, C., Leydecker, M.T., Bedu, M., Meyer, C., & Truong, H.N. (2005) Nitrate, a signal relieving seed dormancy in Arabidopsis. Plant, Cell & Environment 28, 500–512. Almagro, A., Lin, S.H., & Tsay, Y.-F. (2008) Characterization of the Arabidopsis nitrate transporter NRT1.6 reveals a role of nitrate in early embryo development. The Plant Cell 20, 3289–3299. Aslam, M., Travis, R.L., & Rains, D.W. (1996) Evidence for substrate induction of a nitrate efflux system in barley roots. Plant Physiology 112, 1167–1175. Cerezo, M., Tillard, P., Filleur, S., Munos, S., DanielVedele, F., & Gojon, A. (2001) Major alterations of the regulation of root NO3− uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis. Plant Physiology 127, 262–271. Chen, Y.F., Wang, Y., & Wu, W.H. (2008) Membrane transporters for nitrogen, phosphate and potassium uptake in plants. Journal of Integrative Plant Biology 50, 835–848. Chiu, C.-C., Lin, C.-S., Hsia, A.-P., Su, R.-C., Lin, H. -L., & Tsay, Y.-F. (2004) Mutation of a nitrate transporter, AtNRT1:4, results in a reduced petiole nitrate content and altered leaf development. Plant & Cell Physiology 45, 1139–1148.

Chopin, F., Orsel, M., Dorbe, M.-F., et al. (2007a) The Arabidopsis AtNRT2.7 nitrate transporter controls nitrate content in seeds. The Plant Cell 19, 1590–1602. Chopin, F., Wirth, J., Dorbe, M.F., et al. (2007b) The Arabidopsis nitrate transporter AtNRT2.1 is targeted to the root plasma membrane. Plant Physiology and Biochemistry 45, 630–635. Crawford, N.M. & Glass, A.D.M. (1998) Molecular and physiological aspects of nitrate uptake in plants. Trends in Plant Science 3, 389–395. De Angeli, A., Monachello, D., Ephritikhine, G., et al. (2006) The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. Nature 442, 939–942. De Angeli, A., Monachello, D., Ephritikhine, G., et al. (2009a) CLC-mediated anion transport in plant cells. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 364, 195–201. De Angeli, A., Moran, O., Wege, S., et al. (2009b) ATP binding to the C terminus of the Arabidopsis thaliana nitrate/proton antiporter, AtCLCa, regulates nitrate transport into plant vacuoles. Journal of Biological Chemistry 284, 26526–26532. Dietrich, D., Hammes, U., Thor, K., et al. (2004) AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis. Plant Journal 40, 488–499. Fan, S.-C., Lin, C.-S., Hsu, P.-K., Lin, S.-H., & Tsay, Y.-F. (2009) The Arabidopsis nitrate transporter NRT1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate. The Plant Cell 21, 2750–2761. Ferrario-Méry, S., Meyer, C., & Hodges, M. (2008) Chloroplast nitrite uptake is enhanced in Arabidopsis PII mutants. FEBS Letters 582, 1061–1066. Fischer, W.-N., Loo, D.D.F., Koch, W., et al. (2002) Low and high affinity amino acid H+-cotransporters for cellular import of neutral and charged amino acids. Plant Journal 29, 717–731. Forde, B.G. (2000) Nitrate transporters in plants: structure, function and regulation. Biochimica et Biophysica Acta 1465, 219–235. Fraisier, V., Gojon, A., Tillard, P., & Daniel-Vedele, F. (2000) Constitutive expression of a putative highaffinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source. Plant Journal 23, 489–496. Gansel, X., Muños, S., Tillard, P., & Gojon, A. (2001) Differential regulation of the NO3− and NH4+ transporter genes AtNrt2.1 and AtAmt1.1 in Arabidopsis: relation with long-distance and local controls by N status of the plant. Plant Journal 26, 143–155.

TRANSPORTERS INVOLVED IN NITROGEN UPTAKE AND MOVEMENT

Girin, T., Lejay, L., Wirth, J., et al. (2007) Identification of a 150 bp cis-acting element of the AtNRT2.1 promoter involved in the regulation of gene expression by the N and C status of the plant. Plant, Cell & Environment 30, 1366–1380. Guo, F.-Q., Wang, R.C., Chen, M.S., & Crawford, N.M. (2001) The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1. (CHL1) is activated and functions in nascent organ development during vegetative and reproductive growth. The Plant Cell 13, 1761–1777. Guo, F.-Q., Young, J., & Crawford, N.M. (2003) The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis. the Plant Cell 15, 107–117. Gutiérrez, R.A., Lejay, L.V., Dean, A., Chiaromonte, F., Shasha, D.E., & Coruzzi, G.M. (2007) Qualitative network models and genome-wide expression data define carbon/nitrogen-responsive molecular machines in Arabidopsis. Genome Biology 8, 13. Ho, C.-H., Lin, S.-H., Hu, H.-C., & Tsay, Y.-F. (2009) CHL1 functions as a nitrate sensor in plants. Cell 138, 1184–1194. Hu, H.-C., Wang, Y.-Y., & Tsay, Y.-F. (2009) AtCIPK8, a CBL-interacting protein kinase, regulates the lowaffinity phase of the primary nitrate response. Plant Journal 57, 264–278. Huang, N.C., Liu, K.H., Lo, H.J., & Tsay, Y.-F. (1999) Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low affinity uptake. The Plant Cell 11, 1381–1392. Jahn, T.P., Møller, A.L.B., Zeuthen, T., et al. (2004) Aquaporin homologues in plants and mammals transport ammonia. FEBS Letters 574, 31–36. Jeong, J., Suh, S., Guan, C., et al. (2004) A nodulespecific dicarboxylate transporter from alder is a member of the peptide transporter family. Plant Physiology 134, 969–978. Jonassen, E.M., Lea, U.S., & Lillo, C. (2008) HY5 and HYH are positive regulators of nitrate reductase in seedlings and rosette stage plants. Planta 227, 559–564. Kaiser, B.N., Rawat, S.R., Siddiqi, M.Y., Masle, J., & Glass, A.D.M. (2002) Functional analysis of an Arabidopsis T-DNA “knockout” of the high-affinity NH4+ transporter AtAMT1;1. Plant Physiology 130, 1263–1275. Kolukisaoglu, U., Weinl, S., Blazevic, D., Batistic, O., & Kudla, J. (2004) Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks. Plant Physiology 134, 43–58.

207

Komarova, N.Y., Thor, K., Gubler, A., et al. (2008) AtPTR1 and AtPTR5 transport dipeptides in planta. Plant Physiology 148, 856–869. Krapp, A., Fraisier, V., Scheible, W.R., et al. (1998) Expression studies of Nrt2:1Np, a putative highaffinity nitrate transporter: evidence for its role in nitrate uptake. Plant Journal 14, 723–731. Krouk, G., Tillard, P., & Gojon, A. (2006) Regulation of the high-affinity NO3− uptake system by NRT1.1mediated NO3− demand signaling in Arabidopsis. Plant Physiology 142, 1075–1086. Krouk, G., Lacombe, B., Bielach, A., et al. (2010) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Developmental Cell 18, 927–937. Lalonde, S., Wipf, D., & Frommer, W.B. (2004) Transport mechanisms for organic forms of carbon and nitrogen between source and sink. Annual Review of Plant Biology 55, 341–372. Lee, Y.-H., Foster, J., Chen, J., Voll, L.M., Weber, A.P.M., & Tegeder, M. (2007) AAP1 transports uncharged amino acids into roots of Arabidopsis. Plant Journal 50, 305–319. Lee, R.B., Purves, J.V., Ratcliffe, R.G., & Saker, L. (1992) Nitrogen assimilation and the control of ammonium and nitrate absorption by maize roots. Journal of Experimental Botany 43, 1385–1396. Leifert, C. & Golden, M. (2000) A re-evaluation of the benficial and other effects of dietary nitrate. Proceedings of the International Fertiliser Society, International Fertiliser Society, 456. ISBN 978-0-85310-092-8. Lejay, L., Tillard, P., Lepetit, M., et al. (1999) Molecular and functional regulation of two NO3− uptake systems by N- and C-status of Arabidopsis plants. Plant Journal 18, 509–519. Lejay, L., Gansel, X., Cerezo, M., et al. (2003) Regulation of root ion transporters by photosynthesis: functional importance and relation with hexokinase. The Plant Cell 15, 2218–2232. Li, W., Wang, Y., Okamoto, M., Crawford, N.M., Siddiqi, M.Y., & Glass, A.D.M. (2007) Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate transporter gene cluster. Plant Physiology 143, 425–433. Lin, C.M., Koh, S., Stacey, G., Yu, S.M., Lin, T.Y., & Tsay, Y.F. (2000) Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice. Plant Physiology 122, 379–388. Lin, S.-H., Kuo, H.-F., Canivenc, G., et al. (2008) Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. The Plant Cell 20, 2514–2528.

208

NUTRIENT USE EFFICIENCY IN CROPS

Linka, N. & Weber, A.P.M. (2010) Intracellular metabolite transporters in plants. Molecular Plant 3, 21–53. Little, D.Y., Rao, H.Y., Oliva, S., Daniel-Vedele, F., Krapp, A., & Malamy, J.E. (2005) The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues. Proceedings of the National Academy of Sciences of the United States of America 102, 13693–13698. Liu, K.H., Huang, C.Y., & Tsay, Y.F. (1999) CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. The Plant Cell 11, 865–874. Liu, K.-H. & Tsay, Y.-F. (2003) Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO Journal 22, 1005–1013. Loqué, D. & von Wirén, N. (2004) Regulatory levels for the transport of ammonium in plant roots. Journal of Experimental Botany 55, 1293–1305. Loqué, D., Tillard, P., Gojon, A., & Lepetit, M. (2003) Gene expression of the NO3− transporter NRT1.1 and the nitrate reductase NIA1 is repressed in Arabidopsis roots by NO2−, the product of NO3− reduction. Plant Physiology 132, 958–967. Loqué, D., Ludewig, U., Yuan, L., & von Wirén, N. (2005) Tonoplast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole. Plant Physiology 137, 671–680. Loqué, D., Yuan, L., Kojima, S., et al. (2006) Additive contribution of AMT1;1 and AMT1;3 to highaffinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots. Plant Journal 48, 522–534. Loqué, D., Lalonde, S., Looger, L.L., von Wirén, N., & Frommer, W.B. (2007) A cytosolic trans-activation domain essential for ammonium uptake. Nature 446, 195–198. Ludewig, U., Wilken, S., Wu, B., et al. (2003) Homoand hetero-oligomerization of ammonium transporter-1 NH4+ uniporters. Journal of Biological Chemistry 278, 45603–45610. Mariscal, V., Moulin, P., Orsel, M., Miller, A.J., Fernández, E., & Galván, A. (2006) Differential regulation of the Chlamydomonas Nar1 gene family by carbon and nitrogen. Protist 157, 421–433. Martin, Y., Navarro, F.J., & Siverio, J.M. (2008) Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha. Plant Molecular Biology 68, 215–224. Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., Chardon, F., Gaufichon, L., & Suzuki, A. (2010)

Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Annals of Botany 105, 1141–1157. Miller, A.J. & Cramer, M.D. (2004) Root nitrogen acquisition and assimilation. Plant and Soil 274, 1–36. Miller, A.J. & Zhen, R.-G. (1991) Measurements of intracellular nitrate concentrations in Chara using nitrate-selective microelectrodes. Planta 187, 47–52. Miller, A.J., Fan, X., Orsel, M., Smith, S.J., & Wells, D.M. (2007) Nitrate transport and signalling. Journal of Experimental Botany 58, 2297–2306. Miller, A.J., Shen, Q., & Xu, G. (2009) Freeways in the plant: transporters for N, P and S and their regulation. Current Opinion in Plant Biology 12, 284–290. Muller, B. & Touraine, B. (1992) Inhibition of NO3− uptake by various phloem-translocated amino acids in soybean seedlings. Journal of Experimental Botany 43, 617–623. Nasholm, T., Kielland, K., & Ganeteg, U. (2009) Uptake of organic nitrogen by plants. New Phytologist 182, 31–48. Nazoa, P., Vidmar, J.J., Tranbarger, T.J., et al. (2003) Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana: responses to nitrate, amino acids and developmental stage. Plant Molecular Biology 52, 689–703. Niemietz, C.M. & Tyerman, S.D. (2000) Channelmediated permeation of ammonia gas through the peribacteroid membrane of soybean nodules. FEBS Letters 465, 110–114. Obermeyer, G. & Tyerman, S.D. (2005) NH4+ currents across the peribacteroid membrane of soybean. Macroscopic and microscopic properties, inhibition by Mg2+, and temperature dependence indicate a subpico Siemens channel finely regulated by divalent cations. Plant Physiology 139, 1015–1029. Okamoto, M., Kumar, A., Li, W., et al. (2006) Highaffinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1. Plant Physiology 140, 1036–1046. Okumoto, S., Schmidt, R., Tegeder, M., et al. (2002) High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis. Journal of Biological Chemistry 277, 45338–45346. Orsel, M., Krapp, A., & Daniel-Vedele, F. (2002) Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression. Plant Physiology 129, 886–896. Orsel, M., Eulenburg, K., & Krapp, A. (2004) Disruption of the nitrate transporter genes AtNRT2.1 and

TRANSPORTERS INVOLVED IN NITROGEN UPTAKE AND MOVEMENT

AtNRT2.2 restricts growth at low external nitrate concentration. Planta 219, 714–721. Orsel, M., Chopin, F., Leleu, O., et al. (2006) Characterisation of a two component high affinity nitrate uptake system in Arabidopsis; physiology and protein-protein interaction. Plant Physiology 142, 1304–1317. Orsel, M., Chopin, F., Leleu, O., et al. (2007) Nitrate signaling and the two component high affinity uptake system in Arabidopsis. Plant Signaling & Behavior 2(4), 260–262. Rawat, S.R., Silim, S.N., Kronzucker, H.J., Siddiqi, M.Y., & Glass, A.D.M. (1999) AtAMT1 gene expression and NH4+ uptake in roots of Arabidopsis thaliana: evidence for regulation by root glutamine levels. Plant Journal 19, 143–152. Remans, T., Nacry, P., Pervent, M., et al. (2006a) The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches. Proceedings of the National Academy of Sciences of the United States of America 103, 19206–19211. Remans, T., Nacry, P., Pervent, M., et al. (2006b) A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis. Plant Physiology 140, 909–921. Reynolds, H., Hartley, A., Vogelsang, K., Bever, J., & Schultz, P. (2005) Arbuscular mycorrhizal fungi do not enhance nitrogen acquisition and growth of old-field perennials under low nitrogen supply in glasshouse culture. New Phytologist 167, 869–880. Segonzac, C., Boyer, J.-C., Ipotesi, E., et al. (2007) Nitrate efflux at the root plasma membrane: identification of an Arabidopsis excretion transporter. The Plant Cell 19, 3760–3777. Stacey, M., Osawa, H., Patel, A., Gassmann, W., & Stacey, G. (2006) Expression analyses of Arabidopsis oligopeptide transporters during seed germination, vegetative growth and reproduction. Planta 223, 291–305. Sugiura, M., Georgescu, M.N., & Takahashi, M. (2007) A nitrite transporter associated with nitrite uptake by higher plant chloroplasts. Plant & Cell Physiology 48, 1022–1035. Svennerstam, H., Ganeteg, U., Bellini, C., & Nasholm, T. (2007) Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids. Plant Physiology 143, 1853–1860. Tong, Y., Zhou, J.-J., Li, Z., & Miller, A.J. (2005) A two-component high-affinity nitrate uptake system in barley. Plant Journal 41, 442–450.

209

Tsay, Y.F., Schroeder, J.I., Feldmann, K.A., & Crawford, N.M. (1993) The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell 72, 705–713. Tsay, Y.-F., Chiu, C.C., Tsai, C.B., Ho, C.H., & Hsu, P.K. (2007) Nitrate transporters and peptide transporters. FEBS Letters 581, 2290–2300. Van der Leij, M., Smith, S.J., & Miller, A.J. (1998) Remobilisation of vacuolar stored nitrate in barley root cells. Planta 205, 64–72. Vidal, E.A. & Gutiérrez, R.A. (2008) A systems view of nitrogen nutrient and metabolite responses in Arabidopsis. Current Opinion in Plant Biology 11, 521–529. Walch-Liu, P. & Forde, B.G. (2008) Nitrate signalling mediated by the NRT1.1 nitrate transporter antagonises L-glutamate-induced changes in root architecture. Plant Journal 54, 820–828. Wendehenne, D., Lamotte, O., Frachisse, J.M., BarbierBrygoo, H., & Pugin, A. (2002) Nitrate efflux is an essential component of the cryptogein signaling pathway leading to defense responses and hypersensitive cell death in tobacco. The Plant Cell 14, 1937–1951. Williams, L.E. & Miller, A.J. (2001) Transporters responsible for the uptake and partitioning of nitrogenous solutes. Annual Review of Plant Physiology and Plant Molecular Biology 52, 659–688. Wipf, D., Benjdia, M., Tegeder, M., & Frommer, W.B. (2002) Characterization of a general amino acid permease from Hebeloma cylindrosporum. FEBS Letters 528, 119–124. Wirth, J., Chopin, F., Santoni, F., et al. (2007) Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana. Journal of Biological Chemistry 282, 23541–23552. Wood, C.C., Poree, F., Dreyer, I., Koehler, G.J., & Udvardi, M. (2006) Mechanisms of ammonium transport, accumulation, and retention in oocytes and yeast cells expressing Arabidopsis AtAMT1;1. FEBS Letters 580, 3931–3936. Yin, L.P., Li, P., Wen, B., Taylor, D., & Berry, J.O. (2007) Characterization and expression of a highaffinity nitrate system transporter gene (TaNRT2.1) from wheat roots, and its evolutionary relationship to other NTR2 genes. Plant Science 172, 621–631. Yong, Z., Kotur, Z., & Glass, A.D.M. (2010) Characterization of an intact two-component highaffinity nitrate transporter from Arabidopsis roots. Plant Journal 63, 739–748. Yuan, L., Loqué, D., Kojima, S., et al. (2007a) The organization of high-affinity ammonium uptake in Arabidopsis roots depends on the spatial

210

NUTRIENT USE EFFICIENCY IN CROPS

arrangement and biochemical properties of AMT1type transporters. The Plant Cell 19, 2636–2652. Yuan, L., Loqué, D., Ye, F., Frommer, W.B., & von Wirén, N. (2007b) Nitrogen-dependent posttranscriptional regulation of the ammonium transporter AtAMT1;1. Plant Physiology 143, 732–744. Zhao, X.Q., Li, Y.J., Liu, J.Z., et al. (2004) Isolation and expression analysis of a high-affinity nitrate transporter TaNRT2.3 from roots of wheat. Acta Botanica Sinica 46, 347–354.

Zhou, J.J., Theodoulou, F.L., Muldin, I., Ingemarsson, B., & Miller, A.J. (1998) Cloning and functional characterization of a Brassica napus transporter which is able to transport nitrate and histidine. Journal of Biological Chemistry 273, 12017–12033. Zhou, J.J., Fernández, E., Galván, A., & Miller, A.J. (2000) A high affinity nitrate transport system from Chlamydomonas requires two gene products. FEBS Letters 466, 225–227.

Chapter 11

Crop Improvement for Nitrogen Use Efficiency in Irrigated Lowland Rice Shaobing Peng

Abstract

Introduction

There is an urgent need to develop superior nitrogen-efficient rice varieties because of poor fertilizer nitrogen use efficiency (NUE), associated economic and environmental concerns, and the lack of adoption of improved nitrogen management practices. Genotypic variation in NUE has been reported for rice in many studies. The physiological mechanisms underlying both nitrogen uptake efficiency and utilization efficiency are fairly well understood. Plant traits that are closely associated with NUE have been identified. In general, there is a positive relationship between grain yield and NUE, suggesting that it is possible to develop rice varieties with both high grain yield and high NUE. An empirical breeding method can be used for developing nitrogenefficient varieties using existing genotypic variation in NUE. However, for significant improvement in NUE, new breeding techniques such as the development of F1 hybrids, marker-aided selection, transformation, and genetic engineering should be considered.

Nitrogen plays a key role in rice yield formation because plant nitrogen status affects leaf photosynthesis and the development of components of grain yield (Yoshida, 1981). Leaf nitrogen concentration determines the size of the photosynthetic apparatus by affecting leaf area development and tiller production, and the rate of photosynthesis per unit leaf area by affecting Rubisco content and leaf senescence (Peng and Ismail, 2004). The lack of adequate nitrogen in plant nutrition is a major constraint to rice production around the world (Mikkelsen et al., 1995). Nitrogen deficiency reduces plant height, tillering, leaf area index, leaf area duration, and crop photosynthesis, which lead to lower radiation interception and lower radiation use efficiency (Fageria et al., 2003). Rice crops absorb nitrogen from both indigenous nitrogen sources and nitrogen fertilizers. Soil is the principal source of nitrogen and it can provide more than half of the total nitrogen requirement for the rice crop even when nitrogen fertilizer is applied at high rates (Kundu and

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 211

212

NUTRIENT USE EFFICIENCY IN CROPS

Ladha, 1995). Cassman (1999) stated that increased rice production in the last 40 years was largely attributed to the increased use of nitrogen fertilizers, which was supported by Food and Agriculture Organization (FAO) statistics (Fig. 11.1a, b). In intensive rice production systems, nitrogen fertilizer is the most expensive input for rice farming (Mae, 1997). In 2006, annual nitrogen fertilizer consumption for rice production was 15.8 million metric tons or 16.1% of the global nitrogen consumption (Heffer, 2008). Fertilizer nitrogen use efficiency (NUE) is relatively low in irrigated lowland rice due to rapid nitrogen losses from ammonia volatilization, denitrification, surface runoff, and leaching in the soil–floodwater system (De Datta and Buresh, 1989). In general, ammonia volatilization is the major pathway of nitrogen loss in irrigated lowland rice (Zhu, 1997). The magnitude and nature of nitrogen losses vary depending on the timing, rate, and method of nitrogen application, source of nitrogen fertilizer, soil chemical, and physical properties, climatic conditions, and crop status. The nitrogen losses in crop production cause severe environmental consequences such as groundwater contamination (caused by nitrate leaching from soil), eutrophication of lakes and rivers (due to surface runoff and seepage of nitrogen from rice fields), and acid rain (caused by ammonia volatilization). Denitrification contributes to global warming by emitting greenhouse gases such as nitrous oxide (N2O). Improving fertilizer NUE will play an important role in protecting the environment. A further increase in rice production has to be achieved with less nitrogen fertilizer by improving fertilizer NUE through better nitrogen fertilizer management and developing new rice varieties. In the past, research on improving fertilizer NUE of the rice crop has focused on the development of fertilizer management strategies to reduce nitrogen losses and increase nitrogen uptake (Cassman

et al., 1998). Great progress was achieved to reduce nitrogen losses by new application methods and modified nitrogen sources (De Datta, 1986). Nitrification and urease inhibitors (De Datta, 1986; Zhu, 1997), balanced fertilization (Zhu, 1997), and computerbased decision support systems such as MANAGE RICE (Angus et al., 1996) and MANAGE-N (ten Berge et al., 1997) were also effective in increasing fertilizer NUE. Another important research area is optimizing the timing and rate of nitrogen application for better synchronization between the supply and demand of nitrogen by the crop (Cassman et al., 1998). Sitespecific nitrogen management (Dobermann et al., 2002) and real-time nitrogen management (Peng et al., 1996) were developed to achieve a balance between crop nitrogen demand and fertilizer nitrogen supply. Across many sites in Asia, these two nitrogen management techniques save nitrogen fertilizer and increase grain yield and fertilizer NUE compared with the farmers’ fertilizer practices (Dobermann et al., 2002; Peng et al., 2006). NUE should be considered both when developing new varieties and when making fertilizer nitrogen recommendations (Samonte et al., 2006). Some efforts have been devoted to germplasm improvement for NUE, but the impact has been relatively small compared with the management approaches for increasing NUE. This is partially because NUE is seldom evaluated in breeding programs, although efficient nitrogen use is an important factor in the yielding capacity of varieties. This chapter will summarize the constraints and opportunities for increasing rice NUE through germplasm improvement. Definition of NUE and its components NUE can be separated into different component indices (Novoa and Loomis, 1981).

4.5

Grain yield (t ha–1)

4.0 3.5 3.0 2.5 2.0

A 1.5 1960

1970

1980

1990

2000

2010

120

N rate (kg ha–1)

100 80 60 40 20

B 0 1960

1970

1980

1990

2000

2010

140

PFP (kg kg–1)

120 100 80 60 40

C 20 1960

1970

1980

1990

2000

2010

Year Fig. 11.1. (a) Grain yield, (b) the rate of fertilizer nitrogen (N) input, and (c) partial factor productivity (PFP) of

applied fertilizer nitrogen of rice crop in the world from 1961 to 2007 based on FAO Statistics (2010). The nitrogen rate was calculated based on the total nitrogen consumption and planting area of world rice. Rice nitrogen consumption was estimated as 16% of the world’s total nitrogen consumption.

213

214

NUTRIENT USE EFFICIENCY IN CROPS

Table 11.1.

Component indices of nitrogen use efficiency and their typical ranges for a well-managed rice crop

Parameter

Symbol

Definition

Typical Range

Nitrogen recovery efficiency

RE

40–60%

Nitrogen utilization efficiency for grain production Nitrogen utilization efficiency for biomass production Agronomy nitrogen use efficiency Partial factor productivity of applied fertilizer nitrogen

NUEg

100 × Nitrogen uptake from fertilizer nitrogen/Fertilizer nitrogen input Grain yield/Total nitrogen uptake Aboveground dry matter/Total nitrogen uptake Grain yield increase/Fertilizer nitrogen input Grain yield/Fertilizer nitrogen input

NUEb AE PFP

Table 11.1 summarizes the most common NUE component indices with their typical ranges for a well-managed rice crop. All indices that have been used for studying NUE of rice genotypes can be divided into three groups: uptake efficiency, utilization efficiency, and NUE-related traits (Ladha et al., 2005). Different NUE indices can be used for different purposes. Recovery efficiency (RE, i.e., uptake efficiency) of applied nitrogen fertilizer is the percentage of nitrogen fertilizer recovered in aboveground plant biomass at the end of the cropping season. Both the N-difference and 15 N-dilution methods can be used to quantify RE, but the estimate is typically higher with the difference method than with the 15 N-dilution method (Ladha et al., 2005). This is because of the priming effect of nitrogen fertilization, stimulation of nitrogen mineralization in the presence of nitrogen fertilizer, and greater root exploration in fertilized plots (Fageria et al., 2003). The measurement of nitrogen fertilizer recovery in the aboveground biomass at maturity may underestimate RE because of gaseous nitrogen losses from the senescing leaves (Norman et al., 1992). RE is usually 30– 50% in the tropics (De Datta, 1986). The average RE is only 30% for irrigated rice in Asia (Dobermann and Fairhurst, 2000). At panicle initiation stage, when the root system

50–60 kg kg−1 90– 110 kg kg−1 25–30 kg kg−1 60–70 kg kg−1

is well developed and crop demand for nitrogen and the nitrogen uptake ability are high, the RE of topdressed nitrogen fertilizer could reach as high as 78% in the tropics (Peng and Cassman, 1998). Nitrogen utilization efficiency can be expressed as biomass or grain production per unit nitrogen uptake or application. The most widely used method for comparing different genotypes is nitrogen utilization efficiency for grain production (NUEg), which is the ratio of grain yield per unit nitrogen uptake at maturity. The quantification of NUEg does not need a zero-N plot. The average NUEg in lowland rice in the tropics is about 50 kg grain produced per kg nitrogen absorbed (Yoshida, 1981). It is generally believed that NUEg is about 20% higher in temperate regions than in the tropics when nitrogen rate is optimum (De Datta, 1986). When fertilizer nitrogen application is excessive and the rice crop is in a state of luxuriant consumption, NUEg decreases drastically. Other parameters of nitrogen utilization efficiency include nitrogen utilization efficiency for biomass production (NUEb) and photosynthetic NUE, which is the ratio of leaf photosynthetic rate to leaf nitrogen concentration per unit leaf area. Nitrogen harvest index (NHI) is the proportion of total plant nitrogen partitioned to the grain.

NITROGEN USE EFFICIENCY IN IRRIGATED LOWLAND RICE

Agronomic nitrogen use efficiency (AE) and partial factor productivity of applied nitrogen (PFP) are mainly used by agronomists to evaluate the NUE of different crop management practices. AE is calculated by the increase in grain yield per unit of applied nitrogen. The determination of AE requires a control plot to estimate the grain yield without fertilizer nitrogen application. Yoshida (1981) estimated AE to be 15–25 kg rough rice per kg applied nitrogen in the tropics. Cassman et al. (1996) reported that AE was 15–18 kg kg−1 N in the dry season in farmers’ fields in the Philippines. Peng et al. (2006) reported that AE of farmers’ nitrogen fertilizer practice was only 5–10 kg kg−1 N in China. PFP is calculated as the ratio of total grain output to fertilizer nitrogen input. The PFP reflects both the marginal efficiency from applied nitrogen and the ability to utilize indigenous nitrogen resources from the soil-floodwater system to produce grain (Cassman et al., 1996). In general, PFP decreases as nitrogen rate increases over the years (Fig. 11.1b, c). Compensation takes place among different components of NUE because of the interactions among traits related to NUE (Ladha et al., 2005). Interrelationships among components of NUE are not clearly understood (Tirol-Padre et al., 1996). Indigenous nitrogen supply capacity and nitrogen fertilizer input rate have a large influence on NUE. Growing conditions that govern plant growth and crop yield such as solar radiation, temperature, and availability of water and other nutrients also affect NUE. There is no common standard system for evaluating NUE of different genotypes, making it very difficult to compare results across studies. Physiological mechanisms underlying NUE Several factors can cause differences in nitrogen acquisition among rice genotypes

215

(Ladha et al., 1998). Genotypic differences existed in nitrogen uptake from various soil depths (Kundu and Ladha, 1995). Rice genotypes differed in their ability to stimulate plant associative N2 fixation (Ladha et al., 1998). Rice genotypes had different rhizosphere effects on the extent and pattern of soil nitrogen mineralization (Kundu and Ladha, 1997). These differences could be related to differences in rooting characteristics such as root surface area, root mass, root depth, root number, and root length density (Kirk and Bouldin, 1991). Genotypes with more rapid leaf area development that increases biomass production during the early vegetative growth phase are likely to improve NUE by increasing nitrogen uptake during the period of greatest soil nitrogen supply immediately after planting (Dobermann and Cassman, 2002). On the other hand, many physiological processes affect nitrogen utilization efficiency (Ladha et al., 1998). NUEb is largely affected by critical nitrogen concentrations (internal nitrogen requirement) for leaf area expansion and tiller formation, nitrogen distribution between leaves and stems, vertical nitrogen distribution in the canopy, efficiency of nitrogen use in converting CO2 to carbohydrate through photosynthesis, Rubisco content, and leaf senescence. Grain nitrogen concentration, sink capacity, unproductive tillers, harvest index (HI), and the ability to remobilize the absorbed nitrogen from straw to grain determine NHI and NUEg (Tirol-Padre et al., 1996). The amount of nitrogen uptake at each growth stage affects the formation of each yield component of the rice crop (Mae, 1997). The amount of nitrogen uptake during the vegetative period mainly promotes the early growth of the plant by accelerating leaf area expansion and tiller production. Final panicle number is largely influenced by tiller number. The amount of nitrogen uptake during the early and late phases of panicle

216

NUTRIENT USE EFFICIENCY IN CROPS

formation determines spikelet number per panicle by affecting spikelet differentiation and degeneration, respectively. Higher nitrogen in the plant tissue favors higher differentiation of spikelets, and a higher supply of photosynthates would be required to minimize spikelet degeneration during the reproductive stage (Akita, 1989). Spikelet formation efficiency defined as spikelet number per unit nitrogen absorbed before heading is an important trait in tropical rice for yield improvement (Akita, 1989); consequently, it would have a positive correlation with NUEg. During the ripening phase, a large amount of nitrogen is required for grain filling. Nitrogen absorbed during the grain-filling period accounts for 10–30% of the total panicle nitrogen, while remobilized nitrogen from the straw accounts for 70–90% of the total panicle nitrogen (Mae and Shoji, 1984). The grain-filling percentage can be improved with delayed leaf senescence. Meeting the nitrogen demand and avoiding luxury nitrogen uptake in various growth stages are essential for enhancing nitrogen utilization efficiency. A large part of the nitrogen in the rice plant is allocated to the leaves throughout the life of the plant (Mae and Ohira, 1981). Leaf nitrogen plays a major role in biomass production through photosynthesis. Nitrogen nutrition affects both leaf area index and leaf nitrogen content per unit leaf area (Mae, 1997). Leaf nitrogen content per unit leaf area is closely correlated with single leaf photosynthetic rate (Peng et al., 1995). Canopy photosynthetic rate is largely affected by leaf nitrogen through leaf area index. High plant nitrogen content delays leaf senescence and therefore increases photosynthetic duration (Makino et al., 1984). Increasing leaf nitrogen content and delaying nitrogen efflux from the leaf (i.e., delaying leaf senescence), especially in the flag leaf, could improve NUEb if the ratio of photosynthesis to respiration is not decreased.

In leaves with low nitrogen content, the potential photosynthetic NUE is low, and it increases with increasing nitrogen. The low potential photosynthetic NUE is probably due to the large investment of leaf nitrogen in nonphotosynthetic components such as the nucleic acids and proteins associated with cell regulation and respiration (Chapin et al., 1987). Sivasankar et al. (1993) speculated that photosynthesis, crop production, and NUE could be increased by genetically modifying plants to make leaves with higher nitrogen content. The difference in maximum leaf nitrogen content per unit leaf area between wheat and rice suggested that it might be possible to increase the leaf nitrogen content of rice by genetic means to improve yield potential in relation to both leaf photosynthesis and the amount of remobilized nitrogen (Mae, 1997). However, no one has attempted to select a variety that has high nitrogen content in rice leaves. In the leaf, nitrogen is concentrated in the chloroplasts, mainly as the enzyme protein Rubisco. Makino et al. (1997) confirmed that the amount of Rubisco is a rate-limiting factor of photosynthesis under ambient air and light-saturated conditions throughout the life cycle of the rice leaf using an antisense gene for a small subunit of Rubisco in rice plants. Rubisco accounts for more than 50% of total soluble protein and over 25% of total nitrogen of leaves (Makino et al., 1984). Therefore, the leaf is a major storage organ for nitrogen. The major source of nitrogen for developing leaves of mature rice plants is the nitrogen mobilized from older, senescing leaves. Out of total nitrogen translocated from vegetative tissues to the panicle, 64% was from leaf blades, 16% from leaf sheaths, and 20% from stems (Mae and Ohira, 1981). Therefore, efficiency in nitrogen remobilization from old to new leaves and from straw to grain will affect both NUEb and NUEg.

NITROGEN USE EFFICIENCY IN IRRIGATED LOWLAND RICE

During grain filling in rice, nitrogen is remobilized from leaves to grains along with photo-assimilates to meet the demand of grain development. At the same time, enough nitrogen must remain in the leaves to maintain the rate of leaf photosynthesis. A balance between these two processes is necessary for rice plants to achieve maximum grain yield and NUE. One strategy is to increase nitrogen remobilization from the lower leaves and reduce nitrogen remobilization from the upper leaves so that the gradient of leaf nitrogen concentration in the canopy is steep (Shiratsuchi et al., 2006). Simulation modeling suggests that a steeper slope of the vertical nitrogen concentration gradient in the leaf canopy with more nitrogen present in the uppermost stratum enhances canopy photosynthesis (Dingkuhn et al., 1991). Maintaining a steeper gradient of leaf nitrogen concentration in the rice canopy during grain filling could increase canopy photosynthesis by as much as 13% (Shiratsuchi et al., 2006). A large proportion of solar radiation is intercepted by the top layers of the canopy, especially in a rice crop with a high leaf area index. Optimum leaf nitrogen distribution in the canopy should match the irradiance gradient so that radiation use efficiency for canopy photosynthesis is maximized in rice plants. Therefore, matching vertical nitrogen distribution and light distribution in the canopy is another approach to achieving high NUEb. Unproductive tillers could reduce NUEg, especially when mutual shading occurs. Zhang et al. (2009) observed that varieties with low NUE had more unproductive tillers than varieties with high NUE. Unproductive tillers capture solar radiation and absorb soil nitrogen during the early stages of growth. Genotypic variation in tillering capacity and unproductive tiller percentage exists in rice. The nitrogen and carbon of unproductive tillers can be mobilized to the productive tillers before they die (Thorne and Wood,

217

1987), although the efficiency of such transport needs to be quantified. If the majority of nitrogen in unproductive tillers could be mobilized before they die, the negative impact of unproductive tillers on NUEg could be minimized. Varietal difference in NUE Genotypic variation in NUE has been reported in rice by many researchers using different NUE indices and under different conditions. Wada and Sta Cruz (1990) reported that varietal differences in nitrogen absorption were mainly observed at the tillering stage. The amount of nitrogen uptake during the early growth stage contributed to large sink size and high grain yield, especially in short-duration varieties. The nitrogen absorption ability of rice varieties during the early vegetative stage is a genetically stable character. Varietal differences in nitrogen absorption were not apparent after the maximum tiller number stage. A varietal difference in ability to absorb native soil nitrogen and applied fertilizer nitrogen was reported by Saleque et al. (2004). Although genotypic variation in nitrogen uptake kinetics has been reported for rice (Teo et al., 1995), Dobermann and Cassman (2002) argued that nitrogen uptake capacities of root systems in high-yielding rice varieties are unlikely to be a significant constraint to increasing NUE in favorable rice-growing environments. Hybrid rice is generally believed to have higher NUE than inbred varieties (Fageria et al., 2003). Its higher NUE is associated with greater root nitrogen uptake potential and greater nitrogen remobilization efficiency. Shi et al. (1999) reported that a hybrid had higher root dry weight and greater nitrogen uptake than its parents, especially under low-nitrogen treatment. Suzuki et al. (1988) observed that there was a considerable heterosis for the nitrogen

218

NUTRIENT USE EFFICIENCY IN CROPS

uptake rate per plant regardless of the source of nitrogen (i.e., ammonium vs. nitrate). The heterosis in nitrogen uptake was closely associated with the heterosis in dry matter accumulation, suggesting that hybrid rice did not have a higher nitrogen uptake rate per unit dry weight than conventional varieties. Broadbent et al. (1987) studied NUEg of 24 genotypes with and without fertilizer nitrogen application. They estimated NUEg using the ratio of panicle weight to total nitrogen uptake (WP/TN) and reported that there were significant differences in WP/TN among the genotypes. WP/TN was well correlated with other NUE-related parameters and provided genotypic rankings that did not differ greatly from multiple-parameter rankings. Tirol-Padre et al. (1996) compared NUEg of 180 genotypes without fertilizer nitrogen application. There were significant differences in NUEg among the 180 genotypes, ranging from 38 to 82 kg kg−1. These authors also confirmed the presence of genetic variability for nitrogen acquisition within a maturity group. However, the genotypic difference in NUEg was more stable than that of total nitrogen uptake across trials. Singh et al. (1998) compared the nitrogen responses of 20 genotypes under fertilizer nitrogen rates of 0, 50, 100, 150, and 200 kg ha−1. They identified nitrogenefficient genotypes that produced high grain yield at both low and high levels of nitrogen application, nitrogen-inefficient genotypes that produced low yields at low nitrogen levels but responded well to nitrogen application, and nitrogen-inferior genotypes that gave low yields at both low and high nitrogen levels. Although 75% of the variation in grain yield was explained by total nitrogen uptake, genotypic differences in NUEg were observed (Singh et al., 1998). Based on NUEg and grain yield at low soil nitrogen, rice genotypes have been clas-

sified into four groups (Fageria and Baligar, 2003). The efficient and responsive genotypes produced above-average yield at low nitrogen and had NUEg above the average of all genotypes. The efficient and nonresponsive genotypes produced more than the average yield, but NUEg was lower than the average of all genotypes. The inefficient and responsive genotypes produced less than the average grain yield of all genotypes, but NUEg was above the average of all genotypes. The last group was inefficient and nonresponsive genotypes, which had lower grain yield and NUEg than the average of all genotypes. The genotypes in the first group are the most desirable because they yield well at low soil nitrogen and also respond well to applied nitrogen. Inthapanya et al. (2000) determined the differences in NUEg among 16 genotypes grown under rain-fed lowland conditions at three locations with and without nitrogen application. There was a significant effect of genotype and an insignificant effect of genotype × location interaction for NUEg. Mean NUEg of the 16 genotypes ranged from 55 to 84 kg kg−1. Koutroubas and Ntanos (2003) studied NUEg of five varieties with nitrogen application under Mediterranean conditions. They reported that indica varieties generally had higher NUEg than japonica varieties. In a recent study (Samonte et al., 2006), NUEg of Lemont, Teqing, and 13 advanced recombinant inbred lines obtained from a Lemont × Teqing cross was determined under high-nitrogen application. There was a significant variation in NUEg among the 15 genotypes, ranging from 25 to 64 kg kg−1. The large genotypic variation in NUEg was probably caused by some genotypes with low yield potential. When only high-yielding varieties are compared, the genotypic variation in NUEg is much smaller. Ladha et al. (2005) argued that most modern rice varieties have similar NUEg when grown under

NITROGEN USE EFFICIENCY IN IRRIGATED LOWLAND RICE

comparable conditions, perhaps because of their narrow parent base. Plant traits related to NUEg Many plant traits have been studied to improve the mechanistic understanding of NUEg. Genotypes with higher NUEg had lower percentage straw nitrogen (Ns) at maturity (Tirol-Padre et al., 1996; Singh et al., 1998; Koutroubas and Ntanos, 2003). Low grain nitrogen concentration (Ng) was also associated with high NUEg (Tirol-Padre et al., 1996; Singh et al., 1998; Inthapanya et al. 2000; Koutroubas and Ntanos, 2003). Koutroubas and Ntanos (2003) reported that 76% and 70% of the variation in NUEg among varieties could be explained by the variation in Ng and Ns, respectively. Genotypes with high HI are more efficient in nitrogen use (Inthapanya et al. 2000). Factors affecting HI also affect the utilization efficiency of nitrogen (Janssen, 1998). The higher NUEg for indica varieties could be attributed to high HI and high dry matter remobilization to the grain compared with japonica varieties (Koutroubas and Ntanos, 2003). Ladha et al. (2005) stated that much of the progress in improving NUEg has been associated with improvement in HI rather than in photosynthetic efficiency. Inthapanya et al. (2000) stated that HI, Ns, and Ng determine NUEg. At HI of 0.5, Ns and Ng contribute equally to NUEg. Rice generally has greater NUEb and NUEg than some other C3 crops such as soybean and wheat because of low Ns and Ng. Further decreasing Ng could theoretically contribute to more efficient nitrogen utilization. However, genotypic variation in Ng is relatively small compared with the variation caused by management practices. TirolPadre et al. (1996) studied genotypic variation in Ng of 180 lines grown without fertilizer nitrogen input. About 93% of the genotypes had Ng of 0.95 to 1.20%. It may

219

not be feasible to improve NUEg by reducing Ng using conventional breeding because Ng is affected more by environments than by genotypes (Ladha et al., 1998). Genetic variability for NHI exists within genotypes of cereal crops and high NHI is associated with efficient utilization of nitrogen (Fageria et al., 2003). Fageria and Barbosa Filho (2001) reported a positive relationship between NHI and grain yield across rice genotypes. Therefore, the variation in NHI is a character of genotype, and this trait may be useful for selecting rice genotypes with higher grain yield and NUE. Koutroubas and Ntanos (2003) reported that a difference in NHI explained genotypic variation in NUEg, and a difference in NUEg between indica and japonica varieties. There was a significant positive correlation between NUEg and nitrogen translocation ratio (NTR), which was calculated as the ratio of grain nitrogen content at maturity to plant nitrogen content at flowering (Samonte et al., 2006). Greater dry matter and nitrogen translocation to the grains increased HI and NHI, which in turn favored high NUEg (Koutroubas and Ntanos, 2003). Crop growth duration affects NUEg by influencing crop yield and nitrogen uptake. Short-duration varieties are more dependent on nitrogen fertilizer than varieties with long growth duration (Mikkelsen et al., 1995). Medium-duration genotypes had higher NUEg than short-duration genotypes (Broadbent et al., 1987; De Datta and Broadbent, 1990). Long-duration varieties accumulated more nitrogen, perhaps because of increased nitrogen mineralization and plant associative N2 fixation (Inthapanya et al. 2000). Longer duration could result in a decrease in NUEg due to a reduction in NTR (Samonte et al., 2006). Breeding for higher NUE Although genotypic variation in NUE has been observed for a long time, improving

220

NUTRIENT USE EFFICIENCY IN CROPS

NUE in rice has not been an objective in plant breeding (Samonte et al., 2006). The possibility of breeding for genotypes with high NUEg was questioned by Burns et al. (1997) because it is difficult to further increase HI and because crop growth rates and internal nitrogen requirements appear to be tightly conserved. Little effort has been made to explore the potential genotypic variability in nitrogen uptake and utilization efficiency in crop improvement programs (Tirol-Padre et al., 1996). This is partially due to the lack of simple and quick methods for estimating NUEg, which could be used for screening large numbers of progenies in a breeding program. The G × E interaction for NUEg also makes the improvement of NUE more difficult. Furthermore, there is a danger of selecting for plants with low total nitrogen uptake and low grain yield if selection is based solely on NUEg. Moll et al. (1982) stated that an ideal genotype would absorb more nitrogen from soil and fertilizer, produce a high grain yield per unit of absorbed nitrogen, and store little nitrogen in the straw. In breeding for improved NUE, it is desirable for both uptake and utilization efficiencies to be improved simultaneously (Moll et al., 1982). The relative importance of uptake efficiency and utilization efficiency for NUE is affected by the level of applied nitrogen. Under low nitrogen, uptake efficiency is the predominant component contributing to NUE. As the amount of applied nitrogen increases, the relative importance to uptake efficiency decreases and that of utilization efficiency increases (Ortiz-Monasterio et al., 1997). Moll et al. (1982) reported that nitrogen utilization efficiency is more important than nitrogen uptake efficiency when evaluating the genetic potential among varieties for efficient grain production, especially on soils that require high rates of nitrogen to maximize yield (Moll et al., 1982). TirolPadre et al. (1996) and Inthapanya et al.

(2000) observed that varietal difference in NUEg was more consistent across environments than varietal difference in total nitrogen uptake, which suggests that selection for total nitrogen uptake is more difficult than selection for NUEg in a breeding program. In general, there is a positive relationship between grain yield and NUE (Fageria and Baligar, 2005). Total nitrogen uptake and NUE were not significantly correlated with each other, but both had a significant and positive relationship with grain yield (Samonte et al., 2006). This suggests that a genotype selected based on grain yield may utilize nitrogen efficiently or take up high amounts of nitrogen. Furthermore, rice genotypes with high nitrogen uptake do not necessarily convert the absorbed nitrogen into grain efficiently. A rice breeder should select for a genotype with both high yield and NUE, rather than select for high yield alone because it is possible that the high yield is due to high nitrogen uptake. However, Fischer and Wall (1976) suggested that breeding efforts to increase grain yield would continue to provide indirect selection pressure on nitrogen utilization efficiency. Rice breeders could develop new rice varieties that not only produce high yield but also use nitrogen efficiently. To achieve these objectives, plant traits that are associated with high grain yield and high NUE should be selected concomitantly, and breeders should be able to use these traits easily as selection criteria in their breeding programs. In actual rice breeding, one could select high-yielding genotypes in a yield trial first and then screen for high NTR and NUE so that selected genotypes are both high yielders and efficient users of nitrogen (Samonte et al., 2006). Although NUEg depends on the level of available nitrogen, the genotype with high NUEg at low nitrogen levels also had a high NUEg at high nitrogen levels (Isfan et al., 1991). De Datta and Broadbent (1990)

NITROGEN USE EFFICIENCY IN IRRIGATED LOWLAND RICE

reported that genotypes with high NUEg performed well on nitrogen-deficient soils as well as those where nitrogen supply was more adequate. Furthermore, NUEg was positively correlated with grain yield in both a zero-nitrogen control and nitrogenfertilized treatment. This suggests that NUEg can be used in a breeding program as a selection criterion for identifying high-yielding genotypes capable of exploiting nitrogen inputs more efficiently (Isfan et al., 1991). Koutroubas and Ntanos (2003) argued that using single selection criteria for improving NUE may have negative implications for grain yield because NUE is a complex trait that results from an interaction of several component traits. Broadbent et al. (1987) stated that the evaluation of NUE of different genotypes should be based on multiple parameters rather than just one parameter because an evaluation based on multiple parameters would represent cumulative values rather than a situation in which one parameter cancels out the effects of another. The sum of Z-transformed values of grain yield, NUEg, panicle weight, panicle weight/ total nitrogen uptake, and total dry matter/ total nitrogen uptake, putting equal weights on each parameter, was used to rank genotypes (Broadbent et al., 1987; De Datta and Broadbent, 1988; De Datta and Broadbent 1990). Genotypes with high NUEg had high rankings consistently across seasons and NUE can be satisfactorily evaluated without the use of isotopically labeled fertilizer (De Datta and Broadbent, 1988). Singh et al. (1998) developed a nitrogen productivity index (NPI), which is the product of grain yield at zero nitrogen and NUEg in nitrogenfertilized treatment, for evaluating the NUE of genotypes. Quantifying and ranking nitrogen-efficient genotypes based on NPI was most consistent, whereas NUEg, AE, and RE had biases either toward soil nitrogen or fertilizer nitrogen supply (Singh et al., 1998). However, determination of NPI

221

requires field evaluation of genotypes at low nitrogen (or zero-nitrogen input) and at high nitrogen. The high nitrogen level should not be too high to induce lodging, pest, and disease damage, which affect crop nitrogen response and NUE (Tirol-Padre et al., 1996; Singh et al., 1998). Moreover, Ladha et al. (1998) demonstrated the value of selecting rice germplasm at moderate and suboptimal levels of nitrogen, at which maximum genotypic differences in NUE are expressed. Field screening of genotypes for NUE can be performed under two (low and high) or three (low, medium, and high) nitrogen levels (Fageria and Baligar, 2003). Nitrogenefficient and nitrogen-inefficient varieties and a local check variety should be included in the screening. Grain yield is the best measure of genotype performance across nitrogen levels and grain yield efficiency index (GYEI) was proposed by Fageria and Baligar (2003) to separate genotypes into high-yielding, stable, nitrogen-efficient and low-yielding, unstable, and nitrogeninefficient. GYEI is equal to the product of yield at low nitrogen and yield at high nitrogen divided by the product of experimental mean yield at low nitrogen and experimental mean yield at high nitrogen. Efficient genotypes have a GYEI of 1 or higher. Inefficient genotypes have a GYEI in the range of 0–.50. Molecular approaches to improving NUE Recent developments in molecular biology provide a new opportunity to increase NUE through crop improvement. However, more efforts are needed to identify genes that would be practically useful for improving NUE in rice breeding programs (Zhang, 2007). Hoque et al. (2006) reported that transgenic rice plants with overexpression of a rice ammonium transporter (OsAMT11) gene showed increased ammonium

222

NUTRIENT USE EFFICIENCY IN CROPS

uptake and content, but decreased biomass production, during the early vegetative stage compared with wild-type plants. Transforming a C3 rice plant into a C4 rice plant has resulted in a 10–35% increase in grain yield compared with control plants (Ku et al., 2000). The C4 plant is assumed to have higher NUE than the C3 plant (Ladha et al., 2005). Mae (1997) suggested that an increase in the proportion of Rubisco to total leaf nitrogen, a higher Vmax value, or a lower Km (CO2) value would result in greater leaf photosynthesis and NUE. Makino et al. (1988) reported that the Vmax value of Rubisco was 1.5 times larger in wheat than in rice. If the Vmax value of Rubisco in rice could be increased to the level of that in wheat and spinach, photosynthesis and NUE of rice might be greatly improved. However, there was very little variation in the proportion of Rubisco to soluble nitrogen or in the kinetic properties of Rubisco among rice varieties (Makino et al., 1987), suggesting that there is little scope to modify the kinetic properties of Rubisco by conventional crossbreeding using existing rice germplasm. With the technique of genetic manipulation of the chloroplast gene, it may be possible to attempt modification of the Vmax value of rice Rubisco in the near future (Mae, 1997). Obara et al. (2001) used a backcross inbred line population to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1) and NADH–glutamate synthase (NADH–GOGAT). GS1 is a key enzyme in the mobilization of nitrogen from senescing leaves, and its activity in senescing leaves is positively related to yield. NADH–GOGAT is important in the utilization of nitrogen in grain filling, and its activity in developing grains is positively correlated with yield. Seven chromosomal QTL regions for GS1 and six for NADH–

GOGAT were detected. Some of these QTLs were related to nitrogen recycling from senescing organs to developing organs. A structural gene for GS1 on chromosome 2 was colocated in the QTL region for seed weight. A structural gene for NADH– GOGAT on chromosome 1 was colocated in the QTL region for soluble protein in developing leaves. Yamaya et al. (2002) developed transgenic lines overproducing NADH–GOGAT, and two of these lines showed an increase in grain weight. These studies suggest that genotypes obtained from genetically manipulated populations or genetic resources with high GS1 in senescing leaves and high NADH–GOGAT in developing grains should promote nitrogen remobilization from straw to grain and consequently improve NUEg (Andrews et al., 2004). However, for significant improvement in NUE, more research work is needed to better understand the physiological and biochemical mechanisms responsible for high NUE (Fageria and Baligar, 2003). Summary Practices of improved nitrogen management strategies are often associated with high labor requirements, although their impact on NUE is high. The development of new varieties with high NUE will further improve overall agronomic NUE. However, breeding for high NUE is not an easy task because NUE is a complex trait with multiple components and it is influenced by many factors. That is why NUE is seldom evaluated in breeding programs. Numerous studies have demonstrated that variation in NUE, particularly in NUEg, exists among rice varieties. The genotypic variation in NUEg is fairly consistent across seasons and different levels of soil nitrogen. Plant nitrogen nutrition and its effects on photosynthesis and tiller and leaf area production

NITROGEN USE EFFICIENCY IN IRRIGATED LOWLAND RICE

determine rice yield potential and NUE. Both nitrogen uptake and utilization are affected by crop growth rate, root function, nitrogen partitioning, and translocation. Plant traits such as straw and grain nitrogen concentration, HI, NHI, NTR, and growth duration are closely associated with NUE. More importantly, there is a positive relationship between grain yield and NUE. This suggests that it is possible to develop rice varieties with both high grain yield and NUE. Simple plant traits that are associated with high grain yield and high NUE should be identified so that breeders can use these traits easily as selection criteria in a breeding program to develop nitrogen-efficient varieties without sacrificing rice yield potential. New breeding techniques such as the development of F1 hybrids, markeraided selection, transformation, and genetic engineering should be combined effectively with empirical breeding methods in order to increase rice grain yield with limited nitrogen supply.

References Akita, S. (1989) Improving yield potential in tropical rice. In: Progress in Irrigated Rice Research, pp. 41–73, International Rice Research Institute, Los Baños, Philippines. Andrews, M., Lea, P.J., Raven, J.A., et al. (2004) Can genetic manipulation of plant nitrogen assimilation enzymes result in increased crop yield and great N-use efficiency? An assessment. Annals of Applied Biology 145, 25–40. Angus, J.F., Williams, R.L., & Durkin, C.O. (1996) Manage rice: decision support for tactical crop management. In: Crop Research in Asia: Achievements and Perspective. Proceedings of the 2nd Asian Crop Science Conference (eds. Ishii, R. & Horie, T.), pp. 274–279. 21–23 August, 1995. Fukui, Japan. Broadbent, F.E., De Datta, S.K., & Laureles, E.V. (1987) Measurement of nitrogen utilization efficiency in rice genotypes. Agronomy Journal 79, 786–791. Burns, I.G., Walker, R.L., & Moorby, J. (1997) How do nutrients drive growth? Plant and Soil 196, 321–325.

223

Cassman, K.G. (1999) Ecological intensification of cereal production systems: yield potential, soil quality, and precision agriculture. Proceedings of the National Academy of Sciences of the United States of America 96, 5952–5959. Cassman, K.G., Gines, G.C., Dizon, M.A., et al. (1996) Nitrogen-use efficiency in tropical lowland rice systems: contributions from indigenous and applied nitrogen. Field Crops Research 47, 1–12. Cassman, K.G., Peng, S., Olk, D.C., et al. (1998) Opportunities for increased nitrogen-use efficiency from improved resource management in irrigated rice systems. Field Crops Research 56, 7–39. Chapin, F.S. III, Bloom, A.J., Field, C.B., et al. (1987) Plant responses to multiple environmental factors. Bioscience 37, 49–57. De Datta, S.K. (1986) Improving nitrogen fertilizer efficiency in lowland rice in tropical Asia. Fertilizer Research 9, 171–186. De Datta, S.K. & Broadbent, F.E. (1988) Methodology for evaluating nitrogen utilization efficiency by rice genotypes. Agronomy Journal 80, 793–798. De Datta, S.K. & Broadbent, F.E. (1990) Nitrogen-use efficiency of 24 rice genotypes on an N-deficient soil. Field Crops Research 23, 81–92. De Datta, S.K. & Buresh, R.J. (1989) Integrated nitrogen management in irrigated rice. Advances in Soil Science 10, 143–169. Dingkuhn, M., Penning de Vries, F.W.T., De Datta, S.K., et al. (1991) Concepts for a new plant type for direct seeded flooded tropical rice. In: Direct Seeded Flooded Rice in the Tropics, pp. 17–38. International Rice Research Institute, Los Baños, Philippines. Dobermann, A. & Cassman, K.G. (2002) Plant nutrient management for enhanced productivity in intensive grain production systems of the United States and Asia. Plant and Soil 247, 153–175. Dobermann, A. & Fairhurst, T.H. (2000) Rice: Nutrient Disorders and Nutrient Management. Potash and Phosphate Institute, Singapore, and International Rice Research Institute (IRRI), Los Baños, Philippines. Dobermann, A., Witt, C., Dawe, D., et al. (2002) Sitespecific nutrient management for intensive rice cropping systems in Asia. Field Crops Research 74, 37–66. Fageria, N.K. & Baligar, V.C. (2003) Methodology for evaluation of lowland rice genotypes for nitrogen use efficiency. Journal of Plant Nutrition 26, 1315–1333. Fageria, N.K. & Baligar, V.C. (2005) Enhancing nitrogen use efficiency in crop plants. Advances in Agronomy 88, 97–185. Fageria, N.K. & Barbosa Filho, M.P. (2001) Nitrogen use efficiency in lowland rice genotypes.

224

NUTRIENT USE EFFICIENCY IN CROPS

Communications in Soil Science and Plant Analysis 32, 2079–2089. Fageria, N.K., Slaton, N.A., & Baligar, V.C. (2003) Nutrient management for improving lowland rice productivity and sustainability. Advances in Agronomy 80, 63–152. FAO Statistics (2010) FAO statistical databases. Available at: http://www.fao.org. Food and Agriculture Organization (FAO) of the United Nations, Rome. Fischer, R.A. & Wall, P.C. (1976) Wheat breeding in Mexico and yield increases. Journal of the Australian Institute of Agricultural Sciences 42, 139–148. Heffer, P. (2008) Assessment of Fertilizer Use by Crop at the Global Level. International Fertilizer Industry Association (IFA), Paris, France. Hoque, M., Masle, J., Udvardi, M.K., et al. (2006) Over-expression of the rice OsAMT1-1 gene increases ammonium uptake and content, but impairs growth and development of plants under high ammonium nutrition. Functional Plant Biology 33, 153–163. Inthapanya, P., Sipaseuth, Sihavong, P., et al. (2000) Genotype differences in nutrient uptake and utilization for grain yield production of rainfed lowland rice under fertilized and non-fertilized conditions. Field Crops Research 65, 57–68. Isfan, D., Cserni, I., & Tabi, M. (1991) Genetic variation of the physiological efficiency index of nitrogen in triticale. Journal of Plant Nutrition 14, 1381–1390. Janssen, B.H. (1998) Efficient use of nutrients: an art of balancing. Field Crops Research 56, 197–201. Kirk, G.J.D. & Bouldin, D.R. (1991) Speculations on the operation of the rice root system in relation to nutrient uptake. In: Simulation and Systems Analysis for Rice Production (eds. F.W.T. Penning de Vries, H.H. van Laar & M.J. Kroff), pp. 195–203. Pudoc, Wageningen, The Netherlands. Koutroubas, S.D. & Ntanos, D.A. (2003) Genotypic differences for grain yield and nitrogen utilization in Indica and Japonica rice under Mediterranean conditions. Field Crops Research 83, 251–260. Ku, M.S.B., Cho, D., Ranade, U., et al. (2000) Photosynthetic performance of transgenic rice plants overexpressing maize C4 photosynthesis enzymes. In: Redesigning Rice Photosynthesis to Increase Yield (eds. J.E. Sheehy, P.L. Mitchell & B. Hardy), pp. 193–204. Elsevier Science, Amsterdam, The Netherlands. Kundu, D.K. & Ladha, J.K. (1995) Efficient management of soil and biologically fixed nitrogen in intensively cultivated rice fields. Soil Biology and Biochemistry 27, 431–439.

Kundu, D.K. & Ladha, J.K. (1997) Effect of growing rice on nitrogen mineralization in flooded soil. Soil Science Society of America Journal 61, 839–845. Ladha, J.K., Kirk, G.J.D., Bennett, J., et al. (1998) Opportunity for increased nitrogen-use efficiency from improved lowland rice germplasm. Field Crops Research 56, 41–71. Ladha, J.K., Pathak, H., Krupnik, T.J., et al. (2005) Efficiency of fertilizer nitrogen in cereal production: retrospects and prospects. Advances in Agronomy 87, 85–156. Mae, T. (1997) Physiological nitrogen efficiency in rice: nitrogen utilization, photosynthesis, and yield potential. Plant and Soil 196, 201–210. Mae, T. & Ohira, K. (1981) The remobilization of nitrogen related to leaf growth and senescence in rice plants (Oryza sativa L.). Plant and Cell Physiology 22, 1067–1074. Mae, T. & Shoji, S. (1984) Studies on the fate of fertilizer nitrogen in rice plants and paddy soils by using 15 N as a tracer in northeastern Japan. In: Soil Science and Plant Nutrition in Northeastern Japan (Special issue), pp. 77–94. Northeastern Section of the Japanese Society of Soil Science and Plant Nutrition, Sendai, Japan. Makino, A., Mae, T., & Ohira, K. (1984) Relation between nitrogen and ribulose-1,5-bisphosphate carboxylase in rice leaves from emergence through senescence. Plant and Cell Physiology 25, 429–437. Makino, A., Mae, T., & Ohira, K. (1987) Variation in the contents and kinetic properties of ribulose-1, 5-bisphosphate carboxylase among rice species. Plant and Cell Physiology 28, 799–804. Makino, A., Mae, T., & Ohira, K. (1988) Differences between wheat and rice in the enzymic properties of ribulose-1, 5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange. Planta 174, 30–38. Makino, A., Shimada, T., Takumi, S., et al. (1997) Does decrease in ribulose-1, 5-bisphosphate carboxylase by antisense rbcS lead to a higher N-use efficiency of photosynthesis at saturating CO2 and light in rice plants? Plant Physiology 114, 483–491. Mikkelsen, D.S., Jayaweera, G.R., & Rolston, D.E. (1995) Nitrogen fertilization practices of lowland rice culture. In: Nitrogen Fertilization in the Environment (ed. P.E. Bacon), pp. 171–223. Marcel Dekker, Inc, New York. Moll, R.H., Kamprath, E.J., & Jackson, W.A. (1982) Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agronomy Journal 74, 562–564. Norman, R.J., Guindo, D., Wells, B.R., et al. (1992) Seasonal accumulation and partitioning of

NITROGEN USE EFFICIENCY IN IRRIGATED LOWLAND RICE

nitrogen-15 in rice. Soil Science Society of America Journal 56, 1521–1527. Novoa, R. & Loomis, R.S. (1981) Nitrogen and plant production. Plant and Soil 58, 177–204. Obara, M., Kajiura, M., Fukuta, Y., et al. (2001) Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice (Oryza sativa L.). Journal of Experimental Botany 52, 1209–1217. Ortiz-Monasterio, R.J.I., Sayre, K.D., Rajaram, S., et al. (1997) Genetic progress in wheat yield and nitrogen use efficiency under four nitrogen rates. Crop Science 37, 898–904. Peng, S. & Cassman, K.G. (1998) Upper thresholds of nitrogen uptake rates and associated nitrogen fertilizer efficiencies in irrigated rice. Agronomy Journal 90, 178–185. Peng, S. & Ismail, A. (2004) Physiological basis of yield and environmental adaptation in rice. In: Physiology and Biotechnology Integration for Plant Breeding (eds. H.T. Nguyen & A. Blum), pp. 83– 140. Marcel Dekker, Inc., New York. Peng, S., Cassman, K.G., & Kropff, M.J. (1995) Relationship between leaf photosynthesis and nitrogen content of field-grown rice in the tropics. Crop Science 35, 1627–1630. Peng, S., Garcia, F.V., Laza, R.C., et al. (1996) Increased N-use efficiency using a chlorophyll meter on high yielding irrigated rice. Field Crops Research 47, 243–252. Peng, S., Buresh, R.J., Huang, J., et al. (2006) Strategies for overcoming low agronomic nitrogen use efficiency in irrigated rice systems in China. Field Crops Research 96, 37–47. Saleque, M.A., Naher, U.A., Choudhury, N.N., et al. (2004) Variety-specific nitrogen fertilizer recommendation for lowland rice. Communications in Soil Science and Plant Analysis 35, 1891–1903. Samonte, S.O.P.B., Wilson, L.T., Medley, J.C., et al. (2006) Nitrogen utilization efficiency: relationship with grain yield, grain protein, and yield-related traits in rice. Agronomy Journal 98, 168–176. Shi, Q., Li, M., & Tu, Q. (1999) Preliminary study on the growth superiority and the characteristics of nitrogen uptake of hybrid rice roots. Acta Agriculturae Universitatis Jiangxiensis 21, 145–148. Shiratsuchi, H., Yamagishi, T., & Ishii, R. (2006) Leaf nitrogen distribution to maximize the canopy photosynthesis in rice. Field Crops Research 95, 291–304. Singh, U., Ladha, J.K., Castillo, E.G., et al. (1998) Genotypic variation in nitrogen use efficiency in

225

medium- and long-duration rice. Field Crops Research 58, 35–53. Sivasankar, A., Bansal, K.C., & Abrol, Y.P. (1993) Nitrogen in relation to leaf area development and photosynthesis. In: Nitrogen: Soils, Physiology, Biochemistry, Microbiology, Genetics (eds. Y.P. Abrol, K.V.B.R. Tilak, S. Kumar, et al.), pp. 75–84. Indian National Science Academy, New Delhi, India. Suzuki, Y., Yoshida, H., & Morooka, M. (1988) Heterosis for rate of nitrogen uptake in F1 rice hybrids. Soil Science and Plant Nutrition 34, 87–95. ten Berge, H.F.M., Thiyagarajan, T.M., Shi, Q., et al. (1997) Numerical optimization of nitrogen application to rice. Part I. Description of MANAGE-N. Field Crops Research 51, 29–42. Teo, Y.H., Beyrouty, C.A., Norman, R.J., et al. (1995) Nutrient uptake relationship to root characteristics of rice. Plant and Soil 171, 297–302. Thorne, G.N. & Wood, D.W. (1987) The fate of carbon in dying tillers of winter wheat. Journal of Agricultural Science, Cambridge 108, 515–522. Tirol-Padre, A., Ladha, J.K., Singh, U., et al. (1996) Grain yield performance of rice genotypes at suboptimal levels of soil N as affected by N uptake and utilization efficiency. Field Crops Research 46, 127–143. Wada, G. & Sta Cruz, P.C. (1990) Nitrogen response of rice varieties with reference to nitrogen absorption at early growth stage. Japanese Journal of Crop Science 59, 540–547. Yamaya, T., Obara, M., Nakajima, H., et al. (2002) Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice. Journal of Experimental Botany 53, 917–925. Yoshida, S. (1981) Fundamentals of Rice Crop Science. International Rice Research Institute, Los Baños, Philippines. Zhang, Q. (2007) Strategies for developing green super rice. Proceedings of the National Academy of Sciences of the United States of America 104, 16402–16409. Zhang, Y., Fan, J., Wang, D., et al. (2009) Genotypic differences in grain yield and physiological nitrogen use efficiency among rice cultivars. Pedosphere 19, 681–691. Zhu, Z. (1997) Fate and management of fertilizer nitrogen in agro-ecosystems. In: Nitrogen in Soils of China (eds. Z. Zhu, Q. Wen & J.R. Freney), pp. 239–279. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Part III

Other Critical Macro- and Micronutrients

Chapter 12

Phosphorus as a Critical Macronutrient Carroll P. Vance

Abstract

Introduction

Phosphorus (P) is required for plant growth and development, but its availability is frequently limiting. Plants have evolved numerous adaptive mechanisms for acclimation to phosphorus deﬁciency. These mechanisms involve activation of metabolic, molecular, developmental, and regulatory processes that modify root architecture to increase soil volume exploration and recycling of internal phosphorus. Modiﬁcation of root architecture is frequently accompanied by increased exudation of organic acids, protons, and enzymes to increase phosphorus availability. Recent advances in genomics and genetics suggest that plant acclimation to phosphorus deﬁciency involves crosstalk between sugars and gene expression, including expression of miRNA399. The development of recombinant inbred lines and near inbred lines having phosphorus tolerance coupled to next-generation sequencing will lead to identiﬁcation of genes regulating adaptation to phosphorus stress.

Phosphorus (P) is one of 17 essential elements (nutrients) required for plant growth (Tiessen, 2008; Cordell et al., 2009). The phosphorus concentration in plants ranges from 0.05% to 0.50% dry weight. It plays a role in a wide array of processes including energy generation, nucleic acid synthesis, photosynthesis, glycolysis, respiration, membrane synthesis and stability, enzyme activation/inactivation, redox reactions, signaling, carbohydrate metabolism, and nitrogen (N) ﬁxation. The concentration gradient from the soil solution phosphorus to plant cells exceeds 2000-fold, with an average free phosphorus of 1–5 μM in the soil solution (Bieleski, 1973; Schachtman et al., 1998; Raghothama and Karthikeyan, 2005). This concentration is well below the Km for plant uptake. Thus, although bound phosphorus is quite abundant in many soils, it is largely unavailable for uptake. As such, phosphorus is frequently the most limiting element for plant growth and development.

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 229

230

NUTRIENT USE EFFICIENCY IN CROPS

Crop yield on 40–60% of the world’s arable land is limited by phosphorus availability (Tiessen, 2008; Cordell et al., 2009). Phosphorus is unavailable because it rapidly forms insoluble complexes with cations, particularly calcium, aluminum, and iron under acid conditions. The acid-weathered soils of the tropics and subtropics are particularly prone to phosphorus deﬁciency. Moreover, some 30–70% of the phosphorus can be bound in insoluble organic forms, such as phytate, due to microbial activity. Application of phosphorus-containing fertilizers is the recommended treatment for enhancing soil phosphorus availability and stimulating crop yields. Mined rock phosphate is the primary source of phosphorus fertilizer. Approximately 90% of all mined rock phosphate is used for agriculture (Tiessen, 2008; Cordell et al., 2009). However, rock phosphate is a nonrenewable resource (Steen, 1998; Cordell et al., 2009), and easily mined, high-quality rock phosphate sources are projected to be depleted within 30–50 years (Steen, 1998; Tiessen, 2008; Cordell et al., 2009). In addition, the world’s major reserves of rock phosphate are located in Morocco and China; uncertain political issues could limit access to the world’s phosphorus resources. Peak phosphorus production is projected to occur in 2035–2040 (Cordell et al., 2009). Coalescence of these factors as well as production of crops for energy has seen phosphorus fertilizer cost increase six- to ninefold in the past few years (Tiessen, 2008; Cordell et al., 2009). A potential phosphate crisis looms for agriculture in the 21st century (Abelson, 1999; Vance et al., 2003; Tiessen, 2008). Application of phosphorus fertilizer, however, is problematic for both the intensive and extensive agriculture of the developed and developing worlds, respectively. In intensive agriculture, a grain crop yield of 7 metric tons ha−1 requires the addition of 90– 120 kg P ha−1 (White and Hammond, 2008).

But even under adequate phosphorus fertilization, only 20% or less of that applied is removed in the ﬁrst year ’s growth. This results in phosphorus loading of prime agricultural land. Runoff from phosphorusloaded soils is a primary factor in eutrophication and hypoxia of lakes and marine estuaries of the developed world (Vance et al., 2003; White and Hammond, 2008). An even greater concern than the overabundant use of phosphorus fertilizers by intensive agriculture is the lack of available phosphorus fertilizers for extensive agriculture in the tropics and subtropics, where the majority of Earth’s people live. Lack of fertilizer infrastructure, money for purchase, and transportation make phosphorus fertilization unattainable for these areas. Sustainable management of phosphorus in agriculture requires that plant biologists discover mechanisms that enhance phosphorus acquisition and exploit these adaptations to make plants more efﬁcient at acquiring phosphorus, develop phosphorus -efﬁcient germplasm, and advance crop management schemes that increase soil phosphorus availability. Although mycorrhizal symbioses (see Chapters 2 and 3) are the most important adaptation for angiosperms to acquire scarce phosphorus (Harrison, 2005), many plant families have species that either do not form or rarely form this pivotal association (Skene, 1998; Miller et al., 1999; Cripps and Eddington, 2005; Miller, 2005). This chapter will address common adaptations and mechanisms for acquisition and use of scarce phosphorus in plants lacking effective mycorrhizal symbioses. The primary focus will be on root adaptations in species as they acclimate to phosphorus deﬁciency. Data from Arabidopsis and cluster root species will be considered because they do not form mycorrhizal symbioses and are model species for evaluating plant adaptations to phosphorus stress.

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

Plants have evolved two broad strategies for improved phosphorus acquisition and use in nutrient-limiting environments: (1) those aimed at conservation of use, and (2) those directed toward enhanced acquisition or uptake (Vance et al., 2003; Ticconi and Abel, 2004; Misson et al., 2005; Morcuende et al., 2007). Processes that conserve the use of phosphorus involve decreased growth rate, increased growth per unit of phosphorus uptake, remobilization of internal phosphorus, modiﬁcations in carbon (C) metabolism that bypass phosphorus -requiring steps, alternative respiratory pathways, and alterations in membrane biosynthesis requiring less phosphorus (Plaxton and Carswell, 1999; Uhde-Stone et al., 2003a,b; Wasaki et al., 2003; Misson et al., 2005; Lambers et al., 2006). In comparison, processes that lead to enhanced phosphorus uptake include modiﬁed root architecture and greater root growth, proliﬁc development of root hairs leading to expanded root surface area, enhanced expression of phosphorus transporter genes, and increased production and exudation of phosphatases and organic acids (Marschner et al., 1986; López-Bucio et al., 2002; Shane and Lambers, 2005). These numerous responses to acclimate to phosphorus deﬁciency are not mutually exclusive and all may occur within a single species. Phenotypic response to phosphorus deﬁciency Plant responses to phosphorus stress conditions involve changes in root morphology and architecture (Lynch, 1995; Lynch and Brown, 2001; López-Bucio et al., 2003; Vance et al., 2003; Lambers et al., 2006; Ticconi et al., 2009), as well as changes in shoot and ﬂower development (Nacry et al., 2005; Bucciarelli et al., 2006; Hernández et al., 2007; Huang et al., 2008). Studies of Medicago truncatula, a model legume for

231

plant biology research, showed that phosphorus stress delayed (1) leaf development and expansion along the main and axillary shoot; (2) axillary shoot emergence and elongation, resulting in stunted plants; and (3) timing of ﬂower emergence (Bucciarelli et al., 2006). Leaves were darker green, anthocyanin accumulation increased, and root : shoot ratio increased. In common bean, phosphorus stress resulted in a fourfold reduction in leaf area, an increase in root : shoot ratio, and a reduction in net photosynthetic rate (Hernández et al., 2007). Similarly, shoot growth of phosphorusdeﬁcient barley was reduced as compared to the controls while root growth was enhanced (Huang et al., 2008). Arabidopsis phosphorus deﬁciency symptoms are quite similar to those described above (López-Bucio et al., 2003; Nacry et al., 2005). The timing of phosphorus deﬁciency symptoms to appear varied according to species, but phosphorus deﬁciency symptoms appeared later in species with large seeds as compared to small seeds. Because of the subterranean nature of growth, plant roots have been recalcitrant to phenotypic studies (see Chapter 2). However, in the past decade, root responses to phosphorus deﬁciency have become a rich topic for developmental and genomic studies. Root adaptations to phosphorus limitations include reduced extension of primary roots, highly branched roots with increased lateral roots, and an increased density of root hair formation (Figs. 12.1 and 12.2). Consistent with a general stress response by plants, however, phosphorus-stressed plants tend to allocate a greater proportion of biomass to root dry matter compared with phosphorussufﬁcient plants (López-Bucio et al., 2002, 2003; Hammond et al., 2004; Hill et al., 2006). Most pasture species studied showed reduced total root mass as a response to phosphorus stress conditions. On average, a 32–86% reduction in root mass was observed

232

NUTRIENT USE EFFICIENCY IN CROPS

Fig. 12.1. Phosphorus deﬁciency phenotype of Arabidopsis thaliana. Phosphorus-sufﬁcient A. thaliana (+P) has a vigorous shoot, elongate primary root having few lateral roots, and short root hairs. By comparison, phosphorusdeﬁcient A. thaliana (–P) has a darker green (anthocyanin-containing) smaller shoot, primary root elongation has ceased growth, but lateral roots with numerous long root hairs have developed. (Photo courtesy of B. Bucciarelli and Carroll Vance).

in most pasture species with decreasing phosphorus concentrations. Soil phosphorus limitation is a primary effector of root architecture (Dinkelaker et al., 1995; Borch et al., 1999; Williamson et al., 2001; López-Bucio et al., 2003; Lambers et al., 2006; de Dorlodot et al., 2007) and is known to impact on all aspects of root growth and development. Phenotypic and genotypic adaptations to phosphorus deﬁciency involve changes in root architecture that facilitate acquisition of phosphorus from the topsoil (Lamont, 1982; Lynch and Brown, 2001; López-Bucio et al., 2003). Adaptations that enhance acquisition of phosphorus from topsoil are important because of the relative immobility of phosphorus in soil, with the highest concentrations usually found in the topsoil and little movement of phosphorus into the lower soil proﬁles. Lynch and Brown (2001) refer to phosphorus deﬁciency-induced modiﬁcation

of root architecture as adaptations for topsoil foraging. Root characteristics associated with improved topsoil foraging for scarce phosphorus are a more horizontal basal root growth angle resulting in more shallow roots, increased adventitious root formation, enhanced lateral root proliferation, and increased root hair density and length. Such modiﬁcations in root architecture in response to phosphorus deﬁciency are well documented in Arabidopsis (Fig. 12.1) and in those species forming either cluster or dauciform roots (Fig. 12.2). Genomic, genetic, biochemical, and developmental responses of plants subjected to phosphorus stress have been the subject of many recent reviews (Lambers et al., 2006; Lambers and Shane, 2007; Tesfaye et al., 2007; Doerner, 2008; Hammond and White, 2008; Lin et al., 2009; Hirayama and Shinozaki, 2010). This chapter summarizes research aimed at understanding phosphorus stress acclimation responses in

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

Fig. 12.2. Phosphorus deﬁciency phenotype of white

lupin roots. Phosphorus-sufﬁcient (+P) roots of white lupin have few to no cluster roots. By comparison, phosphorus-deﬁcient (–P) roots of white lupin have numerous cluster roots, which develop in waves along the root axis. (Photo courtesy of B. Bucciarelli and Carroll Vance).

plants with special reference to genomic and genetic control of root traits. Phosphorus uptake Phosphorus is taken up by plants in the orthophosphate (P) forms H2PO4− and HPO42–, which occur in soil solution at very low concentrations (0.1–10 μM; Hinsinger, 2001). A pH optimum for phosphorus uptake of 4.5–5.0 indicates preferential plant uptake of H2PO4 over HPO42– (Schachtman et al., 1998; Raghothama and Karthikeyan, 2005). Because of its strong reactions with soil components, phosphorus is principally supplied to plant roots by diffusion rather than by mass ﬂow (Hinsinger, 2001; Smith et al., 2003). Plant roots can alter soil solution

233

phosphorus availability by acidiﬁcation of the rhizosphere, exudation of organic acids, and secretion of extracellular phosphatases (Marschner, 1995; Hinsinger, 2001; Vance et al., 2003; Lambers et al., 2006). The rapid uptake of phosphorus at the root surface results in a phosphorus depletion shell of 0.2–1.0 mm around the root (Barber et al., 1963; Holford, 1997; Smith et al., 2003). For plants to surmount the concentration difference from soil solution to the internal plant cell as well as the negative membrane potential, active transport across the plasmalemma is required. Moreover, the striking reduction in phosphorus uptake and accumulation in plant cells treated with metabolic inhibitors reﬂects a signiﬁcant energy requirement for uptake (Smith et al., 2003; Raghothama and Karthikeyan, 2005). Over the past 10 years, genomic molecular physiology studies have shown that plants contain a multitude of membranelocalized phosphorus transporters that function as either low- or high-afﬁnity transporters (Paszkowski et al., 2002; Smith et al., 2003; Shin et al., 2004; Bucher, 2007; Liu et al., 2008). Functional complementation of yeast mutants defective in phosphorus transport was used to isolate and characterize phosphorus transporters from a diverse array of plants (Leggewie et al., 1997; Liu et al., 1998a,b). A search of gene indices shows at least 100 proteins from monocotyledons and eudicotyledons annotate as plant phosphorus transporters (Bucher, 2007). Multigene families have been demonstrated in Arabidopsis (9 genes), rice Oryza (13 genes), barley (8 genes), maize (6 genes), potato and tomato (5 genes), and Medicago (6 genes). The majority of these genes are highly expressed in root epidermal and root hair cells of plants grown under phosphorus deﬁciency (Paszkowski et al., 2002; Smith et al., 2003; Shin et al., 2004; Bucher, 2007; Liu et al., 2008) as compared with low or modest

234

NUTRIENT USE EFFICIENCY IN CROPS

expression under phosphorus-sufﬁcient conditions. Interestingly, in species supporting mycorrhizal symbioses, at least one member of the plant phosphorus transporter gene family is found to be speciﬁcally expressed in arbuscule-containing cells (Paszkowski et al., 2002; Grunwald et al., 2004; Bucher, 2007; Liu et al., 2008). Moreover, knockdown of phosphorus transporter expression has been shown to impair phosphorus accumulation in both Arabidopsis and rice (Shin et al., 2004; Ai et al., 2009). Proteins encoded by phosphorus transport genes have Mrs in the range of 56 kDa having 530–550 amino acid residues and 12 transmembrane domains (Smith et al., 2003; Bucher, 2007; Chen et al., 2008b). It is noteworthy that plant phosphorus transporter genes are frequently clustered in the genomes of Arabidopsis, rice, and Medicago (Okumura et al., 1998; Paszkowski et al., 2002; Liu et al., 2008). Such clustering suggests that plant phosphorus transporters arose through gene duplication and have evolved subfunctionalization roles (Liu et al., 2008). Phosphorus deﬁciency alters root biology Phosphorus stress mediates determinate growth of the primary root Phosphorus availability has a marked effect on the root system architecture of Arabidopsis (Narang et al., 2000; Williamson et al., 2001; López-Bucio et al., 2002; Al-Ghazi et al., 2003; Chevalier et al., 2003; Müller and Schmidt, 2004; Nacry et al., 2005). Growth under phosphorus-limiting conditions results in determinate growth of the primary root and redistribution of root growth from the primary root to lateral roots (Ticconi and Abel, 2004; Nacry et al., 2005; Fang et al., 2009). Reduced primary root

growth under low phosphorus was accompanied by increased lateral root density along with increased root hair length and number (Fig. 12.1). Arabidopsis root biomass was concentrated near the soil surface, suggesting topsoil foraging by roots. Moreover, accessions with enhanced phosphorus acquisition also appear to have strengthened root penetration capacity (Narang et al., 2000). Decreased primary root elongation during phosphorus stress is thought to involve localized sensing of phosphorus in the rhizosphere by root meristem and cap cells, which results in cessation of cell division in the primary root meristem (SánchezCalderón et al., 2005; Li et al., 2006; Lai et al., 2007; Svistoonoff et al., 2007). This cessation of cell division has been called root meristem exhaustion (Sánchez-Calderón et al., 2005). Svistoonoff et al. (2007) identiﬁed a multicopper oxidase gene (MCO) that is expressed in the root cap and root meristem, which mediates signaling between the primary root tip and phosphorus-deﬁcient media. The gene conditions primary root determinancy. This MCO gene appears to be the locus for the quantitative traits locus (QTL)-designated low phosphate root (LPR) (Reymond et al., 2006). Ticconi et al. (2004, 2009) identiﬁed an Arabidopsis mutant, phosphorus deﬁciency response 2 (pdr2), which has a disruption in phosphorus sensing. This mutant shows a short–root phenotype that is speciﬁc for phosphorus deﬁciency. The short–root phenotype of pdr2 is the result of inhibition of primary root cell division at phosphorus concentrations below 0.1 mM external phosphorus. Ticconi et al. (2009) proposed that pdr2 monitors external environmental phosphorus and regulates primary root meristem activity to adjust root system architecture to maximize phosphorus acquisition. In followup studies, Ticconi et al. (2009) show that PDR2 is a P5-type ATPase and is required for appropriate expression of the

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

SCARECROW GRAS transcription factor, which is a key regulator of root cell patterning. They also show that the MCO LPR1 and PDR2 interact, resulting in the adjustment of the root meristem to phosphorus status. LPR1 interacting with PDR2 may modify hormone sensitivity in the root cap which signals primary root determinancy. Root hairs The abundant development of lateral roots following cassation of primary root growth in low phosphorus environments is almost invariably accompanied by increased root hair density and length (Fig. 12.1). Root hairs are tubular cells specialized for nutrient uptake (Peterson and Farquhar, 1996; Gahoonia and Nielsen, 1998; Gilroy and Jones, 2000). They arise from root epidermal cells known as trichoblasts and undergo tip growth, thereby extending the root surface area in contact with the soil matrix (Ridge, 1995; Dolan, 2001). Root hairs can form as much as 77% of the root surface area of ﬁeld crops (Peterson and Farquhar, 1996). For plants lacking mycorrhizal associations, they are the primary site of nutrient uptake (Gahoonia and Nielsen, 1998; Gilroy and Jones, 2000; Jungk, 2001). Root hair formation and growth is regulated largely by the supply of mineral nutrients, particularly phosphorus and NO3− (Bates and Lynch, 1996, 2000; Gilroy and Jones, 2000; Ma et al., 2001). In Arabidopsis, trichoblasts are located over the junction of two underlying cortical cells. Trichoblasts can be distinguished by the late stages of embryogenesis. In recent years, root hair formation in Arabidopsis has become a model system for evaluating cell fate and hormonal interactions. Grierson et al. (2001) report that at least 40 genes in Arabidopsis affect root hair initiation and development. Five loci identiﬁed early on involved in root hair formation encode trans-

235

parent testa glabra (TIG), glabra2 (GL2), constitutive triple response1 (CTR1), root hair defective6 (RHD6), and auxin-resistant2 (AXR2) (Masucci and Schiefelbein, 1996). Analysis of these genes in Arabidopsis mutants demonstrated that a network of hormone interactions involving auxin and ethylene regulates root epidermal cell fate and root hair initiation (Tanimoto et al., 1995). Likewise, Parker et al. (2000) found eight genes whose loss-of-function mutants showed root hair defects. Although the function of these genes was not established, they were mapped to Arabidopsis chromosomes. More recently, Jones et al. (2006) showed that in addition to previously reported genes, some 606 novel genes had enhanced expression in Arabidopsis root hairs. They identiﬁed several gene families that appeared to be important in root hair morphogenesis, including cell wall synthesis enzymes, glycosylphosphatidylinositol (GPI)-anchored proteins associated with lipid rafts, armadillorepeat proteins, and leucine-rich receptor kinases. Importantly, a basic helix–loop– helix (bHLH) transcription factor has been shown to be the ancestral regulator of root hair development in Arabidopsis, controlling both postmitotic growth and length of root hairs (Menand et al., 2007; Yi et al., 2010). Phosphate availability regulates root hair elongation (Bates and Lynch, 1996; Yi et al., 2010) and root hair density (Ma et al., 2001). The average length of root hairs on phosphorus-deﬁcient plants was threefold greater than on phosphorus-sufﬁcient plants. Root hair density was ﬁvefold greater in low-phosphorus than in high-phosphorus plants. Trichoblast ﬁles increased from 8 to 12 on phosphorus-deﬁcient plants. Analysis of phosphorus acquisition in Arabidopsis wild type, and root hair mutants rhd6 and rhd2 showed that wild-type plants were more efﬁcient than root hair mutants in obtaining phosphorus when plants were grown at low phosphorus. However, there

236

NUTRIENT USE EFFICIENCY IN CROPS

was no difference in growth when grown at high phosphorus (Bates and Lynch, 2000). Zhu et al. (2010) found genetic plasticity in root hair number and length is an important component of nutrient acquisition for maize grown in low-phosphorus soils. Phosphate stress altered radical cell pattern formation in roots by increasing cortical number and reduced epidermal cell elongation, resulting in increased trichoblasts (Ma et al., 2001, 2003; Müller and Schmidt, 2004). Modiﬁed root hair patterning was most evident in mutant genotypes having defects in the ﬁrst stage of epidermal cell differentiation, suggesting phosphorus deﬁciency signals are perceived very early in epidermal cell development (Yi et al., 2010). Modiﬁed root cell patterning in phosphorus-deﬁcient plants appears to be regulated by auxin–ethylene interactions through a bHLH transcription factor (Dolan, 2001; Stepanova et al., 2007; Yi et al., 2010). Lateral roots The prototype root architecture modiﬁcation to occur in Arabidopsis in response to phosphorus deﬁciency is the promotion of lateral root development at the expense of primary root growth (Williamson et al., 2001; LópezBucio et al., 2002; Jain et al., 2007; PerezTorres et al., 2008). Phosphate deﬁciency appears to impair cell division in the primary root meristem but stimulates initiation of lateral root primordia and elongation of newly emerged lateral roots (Al-Ghazi et al., 2003; Malamy, 2005; Nacry et al., 2005; Sánchez-Calderón et al., 2005; Fukaki and Tasaka, 2009). Root architecture changes induced by phosphorus deﬁciency are strikingly similar to those induced by either auxin addition or overproduction (Al-Ghazi et al., 2003; Malamy, 2005; Nacry et al., 2005; Perez-Torres et al., 2008). The plant hormone auxin (primarily indole-3-acetic acid [IAA]) has long been known to stimu-

late lateral root formation (Boerjan et al., 1995; Reed et al., 1998; Casimiro et al., 2001; Marchant et al., 2002; Fukaki et al., 2007). Nacry et al. (2005) showed that during phosphorus starvation IAA increases in the whole primary root and young lateral roots of Arabidopsis. Without IAA, only primary root growth was observed in Arabidopsis (Karthikeyan et al., 2007). Regardless of phosphorus status, exogenous auxin application enhanced lateral root formation while suppressing primary root elongation in Arabidopsis (Al-Ghazi et al., 2003; Nacry et al., 2005; De Smet et al., 2007; Stepanova et al., 2007). Utilizing Arabidopsis mutants with lesions in auxin and ethylene signaling, a growing body of evidence indicates that phosphorus deﬁciency-induced root architecture changes result from modiﬁcations in auxin and ethylene synthesis and/or signaling. The current working hypothesis for lateral root induction under phosphorussufﬁcient conditions is that auxin is transported acropetally to the root tip and then is redirected basipetally through root outer cell layers (Fukaki and Tasaka, 2009). Inhibition of auxin transport either chemically or through mutant selection results in inhibition of lateral root formation (De Smet et al., 2007; Swarup et al., 2008; Peret et al., 2009). Auxin is transported to pericycle cells opposite xylem points, and upon reaching a threshold level pericycle cells become founder cells for lateral root primordia (Perez-Torres et al., 2008; Fukaki and Tasaka, 2009). Upon attaining a threshold level, auxin appears to be perceived by F-box auxin receptors, which then results in degradation of auxin/indole-3-acetic acid (AUX/IAA) repressor proteins through the ubiquitin protein ligase (SCFTIR) and 26S proteosomes (Dharmasiri et al., 2005; Fukaki and Tasaka, 2009). Degradation of AUX/IAA repressor proteins allows auxinresponsive transcription, activating cell

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

cycle gene expression in the primed pericycle. Perez-Torres et al. (2008) report that increased lateral root formation occurring under phosphorus deﬁciency is mediated by increased auxin sensitivity of root pericycle cells. They show tir-1 mutants lacking TIR have impaired lateral root induction under P-stress. Overexpression of TIR results in induced lateral root formation under phosphorus-sufﬁcient conditions. It appears that phosphorus deﬁciency induces an increase in lateral roots by enhancing TIR-1 in pericycle cells, thereby activating auxinresponse genes involved in lateral root primordia formation. Root architectural changes in response to phosphorus stress have also been shown to be related to ethylene in Arabidopsis (LópezBucio et al., 2002; Ma et al., 2003). Low phosphorus conditions appear to enhance the roots’ sensitivity to ethylene. Ethylene is known to be an effector of primary and lateral root cell elongation. Several recent reports document the interrelationship between auxin and ethylene in regulating root growth and development (OrtegaMartínez et al., 2007; Ruzicka et al., 2007; Stepanova et al., 2007; Swarup et al., 2007). Ethylene appears to stimulate auxin biosynthesis and increase auxin transport (Ivanchenko et al., 2008). Moreover, auxin must be present for ethylene inhibition of cell expansion. Ethylene may also interact with auxin to release lateral root meristems for growth. In contrast, a recent study by Negi et al. (2008) suggest that ethylene negatively regulates lateral root formation in Arabidopsis by altering auxin transport. Exogenous application of ethylene precursor or overproduction of ethylene gave a decrease in lateral roots. It is apparent that the auxin–ethylene interaction in lateral root formation is complex and deserves further attention and the role of ethylene in phosphorus stress lateral root formation is unresolved.

237

Traditionally, cytokinins are thought to be negative regulators of root growth while having positive effects on shoot growth (Christianson and Warnick, 1985; Werner et al., 2001, 2003; Aloni et al., 2006; Chapman and Estelle, 2009). However, recent work by Müller and Sheen (2008) has demonstrated that root stem cell differentiation requires antagonistic interactions between auxin and cytokinin. Cytokinin is also required for correct differentiation of the root vascular system (Mahonen et al., 2006; Schmülling, 2009). Increased cytokinin biosynthesis in protoxylem pericycle cells inhibits lateral root formation, while increased cytokinin in protoxylem pericycle cells inhibits lateral root formation (Laplaze et al., 2007). Phosphorus deﬁciency results in a decrease in cytokinin content (Wagner and Beck, 1993; López-Bucio et al., 2003) accompanied by increased lateral root formation. Application of exogenous cytokinin inhibits root development and abolishes auxin-induced lateral root development (Franco-Zorrilla et al., 2005; Werner and Schmülling, 2009). Cytokinin inhibition of primary root growth appears to occur through its effect on the vascular transition zone and regulation of root meristem size (Dello Iolo et al., 2007). Arabidopsis plants that overexpress cytokinin oxidase (CKX), the key enzyme regulating cytokinin degradation, have reduced cytokinin concentrations accompanied by increased lateral and adventitious root formation (Werner et al., 2003; Lohar et al., 2004). Li et al. (2006) have shown that cytokinins inhibit lateral root formation by blocking the pericycle founder cells transition at the G2 phase. Cytokinins appear to suppress the expression of phosphorus deﬁciency-induced genes (Martin et al., 2000; Hou et al., 2005). Consistent with these observations, the mutant Arabidopsis histidine kinase 3 (ahk3) has reduced sensitivity to cytokinin and also

238

NUTRIENT USE EFFICIENCY IN CROPS

shows impaired expression of several phosphorus deﬁciency-induced genes (FrancoZorrilla et al., 2005). It is quite apparent that modiﬁcation of root architecture by phosphorus deﬁciency in Arabidopsis is regulated by the subtle interaction of auxin, ethylene, and cytokinin. Localized changes in hormone transport, synthesis, and cellular concentration shut down primary root growth, activate lateral root development, and increase trichoblast frequency, giving rise to the root architecture favorable for topsoil foraging for phosphorus. Cluster roots Recent reviews (Shane and Lambers, 2005; Lambers et al., 2006) have documented the striking diversity in cluster root morphology. Cluster roots form through synchronized development of densely packed determinate rootlets (Fig. 12.2). They can comprise single clusters formed on the parent axis, as found in Proteaceae members such as Hakeae spp., Leucadendron spp., Grevillea robusta, and in the Fabaceae white lupin (Lamont, 1982, 2003; Skene et al., 1996; Diem et al., 2000; Lambers et al., 2006). However, some Protea species like Banksia grandis form more complex compound clusters that form embedded mats in the soil proﬁle (Lamont, 1993, 2003; Adams and Pate, 2002; Shane and Lambers, 2005). Several features of dicotyledonous cluster root development and morphology are distinguished from that of typical lateral roots. First, lateral roots are initiated usually in an alternating pattern from the pericycle of primary roots near the zone of metaxylem differentiation (Celenza et al., 1995; Charlton, 1996), while cluster rootlets are initiated in waves along the axis of secondand third-order lateral roots (Skene and James, 2000; Pate and Watt, 2001; Lamont, 2003). Second, typical lateral roots are initi-

ated singularly opposite a protoxylem point, unlike cluster roots, which are in multiples opposite every protoxylem point within the wave of differentiation (Fig. 12.2). Third, cluster rootlets produce a superabundance of root hairs due to an apparent loss of regulation of trichoblast differentiation, while root hair development in typical roots is highly regulated and occurs from a discrete number of trichoblasts (Dolan, 2001; Müller and Schmidt, 2004). The length of cluster root segments varies from a few millimeters upward to 2–4 cm. Under phosphorusdeﬁcient conditions, cluster roots can comprise more than 70% of the plant root mass (Lamont, 1982; Pate and Watt, 2001; Lamont, 2003; Lambers et al., 2006). The frequency of cluster roots in phosphorusdeﬁcient soil and the accompanying increase in root hair density results in an increase in root surface area of greater than 100-fold (Lamont, 2003; Vance et al., 2003; Shane and Lambers, 2005). Another notable feature of cluster rootlets is that their growth is determinate, ceasing shortly after emergence, as contrasted with the indeterminate growth of lateral roots (Neumann and Martinoia, 2002). This highly synchronous developmental pattern reﬂects that cluster root formation is an elegant, ﬁnely tuned process. Moreover, because root pericycle cells are arrested in the G2 phase of the cell cycle (Skene, 1998; Skene and James, 2000), cluster root initiation likely involves a hormone-mediated concerted release of multiple pericycle cells from the G2 phase in a wave-like pattern along second-order lateral roots. Recently, Sbabou et al. (2010) found that silencing of white lupin SCARECROW gene expression impaired white lupin cluster root formation, and SCARECROW has been implicated in regulating Arabidopsis response to phosphorus (Ticconi et al., 2009). Root architectural changes classiﬁed as cluster root types also occur in the sedges

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

239

Fig. 12.3. Biochemical adaptations occurring in proteoid roots involve extrusion of protons and carboxylates into

the rhizosphere via a plasmalemma H+ ATPase and anion channels, respectively. Release of protons and carboxylates solubilize phosphorus from unavailable soil-bound forms. Acid phosphatases and perhaps phytases exuded from root hairs solubilize phosphorus from organic esters. Carboxylates and protons extruded from root hairs may also make organic phosphorus esters (P-esters) more soluble and susceptible to acid phosphatases. Phosphorus released from insoluble ligands and organic P-sters is taken up through phosphorus transporters in the plasmalemma.

(Cyperaceae) and the rushes (Restionaceae). Lamont (1974) characterized the cluster root morphology found in the Cyperaceae as dauciform due to the carrot-like developmental pattern along the root axis. As dauciform roots mature they develop large numbers of long (3–5 mm) root hairs (Shane and Lambers, 2005; Lambers et al., 2006; Vance, 2008). The development of dauciform roots in Schoenus unispiculatus was directly related to phosphorus availability, with their formation being suppressed as phosphorus availability increased (Shane et al., 2005). Recently, Shane et al. 2006 have shown that dauciform roots of S. unispiculatus are structurally distinct from typical cluster roots but functionally analogous to them. In the Restionaecae, the cluster root morphology has been characterized as

capillaroid (Lamont, 1982) due to the sponge-like, water-holding capacity of the root–soil aggregate. Capillaroid species have root clumps with exceptionally long root hairs (Lambers et al., 2006). Cluster root function Similar to mycorrhizal association, cluster roots increase the root surface area and soil volume exploited for mining nutrients. In contrast to mycorrhizae, which grow over much of the entire root surface, the hairy and densely packed lateral rootlets in cluster root zones bind tightly to trapped soil particles and organic matter in localized soil volumes. Cluster root aggregates are most prominent in the upper layers of the soil proﬁle where phosphorus is most abundant (Lamont,

240

NUTRIENT USE EFFICIENCY IN CROPS

2003; Shane and Lambers, 2005; Lambers et al., 2006). The dense aggregation of cluster roots and soil facilitates the acquisition and uptake of nutrients, particularly phosphorus (Lamont, 2003; Vance et al., 2003; Shane and Lambers, 2005; Lambers et al., 2006; Vance, 2008). Enhanced capacity for nutrient acquisition and uptake by cluster roots occurs by effectively concentrating root exudates and plant nutrient uptake systems in localized patches of soil (Grierson and Attiwill, 1989). Cluster roots exude organic acids (Gardner et al., 1983; Dinkelaker et al., 1995; Johnson et al., 1996a,b; Shane et al., 2006), protons (H+) (Dinkelaker et al., 1995; Neumann and Martinoia, 2002; Yan et al., 2002; Shane et al., 2006), phenolics (Neumann et al., 1999; Weisskopf et al., 2006a,b), acid phosphatases (Dinkelaker et al., 1995; Gilbert et al., 1999; Miller et al., 2001), and iron chelate reductase (Dinkelaker et al., 1995; Neumann and Martinoia, 2002). Cluster roots have enhanced expression of phosphate transporters (Liu et al., 2001) and increased phosphorus uptake (Keerthisinghe et al., 1998; Neumann et al., 2000; Sousa et al., 2007). Exudation of carboxylates, protons, phenolics, and acid phosphatases releases phosphorus bound to inorganic and organic particles, making it available for rapid uptake by phosphorus transporters (Fig. 12.3). Cluster root development accompanied by corresponding changes in cluster root metabolism and membrane uptake systems reﬂect an elegant, highly coordinated molecular response to phosphorus deﬁciency. Although the development of cluster roots in Proteaceae and Cyperaceae has received growing attention, white lupin has served as the model for analysis of biochemical and molecular adaptations contributing to enhanced phosphorus acquisition and use by cluster roots (Dinkelaker et al., 1995; Watt and Evans, 1999; Neumann and

Martinoia, 2002; Uhde-Stone et al., 2003a, b; Liu et al., 2005). At the onset of phosphorus deﬁciency stress in white lupin, a cascade of changes in gene expression occurs, resulting in synchronous development of cluster roots having proliﬁc root hair density; exudation of carboxylates, protons, and enzymes; enhanced expression of membrane transporters; and apparent heightened sensitivity in roots to hormonal signals. Neumann et al. (2000) have staged cluster root development into four phases distinguished by rootlet emergence and biochemical response. In the juvenile and premature stages, cluster rootlets have emerged from the cortex and are actively elongating. At these stages rootlets are exuding malate and citrate in fairly equal amounts (300–700 nmol h−1 gfw−1) accompanied by uniform rhizosphere pH (5–6) and little extrusion of protons. As the mature stage is attained, rootlet elongation ceases, citrate exudation exceeds malate by four- to ﬁvefold, and copious amounts of acid phosphatase and phenolics are exuded accompanied by a reduction in rhizosphere pH and H+ extrusion (Neumann and Martinoia, 2002). Mature cluster root zones have increased transcript abundance for phosphorus transporters (Liu et al., 2001), acid phosphatase (Miller et al., 2001), sugar metabolism genes (Uhde-Stone et al., 2003a), and multidrug toxin efﬂux (MATE) transporters (Uhde-Stone et al., 2005). It is noteworthy that total RNA concentration decreases as cluster root zones progress through development into the later phase of maturity, suggesting turnover of nucleic acids to provide phosphorus for remobilization (Johnson et al., 1996a,b). The burst of exudation occurring in mature cluster root zones occurs over a 3- to 4-day period followed by the senescent stages of differentiation in which cluster roots turn brown and physically deteriorate (Watt and Evans, 1999). Recent studies with cluster roots of Hakea, Proteaceae (Shane et al., 2004; Shane

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

and Lambers, 2005), and with dauciform roots of Schoenus, Cyperaceae (Shane et al., 2006) show developmental stages and biochemical modiﬁcations analogous to those that occur in white lupin. Phosphate acquisition from soil is enhanced in cluster root species (Gardner et al., 1982; Lamont, 1982; Grierson, 1992; Dinkelaker et al., 1995; Keerthisinghe et al., 1998; Neumann et al., 2000; Sousa et al., 2007). Enhanced acquisition of soil phosphorus occurs not only because of the biochemical and architectural changes that occur in cluster roots which facilitate increased surface area for explorations and release of exudates to solubilize phosphorus but also because phosphorus uptake is enhanced in cluster roots. Increased phosphorus uptake has been noted in cluster root zones of both white lupin (Keerthisinghe et al., 1998; Neumann et al., 2000) and Hakea (Sousa et al., 2007). The Km for phosphorus uptake in cluste root species ranges from 1.0 μM for the high-afﬁnity uptake in low-phosphorus soils to 40 μM under high phosphorus conditions. Sousa et al. (2007) showed a biphasic phosphorus uptake for Hakea serica, indicating a high- and lowafﬁnity system. Interestingly, Liu et al. (2001) isolated and characterized two phosphorus transporter genes from white lupin cluster roots. One white lupin phosphate transporter, LaPT1, was speciﬁcally expressed in cluster roots under phosphorus deﬁciency and probably corresponds to a high-afﬁnity transporter. In contrast, LaPT2 is expressed in all tissues in fairly high abundance and likely represent a low-afﬁnity transporter. Abundant expression of numerous transporter genes is seen in phosphorus stressinduced cluster roots of white lupin (Uhde-Stone et al., 2003b) ranging from phosphorus transporters to sulfate transporters, amino acid, and sugar transporters. The abundance of these transporters in white lupin cluster roots suggests the cluster root

241

system is geared up for effective transport of many nutrients.

Modiﬁed carbon Under normal conditions of growth and development, plant roots exude a wide variety of organic compounds including simple sugars, organic acids, amino acids, phenolics, quinones, (iso)-ﬂavonoids, growth hormones, proteins, and polysaccharides (Marschner et al., 1986; Kochian et al., 2004). Exudation of organic compounds from roots can alter rhizosphere chemistry, soil microbial populations, competition, and plant growth. Exuded compounds are functionally diverse and can be involved in a wide array of processes ranging from signaling in plant–microbe interactions, to allelopathy and nutrient acquisition (Marschner et al., 1986; Hinsinger, 2001; Ryan et al., 2001; Kochian et al., 2004). During phosphorus deﬁciency stress, roots show enhanced accumulation of sugars, increased synthesis and exudation of carboxylates, and a dependence on sugars or phloem transport for phosphorus stress-induced gene expression in roots (Johnson et al., 1996a; Keerthisinghe et al., 1998; Watt and Evans, 1999; Neumann and Martinoia, 2002; Shane et al., 2004; Liu et al., 2005). Convincing evidence exists for exudation of malate and citrate as a principal mechanism for relieving the edaphic stress of phosphorus deﬁciency (Fig. 12.4). The release of carboxylates allows for the chelation of Al3+, Fe3+, and Ca2+ and the subsequent displacement of phosphorus from bound or precipitated forms (Gardner et al., 1982; Dinkelaker et al., 1995; Ryan et al., 2001; Vance et al., 2003; Shane and Lambers, 2005) and may also make organic phosphorus become more susceptible to acid phosphatase activity. Tricarboxylates such as citrate are more effective than dicarboxylates at displacing

242

NUTRIENT USE EFFICIENCY IN CROPS

Fig. 12.4. Schematic representation of the glycolytic and tricarboxylic acid pathways. Expressed sequence tags (ESTs) with induced expression in phosphorus-deﬁcient roots, compared with phosphorus-sufﬁcient normal roots, are represented by 2-, 3-, 4X-fold increase, and so on. The average of gene induction was estimated from the literature at the corresponding arrows. The majority of ESTs with possible function in the glycolytic and tricarboxylic acid pathways displayed increased expression in phosphorus-deﬁcient roots compared with phosphorus-sufﬁcient normal roots. Notable exceptions are pyruvate kinase (PK), aconitase, and isocitrate dehydrogenase. The metabolic diagram shows (1) sucrose is ﬁrst degraded by sucrose synthase (SS); (2) the phosphoenolpyruvate carboxylase (PEPC) bypass for PK; and (3) the exudation of malate and citrate as representative of phosphorus-stressed roots. ACON, aconitase; ICDH, isocitrate dehydrogenase; KG-DH, alphaketoglutonate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase; CS, citrate synthase; MDH, malate dehydrogenase.

bound phosphorus due to their greater afﬁnity for Fe3+, Al3+, and Ca2+ (Hinsinger, 2001; Ryan et al., 2001; Kochian et al., 2004). While many plant species exude carboxylates under phosphorus-deﬁcient conditions (Marschner et al., 1986; Ryan et al., 2001;

Lambers et al., 2006), this trait achieves maximum effectiveness in cluster root species (Johnson et al., 1996b; Keerthisinghe et al., 1998; Neumann and Martinoia, 2002; Kihara et al., 2003; Shane et al., 2004, 2006). When grown under phosphorus-deﬁcient

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

conditions, cluster roots exude 20- to 40fold more citrate and malate than phosphorussufﬁcient roots (Fig. 12.4). Shane et al. (2004) reported that a signiﬁcant accumulation of carboxylates (75 mol gfw−1) in Hakea cluster roots coincided with the maximum rate of organic acid exudation. Likewise, Watt and Evans (1999) showed that mature lupin cluster roots exude 34 nmol min−1 gfw−1 as compared with nondetectable levels in early stages of development. Malate is frequently detected as the primary component of exudates in juvenile and premature cluster roots, while citrate predominates at peak exudation in mature cluster roots (Johnson et al., 1994, 1996b; Watt and Evans, 1999; Neumann et al., 2000; Shane et al., 2004, 2006). Peak exudation has been referred to as the exudative burst. The amount of carbon exuded in citrate and malate can constitute from 10% to greater than 25% of the apparent current photosynthate of the plant (Johnson et al., 1994, 1996a, Neumann et al., 2000). Cluster roots and phosphorus-stressed roots in general are strong sinks for photosynthate (Johnson et al., 1996a; Watt and Evans, 1999; Neumann et al., 2000; Shane et al., 2004; Hernández et al., 2007; Morcuende et al., 2007). It is noteworthy that sugar signaling is required for cluster root formation (Zhou et al., 2008). In fact, added sucrose stimulates cluster root formation in phosphorus-sufﬁcient white lupin. The striking change that occurs in phosphorus-stressed cluster root organic acid exudation is invariably reﬂected in concurrent changes in RNA expression and activity of enzymes involved in carbon metabolism (Johnson et al., 1994; Neumann et al., 2000; Massonneau et al., 2001; UhdeStone et al., 2003a; Shane et al., 2004). Measurements of shoot and root CO2 ﬁxation with 14CO2 accompanied by measurements of carbon partitioning and exudation show that organic acids exuded from lupin

243

cluster roots are derived from both photosynthetic CO2 ﬁxation and root dark (anapleurotic) CO2 ﬁxation (Johnson et al., 1994, 1996a,b). Some 60–65% of carbon exuded from roots is shoot derived, while 35–40% is root derived. Exudation of carboxylates from roots is accompanied by an increase in the activities of sucrose synthase (SS), enzymes of glycolysis, and organic acids (Fig. 12.4), particularly phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) (Johnson et al., 1994; Massonneau et al., 2001; Uhde-Stone et al., 2003a; Peñaloza et al., 2005). Similar changes in carbon metabolism have been noted for phosphorus-stressed roots of bean (Ciereszko et al., 1998; Hernández et al., 2007) and Arabidopsis (Morcuende et al., 2007). Interestingly, although cluster roots are strong sinks for carbon, there is not a striking increase in respiration. Cluster root carbon metabolism and respiration appear to be relatively efﬁcient due to adaptive changes that occur in response to low phosphorus. For example, phosphorus stress can limit the activity of pyruvate kinase (PK), an enzyme requiring phosphorus and adenosine diphosphate (ADP). The phenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NAD-malic enzyme pathway can bypass the PK entry into the tricarboxylic acid (TCA) cycle, thereby maintaining the ﬂow of carbon while avoiding the use of ADP while generating phosphorus (Plaxton and Carswell, 1999; Morcuende et al., 2007). Likewise, the reduction in cellular ADP and phosphorus in plants undergoing phosphorus stress can result in reduced efﬁciency of respiration by inhibiting the cytochrome pathway of electron transport. Nonphosphorylating pathways that can bypass energy-requiring steps like the alternative oxidase (AOX) system can maintain cellular metabolic integrity (Theodorou and Plaxton, 1993; Vance et al., 2003; Vijayraghavan and Soole, 2010).

244

NUTRIENT USE EFFICIENCY IN CROPS

Recent studies show that AOX is enhanced in Hakea cluster roots (Shane et al., 2004), Arabidopsis (Morcuende et al., 2007), and bean (Hernández et al., 2007). A series of elegant studies has now conclusively demonstrated a tight link between sugars and plant adaptation to phosphorus stress (Liu et al., 2005; Hernández et al., 2007; Karthikeyan et al., 2007; Morcuende et al., 2007; Müller et al., 2007). Sugars are required for the expression of many phosphorus stress-induced genes in both roots and shoots. Liu et al. (2005) and Tesfaye et al. (2007) showed that expression of the phosphorus stress-induced cluster root genes, phosphate transporter (Pt), acid phosphatase (APase), multidrug toxin (MATE), SS, hexokinase (HXK), and fructokinase (FK) required sugar or phloem transport. Dark adaptation or blocking phloem transport reduced the expression of these genes to nondetectable levels in 24 h but reexposure to light activated their transcription within a few hours. The interplay of sugars and phosphorus-stressed gene expression noted in white lupin has been validated with Arabidopsis and common bean. Karthikeyan et al. (2007), Hernández et al. (2009), and Müller et al. (2007) have shown that phosphorus stress gene expression and modiﬁed root architecture in Arabidopsis and common bean is tightly linked to sugar signaling, probably through HXK. Hormonal regulation of cluster roots Because cluster root development involves the synchronized initiation and growth of a large number of tertiary lateral roots in distinct wavelike patterns originating from primary and secondary lateral roots, it would not be surprising that hormone balance plays a role in this phosphorus-adaptive process (Gilbert et al., 2000; Skene and James, 2000). Many of the hormonally controlled developmental responses occurring in

phosphorus-stressed Arabidopsis that give rise to modiﬁed root architecture appear also to be involved in cluster root architecture. A number of plant hormone-related expressed sequence tags (ESTs) have been detected during sequencing of cDNA libraries made to phosphorus deﬁciency-induced cluster roots. As described later for Arabidopsis lateral roots, substantial support for the role of auxin in cluster root formation comes from observations showing that exogenous application of auxin stimulates cluster root formation in phosphorus-sufﬁcient white lupin and Protea species (Gilbert et al., 2000; Skene and James, 2000); auxin transport inhibitors block cluster root formation in phosphorus-deﬁcient plants; and many genes involved in auxin synthesis and signaling are abundantly expressed in developing cluster roots of white lupin (Vance et al., 2003; C.P.Vance, C. Uhde-Stone, and M. Yamagishi, unpublished). These data clearly show that a signiﬁcant component of phosphorus-induced cluster root formation is due to auxin synthesis and transport. Currently, experiments are underway to assess auxin signaling in cluster roots through the transformation of cluster roots with the auxin reporter construct DR5-GUS (Ulmasov et al., 1997). Although the role of ethylene in phosphorus stress-induced cluster root architecture is not clear, the fact that ethylene plays a role in phosphorus-stressed Arabidopsis root hair length, density, and development as well as in lateral root emergency (Ticconi and Abel, 2004) suggests a similar role in cluster roots. As noted previously, cluster roots and dauciform roots have densely packed, unusually long root hairs. Studies with stress-induced cluster roots of white lupin (Gilbert et al., 2000), Casurina (Zaid et al., 2003), and squash (Waters and Blevins, 2000) have correlated ethylene with cluster root formation. Gilbert et al. (2000) demonstrated a two- to threefold

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

increase in ethylene as cluster roots develop in phosphorus-stressed lupin. Zaid et al. (2003) showed that iron deﬁciency stimulated cluster roots in Casurina and inhibition of ethylene synthesis reduced cluster root formation. Waters and Blevins (2000) noted a correlation between ethylene production, root iron reduction, and cluster root formation in squash. A number of genes involved in ethylene biosynthesis are overrepresented in sequencing studies of ESTs derived from phosphorus stress-induced cluster root cDNA libraries, further suggesting a role of ethylene in cluster root architecture (UhdeStone et al., 2003b). In their classical study of the physiology of cluster roots, Neumann et al. (2000) found that addition of cytokinins to lupin signiﬁcantly reduced cluster root formation and cluster rootlet elongation. They also found elevated levels of cytokinin in 4-week-old phosphorus-deﬁcient white lupin roots as compared to P-sufﬁcient roots. They postulated that auxin stimulates emergence of cluster rootlets in P-deﬁcient plants which results in increased production of cytokinin due to the numerous emerged root tips. In mature segments of phosphorus-induced cluster roots we have found numerous ESTs that annotate to CKX (Uhde-Stone et al., 2003b), suggesting that cytokinins may be subject to degradation as cluster rootlets mature. CKX is the key enzyme implicated in cytokinin degradation (Morris et al., 1999). Enhanced degradation of cytokinins in cluster root formation and/or development might be expected because low cytokinin levels favor root growth, and phosphorus deﬁciency reportedly results in reduced xylem sap cytokinin levels (Salama and Wareing, 1979; Martin et al., 2000; Emery and Atkins, 2002). Alternatively, in planta regulation of potentially large quantities of cytokinins that could be released by the mass induction of cluster root meristems may require CKX. As noted earlier, cytoki-

245

nins reduce lateral root initiation in Arabidopsis. While strong correlative physiological and gene expression data suggest a critical role of auxins, cytokinins, and ethylene in phosphorus stress-induced cluster root development, deﬁnitive genetic and biochemical experiments have yet to be performed. Salient questions to be addressed include: What are the internal signals that initiate the cascade of developmental biochemical and genetic changes resulting in cluster roots? How is determinancy in cluster roots regulated? Are reactive oxygen and programmed cell death part of the cluster root developmental phenomenon? Can gene knockdown and overexpression studies be harnessed to deﬁnitively answer questions regarding the role of growth hormones in cluster root development and function? Can the genetic control mechanisms(s) for cluster root formation be identiﬁed and used to enhance phosphorus uptake and use efﬁciency in other plant species? Recent advances in genetic transformation of root tissue via Agobacterium rhizogenes (Boisson-Dernier et al., 2001; Limpens et al., 2004) facilitate high-throughput experiments to evaluate gene function with either knockdown or overexpression approaches. Coupled with metabolomics and proteomics, new insights will be gained into the role of phosphorus stress in the development of complex root architecture.

Phosphorus deﬁciency-induced changes in gene expression Several studies of Arabidopsis (Hammond et al., 2003, 2004; Wu et al., 2003; Misson et al., 2005; Morcuende et al., 2007) and other species (Wang et al., 2001; UhdeStone et al., 2003a,b; Wasaki et al., 2003; Ramírez et al., 2005; Graham et al., 2006; Hernández et al., 2007; Calderon-Vazquez

246

NUTRIENT USE EFFICIENCY IN CROPS

et al., 2008; Li et al., 2010) have now addressed whole genome changes in gene expression in response to phosphorus deﬁciency. Each study has noted that scores of genes respond with a twofold change in expression. Hammond et al. (2004) noted that these changes can be classiﬁed as either early genes that respond rapidly and frequently nonspeciﬁcally to phosphorus stress or late genes that lead to modiﬁcations in root architecture and metabolism. Invariably the studies have shown that early-response genes include transporters, particularly phosphorus transporters. However, numerous other transporters also respond rapidly to phosphorus deﬁciency including sugar, sulfate, iron, amino acid transporters, and aquaporins (Wang et al., 2001; Uhde-Stone et al., 2003b; Misson et al., 2005; Hernández et al., 2007; Morcuende et al., 2007; Calderon-Vazquez et al., 2008; Li et al., 2010). Transcription factor and signal transduction genes also respond quickly to phosphorus stress along with cell wall synthesis genes, hormone homeostasis genes, general stress genes, and acid phosphatases (FrancoZorrilla et al., 2004; Hammond et al., 2004; Misson et al., 2005; Ramírez et al., 2005; Morcuende et al., 2007; Calderon-Vazquez et al., 2008). Genes induced for later responses include those encoding enzymes of glycolysis, carboxylate and amino acid metabolism, secondary metabolism, pyrimidine salvage, and nucleoside hydrolases (Wasaki et al., 2003; Uhde-Stone et al., 2003b; Hammond et al., 2004; Misson et al., 2005; Ramírez et al., 2005; Morcuende et al., 2007; Calderon-Vazquez et al., 2008; Li et al., 2010). A large number of genes involved in phospholipid degradation and modiﬁcation along with genes affecting synthesis of galacto- and sulfolipid synthesis also have strikingly enhanced expression within 24–48 h of phosphorus stress (Essigmann et al., 1998; Andersson et al., 2005; Misson et al., 2005; Morcuende

et al., 2007; Hernández et al., 2009; Li et al., 2010). Modiﬁcations in gene expression in response to phosphorus stress are reﬂected in noteworthy shifts in metabolic activity (Uhde-Stone et al., 2003a; Hammond et al., 2004; Misson et al., 2005; Hernández et al., 2007; Morcuende et al., 2007; CalderonVazquez et al., 2008). For example, the increased transcript abundance of lipidrelated genes mirrors shifts in membrane biogenesis from incorporation of phospholipids to a shift involving galacto- and sulfolipids as membrane components (Essigmann et al., 1998; Andersson et al., 2005; Misson et al., 2005; Calderon-Vazquez et al., 2008). Altered transcript abundance of genes involved in carbon metabolism is echoed in increased sucrose and organic acid metabolism (Uhde-Stone et al., 2003a; Hernández et al., 2007; Morcuende et al., 2007). Carbon also shifts toward starch biosynthesis at low phosphorus and toward starch catabolism under high phosphorus. Likewise, the increase in transcripts involved in phenylpropanoid secondary metabolism is visible in anthocyanin biosynthesis under phosphorus stress conditions (Hernández et al., 2007; Morcuende et al., 2007; Calderon-Vazquez et al., 2008). Interestingly, Morcuende et al. (2007) and Li et al. (2010) found a striking increase in glycerophosphodiesterases (GPDEs) gene expression. Selected GPDE genes have been recently shown to be important in root hair development (Jones et al., 2006), and the phenomenon of increased root hair length and density is a common phosphorus deﬁciency stress response. Whole-genome studies have also led to the discovery of many transcription factors that appear to be important in the phosphorus deﬁciency response. Current evidence suggests that control of phosphorus signaling during stress is complex, involving both positive and negative regulation of gene

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

expression. Some of the transcription factors proven to be involved in phosphorus signal transduction include MYBs, WRKYs, bHLH, zinc ﬁnger proteins, and HD-Zip factors (Rubio et al., 2001; Hammond et al., 2004; Yi et al., 2005; Devaiah et al., 2007; Tesfaye et al., 2007; Hernández et al., 2009). Within recent years, proteomic analyses of phosphorus-stressed plants have substantiated some metabolic aspects of the phosphorus stress response interpreted from whole-genome transcript analysis. In studies of both rice (Fukuda et al., 2007; Torabi et al., 2009) and maize (Li et al., 2007, 2008), between 600 and 1500 protein spots were reproducibly detected on twodimensional gels. Although 100–200 spots appeared to respond to the phosphorus status of the plant, many fewer could be conclusively identiﬁed. Consistent patterns of protein expression noted in maize and rice include the following: (1) Proteins related to glycolysis and organic acid production showed increased abundance in P-deﬁcient plants. Enzymes involved in malate and citrate synthesis increased, while those involved in degradation of citrate and malate decreased. Similarly, SS peptides were found to increase. These ﬁndings support the interpretation that increased sucrose degradation and metabolism during phosphorus deﬁciency provide additional carbon needed for the synthesis of malate and citrate, which are secreted into the rhizosphere to improve phosphorus availability. (2) The phosphorus stress proteome also shows increased abundance of proteins associated with protection against stress such as peroxidases, ascorbate reductases, superoxide dismutases, glutathione reductase, and chaperonins (Li et al., 2008; Tran and Plaxton, 2008; Torabi et al., 2009). This group of proteins protects against reactive oxygen species generated during nutrient stress and protect critical protein folding necessary in the stress response.

247

Multiple mechanisms regulate gene expression during acclimation to phosphorus deﬁciency Transcription factors Regulation of gene expression during plant stress is frequently controlled by the transcriptional activation or repression of genes (Chen et al., 2002; Hammond et al., 2004; Valdés-López et al., 2008; Valdés-López and Hernández, 2008). Transcription factors are key global regulators of gene expression and are known to play central roles in most biological processes including regulation of plant gene expression in response to numerous biotic and abiotic stresses (Sreenivasulu et al., 2007; Century et al., 2008; Hirayama and Shinozaki, 2010). In Arabidopsis, approximately 6% (about 1800) of the total number of genes are composed of transcription factors, including about 72 WRKY family of genes, more than 600 zinc ﬁnger proteins, and 133 MYB transcription factors (Eulgem et al., 2000; Riechmann et al., 2000; Stracke et al., 2001; Guo et al., 2005). In a microarray analysis, approximately 30% of the 333 transcription factor genes included in the array was up- or downregulated twofold or more during phosphorus stress in Arabidopsis (Wu et al., 2003). Misson et al. (2005) and Müller et al. (2007) also reported up to 80 phosphorus stressresponsive transcription factor genes in Arabidopsis. Graham et al. (2006) discovered through bioinformatic analysis of phosphorus stress-induced genes in Arabidopsis, Medicago, Glycine, Phaseolus, and Lupinus that they share in common, transcription factor families encoding WRKYs, MYBs, GRAS, zinc ﬁnger proteins, and bHLH proteins, which respond to plant phosphorus status. For details on speciﬁc transcription factors implicated in acclimation to phosphorus deﬁciency, the reader is

248

NUTRIENT USE EFFICIENCY IN CROPS

referred to several comprehensive reviews that cover the primary literature (Doerner, 2008; Yuan and Liu, 2008; Yang and Finnegan, 2010). The classic example of transcriptional regulation of phosphorus-responsive genes was delineated through studies of the MYB transcription factor PHR1 (Rubio et al., 2001; Miura et al., 2005; Nilsson et al., 2007; Valdés-López et al., 2008). Rubio et al. (2001) identiﬁed an Arabidopsis mutant phr1 that when grown under phosphorus deﬁciency had reduced accumulation of anthocyanin and defective expression phosphorus deﬁciency response genes. These results indicated that PHR1 was a positive regulator of phosphorus response gene expression. PHR1 protein binds to an imperfect palindromic consensus ciselement (5′-GNATATNC-3′) found in the promoter of numerous but not all phosphorus deﬁciency response genes (Hammond et al., 2003; Misson et al., 2005; Morcuende et al., 2007). Knockdown of PHR1 expression mimics the phr1 mutant, while overexpression of PHR1 results in increased phosphorus concentration and enhanced expression of phosphorus deﬁciency response genes (Nilsson et al., 2007). Homologs of PHR1 have been found in numerous species including rice, bean, and lupin (Valdés-López et al., 2008; Zhou et al., 2008; Zinn et al., 2009). The observation that PHR1 expression and nuclear localization did not change in response to phosphorus led to the hypothesis that PHR1 expression was also regulated at the posttranslational level. Miura et al. (2005) noted that the Arabidopsis mutant siz1showed an exaggerated phosphorus deﬁciency response including inhibition of primary root growth, more lateral roots and root hairs, and increased anthocyanin accumulation. Moreover, the SIZ1 protein had Sumo E3 ligase activity and PHR1 was a target of SIZ1 sumolyation. The enhanced sensitivity

of the siz1 mutant to phosphorus deﬁciency was due to Sumo E3 ligase-mediated degradation of PHR1 and could result in both positive and negative regulation of phosphorus-responsive gene expression.

QTLs associated with phosphorus stress tolerance and acclimation Genetic variability for the complex root and shoot responses to phosphorus-limiting conditions has been demonstrated for a wide range of species. Variation for complex phenotypic traits are frequently controlled by many genetic loci, QTLs, scattered throughout the genome (Price, 2006). QTLs for traits related to phosphorus deﬁciency tolerance have been found in rice (Wissuwa et al., 2002; Heuer et al., 2009; Chin et al., 2010), wheat (Su et al., 2009), common bean (Beebe et al., 2006; Ochoa et al., 2006; Cichy et al., 2009), Arabidopsis (Reymond et al., 2006), soybean (Li et al., 2005; Zhang et al., 2009; Wang et al., 2010), barley (Gahoonia and Nielsen, 2004), and maize (Zhu et al., 2005; Chen et al., 2009; Hochholdinger and Tuberosa, 2009). Utilizing a mapping population derived from a cross of the intolerant “Nipponbare” cultivar with the tolerant landrace “Kasalath,” Wissuwa et al. (2002) identiﬁed a QTL for tolerance to low phosphorus in rice designated (Pup1). Introgression of Pup1 into near isogenic lines (NILs) allowed ﬁne mapping of the Pup1 locus to the long arm of chromosome 12 (15.31–15.47 Mb) on the basis of the “Nipponbare” reference genome (Heuer et al., 2009). Next generation sequencing was used to characterize the genomic (278 kb) introgression region. Although the gene regulating the Pup1 phenotype has yet to be identiﬁed, it is worthwhile to note that the Pup1 region is found in 50% of the rice accessions adapted to stress-prone environments (Chin et al., 2010).

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

In common bean, recombinant inbred lines (RILs) were developed for shallowrooted and deep-rooted phenotypes (Rubio et al., 2003). Under ﬁeld conditions when available phosphorus was concentrated in the top soil layer, the shallow-rooted RILs were more productive and had a competitive advantage over deep-rooted RILs. Further analysis of the RILs by Liao et al. (2004) showed 16 QTLs controlling the root traits. Adventitious root formation in 84 RILs grown under limiting phosphorus conditions was shown to be important for phosphorus acquisition in common bean (Ochoa et al., 2006). The QTLs for root traits related to low phosphorus tolerance were mainly located on linkage groups B2 and B9. One QTL on linkage group B9 accounted for 61% of the total phenotypic variation. Beebe et al. (2006) evaluated 71 RILs grown under either low or high phosphorus and identiﬁed 26 QTLs affecting phosphorus accumulation and root architecture. Enhanced phosphorus uptake was associated with basal root development. The 26 QTLs were scattered across the genome. Development of NILs of common bean having enhanced phosphorus tolerance and shallow basal roots will provide the germplasm resources for further ﬁne mapping of important traits. The common bean genome is currently being sequenced. Having a reference genome and NILs genetic resources for common bean coupled with next-generation high-throughput RNA sequencing (RNA-seq) will facilitate ﬁne mapping and candidate gene identiﬁcation regulating phosphorus tolerance and root traits in common bean. Tuberosa and Salvi (2007) developed a library of maize-introgressed lines from B73 (recurrent parent) crossed with Gaspe Flint (donor parent) to identify major QTLs for root architecture and growth which map to maize bin 1.06. Utilizing other maize RILs grown under low and high phosphorus, Chen et al. (2008a) identiﬁed a QTL at bin 1.06 for phosphorus efﬁciency and topsoil root dry

249

weight. They found several QTLs for interactions (epistasis) located near bin 1.06. The bin 1.06 region on maize chromosome 1 has been reported to control QTL for root architecture in ﬁve maize genetic backgrounds (Hochholdinger and Tuberosa, 2009). The QTL at bin 1.06 has also been associated with nitrogen use efﬁciency. This region of the maize genome is currently under evaluation for candidate genes. Building upon a number of approaches, a gene has been identiﬁed in Arabidopsis which is involved in a QTL controlling root growth response (Reymond et al., 2006; Svistoonoff et al., 2007). As noted earlier, when grown on low phosphorus, Arabidopsis primary root growth is inhibited while lateral root growth is stimulated. Reymond et al. (2006) generated NILs by segregating F6 RILs mapped for root growth response to low phosphorus. Fine mapping of the low phosphate root (LPR1) QTL trait located it to a 2.5-Mb region at the top of chromosome 1. Further ﬁne mapping by Svistoonoff et al. (2007) reﬁned the LPR1 trait to a 36-kb region. They then mutagenized the LPR1 line and screened for progeny with long roots on low phosphorus media. Moreover, Svistoonoff et al. (2007) also developed T-DNA insertion mutants for LPR1. Utilizing ﬁne mapping of the locus and mutagenesis, they deﬁned the LPR1 QTL locus as encoding an MCO enzyme. Transcripts for LPR1 were most abundant in the root meristem and root cap. They suggested that the root cap played a critical role in local phosphorus sensing. As evidenced by the cloning of LPR1 in Arabidopsis, complementary approaches will lead to cloning genes controlling QTLs involved in tolerance to phosphorus deﬁciency. Progress on cloning these QTLs will be dependent on several factors including (1) the development of RILs segregating for the desired trait followed by development of NILs having the introgressed trait; (2) ﬁne mapping of the trait utilizing molecular

250

NUTRIENT USE EFFICIENCY IN CROPS

markers derived preferably from single nucleotide polymorphisms (SNPs) generated by RNA-seq comparisons of the NILs; (3) mutations in or near the locus; and (4) a reference genome. In the near future, lowcost next-generation sequencing will rapidly advance cloning of QTLs not only for phosphorus deﬁciency tolerance but also for other traits. Acclimation to phosphate stress involves epigenetic changes? Acclimation and resistance to abiotic and biotic stresses involve signiﬁcant biochemical and developmental plasticity. Although much of this plasticity is the direct result of either increased or decreased transcription of several suites of genes, some may be derived from epigenetic modiﬁcations that alter gene expression (Lukens and Zhan, 2007; Boyko and Kovalchuk, 2008; Saze, 2008; Zhang, 2008; Chinnusamy and Zhu, 2009). In recent years, epigenetic changes have been noted as salient features in adaptation to abiotic and biotic stresses. Changes in DNA methylation are a hallmark of epigenetic regulation of gene expression (Boyko and Kovalchuk, 2008; Zhang, 2008; Zilberman et al., 2008). In plants, DNA hypermethylation is generally linked with repressive chromatin in gene promoters and repression of gene expression, while hypomethylation leads to enhanced transcription. Enhanced expression of a glycerophodiesterase (GPXPD) gene in tobacco in response to aluminum, salt, and cold stresses has been associated with demethylation in the coding region of the GPXPD gene (Choi and Sano, 2007). Choi and Sano did not, however, ﬁnd demethylation in the promoter region of the gene. Many genes showing enhanced transcription in response to aluminum stress are also induced during phosphorus stress. As with aluminum stress, GPXPDs are known to be highly expressed in response to phos-

phorus starvation and return to basal levels as phosphorus stress is relieved (Misson et al., 2005; Morcuende et al., 2007). The methylation status of GPXPDs has not been evaluated during phosphorus stress, but it, as well as many other genes that respond quickly to phosphorus status, may be under a similar form of epigenetic regulation. With the soon to be available single molecule real-time DNA sequencing capabilities, laboratories will be able to do direct sequencing for methylated DNA rather than bisulﬁte sequencing (Flusberg et al., 2010). This will allow for immediate and direct evaluation of the epigenetic status of plants grown under any stress condition. Smith et al. (2010) have implicated the actin-related protein 6 (APR6) as an epigenetic modulator of some phosphorus starvation response (PSR) genes. APR6 is a key component of the SWR1 complex involved in chromatin remodeling and is required for histone H2A.Z incorporation into chromatin (Jarillo et al., 2009). The physiological and molecular phenotypes of apr6 mutants were noticeably similar to those displayed by phosphorus-starved Arabidopsis plants (Smith et al., 2010). Loss of function of APR6 resulted in a dramatic decrease in H2A.Z abundance at several PSR gene sites accompanied by an increase in gene transcription. Chromatin remodeling is an integral mechanism in regulating yeast structural phosphate deﬁciency (PHO) gene expression (Barbaric et al., 2007; Wippo et al., 2009). In addition, chromatin remodeling has also been implicated as a component of adaptation to pathogen stress in Arabidopsis (March-Diaz et al., 2008). Another epigenetic mechanism involved in adaptation to stress appears to involve posttranslational modiﬁcations to the N-terminal region of nucleosome core complex histones through acetylation, phosphorylation, ubiquitination, and sumolyation (Boyko and Kovalchuk, 2008;

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

Chinnusamy and Zhu, 2009). The HOS15 gene of Arabidopsis has been shown to be important in histone deacetylation and is crucial for repression of genes associated with plant acclimation and tolerance to cold stress (Zhu et al., 2008). HOS15 mutants accumulate higher amounts of stress-related transcripts and are hypersensitive to cold temperatures. Phosphorylation of histone H3 Serine10 and acetylation of histone H4 is correlated with increased abundance of salt tolerance transcripts in tobacco and Arabidopsis (Sokol et al., 2007). The limited information available on epigenetic regulation of plant response to phosphorus deﬁciency makes this topic rich for exploration. Signaling of phosphate stress: sugars and microRNAs crosstalk? The plethora of biochemical and developmental adaptations displayed in plants subjected to phosphorus deﬁciency result from both local and systemic signaling that activates the coordinated expression of a medley of genes (Franco-Zorrilla et al., 2004; Tesfaye et al., 2007; Hammond and White, 2008). Sucrose derived from photosynthate and microRNA (miRNAs) have been implicated as critical molecules signaling the phosphorus status of the plant. Chiou and Bush (1998) showed sucrose could act as a signal molecule in assimilate partitioning. A growing body of evidence now supports sucrose derived from photosynthate as part of the systemic signaling leading to phosphorus deﬁciency-induced increase in lateral root formation and increased root hair density (Hermans et al., 2006; Jain et al., 2007; Karthikeyan et al., 2007; Zhou et al., 2008). Moreover, sucrose has been shown to be required for enhanced expression of phosphorus starvation-induced genes (Franco-Zorrilla et al., 2005; Liu et al., 2005; Karthikeyan et al., 2007; Müller et al., 2007). To test the role of photosynthate and

251

phloem sucrose on phosphorus stress transcript induction, shoots of white lupin plants were either darkened or stems girdled to block phloem transport, and the starvationenhanced expression of genes in roots was evaluated (Liu et al., 2005; Tesfaye et al., 2007). Both treatments reduced gene expression in phosphorus-stressed roots to nondetectable levels within a few hours. Returning darkened plants to light restored phosphorus starvation-induced gene expression in roots. In phosphorus-stressed Arabidopsis roots, phosphorus starvation-induced genes showed further enhanced expression when supplemented with 3% sucrose (FrancoZorrilla et al., 2005; Karthikeyan et al., 2007). Müller et al. (2007) evaluated the interaction between phosphorus and sucrose in Arabidopsis leaves. Using a twofold cutoff, they found 187 transcripts responded to phosphorus starvation while 644 responded to sucrose. They identiﬁed 149 transcripts that were regulated by the interaction between phosphorus starvation and sucrose availability. One group of 47 genes having increased expression in response to phosphorus deﬁciency was further enhanced by sucrose. Many of the transcripts in this group encode proteins involved in phosphorus remobilization and carbohydrate metabolism. Although sucrose appears to be important in signaling phosphorus status and full expression of phosphorus starvation genes, the mechanism remains elusive. The sucrose–nonfermenting-1-kinase (SNF1) : calcineurin B-like protein kinase (CIPK) pathway has been implicated as the transduction system for sugar signaling (Hummel et al., 2009; Rosa et al., 2009; Meyer et al., 2010). Whether the SNF1 : CIPK pathway regulates sugar signaling during phosphorus starvation deserves attention. Computational and molecular cloning approaches revealed a group of endogenous noncoding small RNAs that may play important roles in the control of many

252

NUTRIENT USE EFFICIENCY IN CROPS

developmental processes in plants and animals (Bartel, 2004; Jones-Rhodes and Bartel, 2004; Xie et al., 2005; Chen et al., 2006). miRNAs are noncoding small RNA, about 20–24 nucleotides in length in plants, which function as posttranscriptional negative regulators or repressors through base pairing to complementary or partially complementary sequences in target mRNAs leading to cleavage of that RNA (Reinhart et al., 2002; Rhoades et al., 2002; Bartel, 2004; Sunkar and Zhu, 2004). Most known miRNAs in plants are predicted to target the expression of several classes of genes including transcription factors, indicating their importance in regulating various plant developmental aspects (Bartel and Bartel, 2004; Chiou, 2007). Recently, miR399, ﬁrst identiﬁed in Arabidopsis and rice (Sunkar and Zhu, 2004; Sunkar et al., 2007), was shown to be induced by phosphorus stress after 24 and 48 h of phosphorus starvation (Fujii et al., 2005; Bari et al., 2006; Chiou et al., 2006). Transcript abundance of miR399 declines rapidly following the addition of phosphorus to the medium (Bari et al., 2006) and is not detected at all under phosphorus-sufﬁcient conditions (Fujii et al., 2005; Aung et al., 2006; Bari et al., 2006; Chiou et al., 2006). Chiou et al. (2006) found that while miR399 was highly upregulated during phosphorus stress, transcripts for a ubiquitin-conjugating E2 enzyme (UBC24) were reduced by ﬁvefold. By comparison, plants grown under phosphorus sufﬁciency had signiﬁcantly reduced miR399, but UBC24 was abundant. Computational analysis of the 5′-upstream region of UBC24 showed several target-binding sites for miR399. Overexpression of miR399 suppressed accumulation of UBC24 transcripts and resulted in enhanced accumulation of phosphorus in the shoot. Moreover, phosphorus remobilization was impaired in plants overexpressing miR399. Enhanced accumulation of phosphorus in shoots and

impaired phosphorus remobilization in plants overexpressing miR399 phenocopied the Arabidopsis pho2 mutant (Dong et al., 1998). In concurrent studies, Bari et al. (2006) using map-based cloning identiﬁed the impaired gene in the pho2 mutant as the ubiquitin-conjugating enzyme UBC24. They also found that UBC24 had ﬁve complementary miR399 binding sites in the 5′-upstream region. Bari et al. (2006) extended the understanding of the PHO2 miR399 interaction by showing that plants with the phr1-MYB protein mutation failed to accumulate miR399 transcripts. Their observations showed that phr1-MYB was required for miR399 expression. Thus, PHO2, miR399, and PHR1 deﬁne a critical phosphate signaling pathway (Doerner, 2008; Lin et al., 2008). The MYB transcription factor PHR1 regulates miR399 expression, which in turn regulates the UBC24 E2 ligase PHO2. PHO2 then regulates a subset of phosphorus starvation genes. Studies have now clearly shown that miR399 can move in the phloem sap and serve as a longdistance signal for phosphate homeostasis (Liu et al., 2008; Pant et al., 2008). A novel genetic concept superimposed on miR399 and phosphorus signaling has been demonstrated by Franco-Zorrilla et al., 2007). They found that, along with transcriptional control, miR399 activity is also regulated by “target mimicry.” The nonprotein coding gene Induced by Phosphate Starvation (IPS1) contains a motif with sequence complementarity to miR399. IPS1 is induced along with miR399 during phosphorus stress. However, rather than being cleaved by miR399, IPS1 transcripts can bind to and sequester miR399, thereby acting to attenuate the inhibitory activity of miR399. Target mimicry by IPS1 and other nonproteincoding genes may be a mechanism to coregulate numerous miRNAs. A recently proposed unique aspect of phosphorus signaling and miRNA involves

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

potential crosstalk between photosynthate (sucrose) availability and miRNA expression during phosphorus deﬁciency (Liu et al., 2010). Liu et al. found expression of miR399 in either shoots or roots required photosynthetic carbon assimilation. When phosphorus-sufﬁcient bean plants were subjected to phosphorus starvation, miR399 was strongly induced in roots and shoots within 24 h (Fig. 12.5). Surprisingly, miR399 was not expressed in roots of dark-treated and stem-girdled phosphorus-starved plants. Moreover, expression of miR399 transcript

Fig. 12.5. Expression of phosphorus deﬁciency genes in common bean requires crosstalk with photosynthate and sugars. Phosphorus-sufﬁcient common bean (+P) plants have very little to no expression of a serine– threonine protein phosphatase (PvHAD) and miRNA399, while transcripts for both PvHAD and miRNA399 are abundant in phosphorus-deﬁcient (–P) common bean. Dark treatment (dk) and stem girdling (gd) of common bean results in a striking decrease in transcripts of PvHAD and miRNA399 even under phosphorus-deﬁcient conditions (Liu et al., 2010). A, PvHAD expression; B, miR399 expression in roots; C, miR399 expression in leaves.

253

expression was blocked in dark-treated leaves of phosphorus-deﬁcient plants. Whether light and sugars modulate the expression of miR399 and other miRNAs known to be involved in abiotic and biotic stress needs to be addressed. Overview Phosphorus is a critical element for plant growth and is frequently the limiting nutrient in many soils. Continued production and application of phosphorus fertilizer relies on a nonrenewable resource, for which availability may peak in about 2050. This will result in signiﬁcantly increased cost, particularly for developing countries. Signiﬁcant research efforts in genomics of phosphorus stress have shown that many suites of genes regulated in a coordinated fashion are involved in plant acclimation to phosphorus deﬁciency. These genomic studies in conjunction with traditional plant breeding have shown the phosphorus acclimation traits are controlled by multiple genes most probably in QTLs. Future development of NILs and RILs coupled to next-generation sequencing will facilitate mapping of genes and the development of plants having improved phosphorus acquisition and phosphorus use efﬁciency. The development of crop plants with more efﬁcient phosphorus acquisition and use is a necessity for sustainable farming practices. Highly phosphorus-efﬁcient plants could reduce the need for phosphorus fertilizer in the developed world, thereby ameliorating overuse of phosphorus while concurrently enhancing yield in the developing world, where phosphorus is frequently unavailable. References Abelson, P.H. (1999) A potential phosphate crisis. Science 283, 2015. Adams, M.A. & Pate, J.S. (2002) Phosphorus sources and availability modify growth and distribution

254

NUTRIENT USE EFFICIENCY IN CROPS

of root clusters and nodules of native Australian legumes. Plant, Cell & Environment 26, 837–850. Ai, P., Sun, S., Zhao, J., et al. (2009) Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. The Plant Journal 57, 798–809. Al-Ghazi, G., Muller, B., Pinloche, S., et al. (2003) Temporal responses of Arabidopsis root architecture to phosphate starvation: evidence for the involvement of auxin signaling. Plant, Cell & Environment 26(7), 1053–1066. Aloni, R., Aloni, E., Langhans, M., et al. (2006) Role of cytokinin and auxin shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism. Annals of Botany 97, 883–893. Andersson, M.X., Larsson, K.E., Tjellström, H., et al. (2005) Phosphate-limited oat: the plasma membrane and the tonoplast as major targets for phospholipid-to-glycolipid replacement and stimulation of phospholipases in the plasma membrane. Journal of Biological Chemistry 280, 27578–27586. Aung, K., Lin, S.I., Wu, C.C., et al. (2006) Pho2, a phosphate overaccumalator, is caused by a nonsense mutation in a microRNA399 target gene. Plant Physiology 141, 1000–1011. Barbaric, S., Luckenbach, T., Schmid, A., et al. (2007) Redundancy of chromatin remodeling pathways for the induction of the yeast PHO5 promoter in vivo. Journal of Biological Chemistry 282, 27610–27621. Barber, S.A., Walker, J.M., & Vasey, E.H. (1963) Mechanisms for the movement of plant nutrients from the soil and fertilizer to the plant root. Journal of Agricultural and Food Chemistry 11, 204–207. Bari, R., Pant, B.D., Stitt, M., et al. (2006) Pho2, microRNA399, and PHR1 deﬁne a phosphate-signaling pathway in plants. Plant Physiology 141, 988–999. Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism and function. Cell 116, 281–297. Bartel, B. & Bartel, D.P. (2004) Micro RNAs: at the root of plant development. Plant Physiology 132, 709–717. Bates, T.R. & Lynch, J.P. (1996) Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability. Plant, Cell & Environment 19, 529–538. Bates, T.R. & Lynch, J.P. (2000) The efﬁciency of Arabidopsis thaliana (Brassicaceae) root hairs in phosphorus acquisition. American Journal of Botany 87, 964–970.

Beebe, S.E., Rojas-Pierce, M., Yan, X., et al. (2006) Quantitative trait loci for root architecture traits correlated with phosphorus acquisition in common bean. Crop Science 46, 413–423. Bieleski, R.L. (1973) Phosphate pools, phosphate transport, and phosphate availability. Annual Review of Plant Physiology 24, 225–252. Boerjan, W., Cervera, M.T., Delarue, M., et al. (1995) Superroot, a recessive mutation in Arabidopsis, confers auxin overproduction. The Plant Cell 7, 1405–1419. Boisson-Dernier, A., Chabaud, M., Garcia, F., et al. (2001) Agrobacterium-transformed roots of Medicago truncatula for the study of nitrogenﬁxing and endomycorrhizal symbiotic associations. Molecular Plant-Microbe Interactions 14, 695–700. Borch, K., Bouma, T.J., Lynch, J.P., et al. (1999) Ethylene: a regulator of root architectural responses to soil phosphorus availability. Plant, Cell & Environment 22, 425–431. Boyko, A. & Kovalchuk, I. (2008) Epigenetic control of plant stress response. Environmental and Molecular Mutagenesis 49, 61–72. Bucciarelli, B., Hanan, J., Palmquist, D., et al. (2006) A standardized method for analysis of Medicago truncatula phenotype development. Plant Physiology 142, 207–219. Bucher, M. (2007) Functional biology of plant phosphate uptake at root and mycorrhiza interfaces. The New Phytologist 173, 11–26. Calderon-Vazquez, C., Ibarra-Laclette, E., CaballeroPerez, J., et al. (2008) Transcript proﬁling of Zea mays roots reveals gene responses to phosphate deﬁciency at the plant- and species-speciﬁc levels. Journal of Experimental Botany 59(9), 2479–2497. Casimiro, I., Marchant, A., Bhalerao, R.P., et al. (2001) Auxin transport promotes Arabidopsis lateral root initiation. The Plant Cell 13, 843–852. Celenza, J.L. Jr, Grisaﬁ, P.L., & Fink, G.R. (1995) A pathway for lateral root formation in Arabidopsis thaliana. Genes & Development 9, 2131–2142. Century, K., Reuber, T.L., & Ratcliffe, O.J. (2008) Regulating the regulators: the future prospects for transcription-factor-based agricultural biotechnology products. Plant Physiology 147, 20–29. Chapman, E.J. & Estelle, M. (2009) Cytokinin and auxin intersection in root meristems. Genome Biology 10, 210. (doi: 10.1186/gb-200910-2-210). Charlton, W.A. (1996) Lateral root initiation. In: Plant Roots: The Hidden Half, 2nd ed. (eds. Y. Waisel, A. Eshel & U. Kafkaﬁ), pp. 149–173. Marcel Dekker, New York.

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

Chen, W., Provart, N.J., Glazebrook, J., et al. (2002) Expression proﬁle matrix of Arabidopsis transcription factor genes suggest their putative function in response to environmental stresses. The Plant Cell 14, 559–574. Chen, Z., Zhang, J., Kong, J., et al. (2006) Diversity of endogenous small non-coding RNAs in Oryza sativa. Genetica 128, 21–31. Chen, J., Xu, L., Cai, Y., et al. (2008a) QTL mapping of phosphorus efﬁciency and relative biologic characteristics in maize (Zea mays L.) at two sites. Plant and Soil 313, 251–266. Chen, Y.-F., Wang, Y., & Wu, W.-H. (2008b) Membrane transporters for nitrogen, phosphate and potassium uptake in plants. Journal of Integrative Plant Biology 50, 835–848. Chen, J., Xu, L., Cai, Y., et al. (2009) Identiﬁcation of QTLs for phosphorus utilization efﬁciency in maize (Zea mays L.) across P levels. Euphytica 167, 245–252. Chevalier, F., Pata, M., Nacry, P., et al. (2003) Effects of phosphate availability on the root system architecture: large-scale analysis of the natural variation between Arabidopsis accessions. Plant, Cell & Environment 26, 1839–1850. Chin, J.H., Haefele, S.M., Gamuyao, R., et al. (2010) Development and application of gene based markers for the major rice QTL phosphorus uptake 1. Theoretical and Applied Genetics 120, 1073–1086. Chinnusamy, V. & Zhu, J.-K. (2009) Epigenetic regulation of stress responses in plants. Current Opinion in Plant Biology 12, 1–7. Chiou, T.-J. (2007) The role of microRNAs in sensing nutrient stress. Plant, Cell & Environment 30, 323–332. Chiou, T.-J. & Bush, D.R. (1998) Sucrose is a signal molecule in assimilate partitioning. Proceedings of the National Academy of Sciences of the United States of America 95, 4784–4788. Chiou, T.J., Aung, K., Lin, S.L., et al. (2006) Regulation of phosphate homeostatis by microRNA in Arabidopsis. The Plant Cell 18, 412–421. Choi, C.-H. & Sano, H. (2007) Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants. Molecular Genetics and Genomics 277, 589–600. Christianson, M.L. & Warnick, D.A. (1985) Temporal requirements for phytohormone balance in the control of organogenesis in vitro. Developmental Biology 112, 494–497. Cichy, K.A., Blair, M.W., Mendoza, C.H.G., et al. (2009) QTL analysis of root architecture traits and low phosphorus tolerance in an Andean bean population. Crop Science 49, 59–68.

255

Ciereszko, I., Zambrzycka, A., & Rychter, A. (1998) Sucrose hydrolysis in bean roots (Phaseolus vulgaris L.) under phosphate deﬁciency. Plant Science 133, 139–144. Cordell, D., Drangert, J.O., & White, S. (2009) The story of phosphorus: global food security and food for thought. Global Environmental Change 19, 292–305. Cripps, C.L. & Eddington, L.H. (2005) Distribution of mycorrhizal types among alpine vascular plant families on the Beartooth Plateau, Rocky Mountains, U.S.A., in reference to large-scale patterns in arcticalpine habitats. Arctic Antarctic and Alpine Research 37, 177–188. de Dorlodot, S., Forster, B., Pages, L., et al. (2007) Root system architecture: opportunities and constraints for genetic improvement of crops. Trends in Plant Science 12, 474–481. De Smet, I., Tetsurmura, T., De Rybel, B., et al. (2007) Auxin dependent regulation of lateral root positioning in the basal meristem of Arabidopsis. Development 134, 681–690. Dello Iolo, R., Linhares, F.S., Scacchi, E., et al. (2007) Cytokinins determine Arabidopsis root-meristem size by controlling cell differentiation. Current Biology 17, 678–682. Devaiah, B.N., Karthikeyan, A.S., & Raghothama, K.G. (2007) WRKY75 transcription factor is modulated by phosphate acquisition and root development in Arabidopsis. Plant Physiology 143, 1789–1801. Dharmasiri, N., Dharmasiri, S., & Estelle, M. (2005) The F-box protein TIR1 is an auxin receptor. Nature 435, 441–445. Diem, H.G., Duhoux, E., Zaid, H., et al. (2000) Cluster roots in Casuarinaceae: role and relationship to soil nutrient factors. Annals of Botany 85, 929–936. Dinkelaker, B., Hengeler, C., & Marschner, H. (1995) Distribution and function of proteoid roots and other root clusters. Acta Botanica 108, 169–276. Doerner, P. (2008) Phosphate starvation signaling: a threesome controls systemic Pi homeostasis. Current Opinion in Plant Biology 11, 536–540. Dolan, L. (2001) The role of ethylene in root hair growth in Arabidopsis. Journal of Plant Nutrition and Soil Science 164, 141–145. Dong, B., Rengel, Z., & Delhaize, E. (1998) Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of Arabidopsis thaliana. Planta 205(2), 251–256. Emery, N.R.J. & Atkins, C.A. (2002) Cytokinins and roots. In: Plant Roots: The Hidden Half (eds. Y. Waisel, A. Eshel & U. Kafkaﬁ), pp. 417–434. Marcel Dekker, New York. Essigmann, B., Güler, S., Narang, R.A., et al. (1998) Phosphate availability affects the thylakoid lipid

256

NUTRIENT USE EFFICIENCY IN CROPS

composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 95, 1950–1955. Eulgem, T., Rushton, P.J., Robatzek, S., et al. (2000) The WRKY superfamily of plant transcription factors. Trends in Plant Science 5, 199–206. Fang, S., Yan, X., & Liao, H. (2009) 3D reconstruction and dynamic modeling of root architecture in situ and its application to crop phosphorus research. Plant Journal 60, 1096–1108. Flusberg, B.A., Webster, D.R., Lee, J.H., et al. (2010) Direct detection of DNA methylation during singlemolecular, real-time sequencing. Nature Methods 7, 461–465. Franco-Zorrilla, J.M., González, E., Bustos, R., et al. (2004) The transcriptional control of plant responses to phosphate limitation. Journal of Experimental Botany 55, 285–293. Franco-Zorrilla, J.M., Martín, A.C., Leyva, A., et al. (2005) Interaction between phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3. Plant Physiology 138, 847–857. Franco-Zorrilla, J.M., Valli, A., Todesco, M., et al. (2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genetics 39, 1033–1037. Fujii, H., Chiou, T.Z., Lin, S.I., et al. (2005) A miRNA involved in phosphate starvation response in Arabidopsis. Current Biology 15, 2038–2043. Fukaki, H. & Tasaka, M. (2009) Hormone interactions during lateral root formation. Plant Molecular Biology 69, 437–449. Fukaki, H., Okushima, Y., & Tasaka, M. (2007) Auxinmediated lateral root formation in higher plants. International Review of Cytology 256, 111–137. Fukuda, T., Saito, A., Wasaki, J., et al. (2007) Metabolic alterations proposed by proteome in rice roots grown under low P and high Al concentration under low pH. Plant Science 172, 1157–1165. Gahoonia, T.S. & Nielsen, N.E. (1998) Direct evidence on participation of root hairs in phosphorus (32P) uptake from soil. Plant and Soil 198, 147–152. Gahoonia, T.S. & Nielsen, N.E. (2004) Root traits as tools for creating phosphorus efﬁcient crop varieties. Plant and Soil 260, 47–57. Gardner, W.K., Parbery, D.G., & Barber, D.A. (1982) The acquisition of phosphorus by Lupinus albus L. I. Some characteristics of the soil/root interface. Plant and Soil 68, 19–32. Gardner, W.K., Barber, D.A., & Parbery, D.G. (1983) The acquisition of phosphorus by Lupinus albus L.

III. The probable mechanism by which phosphorus movement in the soil/root interface is enhanced. Plant and Soil 70, 107–124. Gilbert, G.A., Knight, J.D., Vance, C.P., et al. (1999) Acid phosphatase activity in phosphorus-deﬁcient white lupin roots. Plant, Cell & Environment 22, 801–810. Gilbert, G.A., Knight, J.D., Vance, C.P., et al. (2000) Proteoid root development of phosphorus deﬁcient lupin is mimicked by auxin and phosphonate. Annals of Botany 85, 921–928. Gilroy, S. & Jones, D.L. (2000) Through form to function: root hair development and nutrient uptake. Trends in Plant Science 5, 56–60. Graham, M.A., Ramirez, M., Valdes-López, O., et al. (2006) Identiﬁcation of candidate phosphorus stress induced genes in Phaseolus vulgaris through clustering analysis across several plant species. Functional Plant Biology 33, 789–797. Grierson, P. (1992) Organic acids in the rhizosphere of Banksia integrifolia L. f. Plant and Soil 144, 259–265. Grierson, P. & Attiwill, P. (1989) Chemical characteristics of the proteoid root mat of Banksia integrifolia L. Australian Journal of Botany 37, 137–143. Grierson, C.S., Parker, J.S., & Kemp, A.C. (2001) Arabidopsis genes with roles in root hair development. Journal of Plant Nutrition and Soil Science 164, 131–140. Grunwald, U., Nyamsuren, O., Tamasloukht, M.-B., et al. (2004) Identiﬁcation of mycorrhiza-regulated genes with arbuscule development-related expression proﬁle. Plant Molecular Biology 55, 553–566. Guo, A., He, K., Liu, D., et al. (2005) DATF: A database of Arabidopsis transcription factors. Bioinformatics 21, 2568–2569. Hammond, J.P. & White, P.J. (2008) Sucrose transport in the phloem: integrating root responses to phosphorus starvation. Journal of Experimental Botany 59, 93–109. Hammond, J.P., Bennett, M.J., Bowen, H.C., et al. (2003) Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants. Plant Physiology 132, 578–596. Hammond, J.P., Broadley, M.R., & White, P.J. (2004) Genetic responses to phosphorus deﬁciency. Annals of Botany 94, 323–332. Harrison, M. (2005) Signaling on the arbuscular mycorrhizal symbiosis. Annual Review of Microbiology 59, 19–42. Hermans, C., Hammond, J.P., White, P.J., et al. (2006) How do plants respond to nutrient shortage by

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

biomass allocation? Trends in Plant Science 11, 610–617. Hernández, G., Ramirez, M., Valdes-Lopez, O., et al. (2007) Phosphorus stress in common bean: root transcript and metabolic responses. Plant Physiology 144, 752–767. Hernández, G., Valdés-López, O., Ramírez, M., et al. (2009) Global changes in the transcript and metabolic proﬁles during symbiotic nitrogen ﬁxation in phosphorus-stressed common bean plants. Plant Physiology 151, 1221–1238. Heuer, S., Lu, X., Chin, J.H., et al. (2009) Comparative sequence analyses of the major quantitative trait locus Phosphorus uptake 1 (Pup1) reveal a complex genetic structure. Plant Biotechnology Journal 7, 456–471. Hill, J.O., Simpson, R.J., Moore, A.D., et al. (2006) Morphology and response of roots of pasture species to phosphorus and nitrogen nutrition. Plant and Soil 286, 7–19. Hinsinger, P. (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant and Soil 237, 173–195. Hirayama, T. & Shinozaki, K. (2010) Research on plant abiotic stress responses in the post-genome era: past, present and future. Plant Journal 61, 1041–1052. Hochholdinger, F. & Tuberosa, R. (2009) Genetic and genomic dissection of maize root development and architecture 2009. Current Opinions in Plant Biology 12, 172–177. Holford, I.C.R. (1997) Soil phosphorus: its measurement and its uptake by plants. Australian Journal of Soil Research 35, 227–239. Hou, W.L., Wu, P., Jiao, F.C., et al. (2005) Regulation of the expression of OsIPS1 and OsIPS2 in rice via systemic and local Pi signaling and hormones. Plant, Cell & Environment 28, 353–364. Huang, C.Y., Roessner, U., Eickmeier, I., et al. (2008) Metabolite proﬁling reveals distinct changes in carbon and nitrogen metabolism in phosphatedeﬁcient barley plants (Hordeum vulgare L.). Plant Cell Physiology 49, 691–703. Hummel, M., Rahamani, F., Smeekens, S., et al. (2009) Sucrose mediated translational control. Annals of Botany 104, 1–7. Ivanchenko, M.G., Muday, G.K., & Dubrovsky, J.G. (2008) Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana. The Plant Journal 55, 335–347. Jain, A., Poling, M.D., Karthikeyan, A.S., et al. (2007) Differential effects of sucrose and auxin on localized phosphate deﬁciency-induced modulation of different traits of root system architecture in Arabidopsis. Plant Physiology 144, 232–247.

257

Jarillo, J.A., Piñeiro, M., Cubas, P., et al. (2009) Chromatin remodeling in plant development. International Journal of Developmental Biology 53, 1581–1596. Johnson, J.F., Allan, D.L., & Vance, C.P. (1994) Phosphorus stress-induced proteoid roots show altered metabolism in Lupinus albus. Plant Physiology 104, 657–665. Johnson, J.F., Allan, D.L., Vance, C.P., et al. (1996a) Root carbon dioxide ﬁxation by phosphorusdeﬁcient Lupinus albus (contribution to organic acid exudation by proteoid roots). Plant Physiology 112, 19–30. Johnson, J.F., Vance, C.P., & Allan, D.L. (1996b) Phosphorus deﬁciency in Lupinus albus. Altered lateral root development and enhanced expression of phosphoenolpyruvate carboxylase. Plant Physiology 112, 31–41. Jones, M.A., Raymond, M.J., & Smirnoff, N. (2006) Analysis of the root-hair morphogenesis transcriptome reveals the molecular identity of six genes with roles in root-hair development. Plant Journal 45, 83–100. Jones-Rhodes, M.W. & Bartel, D.P. (2004) Computational identiﬁcation of plant microRNAs and their targets, including a stress-induced miRNA. Molecular Cell 14, 787–799. Jungk, A. (2001) Root hairs and acquisition of plant nutrients from soil. Journal of Plant Nutrition and Soil Science 164, 121–129. Karthikeyan, A.S., Varadarajan, D.K., Jain, A., et al. (2007) Phosphate starvation responses are mediated by sugar signaling in Arabidopsis. Planta 225, 907–918. Keerthisinghe, G., Hocking, P.J., Ryan, P.R., et al. (1998) Effect of phosphorus supply on the formation and function of proteoid roots of white lupin. Plant, Cell & Environment 21, 467–478. Kihara, T., Wada, T., Suzuki, Y., et al. (2003) Alteration of citrate metabolism in cluster roots of white lupin. Plant Cell Physiology 44, 901–908. Kochian, L.V., Hoekenga, O.A., & Piñeros, M.A. (2004) How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorus efﬁciency. Annual Review of Plant Biology 55, 459–493. Lai, F., Thacker, J., Li, Y., et al. (2007) Cell division activity determines the magnitude of phosphate starvation responses in Arabidopsis. Plant Journal 50, 545–556. Lambers, H. & Shane, M.W. (2007) Role of cluster roots in phosphorus acquisition and increasing biological diversity. In: Scale and Complexity in Plant Systems Research: Gene-Plant-Crop Relations (eds. J.H.J. Spiertz, P.C. Struik & H.H. van Laar), pp. 237–250. Springer, Dordrecht.

258

NUTRIENT USE EFFICIENCY IN CROPS

Lambers, H.Y., Shane, M.W., Cramer, M.D., et al. (2006) Root structure and functioning for efﬁcient acquisition of phosphorus: matching morphological and physiological traits. Annals of Botany 98, 693–713. Lamont, B.B. (1974) The biology of dauciform roots in the sedge Cyathochaete avenacea. The New Phytologist 73, 985–996. Lamont, B.B. (1982) Mechanisms for enhancing nutrient uptake in plants, with particular reference to Mediterranean South Africa and Australia. Botanical Review 48, 597–689. Lamont, B.B. (1993) Why are hairy root clusters so abundant in the most nutrient-impoverished soils of Australia? Plant and Soil 155/156, 269–272. Lamont, B. (2003) Structure, ecology and physiology of root clusters—a review. Plant and Soil 48, 1–19. Laplaze, L., Benkova, E., Casimiro, I., et al. (2007) Cytokinins act directly on lateral root founder cells to inhibit root initiation. The Plant Cell 19, 3889–3900. Leggewie, G., Willmitzer, L., & Riesmeier, J.W. (1997) Two cDNAs from potato are able to complement a phosphate uptake-deﬁcient yeast mutant: identiﬁcation of phosphate transporters from higher plants. The Plant Cell 9, 381–392. Li, Y.D., Wang, Y.J., Tong, Y.P., et al. (2005) QTL mapping of phosphorus deﬁciency tolerance in soybean (Glycine max L. Merr.). Euphytica 142, 137–142. Li, X., Mo, X., Shou, H., et al. (2006) Cytokininmediated cell cycling arrest of pericycle founder cells in lateral root initiation of Arabidopsis. Plant Cell Physiology 47, 1112–1123. Li, K., Xu, C., Zhang, K., et al. (2007) Proteomic analysis of roots growth and metabolic changes under phosphorus deﬁcit in maize (Zea mays L.) plants. Proteomics 7, 1501–1512. Li, K., Xu, C., Li, Z., et al. (2008) Comparative proteome analyses of phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant reveal root characteristics associated with phosphorus efﬁciency. Plant Journal 55, 927–939. Li, L., Liu, C., & Lian, X. (2010) Gene expression proﬁles in rice roots under low phosphorus stress. Plant Molecular Biology 72, 423–432. Liao, H., Yan, X., Rubio, G., et al. (2004) Genetic mapping of basal root gravitropism and phosphorus acquisition efﬁciency in common bean. Functional Plant Biology 31, 959–970. Limpens, E., Ramos, J., Franken, C., et al. (2004) RNA interference in Agrobacterium rhizogenes-

transformed roots of Arabidopsis and Medicago truncatula. Journal of Experimental Botany 55, 983–992. Lin, S.I., Chiang, S.F., Lin, W.Y., et al. (2008) Regulatory network of microRNA399 and PHO2 by systemic signaling. Plant Physiology 147, 732–746. Lin, W.-Y., Lin, S.-I., & Chiou, T.J. (2009) Molecular regulators of phosphate homeostasis in plants. Journal of Experimental Botany 60, 1427–1438. Liu, J., Uhde-Stone, C., Li, A., et al. (2001) A phosphate transporter with enhanced expression in proteoid roots of white lupin (Lupinus albus L.). Plant and Soil 237, 257–266. Liu, J., Samac, D.A., Bucciarelli, B., et al. (2005) Signaling of phosphorus deﬁciency-induced gene expression in white lupin requires sugar and phloem transport. The Plant Journal 41, 257–268. Liu, C., Muchhal, V.S., Mukatira, U., et al. (1998a) Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus. Plant Physiology 116, 91–99. Liu, H., Trieu, A.T., Blaylock, L.A., et al. (1998b) Cloning and characterization of two phosphate transporters from Medicago truncatula roots: regulation in response to phosphate and to colonization by arbuscular mycorrhizal (AM) fungi. Molecular Plant-Microbe Interactions 11, 14–22. Liu, J., Versaw, W.K., Pumplin, N., et al. (2008) Closely related members of the Medicago truncatula PHT1 phosphate transporter gene family encode phosphate transporters with distinct biochemical activities. Journal of Biological Chemistry 283, 24673–24681. Liu, J., Allan, D.L., & Vance, C.P. (2010) Systemic signaling and local sensing of phosphate in common bean: cross-talk between photosynthate and MicroRNA399. Molecular Plant 3, 428–437. Lohar, D.P., Schaff, J.E., Laskey, J.G., et al. (2004) Cytokinins play opposite roles in lateral root formation, and nematode and rhizobial symbioses. Plant Journal 38, 203–214. López-Bucio, J., Hernández-Abreu, E., SánchezCalderón, L., et al. (2002) Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiology 129, 244–256. López-Bucio, J., Cruiz-Ramirez, A., & Herrera-Estrella, L. (2003) The role of nutrient availability in regulating root architecture. Current Opinion in Plant Biology 6, 280–287. Lukens, L.N. & Zhan, S. (2007) The plant genome’s methylation status and response to stress: implications for plant improvement. Current Opinion in Plant Biology 10, 317–322.

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

Lynch, J.P. (1995) Root architecture and plant productivity. Plant Physiology 109, 7–13. Lynch, J.P. & Brown, K.M. (2001) Topsoil foraging— an architectural adaptation of plants to low phosphorus availability. Plant and Soil 237, 225–237. Ma, Z., Bielenberg, D.G., Brown, K.M., et al. (2001) Regulation of root hair density by phosphorus availability in Arabidopsis thaliana. Plant, Cell & Environment 24, 459–467. Ma, Z., Baskin, T.I., Brown, K.M., et al. (2003) Regulation of root elongation under phosphorus stress involves changes in ethylene responsiveness. Plant Physiology 131, 1381–1390. Mahonen, A.P., Bishopp, A., Higuchi, M., et al. (2006) Cytokinin signaling and its inhibitor AHP6 regulate cell fate during vascular development. Science 311, 94–98. Malamy, J.E. (2005) Intrinsic and environmental response pathways that regulate root system architecture. Plant, Cell & Environment 28, 67–77. Marchant, A., Bhalerao, R., Casimiro, I., et al. (2002) AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissue in Arabidopsis seedling. The Plant Cell 14, 589–597. March-Diaz, R., Garcia-Dominguez, M., Lozano-Juste, J., et al. (2008) Histone H2A.Z and homologues of components of the SWR1 complex are required to control immunity in Arabidopsis. The Plant Journal 53, 475–487. Marschner, H. (1995) Mineral Nutrition of Higher Plants, 2nd ed. 889 pp. Academic Press, Boston, MA. Marschner, H., Römheld, V., Horst, W.J., et al. (1986) Root induced changes in the rhizosphere: importance for mineral nutrition of plants. Zeitschrift für Pﬂanzenernährung und Bodenkunde 149, 441–456. Martin, A.C., del Pozo, J.C., Iglesias, J., et al. (2000) Inﬂuence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis. The Plant Journal 24, 559–567. Massonneau, A., Langlade, N., Leon, S., et al. (2001) Metabolic changes associated with cluster root development in white lupin (Lupinus albus L.): relationship between organic acid excretion, sucrose metabolism and energy status. Planta 213, 534–542. Masucci, J.D. & Schiefelbein, J.W. (1996) Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root. The Plant Cell 8, 1505–1517. Menand, B., Yi, K., Jouannic, S., et al. (2007) Ancient mechanism controls the development of cells with

259

a rooting function in land plants. Science 316, 1477–1480. Meyer, A.K., Longin, C.F.H., Klose, C., et al. (2010) A new regulator for energy signaling pathway in plants highlights conservation among species. Science Signaling 3, jc5. Miller, R.M. (2005) The nonmycorrhizal root—a strategy for survival in nutrient impoverished soils. The New Phytologist 165, 655–658. Miller, R.M., Smith, C.I., Jastrow, J.D., et al. (1999) Mycorrhizal status of the genus Carex (Cyperaceae). American Journal of Botany 86, 547–553. Miller, S.S., Liu, J., Allan, D.L., et al. (2001) Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupin. Plant Physiology 127, 594–606. Misson, J., Raghothama, K.G., Jain, A., et al. (2005) A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proceedings of the National Academy of Sciences of the United States of America 102, 11934–11939. Miura, K., Rus, R., Sharkhuu, A., et al. (2005) The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deﬁciency responses. Proceedings of the National Academy of Sciences of the United States of America 102, 7760–7765. Morcuende, R., Bari, R., Gibon, Y., et al. (2007) Genome-wide reprogramming of metabolism and regulatory networks of Arabidopsis in response to phosphorus. Plant, Cell & Environment 30, 85–112. Morris, R.O., Bilyeu, K.D., Laskey, J.G., et al. (1999) Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochemical and Biophysical Research Communications 255, 328–333. Müller, M. & Schmidt, W. (2004) Environmentally induced plasticity of root hair development in Arabidopsis. Plant Physiology 134, 409–419. Müller, B. & Sheen, J. (2008) Cytokinin and auxin interaction in root stem-cell speciﬁcation during early embryogenesis. Nature 453, 1094–1097. Müller, R., Morant, M., Jarmer, H., et al. (2007) Genome-wide analysis of Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiology 143, 156–171. Nacry, P., Canivenc, G., Muller, B., et al. (2005) A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis. Plant Physiology 138, 2061–2074. Narang, A., Bruene, A., & Aitmann, T. (2000) Analysis of phosphate acquisition efﬁciency in Arabidopsis accessions. Plant Physiology 124, 1786–1799.

260

NUTRIENT USE EFFICIENCY IN CROPS

Negi, S., Ivanchenko, M.G., & Muday, G.K. (2008) Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana. The Plant Journal 55, 175–187. Neumann, G. & Martinoia, E. (2002) Cluster roots—an underground adaptation for survival in extreme environments. Trends in Plant Science 7, 162–167. Neumann, G., Massonneau, A., Martinoia, E., et al. (1999) Physiological adaptations to phosphorus deﬁciency during proteoid root development in white lupin. Planta 208, 373–382. Neumann, G., Massonneau, A., Langlade, N., et al. (2000) Physiological aspects of cluster root function and development in phosphorus-deﬁcient white lupin (Lupinus albus L.). Annals of Botany 85, 909–919. Nilsson, L., Muller, R., & Nielsen, T.H. (2007) Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant, Cell & Environment 30, 1499–1512. Ochoa, I.E., Blair, M.W., & Lynch, J.P. (2006) QTL analysis of adventitious root formation in common bean under contrasting phosphorus availability. Crop Science 46, 1609–1621. Okumura, S., Mitsukawa, N., Shirano, Y., et al. (1998) Phosphate transporter gene family of Arabidopsis thaliana. DNA Research 5, 261–269. Ortega-Martínez, O., Pernas, M., Carol, R.J., et al. (2007) Ethylene modulates stem cell division in the Arabidopsis thaliana. Root Science 317, 507–510. Pant, B.D., Buhtz, A., Kehr, J., et al. (2008) MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant Journal 53, 731–738. Parker, J.S., Cavell, A.C., Dolan, L., et al. (2000) Genetic interactions during root hair morphogenesis in Arabidopsis. The Plant Cell 12, 1961–1974. Paszkowski, U., Kroken, S., Roux, C., et al. (2002) Rice phosphate transporters include an evolutionarily divergent gene speciﬁcally activated in arbuscular mycorrhizal symbiosis. Proceedings of the National Academy of Sciences USA 99, 13324–13329. Pate, J. & Watt, M. (2001) Roots of Banksia spp. (Proteacea) with special reference to functioning of their specialized root clusters. In: Plant Roots: The Hidden Half, 3rd ed. (eds. Y. Waisel, A. Eshel & U. Kafkaﬁ), pp. 989–1006. Marcel Dekker Inc, New York, NY. Peñaloza, E., Muñoz, G., Salvo-Garrido, H., et al. (2005) Phosphate deﬁciency regulates phosphoenolpyruvate carboxylase expression in proteoid

root clusters of white lupin. Journal of Experimental Botany 56, 145–153. Peret, B., de Rybel, B., Casimiro, I., et al. (2009) Arabidopsis lateral root development: an emerging story. Trends in Plant Science 14, 399–408. Perez-Torres, C.-A., Lopez-Bucio, J., Cruz-Ramirez, A., et al. (2008) Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor. Plant and Cell 20, 3258–3272. Peterson, R.L. & Farquhar, M.L. (1996) Root hairs: specialized tubular cells extending root surfaces. Botanical Gazette 62, 1–40. Plaxton, W.C. & Carswell, M.C. (1999) Metabolic aspects of the phosphate starvation response in plants. In: Plant Responses to Environmental Stress: From Phytohormones to Genome Reorganization (ed. H.R. Lerner), pp. 349–372. Marcel-Dekker, New York, NY. Price, A.H. (2006) Believe it or not, QTLs are accurate. Trends in Plant Science 11, 213–216. Raghothama, K.G. & Karthikeyan, A.S. (2005) Phosphate acquisition. Plant and Soil 274, 37–49. Ramírez, M., Graham, M.A., Blanco-López, L., et al. (2005) Sequencing and analysis of common bean ESTs. Building a foundation for functional genomics. Plant Physiology 137, 1211–1227. Reed, R.C., Brady, S.R., & Muday, G.K. (1998) Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis. Plant Physiology 118, 1369–1378. Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., et al. (2002) MicroRNAs in plants. Genes & Development 16, 1616–1626. Reymond, M., Svistoonoff, S., Loudet, O., et al. (2006) Identiﬁcation of QTL controlling root growth response to phosphate starvation in Arabidopsis thaliana. Plant, Cell & Environment 29, 115–125. Rhoades, M.W., Reinhart, B.J., Lim, L.P., et al. (2002) Prediction of plant microRNA targets. Cell 110, 513–520. Ridge, R.W. (1995) Recent developments in the cell and molecular biology of root hairs. Journal of Plant Research 108, 399–405. Riechmann, J.L., Heard, J., Martin, G., et al. (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290, 2105–2110. Rosa, M., Prado, C., Podazzo, G., et al. (2009) Soluble sugars—metabolism, sensing, and abiotic stress. Plant Signaling Behavior 4, 388–393.

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

Rubio, V., Linhares, F., Solano, R., et al. (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes & Development 15, 2122–2133. Rubio, G., Liao, H., Yan, X., et al. (2003) Topsoil foraging and its role in plant competitiveness for phosphorus in common bean. Crop Science 43, 598–607. Ruzicka, K., Ljung, K., Vanneste, S., et al. (2007) Ethylene regulates root growth through effects on auxin biosynthesis and transport auxin distribution. The Plant Cell 19, 2197–2212. Ryan, P.R., Delhaize, E., & Jones, D.L. (2001) Function and mechanism of organic anion exudation from plants. Annual Review of Plant Physiology and Plant Molecular Biology 52, 527–560. Salama, A.M.S.A.E.-D. & Wareing, P.F. (1979) Effects of mineral nutrition on endogenous cytokinins in plants of sunﬂower (Helianthus annuus L.). Journal of Experimental Botany 30, 971–981. Sánchez-Calderón, L., Lopéz-Bucio, J., Chacón-Lopéz, A., et al. (2005) Phosphate starvation induces a determinate developmental program in the roots of Arabidopsis thaliana. Plant Cell Physiology 46, 174–184. Saze, H. (2008) Epigenetic memory transmission through mitosis and meiosis in plants. Seminars in Cell and Developmental Biology 6, 527–536. Sbabou, L., Bucciarelli, B., Miller, S., et al. (2010) Molecular analysis of SCARECROW genes expressed in white lupin cluster roots. Journal of Experimental Botany 61, 1351–1363. Schachtman, D.P., Reid, R.J., & Ayling, S.M. (1998) Phosphorus uptake by plants: from soil to cell. Plant Physiology 116, 447–453. Schmülling, W.T. (2009) Cytokinin action in plant development. Current Opinion in Plant Biology 12, 527–538. Shane, M.W. & Lambers, H. (2005) Cluster roots: a curiosity in context. Plant and Soil 274, 101–125. Shane, M.W., Cramer, M.D., Funayama-Noguchi, S., et al. (2004) Developmental physiology of cluster-root carboxylate synthesis and exudation in Harsh Hakea. Expression of phosphoenolpyruvate carboxylase and the alternative oxidase. Plant Physiology 135, 549–560. Shane, M.W., Dixon, K.W., & Lambers, H. (2005) The occurrence of dauciform roots amongst Western Australian reeds, rushes and sedges, and the impact of phosphorus supply on dauciform-root development in Schoenus unispiculatus (Cyperaceae). The New Phytologist 165, 887–898.

261

Shane, M.W., Cawthray, G.R., Cramer, M.D., et al. (2006) Specialized “dauciform” roots of Cyperaceae are structurally distinct but functionally analogous with “cluster” roots. Plant, Cell & Environment 29, 1989–1999. Shin, H., Shin, H.S., Dewbre, G.R., et al. (2004) Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. The Plant Journal 39, 629–642. Skene, K.R. (1998) Cluster roots: some ecological considerations. Journal of Ecology 86, 1060–1064. Skene, K.R. & James, W.M. (2000) A comparison of the effects of auxin on cluster root initiation and development in Grevillea robusta Cunn. ex R. Br. (Proteaceae) and the genus Lupinus (Leguminosae). Plant and Soil 219, 221–229. Skene, K.R., Kierans, M., Sprent, J., et al. (1996) Structural aspects of cluster root development and their possible signiﬁcance for nutrient acquisition in Grevillea robusta (Proteacea). Annals of Botany 77, 443–451. Smith, F.W., Mudge, S.R., Rae, A.L., et al. (2003) Phosphate transport in plants. Plant and Soil 248, 71–83. Smith, A.P., Jain, A., Deal, R.B., et al. (2010) Histone H2A.Z regulates the expression of several classes of phosphate starvation response genes but not as transcriptional activator. Plant Physiology 152, 217–225. Sokol, A., Kwiathowska, A., Jerzmanowski, A., et al. (2007) Up-regulation of stress-inducible genes in tobacco and Arabidopsis cells in response to abiotic stresses and ABA treatment correlates with dynamic changes in histone H3 and H4 modiﬁcations. Planta 227, 245–254. Sousa, M.F., Façanha, A.R., Tavares, R.M., et al. (2007) Phosphate transport by proteoid roots of Hakea sericea. Plant Science 173, 550–558. Sreenivasulu, N., Sopory, S.K., & Kavi Kishor, P.B. (2007) Deciphering the regulatory mechanisms of abiotic stress tolerance in plants by genomic approaches. Gene 388, 1–13. Steen, I. (1998) Phosphorus availability in the 21st century. Management of a non-renewable resource. Phosphorus and Potassium 217, 25–31. Stepanova, A.N., Yun, J., Likhacheva, A.V., et al. (2007) Multilevel interactions between ethylene and auxin in Arabidopsis roots. The Plant Cell 19, 2169–2185. Stracke, R., Werber, M., & Weisshaar, B. (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Current Opinion in Plant Biology 4, 447–456.

262

NUTRIENT USE EFFICIENCY IN CROPS

Su, J.-Y., Zheng, Q., Li, H.-W., et al. (2009) Detection of QTLs for phosphorus use efﬁciency in relation to agronomic performance in wheat grown under phosphorus sufﬁcient and limited conditions. Plant Science 176, 824–836. Sunkar, R. & Zhu, J.K. (2004) Novel and stressregulated microRNAs and other small RNAs from Arabidopsis. The Plant Cell 16, 2001–2019. Sunkar, R., Chinnusamy, V., Zhu, J., et al. (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends in Plant Science 12, 301–309. Svistoonoff, S., Creff, A., Reymond, M., et al. (2007) Root tip contact with low phosphate media reprograms plant root architecture. Nature Genetics 19, 792–796. Swarup, R., Perry, P., Hagenbeek, D., et al. (2007) Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root elongation. The Plant Cell 19, 2186–2196. Swarup, K., Benková, E., Swarup, R., et al. (2008) The auxin inﬂux carrier LAX3 promotes lateral root emergence. Nature Cell Biology 10, 946–954. Tanimoto, M., Roberts, K., & Dolan, L. (1995) Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. The Plant Journal 8, 943–948. Tesfaye, M., Liu, J., Allan, D.L., et al. (2007) Genomic and genetic control of phosphate stress in legumes. Plant Physiology 144, 594–603. Theodorou, M.E. & Plaxton, W.C. (1993) Metabolic adaptations of plant respiration to nutritional phosphate deprivation. Plant Physiology 101, 339–344. Ticconi, C.A. & Abel, S. (2004) Short on phosphate: plant surveillance and countermeasures. Trends in Plant Science 9, 548–555. Ticconi, C.A., Delatorre, C.A., Lahner, B., et al. (2004) Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development. Plant Journal 37, 801–814. Ticconi, C.A., Lucero, R.D., Sakhonwasee, S., et al. (2009) ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability. Proceedings of the National Academy of Sciences of the United States of America 106, 14174–14179. Tiessen, H. (2008) Phosphorus in the global environment. In: Ecophysiology of Plant-Phosphorus Interactions (eds. P.J. White & J.P. Hammond), pp. 1–8. Springer Science & Business Media B.V., Dordrecht. Torabi, S., Wissuwa, M., Heidari, M., et al. (2009) A comparative proteome approach to decipher the mechanism of rice adaptation to phosphorous deﬁciency. Proteomics 9, 159–170.

Tran, H.T. & Plaxton, W.C. (2008) Proteomic analysis of alterations in the secretome of Arabidopsis thaliana suspension cells subjected to nutritional phosphate deﬁciency. Proteomics 8, 4317–4326. Tuberosa, R. & Salvi, S. (2007) From QTLs to genes controlling root traits in maize. In: Scale and Complexity in Plant Systems Research: GenePlant-Crop Relations (eds. J.H.J. Spiertz & H.H. van Laar), pp. 15–24. Springer, Dordrecht. Uhde-Stone, C., Gilbert, G., Johnson, J.M.F., et al. (2003a) Acclimation of white lupin to phosphorus deﬁciency involves enhanced expression of genes related to organic acid metabolism. Plant and Soil 248, 99–116. Uhde-Stone, C., Zinn, K.E., Ramirez-Yáñez, M., et al. (2003b) Nylon ﬁlter arrays reveal differential gene expression in proteoid roots of white lupin in response to phosphorus deﬁciency. Plant Physiology 131, 1064–1079. Uhde-Stone, C., Liu, J., Zinn, K.E., et al. (2005) Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress. The Plant Journal 44, 840–853. Ulmasov, T., Murfett, J., Hagen, G., et al. (1997) Aux/ IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. The Plant Cell 9, 1963–1971. Valdés-López, O., Arenas-Huertero, C., Ramirez, M., et al. (2008) Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deﬁciency signaling in common bean roots. Plant, Cell & Environment 31, 1834–1843. Valdés-López, O. & Hernández, G. (2008) Transcriptional regulation and signaling in phosphorus starvation: what about legumes. Journal of Integrative Plant Biology 50, 1213–1222. Vance, C.P. (2008) Plants without arbuscular mycorrhizae. In: Ecophysiology of Plant-Phosphorus Interactions (eds. P.J. White & J. Hammond), pp. 117–142. Springer Science & Business Media B.V., Dordrecht. Vance, C.P., Uhde-Stone, C., & Allan, D.L. (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. The New Phytologist 157, 423–447. Vijayraghavan, V. & Soole, K. (2010) Effect of shortand long-term phosphate stress on the nonphosphorylating pathway of mitochondrial electron transport in Arabidopsis thaliana. Functional Plant Biology 37, 455–466. Wagner, B. & Beck, F. (1993) Cytokinins in the perennial herb Urtica dioica L. as inﬂuenced by nitrogen status. Planta 190, 511–518.

PHOSPHORUS AS A CRITICAL MACRONUTRIENT

Wang, Y.-H., Garvin, D.F., & Kochian, L.V. (2001) Nitrate-induced genes in tomato roots. Array analysis reveals novel genes that may play a role in nitrogen nutrition. Plant Physiology 127, 345–359. Wang, Z., Libault, M., Joshi, T., et al. (2010) SoyDB: a knowledge database of soybean transcription factors. BMC Plant Biology 10, 14. Wasaki, J., Yonetani, R., Kuroda, S., et al. (2003) Transcriptomic analysis of metabolic changes by phosphorus stress in rice plant roots. Plant, Cell & Environment 26, 1515–1523. Waters, B.M. & Blevins, D.G. (2000) Ethylene production, cluster root formation, and localization of iron (III) reducing capacity in Fe deﬁcient squash roots. Plant and Soil 225, 21–31. Watt, M. & Evans, J.R. (1999) Proteoid roots. Physiology and development. Plant Physiology 121, 317–324. Weisskopf, L., Abou-Mansour, E., Fromin, N., et al. (2006a) White lupin has developed a complex strategy to limit microbial degradation of secreted citrate required for phosphate acquisition. Plant, Cell & Environment 29, 919–927. Weisskopf, L., Tomasi, N., Santelia, D., et al. (2006b) Isoﬂavonoid exudation from white lupin roots is inﬂuenced by phosphate supply, root type and cluster-root stage. The New Phytologist 171, 657–668. Werner, T. & Schmülling, T. (2009) Cytokinin action in plant development. Current Opinion in Plant Biology 12, 1–12. Werner, T., Motyka, V., Strand, M., et al. (2001) Regulation of plant growth by cytokinin. Proceedings of the National Academy of Sciences of the United States of America 98, 10487–10492. Werner, T., Motyka, V., Laucou, V., et al. (2003) Cytokinin-deﬁcient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. The Plant Cell 15, 2532–2550. White, P.J. & Hammond, J.P. (2008) Phosphorus nutrition of terrestrial plant. In: Ecophysiology of PlantPhosphorus Interactions (eds. P.J. White & J.P. Hammond), pp. 51–81. Springer Science & Business Media B.V., Dordrecht. Williamson, L.C., Ribrioux, S.P., Fitter, A.H., et al. (2001) Phosphate availability regulates root system architecture in Arabidopsis. Plant Physiology 126, 875–882. Wippo, C.J., Krstulovic, B.S., Ertel, F., et al. (2009) Differential cofactor requirements for histone eviction from two nucleosomes at the yeast

263

PHO84 promoter are determined by intrinsic nucleosome stability. Molecular Cell Biology 29, 2960–2981. Wissuwa, M., Wegner, J., Ae, N., et al. (2002) Substitution mapping of Pup1: a major QTL increasing phosphorus uptake of rice. Theoretical and Applied Genetics 105, 890–897. Wu, P., Ma, L., Hou, X., et al. (2003) Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves. Plant Physiology 132, 1260–1271. Xie, Z., Allen, E., Fahlgren, N., et al. (2005) Expression of Arabidopsis MIRNA genes. Plant Physiology 138, 2145–2154. Yan, F., Zhu, Y., Mueller, C., et al. (2002) Adaptation of H+ pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deﬁciency. Plant Physiology 129, 50–63. Yang, X.J. & Finnegan, P.M. (2010) Regulation of phosphate starvation responses in higher plants. Annals of Botany 105, 513–526. Yi, K., Wu, Z., Zhou, J., et al. (2005) OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice. Plant Physiology 138, 2087–2096. Yi, K., Menand, B., Bell, E., et al. (2010) A basic helixloop-helix transcription factor controls cell growth and size in root hairs. Nature Genetics 42, 264–267. Yuan, H. & Liu, D. (2008) Signaling components involved in plant responses to phosphate starvation. Journal of Integrative Plant Biology 7, 849–859. Zaid, H., El Morabet, R., Diem, H.G., et al. (2003) Does ethylene mediate cluster root formation under iron deﬁciency? Annals of Botany 92, 673–677. Zhang, X. (2008) The epigenetic landscape of plants. Science 320, 489–492. Zhang, D., Cheng, H., Geng, L., et al. (2009) Detection of quantitative trait loci for phosphorus deﬁciency tolerance at soybean seedling stage. Euphytica 167, 313–322. Zhou, K., Yamagishi, M., Osaki, M., et al. (2008) Sugar signaling mediates cluster root formation and phosphorus starvation-induced gene expression in white lupin. Journal of Experimental Botany 59, 2749–2756. Zhu, J., Kaeppler, S.M., & Lynch, J.P. (2005) Mapping of QTL controlling root hair length in maize (Zea mays L.) under phosphorus deﬁciency. Plant and Soil 270, 299–310. Zhu, J., Jeong, J.C., Zhu, Y., et al. (2008) Involvement of Arabidopsis HOS15 in histone deacetylation and cold tolerance. Proceedings of the National Academy of Sciences USA 105, 4945–4950.

264

NUTRIENT USE EFFICIENCY IN CROPS

Zhu, Y.-Y., Zeng, H.-Q., Dong, C.X., et al. (2010) microRNA expression proﬁles associated with phosphorus deﬁciency in white lupin (Lupinus albus L.). Plant Science 178, 23–29. Zilberman, D., Colemann-Derr, D., Ballinger, T., et al. (2008) Histone H2A.Z and DNA methylation are

mutually antagonistic chromatin marks. Nature 456, 125–129. Zinn, K.E., Liu, J., Allan, D.L., et al. (2009) White lupin (Lupinus albus) response to phosphorus stress: evidence for complex regulation of LaSAP1. Plant and Soil 322, 1–15.

Chapter 13

Uptake, Distribution, and Physiological Functions of Potassium, Calcium, and Magnesium Frans J.M. Maathuis and Dorina Podar

Abstract Plants require at least 14 essential macroand micronutrients. Of the macronutrients, the cationic potassium (K+), calcium (Ca2+), and magnesium (Mg2+) are required in large quantities and play distinct roles in plant growth and development. In the past decades, great progress has been made in identifying and characterizing the molecular components that are responsible for the uptake and distribution of these minerals and as such provides options to manipulate and optimize these processes. This chapter gives a detailed overview of the functions of K+, Ca2+, and Mg2+ in plants, how they are taken up and distributed, and how genetic manipulation of speciﬁc systems may contribute to improved crop production. Introduction Plants require at least 14 essential nutrients. Six of these are required in relatively large quantities and hence named macronutrients. Of the macronutrients, three are cationic, consisting of potassium (K+), calcium (Ca2+), and magnesium (Mg2+). In combination,

these three ions can easily make up 10–20% of plant dry weight. Although K+, Ca2+, and Mg2+ are required in large quantities, their spatial distribution and physiological roles vary widely. This chapter provides a detailed overview of the availability of each cation in the external environment, the mechanisms of their uptake and distribution, and the speciﬁc functions they play. In addition, possibilities to exploit the knowledge gained about transport mechanisms to improve crop production are discussed.

Potassium Potassium occurrence and availability in the soil Around 2.6% of the earth’s crust consists of potassium (K). Most of this potassium (up to 95%) forms structural elements of soil minerals and is as such extremely immobile. This ﬁrst fraction primarily consists of K+, which is dehydrated and covalently coordinated in a hexagonal conformation to the oxygen atoms of soil colloids. A second fraction is tightly adsorbed to soil minerals such

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 265

266

NUTRIENT USE EFFICIENCY IN CROPS

as feldspars, illites, and vermiculites, or to organic material and is therefore also not directly available to plants. The K+ in this fraction remains hydrated and is electrostatically bound to soil particles and organic matter. Part of the electrostatically bound K+ can be released through weathering of minerals and soil colloids; the released K+ fraction is readily exchangeable with the third K+ compartment, the soil solution. Within the soil solution, K+ is extremely mobile. Both content and release capacity for K+ from mineral lattices varies greatly between soils: Soils rich in illite and chlorite minerals contain larger amounts of K+, whereas alluvial and sandy soils have very low K+ contents and, consequently, low cation exchange capacity (CEC). Related to this is the fact that although clay rich soils contain more total K+, the large number of adsorption sites limits K+ release into the soil solution and thus such soils have a low “K+ solubility index” (Mengel and Kirkby, 2001). Equally, the soil K+ buffer capacity will depend on soil composition and is generally lower in sandy soils. The diverse capacities of soils to bind and release K+ culminates in soil solutions with K+ concentrations that greatly ﬂuctuate, although the majority is within the range of 0.1–1 mM K+ and therefore signiﬁcantly higher than many other nutrients. The relatively high levels of K+ and its large mobility mean that severe K+ deﬁciency is rare in most environments. However, in many crops, K+ demand is high; it often equals or outstrips nitrogen demand in intensive farming conditions, and even without the presence of drought or salinity, K+ supply is therefore often insufﬁcient for optimal crop production (Fig. 13.1A). Apart from negative effects on biomass production, K+ deﬁciency also has a large negative effect on general plant health. When K+ supply is insufﬁcient, the incidence of many common crop diseases such as mildew and brown spot increases drastically (Mengel and Kirkby,

A

B

Chloroplast K+ CHX

NHX

AKT2

SHOOT Vacuole H+ K+ K+ TPK1

K+ (NO3-)

K+ (sugar)

XYL PHLO

CNGC

SKOR K+

AKT1 (KC1)

K+

KUP/ HAK5

K+ H+

GORK

K+

K+

ROOT

Potassium nutrition. (A) Potassium (K+) deﬁciency manifests itself as chlorosis, particularly along the edges of older leaves. Deﬁciency reduces crop yield and negatively affects disease and stress tolerance in plants. (B) Main transport systems that participate in K+ uptake, distribution, and compartmentation. At the root soil boundary, high-afﬁnity (KUP/HAK) and lowafﬁnity (AKT) uptake systems are present. Uptake may be supplemented through other systems such as cyclic nucleotide-gated channels (CNGCs). A large K+ efﬂux is likely to be mediated by outward rectifying channels such as GORK. Xylem loading of K+ is primarily carried out by K+ channels such as SKOR, whereas translocation from shoot to root probably involves AKT2. Compartmentation of K+ in vacuoles and other endocompartments is mediated by proton gradient driven systems such as CHX and NHX, and by ion channels such as TPK1. “XYL” and “PHLO” denote xylem and phloem. Fig. 13.1.

POTASSIUM, CALCIUM, AND MAGNESIUM

2001). As a consequence, annual global K+ fertilizer application exceeds 30 megatonnes, incurring a considerable cost to farmers in both developed and developing countries. The efﬁcacy of fertilization again depends to a large extent on soil characteristics; in K+, depleted clay rich soils fertilization may initially have little or no effect on the levels of plant available K+. This happens because the large adsorption capacity of such soils rapidly leads to “K+ ﬁxing,” that is, the replenishment of fraction one and two. In contrast, when depleted substrates with low CEC such as sandy soils are fertilized, they will show a rapid rise in soil solution K+, but their low buffer capacity ensures this is relatively short-lived (Wulff et al., 1998). Physiological functions of K+ The monovalent cation K+ is an essential nutrient for all living organisms. It has irreplaceable functions in many metabolic reactions, particularly via its activation of speciﬁc enzymes. Its high mass : charge ratio also makes K+ well suited as a counter ion for cytoplasmic polyanions, hence the high levels of K+ that are found in the cytosol of all cells. Furthermore, plants need K+ in the vasculature as a counter ion for long-distance transport of sugars and nitrate and K+ is a predominant contributor to cell turgor via its osmotic effects in the vacuole. Not surprisingly, these multiple functions require high cellular K+ concentrations, and K+ can comprise up to 10% of plant dry weight. Biochemical roles of K+ Many of the roles performed by K+ in the cytoplasm are related to biochemical reactions. A large number of enzymes requires K+ as a cofactor, and maximum activation measured in vitro typically occurs when 50–80 mM K+ is present. This value agrees well with cytoplasmic concentrations that

267

have been determined in cells from many different organisms and typically range from 70 to100 mM (Maathuis and Sanders, 1993; Walker et al., 1996). There are probably at least 80 enzymes that require K+ for proper functioning. Many of those are involved in basal C-metabolic pathways (Marschner, 1995). One of the best researched enzymes in this respect is pyruvate kinase (PK), an enzyme that catalyzes transfer of a phosphate group from phosphoenolpyruvate to adenosine diphosphate (ADP), thereby forming adenosine triphosphate (ATP) + pyruvate. In the 1950s, it was established that PK requires K+ for activity with a typical Km (half maximum enzyme activity) of around 10 mM for K+ (Kachmar and Boyer, 1953). Other monovalent cations such as NH4+ and Rb+, which have similar atomic radii, are also capable of activating PK but only with half the efﬁcacy of K+. In contrast, Na+ and Li+ ions show only 5–10% of K+ activation. The reason for this K+ speciﬁcity originates in the way K+ binds to sites on the protein: K+ with an ionic radius of 138 pm binds in its dehydrated form, probably via coordination with six oxygens that can derive from carboxyl, carbonyl, and hydroxyl groups, and from water molecules. This conﬁguration creates steric and energetic conditions that allow only ions of the correct size and dehydration energy to bind. Chemically similar ions such as Na+ and Li+ have smaller radii of 102 and 76 pm, respectively, and also need far greater energies to dehydrate. Apart from enzymes such as PK that are found in all kingdoms of life, various plant enzymes have been scrutinized for their K+ activation properties. For example, several types of vacuolar PPase, which in parallel with the vacuolar H+-ATPase accumulate protons into the vacuolar lumen, are completely inactive in the absence of cytoplasmic K+ (Nilima and Vinay, 2008). As was

268

NUTRIENT USE EFFICIENCY IN CROPS

observed for PK, PPase activity can be stimulated to some degree by other monovalent cations such as Na+ and Li+, but stimulation is 3–15 times higher with K+. Similarly, the plant-speciﬁc enzyme starch synthetase shows virtually no activity even in the presence of 50 mM Li+, whereas half maximum activity is achieved with ∼5 mM K+ (Nitsos and Evans, 1969). Not surprisingly, K+ deﬁciency rapidly produces the accumulation of soluble carbohydrates and reduced starch levels. Protein synthesis, which occurs at ribosomes, is another key mechanism that requires high concentrations of K+. Although this includes the processes of tRNA and mRNA binding, the identity of speciﬁc enzymes has yet to be revealed. Sensitivity of protein synthesis to K+ is believed to be an important factor in the reduction of photosynthetic activity in K+-deﬁcient plants, due to the high protein turnover of the photosynthetic machinery. The lack of reduced carbohydrate that ensues creates the accumulation of soluble nitrogen. Biophysical roles of K+ The predominant biophysical role of K+ is in turgor provision and water homeostasis. As the main tissue cation, K+ is a predominant contributor to cell turgor and thus important for cell expansion and plant growth. Turgor pressure in K+-deﬁcient plants is often lower than that of K+-replete plants, reducing growth rates. Plant movement is normally also greatly dependent on K+-derived turgor. Movement includes many processes such as the rapid closure of the Dionaea muscipula (Venus ﬂytrap) and the nyctinastic reorientation of leaves in response to diurnal rhythms, but it is best represented by opening and closing of stomata. Adjustment of stomatal aperture requires changes in guard cell shape that in turn are the result of changes in cell turgor. Uptake and release of large amounts of K+ and Cl− are responsible for modulation

of guard cell turgor. Thus, K+ provision greatly affects plant water homeostasis, directly by providing cellular turgor and indirectly by modulating stomatal conductance and gas exchange. Although K+ is the predominant cation in these processes for most terrestrial plants, it can to a large extent be replaced by other monovalent cations with similar physicochemical properties. This is well illustrated by the difference between glycophytic and halophytic species. In conditions of water stress (drought), most glycophytes respond by rapidly increasing K+ uptake rates to restore the osmotic potential of their cell sap (e.g., Mahouachi et al., 2006). However, in halophytes, it is often Na+ rather than K+ that is used for this purpose. A further biophysical function of K+ is in cytoplasmic charge balancing. The larger radius and mass : charge ratio of K+ (m/z = 39) compared with, for example, Na+ (m/z = 23) ensures that K+ has a less disruptive effect on water structure; K+ has less tendency to sever hydrogen bonds between water molecules or between water molecules and macromolecules such as nucleic acids and proteins. Thus, K+ is less chaotropic than Na+, even at high ionic strength, and consequently, evolution selected K+ as the main cytoplasmic counterion to balance the multiple anionic groups of nucleic acids and proteins. This role cannot easily be fulﬁlled by other cations and is another reason why relatively high (∼100 mM) cytosolic K+ concentrations are found in all living cells. At the whole-plant level, K+ is also essential as a counterion in the xylem and the phloem to maintain electroneutrality during transport of NO3−, amino acids, and nucleotides. K+dependent charge balancing is also critical in chloroplasts, where stromal H+ are exchanged with cytoplasmic K+ to maintain a steep pH gradient. Hence, K+ deﬁciency negatively affects photosynthesis in this manner as well.

POTASSIUM, CALCIUM, AND MAGNESIUM

Cells have negative membrane potentials, which in plants can reach values in excess of −200 mV. This electrical potential can be used for transport of nutrients and other compounds into and out of the cell, either directly or via H+-coupled mechanisms. The overall plant plasmamembrane conductance is usually dominated by K+ conductance (Maathuis et al., 1997) due to the large number of K+ selective ion channels. Since cytoplasmic K+ levels are believed to be tightly regulated to a value of around 100 mM, plant cell membrane potentials reliably follow the K+ Nernst potential over a large range of external K+ concentrations. As such, natural ﬂuctuations in external K+ can affect the cell polarization and could therefore alter membrane transport functions. In most conditions, however, the cytoplasmic K+ concentration will be considerably higher than the external concentration, and this outwardly directed electrochemical K+ gradient can be used to maintain a negative cell interior. Indeed, speciﬁc K+-selective ion channels may reside in the membrane, and their opening is regulated such that a negative membrane potential prevails. The relationship between K+ gradient, membrane potential, and generic transport functions is difﬁcult to disentangle, but effects of external K+ have been reported to affect K+ uptake and even stelar K+ movement (Ishikawa et al., 1984). An example where K+ mediates membrane repolarization has been described for amino acid uptake: Since this uptake is proton coupled, it depolarizes the membrane, a process that is counteracted by increasing the K+ permeability to clamp the cell potential to the negative K+ Nernst potential (Kinraide et al., 1984). Electrical signaling is generally associated with multicellular animals, where nerval action potentials propagate with high speed from one organ to another. However, there is good evidence that similar processes take place in plants, albeit with kinetics that

269

are 100- to 1000-fold slower (Fromm and Lautner, 2007). Plant “action potentials” have been recorded in many species and in response to many stimuli, but in most cases their biological relevance is unknown. In plants that show rapid movement such as the touch response of Mimosa leaves or the closing of the trap leaves of Venus ﬂytrap, the role of action potentials is less obscure. Plant action potentials are believed to be initiated by Cl− efﬂux (possibly via Ca2+ activation), which leads to rapid depolarization. Depolarizations may trigger outward K+ channels to open and thus lead to a repolarization to the K+ Nernst potential. This role of K+ in action potentials has to some extent been veriﬁed with K+ ion channel inhibitors (Fromm and Lautner, 2007), but the molecular components are unknown. Many of the biophysical functions of K+ can be fulﬁlled by cations such as Rb+ or Na+. This is particularly clear on exposure to salinity when a large proportion of vacuolar K+ is substituted by Na+. Indeed, when K+ is deﬁcient, addition of Na+ to the growth medium can notably improve growth (Maathuis et al., 1996). However, large amounts of Na+ have detrimental effects on K+ nutrition (Maathuis and Amtmann, 1999). For example, high-afﬁnity K+ transporters are inhibited by Na+, affecting K+ uptake and distribution (Santa-Maria et al., 1997). Many glycophytic plants preferably translocate K+ to their shoots. Although this can be beneﬁcial in some circumstances, it easily causes a severe reduction in shoot turgor during salt stress due to a lack of K+ and the inability to utilize Na+ effectively. A critical function of K+ during salt stress is also reﬂected in many species where salt sensitivity is more strongly correlated to reduced K+ content than to increased Na+ content (e.g., Munns and Tester, 2008). Thus, a major challenge for salt-exposed plants is the maintenance of adequate cellular K+ levels in the presence of large concentrations of Na+, and retaining

270

NUTRIENT USE EFFICIENCY IN CROPS

a high K+/Na+ selectivity is a characteristic property of salt tolerance in glycophytes. Uptake and distribution of K+ K+ uptake from the soil K+ uptake into plant roots has high- and lowafﬁnity components, and electrophysiological studies during the 1980s and 1990s suggested that passive transport of K+ through ion channels with a Km in the millimolar range, and active transport of K+ through H+-cotransporters with a Km in the micromolar range, were responsible for lowafﬁnity and high-afﬁnity K+ uptake, respectively (Maathuis and Sanders, 1994, 1995; Maathuis et al., 1997). The molecular identity of many transporters that contribute to K+ uptake is now available (Fig. 13.1B). One of the ﬁrst to be identiﬁed (via complementation of K+ transport-deﬁcient yeast strains) was the Arabidopsis K+ channel, AtAKT1 (Table 13.1) AKT1 is a K+-selective inward rectifying channel, expressed in the root

Table 13.1.

cortex (Lagarde et al., 1996) and one of the main components of low-afﬁnity K+ uptake. However, loss of function mutations in AtAKT1 (Hirsch et al., 1998) have shown that this channel can also mediate K+ uptake in the high-afﬁnity (micromolar) range of external K+ but not to the extent of H+cotransporters (Rodriguez-Navarro and Rubio, 2006). The AKT1 transcript level is largely impervious to ambient K+ conditions, but K+ deﬁciency does instigate a posttranslational response: K+ starvation generates a Ca2+ transient which activates calcineurin B-like (CBL) proteins via Ca2+ binding; speciﬁc CBLs interact with CBL interacting protein kinases (CIPKs); the isoforms CBL1 and CBL9 activate CIPK23 during K+ starvation with CIPK23 phosphorylating the C-terminal region of AKT1 (Xu et al., 2006). The resulting stimulation of AKT1 activity increases K+ uptake (Fig. 13.1B). Another mechanism to regulate uptake may exist via AKT1 interaction with other K+ channel subunits such as KC1, a K+ channel subunit that is not functional when

The abbreviations of different classes and isoforms of transporter proteins discussed in the text

Abbreviation

Name

Generic Function

AKT ACA/ECA CAX CHX CNGC GLR KC KUP/HAK

Arabidopsis potassium channel Arabidopsis calcium pump Calcium exchanger Cation proton exchanger Cyclic nucleotide-gated channel Glutamate-like receptor Potassium channel Potassium uptake protein

MCA MHX MGT/MRS NHX NSCC SKOR

Mechanosensitive calcium channel Magnesium proton exchanger Magnesium transporter Sodium proton exchanger Nonselective cation channel Potassium outward rectifying channel Two-pore channel Two-pore potassium channel

Potassium uptake Calcium extrusion from the cytosol Calcium extrusion from the cytosol Monovalent cation transport Mono- and divalent cation transport Mono- and divalent cation transport Potassium channel regulatory subunit High-afﬁnity potassium uptake and distribution Calcium uptake and signaling Magnesium transport Magnesium uptake and transport Sodium and potassium transport Mono- and divalent cation transport Xylem potassium loading

TPC TPK

Vacuolar cation transport Vacuolar potassium transport

POTASSIUM, CALCIUM, AND MAGNESIUM

expressed on its own. KC1 trafﬁcking from the endoplasmic reticulum (ER) to the plasma membrane occurs during K+ starvation and leads to expression of AKT1:KC1 heteromers (Reintanz et al., 2002). Association of AKT1 and KC1 alters voltage dependence of the channel gating by shifting the activation threshold negative by approximately −70 mV. This prevents cellular K+ loss during conditions where a steep outward K+ gradient prevails (Geiger et al., 2009). Nonselective, ligand gated channels from the cyclic nucleotide-gated channel (CNGC) family are also implicated as K+ uptake pathways. For example, CNGC10, which is relatively highly expressed in root tissue, was able to partially complement the K+ uptake-deﬁcient phenotype of the akt1-1 loss of function mutant, showing it can form a root K+ uptake pathway and considerably augment K+ uptake when overexpressed in the akt1-1 genotype (Li et al., 2005). Several members of the Arabidopsis KUP/HAK gene family have been found to mediate high-afﬁnity uptake in heterologous expression systems (e.g., Kim et al., 1998) and in planta (Gierth et al., 2005). In Arabidopsis and several other species, K+ starvation rapidly increases HAK5 (or its orthologs) transcript levels (Armengaud et al., 2004), and this may be one of the reasons for the observation that in many plants highafﬁnity K+ uptake is inducible by low external K+ (Kochian and Lucas, 1983). Induction of HAK5 expression possibly follows the burst of H2O2 production that is generated in response to low K+ conditions (Shin and Schachtman, 2004). KUP/HAKs are inhibited by external NH4+ (Santa-Maria et al., 1997) and most likely function as H+-coupled systems (Maathuis and Sanders, 1994). They therefore have a greater capacity to accumulate K+ from very dilute external solutions compared with ion channels, and this type of mechanism can deplete external K+ to

271

levels below 1 μM (Rodriguez-Navarro and Rubio, 2006). In Arabidopsis, CHX17 expression rapidly increased in response to abscisic acid (ABA) treatment or K+ deprivation. A loss of function mutant accumulated less K+ in roots in response to salt stress or K+ shortage (Cellier et al., 2004). These ﬁndings indicate that AtCHX17 helps maintain K+ homeostasis by providing extra K+ acquisition capacity, for example, to compensate for loss of K+ uptake through HAK/KUP type systems, which are sensitive to Na+ inhibition (SantaMaria et al., 1997). Interestingly, K+ efﬂux in roots is considerable, especially in K+-replete conditions when it becomes comparable to unidirectional inﬂux (Szczerba et al., 2009). This results in only small net uptake rates and a large degree of what seems to be futile recycling of K+. The exact reasons for this phenomenon are not clear, but similar efﬂuxes have been observed for other nutrients such as NO3− and possibly form some sort of “safety valve” to prevent toxic effects when high levels of ambient K+ are present. K+ efﬂux channels have been characterized in many plant cells, but the molecular identity in most root cells is unknown apart from root hairs where guard cell outward rectifying K+ channel (GORK) (Fig. 13.1B) is believed to mediate K+ efﬂux for osmoregulation and to regulate root hair membrane potentials (Ivashikina et al., 2001). K+ translocation and distribution High levels of K+ are required throughout the plant and sophisticated mechanisms are in place to ensure delivery of this ion to every cell. K+ uptake at the root soil boundary occurs through mechanisms discussed above, and its distribution around the root symplast most likely depends on bulk ﬂow through interconnecting plasmodesmata.

272

NUTRIENT USE EFFICIENCY IN CROPS

Delivery to nonroot tissues requires K+ loading into the xylem apoplast, a process largely controlled by outward rectifying K+ channels originally identiﬁed in Arabidopsis as AtSKOR (Fig. 13.1B; Gaymard et al., 1998). SKOR is a Shaker-type K+ channel activated when the membrane depolarizes. A SKOR knockout mutant showed a reduced shoot K+ content and a reduced K+ content in the xylem sap, providing clear evidence that SKOR plays an important role in the root–shoot partitioning of K+. SKOR transcription is inhibited by ABA (Gaymard et al., 1998) presumably to ensure reduced K+ loading into the xylem to maintain adequate root turgor when soils dry out. Although these properties suggest an important role for SKOR in K+ xylem loading and hence its delivery to shoot tissue, patch clamp studies on xylem parenchyma cells have identiﬁed several further other outward rectifying, K+ conducting channels that may also contribute to xylem K+ loading (Roberts and Tester, 1995; Wegner and De Boer, 1999). Long-distance K+ transport is by no means unidirectional, and a large proportion of shoot K+ is recycled to the root through the phloem (Marschner et al., 1997). One reason for this apparently futile cycle is the role that K+ plays as a counterion for root to shoot translocation of NO3− and for assimilates such as sucrose, malate, and amino acids that move in the phloem from shoot to root and generally from source to sink. The K+ channel AKT2,3 is weakly inward rectifying and has been shown to be expressed in the phloem of Arabidopsis leaves (Fig. 13.1B; Marten et al., 1999). It has been postulated to function as a shunt conductance in phloem cells, and as such AKT2,3 is a prime candidate for the release of K+ into the phloem (Marten et al., 1999) and its recirculation between shoot and root. KUP/HAK transporters are also involved in K+ distribution; KUP2, which is predomi-

nantly expressed in rapidly growing tissues, has been shown to play a role in cell expansion, particularly that of the hypocotyl (Elumalai et al., 2002). KUP4 is important in root hair cell expansion and as such impacts on multiple nutritional aspects. Whether this effect is directly due to K+ transport is not clear since KUP4 also appears to modulate auxin efﬂux (VicenteAgullo et al., 2004). In addition to highafﬁnity KUP/HAKs, there are also isoforms that show low-afﬁnity K+ transport. Many of these are expressed in different tissues throughout the plant and are likely to play roles in K+ distribution between organs and tissues, but the details have yet to be studied. Subcellular K+ partitioning Although K+ is likely to be present at high concentrations in all cellular compartments, its prominent role as turgor provider means the majority is deposited in the vacuole. Nevertheless, vacuolar K+ may need to be released in many conditions, for example, when cytoplasmic K+ becomes deﬁcient (Walker et al., 1996), when osmotic adjustment is necessary, or when turgor driven movement is required such as during stomatal closure. The processes of vacuolar deposition and vacuolar release are mediated by tonoplast transporters involved in the bidirectional transfer of K+. Vacuolar loading of K+ may to some extent be mediated by cation channels such as TPC1 and TPK1 but must rely on energized mechanisms to reach K+ concentrations that are equal or higher than those in the cytoplasm, due to the membrane potential across the tonoplast, which keeps the lumen at a potential ∼20 mV positive with respect to the cytoplasm. It is generally assumed that K+:H+ exchangers, particularly from the CHX and NHX families, drive such ﬂuxes (Fig. 13.1B; Cellier et al., 2004; Sze et al., 2004; Pardo et al.,

POTASSIUM, CALCIUM, AND MAGNESIUM

2006). However, hard evidence for this assumption still remains elusive and further research is urgently needed to identify speciﬁc members of the CHX or other exchanger families that are responsible for this important mechanism. NHX- and CHX-type K+:H+ exchangers may also regulate K+ homeostasis of other endocompartments. For example, the tomato LeNHX2 protein colocalizes with prevacuolar and Golgi markers in both yeast and plants (Rodriguez-Rosales et al., 2008), whereas a translational AtNHX5:GFP fusion localized to prevacuolar compartments of onion cells (Pardo et al., 2006). These systems are possibly responsible for loading and unloading of K+ in cellular endocompartments and thus serve to maintain electrical and pH homeostasis. K+ provision of chloroplasts may rely on CHX antiporters: AtCHX23 (Fig. 13.1B), was found to be targeted to the chloroplast envelope, and its K+:H+ exchange activity was hypothesized to impact on stromal pH and chloroplast development (Song et al., 2004). Release of vacuolar K+ is largely thermodynamically “down hill” and thus likely to be through ion channels. Particularly TPK (two-pore K+ channel)-type channels appear to be a main contributor to this process. TPKs show a four-transmembrane/two-pore structure, GYGD K+ selectivity motifs, and one or two C-terminal EF hands. The Arabidopsis genome encodes ﬁve TPKs, and similarly sized TPK families have been found in genomes of other species such as rice, tobacco, and Physcomitrella. Out of ﬁve TPKs present in Arabidopsis, all but one (TPK4) are expressed at the tonoplast (Czempinski et al., 2002; Becker et al., 2004). Most isoforms do not appear to form functional ion channels in planta or in heterologous expression systems, but AtTPK1 has been characterized in detail regarding membrane and tissue-speciﬁc expression. It is expressed in most plant tissues where

273

it probably forms homomeric channels (Voelker et al., 2006; Gobert et al., 2007). In addition, heterologous expression of TPK1 in yeast vacuoles showed that it constitutes a voltage-independent, Ca2+-dependent conductance with a high selectivity for K+ over Na+. AtTPK1 expression was shown to impact on overall K+ homeostasis and on stomatal closure, in particular giving credence to the notion that it constitutes a major pathway for vacuolar K+ release (Gobert et al., 2007). In seeds, TPK1 may also be involved in the release of K+ from protein storage vacuoles during the initial phases of germination (Gobert et al., 2007). Another K+-conducting tonoplast channel (the fast vacuolar [FV] channel) may also participate in intracellular K+ distribution. The FV channel has a potassium/sodium selectivity of around unity and was originally described in red beet storage tissue (Hedrich and Neher, 1987). The gene(s) encoding the FV channel is not known, and this frustrates in-depth studies regarding its characteristics and in planta role. But since both luminal and cytoplasmic K+ levels impact on FV channel open probability, it has been hypothesized that maintaining cellular K+ homeostasis is one of the physiological roles of this transporter (Pottosin and Martinez-Estevez, 2003). During prolonged K+ starvation, vacuolar + K concentrations become signiﬁcantly lower than those in the cytoplasm (Walker et al., 1996). In such conditions, energized transport may be required for vacuolar K+ release, ruling out ion channels. The transporter(s) that is responsible for this process is unknown, but, interestingly, proteomics studies suggest that several members of the HAK/KUP family are localized at the tonoplast (e.g., Jaquinod et al., 2007; Whiteman et al., 2008). Driven by the transtonoplast proton motive force, such systems could facilitate “up hill” K+ release from the vacuole.

274

NUTRIENT USE EFFICIENCY IN CROPS

Calcium Calcium occurrence and availability in the soil Like potassium, calcium (Ca2+) is one of the most abundant elements in the lithosphere representing 3.64% of the earth’s crust. Being a very reactive element, calcium is never found in elemental form but most prevalently as carbonate (in marble, chalk, limestone, and calcite), sulfate (in anhydrite and gypsum), ﬂuoride (ﬂuorspar or ﬂuorite), phosphate (in apatite) silicate, or borate salt. Although relatively abundant in soils, the availability of calcium for plants is often impeded by other factors such as pH and CEC. In contrast to most other cations, acidity reduces Ca2+ availability, mainly because the increased release of anions leads to deposition of insoluble Ca2+ salts. As such, this process can easily cause deﬁciencies of other minerals such as phosphorus. Acidity also promotes leaching, which can result in Ca2+ depletion. Other soil factors that have detrimental effects on Ca2+ availability include salinity when excess Na+ displaces Ca2+ from exchange sites. Although calcareous soils are relatively widespread, Ca2+ toxicity in plants is rare. Toxicity symptoms include yellow spots on leaves, calyx, or fruit that result from calcium being deposited as calcium–oxalate in these organs (White and Broadley, 2003). However, most plants can accumulate Ca2+ up to 10% of dry weight without symptoms of toxicity (Marschner, 1995). Deposition of calcium–oxalate in leaf mesophyll cells or trichomes is also a strategy of calcareous soil-loving plants called calcicoles, which include members of the Crassulaceae, Brassicaceae, and Fabaceae families. Physiological functions of Ca2+ Plant requirement for calcium, though species-speciﬁc, is generally high and ranges

from 1 to 50 mg g−1 dry weight (Marschner, 1995). For example, in dicotyledonous angiosperms, calcium often constitutes up to 5% of the plant dry weight (Marschner, 1995; Broadley et al., 2003, 2004). At the cellular level, Ca2+ roles can be divided into two main categories: structural and signaling. Structural roles of Ca2+ Calcium plays an important role in maintaining plant structure through its role in strengthening the cell wall. Depending on the plant tissue, up to 90% of the cellular calcium can be found within the cell wall (Marschner, 1995). Being a cation, Ca2+ is readily attracted to negative charges of carboxyl groups that are prevalent in cell wall polymers. Ca2+ links the carboxyl groups of pectins (polygalacturonic acids) within the middle lamella, thus stabilizing the polysaccharide network. The proportion of calcium within the middle lamella depends directly on the Ca2+ availability. Ca2+-depleted tissues therefore will be characterized by weaker cell walls, and this can take place when Ca2+ is deﬁcient or when Na+ and H+ concentrations are raised (Marschner, 1995). The latter has physiological importance during auxin-induced cell extension when wall acidity is increased to replace Ca2+ with H+. Cell wall strength and thickness are important factors that limit pathogen attack, and Ca2+ plays an indirect role in conferring resistance to various infections. Ca2+ also plays a structural role in plasma membranes; electrostatic binding to phosphate and carboxyl groups of phospholipids and proteins impacts greatly on membrane integrity. This mechanism largely occurs at the external face of the plasma membrane, requiring relatively high levels of apoplastic Ca2+. Removal of membrane Ca2+, or its replacement with other cations such as Na+, K+, Rb+, and Mg2+ (Ozaki et al., 2005)

POTASSIUM, CALCIUM, AND MAGNESIUM

rapidly compromises membrane integrity and can cause electrolyte loss and increased inﬂux of potentially damaging ions. It is likely that this phenomenon explains common observations such as stimulation of cation uptake and improved ion selectivity at high ambient Ca2+ (Marschner, 1995; Ozaki et al., 2005). For example, application of Ca2+ markedly limits Na+ uptake and saltinduced K+ efﬂux in plants (Marschner, 1995; Shabala and Cuin, 2008). Apart from cell walls, a large proportion of calcium ends up in the vacuole, where it is likely to contribute to charge balance and turgor provision. Particularly in the mesophyll cells, substantial amounts of Ca2+ are stored in the central vacuole, where it neutralizes organic and inorganic anions such as oxalate, malate, citrate, and NO3− (Karley et al., 2000; Martinoia et al., 2007; Pottosin and Schönknecht, 2007).

275

signals to downstream targets such as gene transcription. Evidence has been reported that Ca2+ acts as a secondary messenger in the responses to a multitude of stimuli such as biotic and abiotic stress and is involved in many physiological processes such as stomatal regulation, nodulation, temperature perception, and pollen tube growth (Sanders et al., 2002; Hetherington and Brownlee, 2004; McAinsh and Pittman, 2009; Saidi et al., 2009; Dodd et al., 2010). An elegant example of how Ca2+ signaling impacts on important physiological processes was recently unraveled for K+ nutritional stress (see “K+ uptake from the soil”) where Ca2+ signals lead to increased K+ uptake via CBL proteins and CIPK protein kinases (Xu et al., 2006). Uptake and distribution of Ca2+ Ca2+ uptake from the soil

2+

Signaling roles of Ca

The cytoplasmic Ca2+ concentration is strictly maintained within the nanomolar range, usually 10–200 nM. Transient changes in the cytoplasmic concentration of Ca2+ ([Ca2+]cyt) have been shown to form signaling events in many different organisms including plants. The spatial and temporal variations of these transients, or Ca2+ signatures, are believed to convey stimulus speciﬁcity (Sanders et al., 2002; Dodd et al., 2010). Increased [Ca2+]cyt is generated by inﬂux of Ca2+ from the apoplast and/or endocompartments through Ca2+-permeable ion channels located in the plasma- and endomembranes (Fig. 13.2A). Poststimulus removal of Ca2+ is mediated by H+-coupled antiporters and ATP-fueled Ca2+-pumps (see “Cellular Ca2+ partitioning”). Upon an increase in [Ca2+]cyt, calcium-binding proteins such as calmodulin (CaM), CBL proteins, or calcium-dependent protein kinases (CDPK) can be activated and transduce

Unlike their animal counterparts, plants do not appear to have Ca2+-selective ion channels. Instead, plants use Ca2+-permeable cation channels located at the root system to take up Ca2+ from the soil (Fig. 13.2A). Ca2+permeable cation channels are part of a large, ill-deﬁned group of nonselective cation channels (NSCCs) that vary in selectivity and gating properties. Whereas some NSCCs are relatively selective for Ca2+, most are able to transport monovalent and other divalent cations. NSCCs subsume hyperpolarization-activated (HA-NSCCs), depolarization-activated (DA-NSCCs), and voltage-independent nonselective cation channels (VI-NSCCs), all of which are likely to conduct Ca2+ (Demidchik and Maathuis, 2007) (Fig. 13.2A). The exact role of most of these transporters is unknown, but to evaluate their possible contribution to Ca2+ uptake and distribution, the main ﬁndings associated with each group are discussed below.

A Voltage

Ca

DA NSCC

2+

H+ [Ca2+]cytosol y

GLR

[Ca2+]vacuole 1 – 10 mM

ADP+ Pi

CNGC VI-NSC CC

Vacuole

ATP

HA NSCC

Ca2+ TPC1 voltage? ?

100 nM

MCA ATP

Ca2+

Ca2+

Ca2+ ADP+ Pi Ca 2+

Na +/H+

ER

?

[Ca 2+] 1–10 mM

B

276

a

b

d

e

c

f

ligand

POTASSIUM, CALCIUM, AND MAGNESIUM

HA-NSCCs have been shown to be present at the plasma membranes of root cells, where they are activated at membrane potentials more negative than around −150 mV. This class of NSCC has been shown to be involved in the inﬂux of Ca2+ into the cytosol of root cells (Demidchik et al., 2002). Many HA-NSCCs have a high Ca2+ selectivity, are more prevalent in growing tissues, and are believed to catalyze the large Ca2+ inﬂux that is required for expansion of root hair cells and of cells in the elongation zone (Kiegle et al., 2000; Véry and Davies, 2000; Demidchik and Maathuis, 2007; Demidchik et al., 2007). Trafﬁcking to the membrane of certain types of HA-NSCCs in Arabidopsis thaliana epidermal protoplasts seems to occur in response to reactive oxygen species (Demidchik et al., 2007). Ca2+ uptake and HA-NSCC function may also be modulated by plant annexins, a family of soluble proteins that can integrate into the membrane, depending on cellular pH, Ca2+ concentration, and membrane voltage (Gerke and Moss, 2002; Laohavisit et al., 2010). The presence of DA-NSCCs within the plasma membrane of root epidermal cells has been debated since evidence for both

277

their existence (Thion et al., 1998; Demidchik et al., 2002) and their absence was published (Kiegle et al., 2000). Miedema et al. (2008) provided evidence that the plasma membrane of the Arabidopsis root hair cells has both DA- and HA-NSCCs, with DA-NSCCs showing maximum activation at membrane potentials less negative than around −80 mV. Resting membrane potentials in these cells are typically more negative than −160 mV, but DA-NSCCs could be important for Ca2+ uptake during periods when root hair cells are depolarized (Mortimer et al., 2008). As is the case for HA-NSSCs, the genes for DA-NSCCs are not known except for one type of DA-NSCC, which was identiﬁed as the two-pore channel TPC1 (Peiter et al., 2005). However, in spite of some initial confusion about membrane localization (Kurusu et al., 2005), it is now clear that TPC isoforms are endomembrane channels (Ranf et al., 2008) and thus would not participate in Ca2+ uptake from the soil. VI-NSCCs are thought to be the ligandactivated channels of the CNGC and/or glutamate receptor-like (GLR) families (Demidchik et al., 2004; Demidchik and Maathuis, 2007; Karley and White, 2009) (Fig. 13.2A). Both CNGCs and GLRs are encoded by large gene families consisting 20

Fig. 13.2. Calcium nutrition. (A) Calcium transporters in plant cells. Plasma membrane localized nonselective Ca2+-permeable channels (NSCCs) are believed to be the main pathways for Ca2+ uptake from the soil and likely to subsume hyperpolarization-dependent (HA-NSCC), depolarization-dependent (DA-NSCC), and voltage-independent channels such as cyclic nucleotide-gated channels (CNGCs), glutamate receptor-like channels (GLRs), and mechanosensitive channels (MCAs). Cytosolic Ca2+ inﬂux can also derive from internal compartments such as the vacuole and endoplasmic reticulum (ER), and various ligand-gated channels have been described that may contribute to this process such as cADPR-, NAADP-, IP3-, and IP6-gated channels. The voltage-gated nonselective channel TPC1 has also been suggested to allow vacuolar Ca2+ release. Ca2+ extrusion from the cytosol is carried out by directly energized systems such as Ca2+–ATPases (P2A- and P2B-type) and Ca2+/H+ antiport at the plasma membrane, tonoplast, ER, and Golgi. (B) Calcium (Ca2+) disorders in cultivated plant species: (a) cracking in tomato fruit; (b); blossom end rot of immature tomato fruit; (c) bitter pit apple; (d) gold spot in tomato fruit with calcium oxalate crystals (top); (e) calcium deﬁciency in celery (f) tipburn of lettuce. Reprinted from White and Broadley, (2003), with permission from Oxford Journals.

278

NUTRIENT USE EFFICIENCY IN CROPS

members in Arabidopsis (Lacombe et al., 2001; Ma et al., 2006). Plant CNGCs have been shown to be involved in ﬂuxes of Ca2+, as well as those of other cations such as K+ and Na+ (Kaplan et al., 2007). AtCNGC1 was shown to be expressed mainly in root cells and impacts on Ca2+ uptake and root growth (Ma et al., 2006). Seedlings of Atcngc1 mutants exhibited 6–22% lower Ca2+ contents in their shoots in comparison with wild-type plants (Ma et al., 2006). The GLR isoform AtGLR3.4 is ubiquitously expressed in plant tissues including roots (Meyerhoff et al., 2005). AtGLR3.4 transcript levels are induced by glutamate, touch, and cold; GLR3.4 is therefore believed to be involved in signal transduction during environmental stresses rather than in nutritional Ca2+ uptake (Meyerhoff et al., 2005). Another root cell-expressed GLR is AtGLR3.3. AtGLR3.3 can mediate Ca2+ inﬂux in response to various amino acids such as glutamate and glycine that occur naturally in the rhizosphere (Qi et al., 2006). Whether AtGLR3.3 is involved in the uptake of Ca2+ as a nutrient remains to be established (Qi et al., 2006). Ca2+ translocation and distribution Calcium mobility within plants is relatively restricted and as such it is considered a rather immobile element (Marschner, 1995). Ca2+ is particularly immobile in the phloem, which limits long-distance transport and distribution. The main driving force for calcium translocation is the negative xylem pressure established by transpiration and positive root pressure derived from osmotically active components. In the xylem sap, the Ca2+ concentration can range from nM to over 20 mM, and calcium can be present in ionic form or complexed with organic acids such as malate and citrate (White and Broadley, 2003; Karley and White, 2009). Since calcium translocation to distant tissues

depends primarily on transpiration, phloemfed tissues such as storage organs and seeds are prone to calcium deﬁciency. The lack of calcium movement in the phloem also hampers (re)distribution between tissues, for example, during calcium starvation. This issue is important for agronomy since partitioning of calcium within edible organs such as fruits, seeds, and tubers is often low, and these organs frequently suffer from calcium deﬁciency (White and Broadley, 2003; Busse and Palta, 2006; Karley and White, 2009) (Fig. 13.2B). Radial transport of Ca2+ through root tissues occurs both via apoplastic and via symplastic pathways. Symplastic uptake is likely mediated by Ca2+-permeable channels and Ca2+-ATPases located at the plasma membranes of epidermal, cortical, and endodermal root cells (Hayter and Peterson, 2004). In addition to the Ca2+-permeable channels described earlier (see Ca2+ uptake from the soil”) it has been suggested that mechanosensitive Ca2+ channels (MCAs) might play a role in the symplastic movement of Ca2+ (Yamanaka et al., 2010). The A. thaliana plasma membrane-localized MCA1 (Nakagawa et al., 2007) and MCA2 (Yamanaka et al., 2010) have both been identiﬁed in the root endodermis and stele but not in the cortex or epidermis. Atmca2-null mutants exhibit reduced Ca2+ uptake compared with wild-type plants. AtMCA2 therefore has been proposed to play a role in Ca2+ uptake and translocation (Yamanaka et al., 2010). The relevance of symplastic Ca2+ movement is not entirely clear (Karley and White, 2009) and the extent to which such a mechanism sufﬁces will depend on the root zone. In mature roots, where the Casparian band of the endodermis is fully developed, apoplastic transport is likely to be restricted. However, in less mature root, the apoplastic route from soil to xylem is likely to dominate. It is also noteworthy that no transporter has yet been identiﬁed that is responsible for

POTASSIUM, CALCIUM, AND MAGNESIUM

Ca2+ loading into the xylem. These factors have led some researchers to conclude that in most conditions, apoplastic transport is the main mechanism for calcium translocation to the shoot (White and Broadley, 2003; Karley and White, 2009). Cellular Ca2+ partitioning The cytoplasmic concentration of Ca2+ is the net effect from inﬂux and efﬂux between the cytoplasmic compartment, the apoplast, and cellular endocompartments. The main storage endocompartment for calcium is the vacuole, but ER, Golgi, chloroplasts, mitochondria, and nuclei are also important components. The concentration of Ca2+ in the vacuole is typically in the millimolar range and easily exceeds that of the cytoplasm by a factor of 105 (Pottosin and Schönknecht, 2007). Vacuolar calcium is partly ionic but mostly complexed to anions such as organic acids (e.g., malate, oxalate, citrate). While Ca2+ inﬂux into the cytosol is passive, sequestration in endocompartments occurs against a steep electrochemical gradient and is therefore energy dependent. Accumulation of Ca2+ into vacuole, ER, and Golgi is mediated by high-afﬁnity P-type ATPases and lower afﬁnity Ca2+/H+ exchangers (CAXs) (Fig. 13.2A). The plant P-type Ca2+-ATPases belong to the P2A- and P2Bgroups. The P2A-group consists of four members in Arabidopsis and contains ERlike pumps (ECAs) that are localized to endomembranes (Liang et al., 1997; Axelsen and Palmgren, 2001; Mills et al., 2008; McAinsh and Pittman, 2009). The P2B-group consists of 10 members in Arabidopsis and includes the ACA-type ATPases (Chung et al., 2000; Axelsen and Palmgren, 2001; Martinoia et al., 2007; Qudeimat and Frank, 2009) that occur in both the plasma membrane (Bonza et al., 2000; Schiøtt et al., 2004; George et al., 2008) and endomembranes (Geisler et al., 2000; Anil et al.,

279

2008). Activation of ACAs is induced after the detachment of an N-terminal autoinhibitory domain upon CaM binding (Bækgaard et al., 2005, 2006). In the case of ECAs, both CaM-dependent and CaM-independent mechanisms have been reported, sometimes for the same enzyme: In tomato, two LCA1 splice variants occur which express either at the tonoplast or at the plasma membrane (Ferrol and Bennett, 1996). The tonoplast version is not modulated by CaM in contrast to the plasma membrane version (NavarroAviñó and Bennett, 2003). H+/Ca2+ antiporters are a second system to extrude cytoplasmic Ca2+. In Arabidopsis, six genes encode H+/Ca2+ exchangers (CAX1-6) that transport Ca2+, Mg2+, and Mn2+, from the cytosol to the vacuole in exchange for protons (Hirschi, 1999; Pittman and Hirschi, 2001; Pittman et al., 2002a, 2004; Mei et al., 2007, 2009) (Fig. 13.2A). Like Ca2+-ATPases, CAXs possess an N-terminal autoinhibitory domain (Pittman and Hirschi, 2001; Pittman et al., 2002b, 2004; Mei et al., 2007), and CAX activity is also regulated by phosphorylation (Cheng et al., 2004a), heteromerization (Cheng et al., 2005; Zhao et al., 2009), and CAX interacting proteins (CXIPs) (Cheng et al., 2003, 2004b). Other genes (e.g., CCXs) may also encode cation/Ca2+ exchangers but their role and Ca2+ selectivity has not been characterized (Morris et al., 2008). Ca2+ release from endocompartments is believed to take place through voltage- and ligand-gated Ca2+channels. Alexandre et al. (1990) showed that IP3 could release Ca2+ from isolated red beet vacuoles through IP3activated ion channels. However, very few reports subsequently showed the existence of such channels, and a genetic identiﬁcation was never made. Similarly, a limited number of reports suggested the presence of cyclic adenosine 5′-diphosphoribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP)-dependent channels in beet

280

NUTRIENT USE EFFICIENCY IN CROPS

vacuoles and cauliﬂower ER (Allen et al., 1995; Navazio et al., 2000, 2001; Krinke et al., 2007) (Fig. 13.2A). The physiological role in either case is unclear, and whether these channels exist in other tissues and/or species also remains unknown. In contrast, the slow-activating vacuolar (SV) channel has been extremely well characterized (e.g., Hedrich et al., 1988) and is now known to be encoded by TPC genes (Kurusu et al., 2005; Peiter et al., 2005). TPC activity is sensitive to many cytoplasmic factors such as kinases, 14-3-3 proteins, redox, and cytoplasmic Ca2+ concentration (Pottosin and Schönknecht, 2007) and TPC channels can conduct Ca2+ (Peiter et al., 2005), but large questions still remain regarding their capability for vacuolar Ca2+ release (Ranf et al., 2008). Vacuolar Ca2+ release may also be induced by inositol hexakiphosphate or phytate (InsP6), as was observed in guard cells of Solanum tuberosum and Vicia faba (Lemtiri-Chlieh et al., 2000, 2003) (Fig. 13.2A). Magnesium

hydrated ionic radius and electrostatic interaction with soil particles is therefore weaker than for most other ions. Leaching is particularly problematic in sandy soils. Mg2+ availability to plants is also inﬂuenced by other cations present at the rhizosphere. For example, high levels of K+, NH4+, Ca2+, or Mn2+ all reduce Mg2+ availability as does Al3+ (Rengel, 1990; Kochian et al., 2005; Deng et al., 2006), although the underlying mechanism is still not fully understood. Physiological functions of Mg2+ Magnesium is abundant in plant cells and involved in numerous physiological and metabolic processes. Mg2+ levels in plants vary widely between species, from around 0.03% to 3.2% dry weight (DW), but typically are around 0.5% DW. (Broadley et al., 2003, 2004; Watanabe et al., 2007). Some of the factors that inﬂuence this interspecies variety include the composition and CEC of the cell wall (Broadley et al., 2003, 2004), where much of the plant Mg2+ resides associated with carboxyl groups of pectins and other polysaccharides (Marschner, 1995).

Magnesium occurrence and availability in the soil

Photosynthesis roles of Mg2+

At around 2%, magnesium (Mg) is the eighth most abundant element in the earth’s crust. As with calcium, magnesium is normally found in the form of salts and minerals, the most abundant being dolomite (CaCO3·MgCO3), carnallite (KCl·MgCl2·6H2O), and magnesite (MgO). Plant available magnesium is in its ionic Mg2+ form and will vary with soil organic matter content, pH, and the presence of other cations. As is the case for calcium, soil acidity ultimately reduces Mg2+ availability for plants due to Mg2+ displacement by H+ from mineral exchange sites coupled with the fact that Mg2+ is very prone to leaching. Mg2+ leaching is common because Mg2+ has a large

Mg2+ plays an important role in photosynthesis and for most plants 15–30% of total plant Mg2+ is found in chloroplasts (Marschner, 1995). As the central ion in the chlorophyll molecule, Mg2+ plays a vital role in capturing light energy during photosynthesis. Biosynthesis and degradation of chlorophylls are processes that involve magnesium-dependent chelatases and dechelatases (Marschner, 1995; Langmeier et al., 2006). In addition to the structural role in chlorophyll, Mg2+ is essential for photosynthesis during the light reactions in the stroma, where Mg2+ dissipates the electrical component of the proton motive force generated by light perception. In this manner,

POTASSIUM, CALCIUM, AND MAGNESIUM

Mg2+ ensures the continued inﬂux of protons into the thylakoid lumen. Stromal Mg2+ regulates enzymes involved in the ﬁxation of CO2: Mg2+ acts as a cofactor for ribuloso1,6-bisphosphate carboxylase (RuBP carboxylase), the ﬁrst enzyme of the Calvin cycle, and for fructose-1,6-bisphosphatase, which regulates the partitioning between starch synthesis and export of triosephosphates to the cytosol.

281

In addition, many of the enzymes responsible for formation and break down of nucleic acids such as polymerases, DNases, and RNases require Mg2+. Thus, gene transcription, gene translation, and therefore cell division and protein synthesis all critically depend on adequate levels of cytoplasmic Mg2+.

Uptake and distribution of Mg2+ Mg

2+

as a cofactor

Magnesium acts as a cofactor or promoter for numerous cellular enzymes including RNA polymerases, ATPases, phosphatases, kinases, carboxylases (phosphoenolpyruvate [PEP] carboxylase), and synthases (e.g., glutathione synthase). Many ATP-dependent proteins, including the plasma membrane H+-ATPases and the vacuolar pyrophosphatases (PPi) use magnesium–ATP as a substrate. During the reaction cycle of these enzymes, Mg2+ bridges the oxygen atom of ATP phosphate groups with nitrogen from the protein and as such promotes catalysis. Mg2+ has structural roles in the stabilization of nucleic acids (DNA and RNA) to which it binds. In DNA, Mg2+ attaches to the major groove via hydrogen bonds with adjacent guanines (Robinson et al., 2000), consequently increasing the DNA melting temperature and hence stability. In RNA, Mg2+ has similar roles, but in addition, it binds to negative phosphate groups and thus is essential for establishing and maintaining secondary structure. For example, tRNAs bind multiple Mg2+ ions, which coordinate and stabilize hairpin and other structural conﬁgurations in both the codon and anticodon areas of the tRNA molecule (Misra and Draper, 2000). Some sections of RNA show enzymatic activity, the so-called ribozymes. These RNA-based enzymes essentially are metalloenzymes and require Mg2+ for their catalytic activity.

The identities of transporters involved in Mg2+ uptake, transport, and intracellular trafﬁcking are currently being established. However, little is still known about the exact function of each of these transporters since their molecular and in planta characterization is still scarce. Apart from the nonselective Mg2+-permeable cation channels (Demidchik and Maathuis, 2007), plant Mg2+ transporters belong to two main families: Mg2+/H+ exchangers (MHXs) (Shaul et al., 1999) and magnesium transporters (MGTs)/mitochondrial RNA splicing 2 (MRS2) (Schock et al., 2000; Li et al., 2001; Shaul, 2002; Gardner, 2003; Fig. 13.3A). The plant MGT/MRS2 transporter family consists of 10 very diverse members in A. thaliana and 9 in Oryza sativa (Schock et al., 2000; Li et al., 2001; Shaul, 2002; Gardner, 2003; Drummond et al., 2006; Gebert et al., 2009). MGT/MRS2s are homologous to the bacterial cobalt resistance (CorA) proteins and MRS2s from yeast. Members of the CorA–MRS2–ALR (aluminium resistance) superfamily are ubiquitous and have been identiﬁed both in prokaryotes and eukaryotes. The ﬁrst plant members of this family were identiﬁed independently as MRS2, which was capable of restoring mitochondrial concentrations of Mg2+ in yeast mrs2 mutants (Schock et al., 2000), and MGT for magnesium transport (Li et al., 2001). Here AtMGT/MRS2

A

Mitochondrium

VI-NSCC

Vacuole [Mg2+]vacuole

MGT?

10 – 100 mM MGT3?

MGT6? MGT5

H+

Mg2+

TPC1? VI-NSCC

[Mg2+]cytosol

MGT1

0.5 – 1 mM

MGT2?

MGT10

MGT6-8? MGT7?

MGT?

B

282

ER

Chloroplast

POTASSIUM, CALCIUM, AND MAGNESIUM

Table 13.2. The Arabidopsis thaliana MGT/MRS2

transporter family. Relationship between the MRS2 (Schock et al., 2000) and MGT (Li et al., 2001) nomenclatures and their chromosomal loci (Gebert et al., 2009) MRS2 Annotation

MGT Annotation

Arabidopsis thaliana Chromosomal Locus

MRS2-1 MRS2-2 MRS2-3 MRS2-4 MRS2-5 MRS2-6 MRS2-7 MRS2-8 MRS2-9 MRS2-10 MRS2-11

MGT2 MGT9 MGT4 MGT6 MGT3 MGT5 MGT7 MGT8 — MGT1 MGT10

At1g16010 At5g64560 At3g19640 At3g58970 At2g03620 At4g28580 At5g09690 At5g09720 — At1g80900 At5g22830

members are referred to according to their MGT nomenclature; however, alternative annotations are given in Table 13.2. Members of the AtMGT/MRS2 family have been proposed to be localized to either plasma membrane or to different endocompartments and therefore involved in the uptake, compartmentation, and homeostasis of Mg2+ and other metals. All AtMGT members mediate uptake of Mg2+ when expressed heterologously in yeast, displaying both high and low afﬁnity for the ion (Schock et al., 2000; Li et al., 2001; Chen et al., 2009; Gebert et al., 2009). MGT/MRS2s appear to be ubiquitously expressed within

283

plant organs and tissues, except for AtMGT5, which showed pollen grain-speciﬁc expression (Li et al., 2001; Gebert et al., 2009), AtMGT6, and 10 that were not found in roots, and AtMGT8, which is not found in shoots (Gebert et al., 2009). However, the expression patterns are likely to be developmentally regulated (Gebert et al., 2009). Mg2+ uptake from the soil Mg2+ uptake by roots appears to be passive and is thought to be mediated by nonselective Mg2+-permeable cation channels (Demidchik and Maathuis, 2007) and members of the MGT/MRS2 family (Gebert et al., 2009). Six AtMGT members have been shown to be expressed in root tissues and as such form prime candidates for Mg2+ uptake and translocation mechanisms (Gebert et al., 2009). AtMGT1 (Li et al., 2001), and possibly AtMGT2 (Schock et al., 2000), are localized to the plasma membrane and are thought to mediate high-afﬁnity Mg2+ uptake at the root (Deng et al., 2006) (Fig. 13.3A). AtMGT1 expression in roots occurs both in early developmental stages and in mature plants. In the main root, AtMGT1 is expressed in the meristematic zone and also in vascular tissues within the hair root zone (Gebert et al., 2009). No growth phenotype was observed for the Atmgt1 knockout mutant in comparison to wild-type plants (Gebert et al., 2009). Nevertheless, overexpression of AtMGT1 in

Fig. 13.3. Magnesium nutrition. (A) Magnesium transporters in plant cells. Mg2+ enters the cells through voltageindependent nonselective Mg2+-permeable cation channels (VI-NSCC) and members of the MGT/MRS2 family. Mg2+ transport to different endocompartments is thought to be mediated by members of the MGT/MRS2 family. In addition, delivery of Mg2+ to the vacuole is accomplished through Mg2+/H+ exchangers (MHX) at the tonoplast. Release of Mg2+ from ER and vacuole is thought to be through nonselective Mg2+-permeable cation channels and may include the vacuolar channel TPC1. Mg2+ export from the cell (e.g., in the case of xylem loading) might be performed by MGT/MRS2 isoforms, but this is still speculative. (B) Magnesium (Mg2+) deﬁciency in Frangula alnus manifested by severe chlorosis of the leaves.

284

NUTRIENT USE EFFICIENCY IN CROPS

tobacco led to signiﬁcantly increased tolerance to Mg2+ deﬁciency and also to Al3+ toxicity, especially within the root (Deng et al., 2006). AtMGT7a (a splice variant of AtMGT7) has been proposed to function as a lowafﬁnity Mg2+ uptake system in roots (Mao et al., 2008). Atmgt7 knockout mutants showed severe growth retardation in media with low levels of Mg2+ (less than 50 μM), a defect that was restored by supplementation of the medium with Mg2+ or by complementation of the mutant with AtMGT7. Yet no differences in the tissue levels of Mg2+ or other ions were detected between Atmgt7 knockout and wild-type plants (Gebert et al., 2009). AtMGT7 is expressed to the vascular tissues of mature roots and in the quiescent zone of the radicle (Gebert et al., 2009). However, since AtMGT7 membrane localization is still unclear, its exact involvement in Mg2+ uptake remains speculative (Mao et al., 2008; Gebert et al., 2009) (Fig. 13.3A).

The nature of transporters involved in Mg2+ translocation and distribution are not yet known. AtMHX (a Mg2+/H+ exchanger) has been proposed to be involved in Mg2+ loading into the xylem since its expression is mainly associated with the xylem parenchyma (Shaul et al., 1999). AtMHX was shown to be located to the tonoplast, where it catalyzes the transport of Mg2+ (Shaul et al., 1999), Zn2+, and Cd2+ (Berezin et al., 2008) across the vacuolar membrane in exchange for protons. AtMHX might therefore create a vacuolar pool of Mg2+ within the xylem parenchyma of the roots, from where it is readily available for long-distance transport to other parts of the plant (Shaul et al., 1999; Metzner et al., 2008). Other AtMGT isoforms (2, 3, 4, and 9) also show expression within the vascular tissues of the root (Gebert et al., 2009) and as such may also be involved in Mg2+ translocation. However, membrane localization is unknown for these proteins and their exact role in planta has yet to be identiﬁed.

Mg 2+ translocation and distribution Within plants, magnesium is transported either in ionic form or complexed with organic acids (Buxton et al., 2007). Magnesium is considered one of the most mobile mineral nutrients in plants, both within the xylem and the phloem. Thus, it is easily translocated from roots to shoots and between tissues, for example, from older leaves to young leaves, fruits, seeds, or tubers (Marschner, 1995; Karley and White, 2009). Much of the magnesium that is initially taken up remains within roots, particularly in the xylem parenchyma. This reserve is released into the xylem should aboveground tissues become magnesium deﬁcient and xylem sap magnesium concentrations can easily exceed 1.0 mM in these conditions. In contrast, magnesium levels in the phloem are relatively low (Metzner et al., 2008).

Cellular Mg 2+ partitioning The cytosolic free Mg2+ concentration is around 0.4 mM and is tightly regulated (Karley and White, 2009). Cytosolic magnesium is the net result of inﬂux, efﬂux, and intracellular compartmentation and occurs almost entirely in the form of MgATP (Marschner, 1995) (Fig. 13.3A). In contrast, vacuolar Mg2+ is predominantly in ionic form and can reach concentrations of up to 100 mM in endodermal cells (Stelzer et al., 1990). Little detail is available with regard to movement of magnesium between cellular compartments. Transport of Mg2+ into the vacuole is thought to be mediated by the tonoplast antiporter AtMHX (see “Mg2+ translocation and distribution”) and possibly by some members of the AtMGT/MRS2 family. AtMHX has also been detected in photosynthetic tissues, suggesting a putative

POTASSIUM, CALCIUM, AND MAGNESIUM

role in cellular metal distribution (DavidAssael et al., 2006). AtMGT3 also has been proposed to be vacuolar localized (Whiteman et al., 2008), but its physiological role is not known. Vacuolar Mg2+ release could also be passive through nonselective tonoplast channels such as the slow vacuolar (SV) channel (Pottosin and Schönknecht, 2007) (Fig. 13.3A). The important role of Mg2+ in photosynthesis requires substantial transport into the chloroplast. So far, the only Mg2+ transporter localized to this organelle is the high-afﬁnity Mg2+ transporter AtMGT10 (Li et al., 2001; Drummond et al., 2006). Whether AtMGT10 is localized at the inner or outer chloroplast envelope has not yet been established, but transcript levels are relatively high in aboveground organs and reach their maximum during the light period. However, the in planta function of AtMGT10 remains unclear; no growth phenotype was observed for the Arabidopsis overexpressing AtMGT10 and the chloroplast Mg2+ concentration did not correlate with AtMGT10 expression levels (Drummond et al., 2006) (Fig. 13.3A). Members of the AtMGT/MRS2 family have been shown to contribute to magnesium deposition in mitochondria. Among the MGT isoforms, AtMGT5 was shown to be a mitochondrium-localized Mg2+ transporter, expressed predominantly in anthers and essential for pollen development (Li et al., 2008). In Salmonella bacterial cells, AtMGT5 was able to transport Mg2+ bidirectionally, depending on the ion concentration, but against the electrochemical gradient (Li et al., 2008). Based on such heterologous expression experiments, AtMGT5 may participate both in maintaining the cytosolic Mg2+ and in preventing the overaccumulation of Mg2+ in mitochondria. Thus, AtMGT5 may actively sequester Mg2+ in mitochondria when Mg2+ in the cytosol is low (Li et al., 2008) (Fig. 13.3A). AtMGT9 has also

285

been proposed to function as an endomembrane low-afﬁnity Mg2+ transporter, although the exact cellular localization has not yet been elucidated. AtMGT9 spatial and temporal expression patterns are very similar to those of AtMGT5, being associated with the tapetum layer of anthers and pollen, but also with the vascular tissues (Chen et al., 2009). The biological function of AtMGT9 within vegetative organs has not yet been discovered, but in pollen it is essential for development: Atmtp9 homozygous knockout mutants were nonviable (Chen et al., 2009). AtMGT6 may also be a candidate for mitochondrial localization since it contains a similar predicted mitochondrial leader sequence as AtMGT5 (Li et al., 2001) (Fig. 13.3A). Improving crops for K+, Ca2+, and Mg2+ nutrition The large body of knowledge regarding molecular components that contribute to the uptake and distribution of K+, Ca2+, and Mg2+ facilitates the possibility of improving these functions in crops via either molecular breeding or via genetic modiﬁcation approaches. However, the rationale for crop improvement will vary for K+, Ca2+, and Mg2+. To optimize growth, fertilization of arable land with potash (K+ fertilizer) is common practice, either to offset low levels of ambient K+ or to increase yield gains from nitrogen and phosphorus fertilization. This constitutes a considerable cost to farmers and justiﬁes efforts to improve K+ use efﬁciency. In contrast, Ca2+ deﬁciency seldom occurs under ﬁeld conditions and Ca2+ fertilization, typically in the form of adding lime, is rare. Soil Mg2+ deﬁciency is also relatively rare but can occur locally (Fig. 13.3B), for example, during periods of high precipitation in sandy soils or when excessive K+ fertilization suppresses Mg2+ availability. Increased nutrient use efﬁciency where Ca2+ and Mg2+ are concerned is therefore less of a priority.

286

NUTRIENT USE EFFICIENCY IN CROPS

There are now efforts in several regions to improve K+ use efﬁciency. A good example occurs in rice production, where K+ exhaustion of intensively farmed rice paddies is becoming more problematic. For example, Yang et al. (2004) studied K+ accumulation and distribution, and dry matter accumulation in low K+ ﬁelds for different rice cultivars and reported large inhibition of growth in these conditions. Biotechnological approaches for increasing crop K+ use efﬁciency could focus on several transportrelated aspects. Many plants show a large K+ efﬂux in roots (Szczerba et al., 2009). Reducing this component, possibly via loss of function in major K+ efﬂux pathways such as GORK, may minimize futile recycling of K+. Arabidopsis loss of function gork mutants showed multiple phenotypes (Ivashikina et al., 2001), but properties regarding K+ use efﬁciency were not investigated. Larger K+ uptake capacity could also increase K+ use efﬁciency, especially when ambient K+ provision is low. In addition, this may enhance water use efﬁciency since in many mesophytes osmotic adjustment involves the uptake and translocation of large amounts of K+ (Wang et al., 2002). The root high-afﬁnity transporter AtHAK5 (Gierth et al., 2005) and its orthologs would form an obvious target for such efforts. Overexpression of TPK1 in Arabidopsis had a modest positive effect on K+ homeostasis in K+-deﬁcient conditions, and similar studies are being carried out in rice to test the impact of endogenous channels on growth of crops. Quantitative trait locus (QTL) analyses carried out in Arabidopsis (Harada and Leigh, 2006) and Brassica oleracea (White et al., 2009) also identiﬁed many potential candidates for manipulation including HAK5, AKT1, TPK1, and SKOR and various members of the CNGC family. Since Ca2+ fertilization is rare, there are no big efforts to increase Ca2+ use efﬁciency.

However, some crop varieties cannot supply sufﬁcient Ca2+ to buds, young leaves, or fruits during periods of rapid growth. The insufﬁcient calcium delivery to fast-growing organs and storage tissues can lower crop yield and the nutritional value of seeds and grains. Colorful names such as “black heart” in celery, “blossom end rot” in tomatoes, or “bitter-pit” in apples describe plant diseases that result from poor delivery of Ca2+ (e.g., through drought) (Fig. 13.2B). These negative effects are mainly due to the low mobility of Ca2+ in the vasculature and not related to levels of soil Ca2+ per se. The low mobility of Ca2+ is related to its physiochemical characteristics and its propensity to bind to a large number of organic groups, a phenomenon that would be hard to alter through genetic engineering. Nevertheless, there are current undertakings to increase Ca2+ deposition in edible parts such as cereal grains (White and Broadley, 2009). If successful, such strategies could help alleviate dietary calcium deﬁciencies and reduce the occurrence of calcium-related disease in humans such as osteoporosis. One potentially successful strategy for such biofortiﬁcation would be to increase the seed sink capacity for Ca2+, for example, via overexpression of aleurone Ca2+ transporters to augment passage of Ca2+ from maternal to ﬁlial tissues. In other systems, Ca2+ distribution relies less on the phloem: In potato tubers, calcium loading occurs through transfer from adjacent roots rather than retranslocation via phloem (Busse and Palta, 2006). In such cases, optimizing calcium xylem loading in source roots could lead to higher levels in tubers. Increased sequestration of Ca2+ within cells could further help in developing plants with higher calcium contents. Expression of AtCAX1 lacking the N-terminal autoinhibitory sequence in potato resulted in a threefold increase in the concentration of calcium in tubers in comparison with wild-type

POTASSIUM, CALCIUM, AND MAGNESIUM

plants (Park et al., 2005a). The trait remained stable over successive generations and did not negatively affect potato growth and development (Park et al., 2005a). Expression of a truncated AtCAX1 in tomatoes similarly promoted sequestration of Ca2+ in both roots and fruits (Park et al., 2005b). However, the transgenic tomatoes required soil supplementation with calcium to prevent growth inhibition. Expression of AtCAX4 in tomatoes also led to high calcium contents in fruits, without the need for extra calcium fertilization (Park et al., 2005b). Efforts for magnesium biofortiﬁcation are underway using a combination of conventional and molecular breeding tools. However, attempts to increase the magnesium content in plants through genetic manipulation of Mg2+ transport have been rare (White and Broadley, 2009), particularly since most of the participating proteins are unknown. One area where Mg2+ transport has been altered has been the vacuolar AtMHX; AtMHX overexpression in tobacco showed increased levels of Mg2+ but also of Zn2+ and Cd2+ and generated leaf tip burn and toxicity symptoms (Berezin et al., 2008). Another area of interest is the reduction of Al3+ toxicity. Al3+ inhibits MGT-mediated transport and is one of the main causative factors for magnesium deﬁciency in plants (Rengel, 1990; Kochian et al., 2005). Most of the above examples pertain to altered expression of single genes, but the multigenic nature of the traits to be optimized is likely to require manipulation of several genes. A further complication is the occurrence of large gene families with the risk of functional redundancy and isoform expression in multiple tissues. Simple overexpression using constitutive promoters and/ or loss of function approaches that affect the entire plant may have to be replaced with methods that rely on epitopic expression and gene silencing according to a precise spatiotemporal pattern to avoid unwanted pleiotro-

287

pic effects. Apart from manipulating transport functions per se, regulation of transport may be another viable strategy to improve crops. For example, transcriptional studies have identiﬁed many putative signaling components that are involved in relaying the K+ status (Amtmann and Blatt, 2009) such as transcription factors, kinases, and phosphatases. Similarly, the role of crucial kinases could be modulated to optimize growth in conditions where nutrients are less abundant (Xu et al., 2006). References Alexandre, J., Lassalles, J.P., & Kado, R.T. (1990) Opening of Ca2+ channels in isolated red beet root vacuole membrane by inositol 1,4,5-triphosphate. Nature 343, 567–570. Allen, G.J., Muir, S.R., & Sanders, D. (1995) Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADP-ribose. Science 268, 735–737. Amtmann, A. & Blatt, M.R. (2009) Regulation of macronutrient transport. The New Phytologist 181, 35–52. Anil, V.S., Rajkumar, P., Kumar, P., & Mathew, M.K. (2008) A plant Ca2+ pump, ACA2, relieves salt hypersensitivity in yeast. Modulation of cytosolic calcium signature and activation of adaptive Na+ homeostasis. The Journal of Biological Chemistry 283, 3497–3506. Armengaud, P., Breitling, R., & Amtmann, A. (2004) The potassium-dependent transcriptome of Arabidopsis reveals a prominent role of jasmonic acid in nutrient signaling. Plant Physiology 136, 2556–2576. Axelsen, K.B. & Palmgren, M.G. (2001) Inventory of the superfamily of P-type ion pumps in Arabidopsis. Plant Physiology 126, 696–706. Bækgaard, L., Fuglsang, A.T., & Palmgren, M.G. (2005) Regulation of plant plasma membrane H+and Ca2+-ATPases by terminal domains. Journal of Bioenergetics and Biomembranes 37, 369–374. Bækgaard, L., Luoni, L., De Michelis, M.I., & Palmgren, M.G. (2006) The plant plasma membrane Ca2+ pump ACA8 contains overlapping as well as physically separated autoinhibitory and calmodulin-binding domains. The Journal of Biological Chemistry 281, 1058–1065. Becker, D., Geiger, D., Dunkel, M., et al. (2004) AtTPK4, an Arabidopsis tandem-pore K+ channel,

288

NUTRIENT USE EFFICIENCY IN CROPS

poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner. Proceedings of the National Academy of Sciences of the United States of America 101, 15621–15626. Berezin, I., Mizrachy-Dagry, T., Brook, E., et al. (2008) Overexpression of AtMHX in tobacco causes increased sensitivity to Mg2+, Zn2+, and Cd2+ ions, induction of V-ATPase expression, and a reduction in plant size. Plant Cell Reports 27, 939–949. Bonza, M.C., Morandini, P., Luoni, L., Geisler, M., Palmgren, M.G., & De Michelis, M.I. (2000) AtACA8 encodes a plasma membrane-localized calcium-ATPase of Arabidopsis with a calmodulinbinding domain at the N terminus. Plant Physiology 123, 1495–1505. Broadley, M.R., Bowen, H.C., Cotterill, H.L., et al. (2003) Variation in the shoot calcium content of angiosperms. Journal of Experimental Botany 54, 1431–1446. Broadley, M.R., Bowen, H.C., Cotterill, H.L., et al. (2004) Phylogenetic variation in the shoot mineral concentration of angiosperms. Journal of Experimental Botany 55, 321–336. Busse, J.S. & Palta, J.P. (2006) Investigating the in vivo calcium transport path to developing potato tuber using 45Ca: a new concept in potato tuber calcium nutrition. Physiologia Plantarum 128, 313– 323. Buxton, K.N., Clearwater, M.J., Giles-Hansen, K., Hewett, E.W., & Ferguson, I.B. (2007) Comparison of xylem sap mineral concentrations between kiwifruit shoot types using spittlebugs for nondestructive sampling of sap. Functional Plant Biology 34, 1029–1037. Cellier, F., Conejero, G., Ricaud, L., et al. (2004) Characterization of AtCHX17, a member of the cation/H+ exchangers, CHX family, from Arabidopsis thaliana suggests a role in K+ homeostasis. The Plant Journal 39, 834–846. Chen, J., Li, L.-G., Liu, Z.-H., et al. (2009) Magnesium transporter AtMGT9 is essential for pollen development in Arabidopsis. Cell Research 19, 887–898. Cheng, N.H., Pittman, J.K., Barkla, B.J., Shigaki, T., & Hirschi, K.D. (2003) The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters. The Plant Cell 15, 347–364. Cheng, N.H., Pittman, J.K., Shigaki, T., et al. (2005) Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis. Plant Physiology 138, 2048–2060. Cheng, N.H., Pittman, J.K., Zhu, J.K., & Hirschi, K.D. (2004a) The protein kinase SOS2 activates the

Arabidopsis H+/Ca2+ antiporter CAX1 to integrate calcium transport and salt tolerance. Journal of Biological Chemistry 279, 2922–2926. Cheng, N.H., Liu, J.Z., Nelson, R.S., & Hirschi, K.D. (2004b) Characterization of CXIP4, a novel Arabidopsis protein that activates the H+/Ca2+ antiporter, CAX1. FEBS Letters 559, 99–106. Chung, W.S., Lee, S.H., Kim, J.C., et al. (2000) Identiﬁcation of a calmodulin-regulated soybean Ca2+-ATPase (SCA1) that is located in the plasma membrane. The Plant Cell 12, 1393–1407. Czempinski, K., Frachisse, J.M., Maurel, C., BarbierBrygoo, H., & Mueller-Roeber, B. (2002) Vacuolar membrane localization of the Arabidopsis “twopore” K+ channel KCO1. The Plant Journal 29, 809–820. David-Assael, O., Berezin, I., Shoshani-Knaani, N., et al. (2006) AtMHX is an auxin and ABA-regulated transporter whose expression pattern suggests a role in ion homeostasis in tissues with photosynthetic potential. Functional Plant Biology 33, 661–672. Demidchik, V. & Maathuis, F.J.M. (2007) Physiological roles of nonselective cation channels in plants: from salt stress to signalling and development. The New Phytologist 175, 387–404. Demidchik, V., Bowen, H.C., Maathuis, F.J.M., et al. (2002) Arabidopsis thaliana root nonselective cation channels mediate calcium uptake and are involved in growth. The Plant Journal 32, 799–808. Demidchik, V., Essah, P.A., & Tester, M. (2004) Glutamate activates cation currents in the plasma membrane of Arabidopsis root cells. Planta 219, 167–175. Demidchik, V., Shabala, S., & Davies, J.M. (2007) Spatial variation in H2O2 response of Arabidopsis thaliana root epidermal Ca2+ ﬂux and plasma membrane Ca2+ channels. The Plant Journal 49, 377–386. Deng, W., Luo, K., Li, D., et al. (2006) Overexpression of an Arabidopsis magnesium transport gene, AtMGT1, in Nicotiana benthamiana confers Al tolerance. Journal of Experimental Botany 57, 4235–4243. Dodd, A.N., Kudla, J., & Sanders, D. (2010) The language of calcium signalling. Annual Review of Plant Biology 61, 593–620. Drummond, R.S.M., Tutone, A., Li, Y.-C., & Gardner, R.C. (2006) A putative magnesium transporter AtMRS2-11 is localized to the plant chloroplast envelope membrane system. Plant Science 170, 78–89. Elumalai, R.P., Nagpal, P., & Reed, J.W. (2002) A mutation in the Arabidopsis KT2/KUP2 potassium trans-

POTASSIUM, CALCIUM, AND MAGNESIUM

porter gene affects shoot cell expansion. The Plant Cell 14, 119–131. Ferrol, N. & Bennett, A.B. (1996) A single gene may encode differentially localized Ca2+-ATPases in tomato. The Plant Cell 8, 1159–1169. Fromm, J. & Lautner, S. (2007) Electrical signals and their physiological signiﬁcance in plants. Plant Cell and Environment 3, 249–257. Gardner, R.C. (2003) Genes for magnesium transport. Current Opinion Plant Biology 6, 263–267. Gaymard, F., Pilot, G., Lacombe, B., et al. (1998) Identiﬁcation and disruption of a plant Shaker-like outward channel involved in K+ release into the xylem sap. Cell 94, 647–655. Gebert, M., Meschenmoser, K., Svidová, S., et al. (2009) A root-expressed magnesium transporter of the MRS2/MGT gene family in Arabidopsis thaliana allows for growth in low-Mg2+ environments. The Plant Cell 21, 4018–4030. Geiger, D., Becker, D., Vosloh, D., et al. (2009) Heteromeric AtKC1.AKT1 channels in Arabidopsis roots facilitate growth under K+-limiting conditions. Journal of Biological Chemistry 284, 21288–21295. Geisler, M., Frangne, N., Gomes, E., Martinoia, E., & Palmgren, M.G. (2000) The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast. Plant Physiology 124, 1814–1827. George, L., Romanowsky, S.M., Harper, J.F., & Sharrock, R.A. (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development and inﬂorescence architecture in Arabidopsis. Plant Physiology 146, 716–728. Gerke, V. & Moss, S.E. (2002) Annexins: from structure to function. Physiological Reviews 82, 331–371. Gierth, M., Maser, P., & Schroeder, J.I. (2005) The potassium transporter AtHAK5 functions in K+ deprivation-induced high-afﬁnity K+ uptake and AKT1 K+ channel contribution to K+ uptake kinetics in Arabidopsis roots. Plant Physiology 137, 1105–1114. Gobert, A., Isayenkov, S., Voelker, C., Czempinski, K., & Maathuis, F.J.M. (2007) The two-pore channel TPK1 gene encodes the vacuolar K+ conductance and plays a role in K+ homeostasis. Proceedings of the National Academy of Sciences of the United States of America 104, 10726–10731. Harada, H. & Leigh, R.A. (2006) Genetic mapping of natural variation in potassium concentrations in shoots of Arabidopsis thaliana. Journal of Experimental Botany 57, 953–960. Hayter, M.L. & Peterson, C.A. (2004) Can Ca2+ ﬂuxes to the root xylem be sustained by Ca2+-ATPases in

289

exodermal and endodermal plasma membranes? Plant Physiology 136, 4318–4325. Hedrich, R. & Neher, E. (1987) Cytoplasmic calcium regulates voltage-dependent ion channels in plant vacuoles. Nature 329, 833–835. Hedrich, R., Barbier-Brygoo, H., Felle, H.H., et al. (1988) General mechanisms for solute transport across the tonoplast of plant vacuoles: a patch clamp survey of ion channels and proton pumps. Botanica Acta 101, 7–13. Hetherington, A.M. & Brownlee, C. (2004) The generation of Ca2+ signals in plants. Annual Review of Plant Biology 55, 401–427. Hirsch, R.E., Lewis, B.D., Spalding, E.P., & Sussman, M.R. (1998) A role for the AKT1 potassium channel in plant nutrition. Science 280, 918–921. Hirschi, K.D. (1999) Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity. The Plant Cell 11, 2113–2122. Ishikawa, H., Yamamura, K., Furukoshi, M., Ohta, E., & Sakata, M. (1984) Effects of K+(Rb-86) transport and the net H+ efﬂux on electrical-properties along bean (Phaseolus-mungo) roots. Plant and Cell Physiology 25, 1045–1051. Ivashikina, N., Becker, D., Ache, P., Meyerhoff, O., Felle, H.H., & Hedrich, R. (2001) K+ channel proﬁle and electrical properties of Arabidopsis root hairs. FEBS Letters 508, 463–469. Jaquinod, M., Villiers, F., Kieffer-Jaquinod, S., et al. (2007) A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture. Molecular and Cellular Proteomics 6, 394–412. Kachmar, J.F. & Boyer, P.D. (1953) Kinetic analysis of enzyme reactions. II. The potassium activation and calcium inhibition of pyruvic phosphotransferase. Journal of Biological Chemistry 200, 669–682. Kaplan, B., Sherman, T., & Fromm, H. (2007) Cyclic nucleotide-gated channels in plants. FEBS Letters 581, 2237–2246. Karley, A.J. & White, P.J. (2009) Moving cationic minerals to edible tissues: potassium, magnesium, calcium. Current Opinion in Plant Biology 12, 291–298. Karley, A.J., Leigh, R.A., & Sanders, D. (2000) Differential ion accumulation and ion ﬂuxes in the mesophyll and epidermis of barley. Plant Physiology 122, 835–844. Kiegle, E., Gilliham, M., Haseloff, J., & Tester, M. (2000) Hyperpolarization-activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots. The Plant Journal 21, 225–229. Kim, E.J., Kwak, J.M., Uozumi, N., & Schroeder, J.I. (1998) AtKUP1: an Arabidopsis gene encoding

290

NUTRIENT USE EFFICIENCY IN CROPS

high-afﬁnity potassium transport activity. The Plant Cell 10, 51–62. Kinraide, T.B., Newman, I.A., & Etherton, B.A. (1984) A quantitative simulation-model for H+-amino acid cotransport to interpret the effects of amino-acids on membrane-potential and extracellular pH. Plant Physiology 76, 806–813. Kochian, L.V. & Lucas, W. (1983) Potassium transport in corn roots. Plant Physiology 73, 208. Kochian, L.V., Piñeros, M.A., & Hoekenga, O.A. (2005) The physiology, genetics and molecular biology of plant aluminium resistance and toxicity. Plant and Soil 274, 175–195. Krinke, O., Novotná, Z., Valentová, O., & Martinec, J. (2007) Inositol trisphosphate receptor in higher plants: is it real? Journal of Experimental Botany 58, 361–376. Kurusu, T., Yagala, T., Miyao, A., Hirochika, H., & Kuchitsu, K. (2005) Identiﬁcation of a putative voltage-gated Ca2+ channel as a key regulator of elicitor-induced hypersensitive cell death and mitogen-activated protein kinase activation in rice. The Plant Journal 42, 798–809. Lacombe, B., Becker, D., Hedrich, R., et al. (2001) The identity of plant glutamate receptors. Science 292, 1486–1487. Lagarde, D., Basset, M., Lepetit, M., et al. (1996) Tissue-speciﬁc expression of Arabidopsis AKT1 gene is consistent with a role in K+ nutrition. The Plant Journal 9, 195–203. Langmeier, M., Ginsburg, S., & Matile, P. (2006) Chlorophyll breakdown in senescent leaves: demonstration of Mg-dechelatase activity. Physiologia Plantarum 89, 347–353. Laohavisit, A., Brown, A.T., Cicuta, P., & Davies, J.M. (2010) Annexins—components of the calcium and reactive oxygen signaling network. Plant Physiology 152, 1824–1829. Lemtiri-Chlieh, F., MacRobbie, E.A.C., & Brearley, C.A. (2000) Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells. Proceedings of the National Academy of Sciences of the United States of America 97, 8687–8692. Lemtiri-Chlieh, F., MacRobbie, E.A.C., Webb, A.A.R., et al. (2003) Inositol hexakisphosphate mobilizes an endomembrane store of calcium in guard cells. Proceedings of the National Academy of Sciences of the United States of America 100, 10091–10095. Li, L.G., Tutone, A.F., Drummond, R.S., Gardner, R.C., & Luan, S. (2001) A novel family of magnesium transport genes in Arabidopsis. The Plant Cell 13, 2761–2775. Li, X.L., Borsics, T., Harrington, H.M., & Christopher, D.A. (2005) Arabidopsis AtCNGC10 rescues potas-

sium channel mutants of E. coli, yeast and Arabidopsis and is regulated by calcium/calmodulin and cyclic GMP in E. coli. Functional Plant Biology 32, 643–653. Li, L.G., Sokolov, L.N., Yang, Y.H., et al. (2008) A mitochondrial magnesium transporter functions in Arabidopsis pollen development. Molecular Plant 1, 675–685. Liang, F., Cunningham, K.W., Harper, J.F., & Sze, H. (1997) ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulumtype Ca2+-ATPase in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 94, 8579–8584. Ma, W., Ali, R., & Berkowitz, G.A. (2006) Characterization of plant phenotypes associated with loss-of-function of AtCNGC1, a plant cyclic nucleotide gated cation channel. Plant Physiology and Biochemistry 44, 494–505. Maathuis, F.J.M. & Amtmann, A. (1999) K+ nutrition and Na+ toxicity: the basis of cellular K+/Na+ ratios. Annals Botany 84, 123–133. Maathuis, F.J.M. & Sanders, D. (1993) Energization of potassium uptake in Arabidopsis thaliana. Planta 191, 302–307. Maathuis, F.J.M. & Sanders, D. (1994) Mechanism of high afﬁnity potassium uptake in roots of Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 91, 9272–9276. Maathuis, F.J.M. & Sanders, D. (1995) Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana. Planta 197, 456–464. Maathuis, F.J.M., Verlin, D., Smith, F.A., Sanders, D., Fernandez, J.A., & Walker, N.A. (1996) The physiological relevance of Na+-coupled K+-transport. Plant Physiology 112, 1609–1616. Maathuis, F.J.M., Ichida, A.M., Sanders, D., & Schroeder, J.I. (1997) Roles of higher plant K+ channels. Plant Physiology 114, 1141–1149. Mahouachi, J., Socorro, A.R., & Talon, M. (2006) Responses of papaya seedlings (Carica papaya L.) to water stress and re-hydration: growth, photosynthesis and mineral nutrient imbalance. Plant and Soil 281, 137–146. Mao, D.D., Tian, L.F., Li, L.-G., et al. (2008) AtMGT7: an Arabidopsis gene encoding a low-afﬁnity magnesium transporter. Journal of Integrative Plant Biology 50, 1530–1538. Marschner, H. (1995) Mineral Nutrition of Higher Plants, 2nd ed. Academic Press, London, UK. Marschner, H., Kirkby, E.A., & Engels, C. (1997) Importance of cycling and recycling of mineral nutrients within plants for growth and development. Botanica Acta 110, 265–273.

POTASSIUM, CALCIUM, AND MAGNESIUM

Marten, I., Hoth, S., Deeken, R., et al. (1999) AKT3, a phloem-localized K+ channel, is blocked by protons. Proceedings of the National Academy of Sciences of the United States of America 96, 7581–7586. Martinoia, E., Maeshima, M., & Neuhaus, H.E. (2007) Vacuolar transporters and their essential role in plant metabolism. Journal of Experimental Botany 58, 83–102. McAinsh, M.R. & Pittman, J.K. (2009) Shaping the calcium signature. The New Phytologist 181, 275–294. Mei, H., Cheng, N.H., Zhao, J., et al. (2009) Root development under metal stress in Arabidopsis thaliana requires the H+/cation antiporter CAX4. The New Phytologist 183, 95–105. Mei, H., Zhao, J., Pittman, J.K., Lachmansingh, J., Park, S., & Hirschi, K.D. (2007) In planta regulation of the Arabidopsis Ca2+/H+ antiporter CAX1. Journal of Experimental Botany 58, 3419–3427. Mengel, K. & Kirkby, E.A. (2001) Principles of Plant Nutrition. Kluwer, Dordrecht. Metzner, R., Schneider, H.U., Breuer, U., & Schroeder, W.H. (2008) Imaging nutrient distribution in plant tissues using time-of-ﬂight secondary ion mass spectrometry and scanning electron microscopy. Plant Physiology 147(4), 1774–1787. Meyerhoff, O., Muller, K., Roelfsema, M.R., et al. (2005) AtGLR3.4, a glutamate receptor channellike gene is sensitive to touch and cold. Planta 222, 418–427. Miedema, H., Demidchik, V., Véry, A.-A., Bothwell, J.H.F., Brownlee, C., & Davies, J.M. (2008) Two voltage-dependent calcium channels co-exist in the apical plasma membrane of Arabidopsis thaliana root hairs. The New Phytologist 179, 378–385. Mills, R.F., Doherty, M.L., López-Marqués, R.L., et al. (2008) ECA3, a Golgi-localized P2A-Type ATPase, plays a crucial role in manganese nutrition in Arabidopsis. Plant Physiology 146, 116–128. Misra, V.K. & Draper, D.E. (2000) Mg(2+) binding to tRNA revisited: the nonlinear Poisson-Boltzmann model. Journal of Molecular Biology 299, 813–825. Morris, J., Tian, H., Park, S., Sreevidya, C.S., Ward, J.M., & Hirschi, K.D. (2008) AtCCX3 is an Arabidopsis endomembrane H+-dependent K+ transporter. Plant Physiology 148, 1474–1486. Mortimer, J.C., Laohavisit, A., Miedema, H., & Davies, J.M. (2008) Voltage, reactive oxygen species and the inﬂux of calcium. Plant Signaling and Behavior 3, 698–699. Munns, R. & Tester, M. (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651–681.

291

Nakagawa, Y., Katagiri, T., Shinozaki, K., et al. (2007) Arabidopsis plasma membrane protein crucial for Ca2+ inﬂux and touch sensing in roots. Proceedings of the National Academy of Sciences of the United States of America 104, 3639–3644. Navarro-Aviñó, J.P. & Bennett, A.B. (2003) Do untranslated introns control Ca2+-ATPase isoform dependence on CaM, found in TN and PM? Biochemical and Biophysical Research Communications 312, 1377–1382. Navazio, L., Bewell, M.A., Siddiqua, A., Dickinson, G.D., Galione, A., & Sanders, D. (2000) Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate. Proceedings of the National Academy of Sciences of the United States of America 97, 8693–8698. Navazio, L., Mariani, P., & Sanders, D. (2001) Mobilization of Ca2+ by cyclic ADP-ribose from the endoplasmic reticulum of cauliﬂower ﬂorets. Plant Physiology 125, 2129–2138. Nilima, K. & Vinay, S. (2008) V-PPase in plants: an overview. Research Journal of Biotechnology 3, 57–63. Nitsos, R.E. & Evans, H.J. (1969) Effects of univalent cations on activity of particulate starch synthetase. Plant Physiology 44, 1260–1269. Ozaki, T., Ambe, S., Abe, T., & Francis, A.J. (2005) Competitive inhibition and selectivity enhancement by Ca in the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, Hf) by carrot (Daucus carota cv. U.S. harumakigosun). Biological Trace Element Research 103, 69–82. Pardo, J.M., Cubero, B., Leidi, E.O., & Quintero, F.J. (2006) Alkali cation exchangers: roles in cellular homeostasis and stress tolerance. Journal of Experimental Botany 57, 1181–1199. Park, S., Kang, T.-S., Kim, C.-K., et al. (2005a) Genetic manipulation for enhancing calcium content in potato tuber. Journal of Agriculture and Food Chemistry 53, 5598–5603. Park, S., Cheng, N.H., Pittman, J.K., et al. (2005b) Increased calcium levels and prolonged shelf life in tomatoes expressing Arabidopsis H+/Ca2+ transporters. Plant Physiology 139, 1194–1206. Peiter, E., Maathuis, F.J.M., Mills, L.N., et al. (2005) The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement. Nature 434, 404–408. Pittman, J.K. & Hirschi, K.D. (2001) Regulation of CAX1, an Arabidopsis Ca2+/H+ antiporter. Identiﬁcation of an N-terminal autoinhibitory domain. Plant Physiology 127, 1020–1029.

292

NUTRIENT USE EFFICIENCY IN CROPS

Pittman, J.K., Shigaki, T., Cheng, N.H., & Hirschi, K.D. (2002a) Mechanism of N-terminal autoinhibition in the Arabidopsis Ca2+/H+ antiporter CAX1. Journal of Biological Chemistry 277, 26452–26459. Pittman, J.K., Sreevidya, C.S., Shigaki, T., UeokaNakanishi, H., & Hirschi, K.D. (2002b) Distinct N-terminal regulatory domains of Ca2+/H+ antiporters. Plant Physiology 130, 1054–1062. Pittman, J.K., Shigaki, T., Marshall, J.L., Morris, J.L., Cheng, N.H., & Hirschi, K.D. (2004) Functional and regulatory analysis of the Arabidopsis thaliana CAX2 cation transporter. Plant Molecular Biology 56, 959–971. Pottosin, I.I. & Martinez-Estevez, M. (2003) Regulation of the fast vacuolar channel by cytosolic and vacuolar potassium. Biophysical Journal 84, 977–986. Pottosin, I.I. & Schönknecht, G. (2007) Vacuolar calcium channels. Journal of Experimental Botany 58, 1559–1569. Qi, Z., Stephens, N.R., & Spalding, E.P. (2006) Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist proﬁle. Plant Physiology 142, 963–971. Qudeimat, E. & Frank, W. (2009) Ca2+ signatures: the role of Ca2+-ATPases. Plant Signaling and Behavior 4, 350–352. Ranf, S., Wünnenberg, P., Lee, J., et al. (2008) Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses. The Plant Journal 53, 287–299. Reintanz, B., Szyroki, A., Ivashikina, N., et al. (2002) AtKC1, a silent Arabidopsis potassium channel alpha-subunit modulates root hair K+ inﬂux. Proceedings of the National Academy of Sciences of the United States of America 99, 4079–4084. Rengel, Z. (1990) Competitive Al3+ inhibition of net Mg2+ uptake by intact Lolium multiﬂorum roots. II. Plant age effects. Plant Physiology 93, 1261–1267. Roberts, S.K. & Tester, M. (1995) Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele. The Plant Journal 8, 811–825. Robinson, H., Gao, Y.G., Sanishvili, R., Joachimiak, A., & Wang, A.H.J. (2000) Hexahydrated magnesium ions bind in the deep major groove and at the outer mouth of A-form nucleic acid duplexes. Nucleic Acids Research 28, 1760–1766. Rodriguez-Navarro, A. & Rubio, F. (2006) Highafﬁnity potassium and sodium transport systems in plants. Journal of Experimental Botany 57, 1149–1160. Rodriguez-Rosales, M.P., Jiang, X.Y., Galvez, F.J., Aranda, M.N., Cubero, B., & Venema, K. (2008) Overexpression of the tomato K+/H+ antiporter

LeNHX2 confers salt tolerance by improving potassium compartmentalization. The New Phytologist 179, 366–377. Saidi, Y., Finka, A., Muriset, M., et al. (2009) The heat shock response in moss plants is regulated by speciﬁc calcium-permeable channels in the plasma membrane. The Plant Cell 21, 2829–2843. Sanders, D., Pelloux, J., Brownlee, C., & Harper, J.F. (2002) Calcium at the crossroads of signaling. The Plant Cell 14, S401–S417. Santa-Maria, G., Rubio, F., Dubcovsky, J., & RodriguezNavarro, A. (1997) The HAK1 gene of barley is a member of a large gene family and encodes a highafﬁnity potassium transporter. The Plant Cell 9, 2281–2289. Schiøtt, M., Romanowsky, S.M., Bækgaard, L., Jakobsen, M.K., Palmgren, M.G., & Harper, J.F. (2004) A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization. Proceedings of the National Academy of Sciences of the United States of America 101, 9502–9507. Schock, I., Gregan, J., Steinhauser, S., Schweyen, R., Brennicke, A., & Knoop, V. (2000) A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant. The Plant Journal 24, 489–501. Shabala, S. & Cuin, T.A. (2008) Potassium transport and plant salt tolerance. Physiologia Plantarum 133, 651–669. Shaul, O. (2002) Magnesium transport and function in plants, the tip of the iceberg. BioMetals 15, 309–323. Shaul, O., Hilgemann, D.W., de-Almeida-Engler, J., Montagu, M.V., Inzé, D., & Galili, D. (1999) Cloning and characterization of a novel Mg2+/H+ exchanger. EMBO Journal 18, 3973–3980. Shin, R. & Schachtman, D.P. (2004) Hydrogen peroxide mediates plant root cell response to nutrient deprivation. Proceedings of the National Academy of Sciences of the United States of America 101, 8827–8832. Song, C.P., Guo, Y., Qiu, Q.S., et al. (2004) A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 101, 10211–10216. Stelzer, R., Lehmann, H., Krammer, D., & Lütge, U. (1990) X-ray microprobe analysis of vacuoles of spruce needle mesophyll, endodermis and transfusion parenchyma cells at different seasons of the year. Botanica Acta 103, 415–423. Szczerba, M.W., Britto, D.T., & Kronzucker, H.J. (2009) K+ transport in plants: physiology and

POTASSIUM, CALCIUM, AND MAGNESIUM

molecular biology. Journal of Plant Physiology 166, 447–466. Sze, H., Padmanaban, S., Cellier, F., et al. (2004) Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K+ homeostasis in pollen development. Plant Physiology 136, 2532–2547. Thion, L., Mazars, C., Nacry, P., et al. (1998) Plasma membrane depolarization-activated calcium channels, stimulated by microtubule-depolymerizing drugs in wild-type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half-life in ton 2 mutant cells affected in the organization of cortical microtubules. The Plant Journal 13, 603–610. Véry, A.A. & Davies, J.M. (2000) Hyperpolarizationactivated calcium channels at the tip of Arabidopsis root hairs. Proceedings of the National Academy of Sciences of the United States of America 97, 9801–9806. Vicente-Agullo, F., Rigas, S., Desbrosses, G., Dolan, L., Hatzopoulos, P., & Grabov, A. (2004) Potassium carrier TRH1 is required for auxin transport in Arabidopsis roots. The Plant Journal 40, 523–535. Voelker, C., Schmidt, D., Mueller-Roeber, B., & Czempinski, K. (2006) Members of the Arabidopsis AtTPK/KCO family form homomeric vacuolar channels in planta. The Plant Journal 48, 296–306. Walker, D.J., Leigh, R.A., & Miller, A.J. (1996) Potassium homeostasis in vacuolate plant cells. Proceeding of the National Academy of Science of United States of America 93, 10510–10514. Wang, S.M., Zheng, W.J., Ren, J.Z., & Zhang, C.L. (2002) Selectivity of various types of salt-resistant plants for K+ over Na+. Journal of Arid Environments 52, 457–472. Watanabe, T., Broadley, M.R., Jansen, S., et al. (2007) Evolutionary control of shoot element composition in plants. The New Phytologist 174, 516–523. Wegner, L.H. & De Boer, A.H. (1999) Activation kinetics of the K+ outward rectifying conductance (KORC) in xylem parenchyma cells from barley

293

roots. Journal of Membrane Biology 170, 103–119. White, P.J. & Broadley, M.R. (2003) Calcium in plants. Annals of Botany 92, 487–511. White, P.J. & Broadley, M.R. (2009) Biofortiﬁcation of crops with seven mineral elements often lacking in human diets—iron, zinc, copper, calcium, magnesium, selenium and iodine. The New Phytologist 182, 49–84. White, P.J., Hammond, J.P., King, G.J., et al. (2010) Genetic analysis of potassium use efﬁciency in Brassica oleracea. Annals of Botany 105, 1199–1210. Whiteman, S.A., Serazetdinova, L., Jones, A.M.E., et al. (2008) Identiﬁcation of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana. Proteomics 8, 3536–3547. Wulff, F., Schulz, V., Jungk, A., & Claasen, N. (1998) Potassium fertilization on sandy soils in relation to soil test, crop yield and K-leaching. Zeitschrift fur Pﬂanzenernahrung und Bodenkunde 161, 591–599. Xu, J., Li, H.D., Chen, L.Q., et al. (2006) A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell 125, 1347–1360. Yamanaka, T., Nakagawa, Y., Mori, K., et al. (2010) MCA1 and MCA2 that mediate Ca2+ uptake have distinct and overlapping roles in Arabidopsis. Plant Physiology 152, 1284–1296. Yang, X.E., Liu, J.X., Wang, W.M., Ye, Z.Q., & Luo, A.C. (2004) Potassium internal use efﬁciency relative to growth vigor, potassium distribution, and carbohydrate allocation in rice genotypes. Journal of Plant Nutrition 27, 837–852. Zhao, J., Shigaki, T., Mei, H., Guo, Y.Q., Cheng, N.H., & Hirschi, K.D. (2009) Interaction between Arabidopsis Ca2+/H+ Exchangers CAX1 and CAX3. The Journal of Biological Chemistry 284, 4605–4615.

Chapter 14

Sulfur Nutrition in Crop Plants Luit J. De Kok, Ineke Stulen, and Malcolm J. Hawkesford

Abstract Sulfur is an essential element for plant growth, and together with nitrogen it is necessary for the synthesis of amino acids, proteins, and various other cellular components, including thiol compounds and so-called secondary sulfur compounds, which play an important role in the protection of plants against stress and pests. Sulfur fertilization is often necessary to obtain optimal crop yield and quality. The timing and form of sulfur fertilizer is of great importance since the rate of remobilization of sulfur from older to young tissue can be low in some crops. Sulfate moving through the soil toward the surface of root by mass/bulk ﬂow and taken up with high afﬁnity by the roots (apparent Km < 10 μM), is the primary sulfur source for plants. The sulfate is reduced in the chloroplasts/plastids prior to its assimilation into organic sulfur compounds. The uptake of sulfate by the root is a primary controlling factor in plant sulfur nutrition and is adjusted to the sulfur demand for growth. The uptake and distribution of

sulfate in the plants is mediated by distinct sulfate transporters. Both the expression and activity of the sulfate transporters and adenosine 5′ phosphosulfate (APS) reductase, the key enzyme of the sulfate reduction pathway, are modulated by the sulfur nutritional status. The signal transduction pathway in the regulation of the sulfate transporters is not well elucidated. It is uncertain to what extent the measurement of changes in concentrations of potential signal compounds, determined at the whole-plant organ level, provides sufﬁcient insight into the actual regulatory control of the sulfate uptake at the cellular level. Furthermore, a key unresolved issue is the signal transduction pathway in the crosstalk between the sulfate reduction pathway in the chloroplasts/plastids and the transcription of sulfate transporters/sulfate-reducing enzymes in the nucleus. With the recent appearance of sulfur deﬁciency in agricultural systems and the awareness of the importance in quality and stress resistance, possibilities for targeting improvements in sulfur use efﬁciency may be required and are discussed.

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 295

296

NUTRIENT USE EFFICIENCY IN CROPS

Introduction Sulfur is an essential macronutrient for plants, which content varies between crop species and ranges from 0.1% to 2 % of the dry weight (De Kok et al., 2002a; Haneklaus et al., 2007b; Zhao et al., 2008). In many areas of the world, sulfur deﬁciency of farmland has been demonstrated and sulfur fertilization is needed to optimize crop yield and quality (De Kok et al., 2002a,b; Haneklaus et al., 2003, 2007a,b; Zhao et al., 2008). Sulfate taken up by the roots is the primary sulfur source for plants, but it needs to be reduced prior to its metabolism into organic sulfur compounds. In polluted areas, dry and wet deposition of atmospheric sulfur gases (i.e., SO2, H2S) may also substantially contribute to plant sulfur nutrition, and may affect the uptake of sulfate by the roots (De Kok et al., 2002a,b, 2005, 2007, 2009). Sulfur and nitrogen metabolism are strongly interrelated since in the majority of crop plants the predominant proportion of the organic sulfur (>90%) is present as cysteine and methionine residues in proteins (De Kok et al., 2002a; Haneklaus et al., 2007a,b; Zhao et al., 2008). On a molar basis, the organic N : S ratio of the vegetative part of crop plants is generally around 20:1. The sulfur residues of cysteine and methionine in proteins are of great signiﬁcance for their structure, conformation, and physiological functioning. For instance, the thiol group of the cysteine residue in proteins is involved in redox/disulﬁde exchange reactions, which have signiﬁcance in DNA and protein synthesis and activation/deactivation of enzymes (De Kok et al., 2002a; Haneklaus et al., 2007b; Zhao et al., 2008). Protein thiols are also imported in substrate binding and in metal-sulfur clusters. Seed storage proteins may contain high levels of cysteine and methionine residues, which are utilized as a sulfur source during germination. Plants contain a wide variety of other organic sulfur compounds, for example, glu-

tathione, sulfolipids, and secondary sulfur metabolites, which are of great signiﬁcance in plant functioning and its tolerance to biotic and abiotic stresses, and protection against herbivory (De Kok et al., 2002a; Bloem et al., 2007; Haneklaus et al., 2007a,b; Hell and Kruse, 2007; Zhao et al., 2008). Glutathione, the predominant water-soluble nonprotein thiol compound present in plant tissue, functions in the reduction of sulfate as an electron donor and metabolic regulator, as the transported form of reduced sulfur, as the precursor of metal-binding phytochelatins, and in the enzymatic detoxiﬁcation of xenobiotics via conjugation, catalyzed by glutathione S-transferases (North and Kopriva, 2007; Sirko and Gotor, 2007). Moreover, via thiol/disulﬁde exchange, glutathione is involved in the enzymatic and direct detoxiﬁcation of reactive oxygen species (De Kok and Stulen, 1993; North and Kopriva, 2007). Sulfoquinovosyl diacylglycerol is a prominent anionic lipid in chloroplast membrane and its content may account for 3–8% of the total organic sulfur content (Stulen and De Kok 1993; Benning, 1998; Benning et al., 2008). Some plant species contain high levels of secondary sulfur metabolites, for example, glucosinolates in Brassica and alliins in Allium, which may have signiﬁcance in plant–herbivore and plant–pathogen interactions (De Kok et al., 2002a; Haneklaus et al., 2007b; Zhao et al., 2008). Humans and animals rely on plants as source of reduced sulfur, and balanced sulfur nutrition of crops is essential for optimal food quality and security (Zhao et al., 1999, 2008). Crop sulfur status combined with levels of sulfur fertilizer will also have effects on selenate and molybdate accumulation (Shinmachi et al., 2010). Crop sulfur nutrition Adequate and balanced sulfur nutrition is essential for optimal crop yield and quality.

SULFUR NUTRITION IN CROP PLANTS

The overall sulfur demand for optimal crop yield and quality varies strongly between species and ranges from 0.3 kg S t−1(sugarcane) to 17 kg S t−1 (oilseed rape, mustard). The total uptake of sulfur by crops, the crop sulfur content times its yield per hectare, ranges from 10–40 kg S ha−1 for grasses and cereals to 50–100 kg S ha−1 for high sulfur-demanding crops such as Brassica (Haneklaus et al., 2003, 2007a,b, Zhao et al., 2008). Groundwater contains generally 0.08–1.6 mM sulfate and is of great signiﬁcance in crop sulfur nutrition at sites where roots have direct access or are irrigated with it (Haneklaus et al., 2007b). In some regions, dry and wet deposition of atmospheric sulfur from salt spray and sulfurous air pollutants may also contribute to the crop sulfur supply since atmospheric sulfur deposition may range from <0.5 in rural areas to >20 kg S ha−1 in heavily polluted areas (Haneklaus et al., 2003). In different regions of the world, incidences of sulfur deﬁciency in agricultural crops and grassland have been reported, which can be corrected by sulfur fertilizer application. The dose of sulfur fertilizer (rate of application) depends on the crop sulfur demand, soil type, and management, and on atmospheric sulfur deposition. Sulfur fertilizer recommendation rates vary between countries and range, for example, for cereals from 10–50 kg S ha−1 and for oilseed rape from 20–100 kg S ha−1 (Walker, 2002; Haneklaus et al., 2007a,b). Balanced sulfur fertilization is also essential for optimal nitrogen use efﬁciency and for preventing negative environmental side effects such as nitrogen leaching (Haneklaus et al., 2007b; Zhao et al., 2008). A fertilizer rate of approximately 0.17 kg kgN−1 (oilseed plants) or 0.1 kg kgN−1 (cereals and seeded grass) appears to be advisable (Walker, 2002). The timing and form of sulfur fertilizer is also of great importance for optimal crop sulfur nutrition. In contrast to nitrogen (and

297

several other minerals), the rate of remobilization of sulfur from older to young tissue appears often to be low in crops. Consequently, a sufﬁcient soil sulfur supply to the root may be required during the entire vegetative and generative growth period for optimal crop production and quality. If crops are fertilized with either elemental or organic sulfur, the rate of mineralization may hinder the sulfur availability required for optimal crop production since generally plants rely on sulfate taken up by the root as the primary sulfur source for growth (Eriksen, 2002; Haneklaus et al., 2007a,b). Sulfate is highly water-soluble and is carried through the soil toward the root surface by mass/bulk ﬂow. The transfer of sulfate from the soil water into the root cells across the plasma-membrane is an active process. Upon its uptake, sulfate may be transported symplastically through plasmodesmata from cell to cell radially through the cortex and endodermis into the root stele (Hawkesford and De Kok 2006). If sulfate is absorbed by the root apoplastic water, then the endodermis will be the selective cell layer for the active uptake of sulfate. In the stele, the sulfate is transferred into the xylem (xylem loading) and transported by the transpiration stream to the shoot (by mass/bulk ﬂow). Distribution of sulfate from the xylem to sink tissue (vascular transport) occurs via active loading/ modulated transfer of the sulfate from the xylem into the phloem via phloem companion/transfer cells (Anderson, 2005). In most crop species, the major proportion of the sulfate taken up is reduced in the plastids (roots) and chloroplasts (shoot), and subsequently further assimilated into organic sulfur compounds (De Kok et al., 2002a; Hawkesford and De Kok, 2006). The remaining sulfate—in some species a substantial proportion of the sulfate taken up—is transferred into the vacuoles of root and shoots cells.

298

NUTRIENT USE EFFICIENCY IN CROPS

In contrast to nitrate, the remobilization and redistribution of the vacuolar sulfate is rather slow, and it may be even too slow to keep pace with the growth of the plant under temporary sulfur-limitations and sulfurdeﬁcient plants may still contain detectable levels of sulfate (Cram, 1990; Hawkesford, 2000). Sulfurous air pollutants may act as both toxin and nutrient for plants, and in addition to sulfate taken up by the roots, plants are also able to utilize sulfur gases, namely SO2, H2S, taken up by shoot as sulfur source for growth (De Kok et al., 2007, 2009). The stomatal opening is generally the limiting factor for the uptake of SO2 by the shoot, since SO2 is highly soluble in the water of the mesophyll cells wherein it is rapidly dissociated upon liberation of H+ ions. The (bi) sulﬁte formed may be directly assimilated into organic sulfur compounds or enzymatically or nonenzymatically oxidized to sulfate and subsequently transferred into the vacuoles (De Kok et al., 2007, 2009). In contrast to SO2, the uptake of H2S by the shoot appears to be determined by the rate of its metabolism into cysteine in the mesophyll cells rather than stomatal opening (De Kok and Tausz, 2001; De Kok et al., 2002b, 2007, 2009). Evidently, atmospheric levels of ≥0.05 μl L−1 SO2 and H2S may substantially contribute to plant sulfur nutrition (De Kok et al., 2007, 2009). Nevertheless, it is unclear as to what extent metabolism contributes to the detoxiﬁcation of the absorbed sulfur gases since there is no clear-cut transition in the level/rate of metabolism of the absorbed sulfur gases and their phytotoxicity (De Kok et al., 2009). Sulfate uptake, distribution, and assimilation in plants The uptake of sulfate by the root, its transport from root to shoot, and the subcellular distribution of sulfate in plants is mediated

by sulfate transporters, generally acting as SO42-/H+ symporters, driven by a proton gradient generated by ATPases (Cram, 1990; Clarkson et al., 1993). Sulfate transporters (see also Chapter 1) are transmembrane proteins containing 12 hydrophobic membrane-spanning domains (MSDs) and a sulfate transport/antisigma-factor antagonists domain (STAS domain) at their C-terminal region (Hawkesford and Smith, 1997; Hawkesford, 2003). The STAS domain possesses a conserved loop with a potentially phosphorylated-conserved serine residue, which may be involved in regulation of the sulfate transporters (Fourcroy et al., 2005). Distinct sulfate transporters are involved in the uptake, transport, and distribution of sulfate in the plant. For instance, Arabidopsis, Brassica, rice, and wheat contain 10–14 different sulfate transporter genes. The sulfate transporters have been classiﬁed in up to ﬁve different groups according to their cellular and subcellular expression and possible functioning in the plant (Hawkesford, 2003, 2007, 2008; Buchner et al., 2004a,b, 2010; Hawkesford and De Kok, 2006; Parmar et al., 2007). The uptake of sulfate by the root plasma membrane is mediated by so-called highafﬁnity Group 1 sulfate transporters, which have an apparent Km for sulfate around 1.5– 10 μM (Clarkson et al., 1993; Smith et al., 1995, 1997; Davidian et al., 2000; Hawkesford and Wray, 2000; Hawkesford, 2003, 2007, 2008; Smith and Diatloff, 2005; Hawkesford and De Kok 2006). Within Group 1 in Arabidopsis, three different sulfate transporters have been characterized. The sulfate transporters Sultr1;1 and Sultr1;2 are responsible for primary sulfate uptake by the roots and are localized in the epidermis, cortex, and root hairs (Takahashi et al., 2000; Shibagaki et al., 2002; Buchner et al., 2004a,b, 2010; Takahashi, 2005; Yoshimoto et al., 2002, 2007). Sultr1;2 appeared also to

SULFUR NUTRITION IN CROP PLANTS

be particularly important for the uptake of selenate in Arabidopsis (El Kassis et al., 2007). Sultr1;3 is presumably involved in phloem loading of sulfate (Yoshimoto et al., 2003). The abundance and expression of the Group 1 sulfate transporters in roots is species speciﬁc. For instance, in roots of dicotyledons (e.g., Arabidopsis, Brassica), Sultr1;2 was the sole constitutively expressed sulfate transporter present, whereas Sultr1;1 was only expressed upon sulfate deprivation (Buchner et al., 2004a,b; Koralewska et al., 2007, 2008, 2009a,b; Parmar et al., 2007). In the monocotyledon wheat, however, the Sultr1;2-type transporter appeared to be absent in the roots, and here the Sultr1;- type sulfate transporter was expressed (Buchner et al., 2010). Group 2 sulfate transporters are so-called low-afﬁnity transporters with an apparent Km for sulfate > 100 μM and are involved in the distribution (vascular transport) of sulfate in the plant (Hawkesford, 2003; Hawkesford et al., 2003a,b). Sultr2;1 is localized in the xylem parenchyma and pericycle cells of roots and in the xylem and parenchyma cells of shoot, whereas Sultr2;2 is localized in the root phloem and leaf vascular bundle sheath cells (Takahashi et al., 2000). Five sulfate transporters (in rice six) belong to the less well-characterized Group 3 (Hawkesford et al., 2003a,b; Buchner et al., 2004a,b, 2010). In Arabidopsis, the Sultr3;1, Sultr3;2, and Sultr3;3 sulfate transporters are localized in the leaves (Takahashi et al., 2000; Hawkesford et al., 2003a,b), whereas in Brassica, the Sultr3;2 appeared exclusively to be present in the root, and Sultr3;3 in leaves, stem, and roots (Buchner et al., 2004a,b). In Brassica, Sultr3;4 appeared only to be present in the stem and Sultr3;5 in the roots (Buchner et al., 2004a). In wheat, however, Sultr3;2 was not expressed, whereas Sultr3;1, Sultr3;3 Sultr3;4, and Sultr3;5 appeared to be present in both root and shoot, although their level

299

of expression was relatively low (Buchner et al., 2010). Until now the role of the Group 3 sulfate transporters is poorly understood, but an enhanced activity of Sultr2;1 and increased sulfate uptake capacity were demonstrated for Sultr3;5 coexpressed with Sultr2;1 in Arabidopis (Kataoka et al., 2004a; Takahashi, 2005). Two Group 4 sulfate transporters have been identiﬁed in roots and shoots of Arabidopsis and Brassica, and only one in rice and wheat (Hawkesford, 2008; Buchner et al., 2010). These transporters are localized in the tonoplast and appear to function in the vacuolar unloading of sulfate (Kataoka et al., 2004b; Takahashi, 2005; Hawkesford, 2007, 2008). The Group 5 transporters are quite distinct from the other sulfate transporter groups since they do not posses the STAS domain (Hawkesford, 2003). Two Group 5 transporters have been identiﬁed in Arabidopsis and rice, and one in Brassica (Hawkesford, 2003, 2008). In Brassica napus, Sultr5;1 is present in the root, stem, and leaves and is localized in the tonoplast (Hawkesford et al., 2003b; Parmar et al., 2007). In Arabidopsis, transporter AtSultr5;2, appears to be a high-afﬁnity molybdenum transporter and has been renamed MOT1 (molybdenum transporter 1; Tomatsu et al., 2007; Baxter et al., 2008). It is evident that sulfate may also be transferred across membranes via so-called rapid activated depolarization-activated R-type anion channels, and sulfate-selective anion channels have been characterized in hypocotyls, the root epidermis, and guard cells (Roberts, 2006). The physiological role of the sulfate selective anion channels in sulfate transport and distribution still needs to be assessed, but they may be involved in sulfate homeostasis and may have signiﬁcance in sulfate efﬂux from cells and roots preventing toxic accumulation of cytosolic levels within the cells (Roberts, 2006).

300

NUTRIENT USE EFFICIENCY IN CROPS

Moreover, sulfate-selective anion channels may also play a role in the radial movement of sulfate from the epidermis to the xylem (Hawkesford, 2007). Sulfate needs to be transported into the stroma of the plastids in roots and chloroplasts in the shoots, where it is reduced to sulﬁde. The transfer of sulfate over the plastid/chloroplast membrane is yet unresolved, but may be mediated by the triose-

phosphate/phosphate translocator and/or an ABC-type transporter (membrane pore proteins; Hawkesford, 2008). Sulfate is ﬁrst activated to adenosine 5′ phosphosulfate (APS) by adenosine triphosphate (ATP)sulfurylase prior to its reduction to sulﬁte by APS reductase (Hell, 1997; De Kok et al., 2002a; Kopriva and Koprivova, 2003, 2004; Saito, 2004; Kopriva et al., 2008; Fig. 14.1). APS may also be further phosphorylated by

Glutathione ADP + Pi glutathione synthetase glycine + ATP g-Glutamyl-cysteine ADP + Pi

g-glutamyl-cysteine synthetase

glutamate + ATP Methionine

Cysteine

Proteins

acetate O-acethlserine (thiol)lyase O-acetylserine H2S

Sulfide 6Fdox sulfite reductase 6Fdred Sulfite

SO2

SQDG

AMP+GSSG OXIDATION

APS reductase 2GSH APS kinase APS

PAPS ATP

SSM

ADP

PPi ATP sulfurylase ATP Sulfate

Sulfur metabolism in plants. APS, adenosine 5′-phosphosulfate; Fdred, Fdox, reduced and oxidized ferredoxin; GSH, GSSH, reduced and oxidized glutathione; PAPS, adenosine 3′-phosphate 5′-phosphosulfate; SQDG, sulfoquinovosyl diacylglycerol; SSM, secondary sulfur metabolites; AMP, adenosine monophosphate; GSSG, oxidized glutathione; PPi, pyrophosphate.

Fig. 14.1.

SULFUR NUTRITION IN CROP PLANTS

APS kinase to adenosine 3′ phosphate 5′ phosphosulfate (PAPS), which is the precursor of secondary sulfur metabolites (e.g., glucosinolates). Sulﬁte is also the sulfur donor for sulfoquinovosyl diacylglycerol (SQDG) biosynthesis, the major sulfolipid present in plants tissue. Sulﬁte is reduced to sulﬁde by sulﬁte reductase and is incorporated into cysteine by cysteine synthase, an enzyme complex consisting of serine acetyltransferase (catalyzes the synthesis of Oacetyl-L-serine) and O-acetyl-L-serine (thiol)lyase (catalyzes the synthesis of cysteine. The synthesis of cysteine is one of the major direct links between sulfur and nitrogen assimilation in plants. Cysteine serves as the sulfur donor for the synthesis of methionine, and both amino acids are incorporated into proteins (Hoefgen and Hesse, 2007). Cysteine is also the precursor for several other sulfur compounds including glutathione (De Kok et al., 2002a, 2005). From localization and genomic organization studies it has become evident that plants may contain different isoforms of the enzymes involved in the sulfate reduction pathway (Kopriva and Koprivova, 2003, 2004; Kopriva et al., 2008). It is evident that APS reductase and sulﬁte reductase are exclusively present in the chloroplasts/plastids, whereas ATP sulfurylase may also be present in the cytosol and mitochondria, and APS kinase also in the cytosol. The biosynthesis of cysteine occurs in the chloroplast, cytosol, and mitochondria; however, the abundance of serine acetyltransferase and O-acetyl-L-serine (thiol)lyase may strongly vary between cellular compartments (Kopriva and Koprivova, 2003; Kopriva et al., 2008). Regulation of uptake, distribution, and assimilation of sulfate in plants The uptake and assimilation of sulfate is controlled by the plant sulfur demand for

301

growth (plant sulfur content times growth rate), which ranges from 10 to 100 μmol g−1 dry weight day−1 for different crop species (Haneklaus et al., 2003, 2007a,b; Hawkesford and De Kok, 2006; Zhao et al., 2008). The sulfate concentration in soil water around the root may be quite variable and ranges from 0.2 to 1600 μM, and crop plants need the ability to regulate the overall sulfate uptake and assimilation efﬁciency in order to keep up with the sulfur demand for growth (Cram, 1990; Clarkson et al., 1993; Haneklaus et al., 2003, 2007a,b: Hawkesford and De Kok 2006; Zhao et al., 2008). The uptake and assimilation of sulfate may be modulated by changing of the activity of the sulfate transporters and the enzymes involved in the sulfur reduction pathway. Evidently, the primary controlling factor in sulfur utilization is the uptake of sulfate by the root. The Group 1 sulfate transporters, which are responsible for the primary uptake of sulfate by the roots, not only have a very high afﬁnity for sulfate, but also their activity is rapidly affected and adjusted to variation in sulfate supply to the roots and the overall sulfur status of the plant. For instance, the uptake of sulfate in Brassica, a species characterized by its high sulfur content and growth, was adjusted to the sulfur demand for growth, even at sulfate concentrations close to the Km of the Group 1 high-afﬁnity sulfate transporters, by an upregulation of sulfate transporter activity of the roots (Koralewska et al., 2007). Plants were able to maintain their growth rate and high sulfur content when grown at 5 and 10 μM sulfate in the root environment (Koralewska et al., 2007). Moreover, if plants were able to utilize foliarly absorbed H2S as a sulfur source, this resulted not only in a downregulation of expression and activity of APS reductase (sulfate reduction) in the shoot but also in a decrease in expression and activity of the sulfate transporters in the root (Westerman et al., 2000, 2001a,b;

302

NUTRIENT USE EFFICIENCY IN CROPS

Koralewska et al., 2008). Sulfate deprivation generally results in a rapid upregulation of the expression of the high-afﬁnity Group 1 sulfate transporters in the roots and, upon prolonged deprivation, also in the shoot (Buchner et al., 2004a,b, 2010; Koralewska et al., 2007, 2008, 2009a; Parmar et al., 2007; Stuiver et al., 2009; Shinmachi et al., 2010). Furthermore, the Group 2 (vascular transport) and Group 4 transporters (vacuolar efﬂux) are highly upregulated in the root and that of Group 4 transporters also in the shoot (Parmar et al., 2007; Stuiver et al., 2009; Koralewska et al., 2007, 2008, 2009a; Buchner et al., 2004a, 2010; Shinmachi et al., 2010). Apparently upon the occurrence of sulfur deﬁciency plants try to remobilize and redistribute all possible available sulfate from, for instance, vacuoles and other resources. The expression of Groups 3 and 5, however, was hardly affected by sulfate deprivation. Evidently, an altered expression and activity of sulfate transporters not only may be the consequence of a changed plant sulfur status and/or demand, but also the consequence of an altered root morphology (e.g., the formation of multiple undeveloped lateral roots) upon prolonged exposure to physiological stress conditions, for example, sulfate deprivation (Koralewska et al., 2009a,b) or elevated copper levels in the root environment (Shahbaz et al., 2010). In general, sulfate deprivation also results in a shift in shoot to root biomass partitioning during growth in favor of that of the root (Hawkesford and De Kok, 2006; Haneklaus et al., 2007a,b; Zhao et al., 2008). However, both an altered shoot to root biomass partitioning and the upregulated expression and activity of the Group 1 sulfate transporters upon sulfate deprivation was determined by the absence of sulfate in the root environment rather than by the sulfur status of the plant itself (Koralewska et al., 2008). For instance, if Brassica was simultaneously exposed to sulfate deprivation and to either

SO2 or H2S, at levels sufﬁcient to maintain normal overall biomass production with time, both the decrease in shoot to root biomass partitioning and the enhanced activity of the sulfate transporters were still quite similar to that of sulfate-deprived nonexposed plants (Yang et al., 2006a,b; Koralewska et al., 2008). The signal transduction pathway involved in the regulation of the uptake, transport, and distribution of sulfate is still largely unsolved. It is presumed that the regulation of expression (transcriptional) and/or activity (translational and/or posttranslational) of the sulfate transporters might be signaled or mediated by sulfate itself or by products of the assimilatory sulfate reduction pathway (e.g., sulﬁde, cysteine and/or glutathione; Hawkesford and De Kok 2006). Furthermore, it has been proposed that the level of the cysteine precursor O-acetylserine would have signiﬁcance in the regulatory control of the sulfate transporters. On the basis of the current knowledge and commonly applied research approaches, it is still unclear as to what extent measuring changes in concentrations of potential signal compounds and expression of the sulfate transporters, both determined at the wholeorgan level, provides sufﬁcient insight into the actual regulatory control of sulfate uptake at the root cellular level. For instance, in Brassica there was generally no clear relation between the overall tissue levels of sulfate, thiols, and O-acetylserine in the root or shoot and the expression and activity of the sulfate transporters upon variation in sulfate supply (Buchner et al., 2004a; Koralewska et al., 2007, 2008, 2009a,b; Stuiver et al., 2009; Shahbaz et al., 2010). First, the fast upregulation of expression and activity of the sulfate transporters in Brassica upon sulfate deprivation usually already occurs without substantial changes in the overall tissue sulfate and/or thiol (viz. glutathione) levels (Buchner et al., 2006;

SULFUR NUTRITION IN CROP PLANTS

Koralewska et al., 2009b), whereas an enhanced level of O-acetylserine in sulfurdeﬁcient plants only reﬂected a resulting imbalance in sulfur and nitrogen assimilation (Buchner et al., 2004a; Hawkesford and De Kok, 2006). Second, exposure of Brassica to elevated and growth-reducing copper levels in the root environment resulted in an upregulated expression and activity of the sulfate transporters in the root and even occurred at normal and enhanced thiol levels (root, shoot) and normal or enhanced sulfate levels (Shahbaz et al., 2010). The reduction of sulfate in the chloroplast (plastid) is also regulated by the sulfur status of the plant. The afﬁnity of ATP sulfurylase appears to be rather low (Km values range from 0.5–3.1 mM; Stulen and De Kok 1993) and the in situ sulfate concentration may be one of the limiting/regulating steps in sulfate reduction (De Kok et al., 2005). Both the gene expression and measured activity of APS reductase change rapidly in response to variation in sulfur nutrition (Kopriva and Koprivova, 2003; Durenkamp et al., 2007). It is presumably the primary regulation points in sulfate reduction since this enzyme has a fast turnover rate and its activity is the lowest of all enzymes of the sulfate reduction pathway. Sulﬁde, O-acetylserine, cysteine, or glutathione are likely regulators of APS reductase and may occur both by allosteric inhibition and by metabolite activation or repression of expression of the genes encoding the APS reductase (Hell, 1997; Leustek and Saito, 1999; Kopriva and Koprivova, 2003). The majority of plant cells, including root cells, have the capacity to reduce and assimilate sulfate in their plastids, presumably facilitating local signaling of sulfate uptake as well as distribution and reduction/ assimilation at the cellular level; it is difﬁcult to distinguish local signaling at the cellular level from that at the integrated tissue

303

level, namely shoot to root interactions (Hawkesford and De Kok, 2006). A key unresolved issue is the signal transduction pathway in the crosstalk between the sulfate reduction pathway in the chloroplasts/ plastids and the transcription of sulfate transporters/sulfate reducing enzymes in the nucleus. Another key issue is the extent to which H2S, the ﬁrst product of the sulfate reduction pathway, may function as an endogenous gaseous transmitter in this crosstalk. It is evident that at the cellular pH, H2S is largely undissociated, and in this form it may easily pass through membranes (De Kok et al., 1998, 2007, 2009). Plants grown under normal conditions may produce (and even emit) minute levels of H2S, which has been presumed to be a regulatory step in the homeostasis of the sulfur pools in plants (Schröder, 1993; Bloem et al., 2007). In prokaryotes, a role of sulﬁde in transcriptional regulation of the cys-operon (for genes involved in sulfur uptake and assimilation) is well documented (Kredich, 1993). Interactions between nitrogen and selenium with sulfur metabolism in plants Similarly to sulfur, plants are able to accomplish the same relative growth rate and plant nitrogen content over a wide range of external nitrate concentrations when grown in nutrient solution (Clement et al., 1978). The net nitrate uptake rate is under the control of an internal regulating mechanism, which adjusts the net nitrate uptake rate to the nitrogen need of the plant, as determined by plant growth and total plant nitrogen content (Ter Steege et al., 1998, 1999). Plants maintain their overall nitrogen and sulfur content within a certain range (Stulen and De Kok, 1993) and this might implicate a mutual regulation of the uptake of nitrogen and sulfur by the root. Upon sulfate deprivation of Brassica, the nitrate uptake rate was

304

NUTRIENT USE EFFICIENCY IN CROPS

decreased (Stuiver et al., 1997; Westerman et al., 2000). When sulfate-deprived plants were exposed to atmospheric H2S, an increase in both biomass production and nitrate uptake was observed. However, when Brassica plants, which had access to sulfate in the root environment, were fumigated with atmospheric H2S, both nitrate uptake rate and relative growth rate were unaffected, while sulfate uptake was decreased (Westerman et al., 2000, 2001a). From these data the conclusion can be drawn that changes in nitrate uptake rate are related to changes in growth rate and that there is no direct linkage between the uptake of nitrate and sulfate. Protein synthesis requires inorganic carbon, and reduced nitrogen and sulfur. Coordination of the assimilatory reduction pathways of nitrate and sulfate is therefore necessary, so that appropriate proportions of both sulfur-containing and other amino acids are available for protein synthesis (Brunold et al., 2003; Hoefgen and Hesse, 2007). For the synthesis of sulfur-containing amino acids such as cysteine and methionine for protein synthesis, sufﬁcient reduced sulfate and nitrogen compounds must be available. Evidently, both the expression and activity of APS reductase and nitrate reductase are affected by either sulfur or nitrogen deﬁciency (Leustek and Saito, 1999). Several compounds might act as signal molecules in the mutual regulation of both pathways. Amino acids and amides such as asparagine and arginine, and also O-acetyl-L-serine, which for instance accumulate under sulfur deﬁciency, may be related to the decrease in nitrate reductase activity (Migge et al., (2000). Selenium is not an essential plant nutrient and high levels are even phytotoxic. However, the uptake and metabolism of selenium and sulfate are strongly interrelated (Anderson, 1993; Hawkesford and Zhao, 2007; Zhu et al., 2009). Selenate is

an analog of sulfate and its uptake and distribution on plants is facilitated by sulfate transporters (and compete with sulfate), and it may be reduced in the chloroplasts/plastid by sulfate-reducing enzymes in yield in the formation of seleno-cysteine and selenomethionine (Anderson, 1993; Hawkesford and Zhao, 2007; Zhu et al., 2009; Shinmachi et al., 2010; Stroud et al., 2010). The selenometabolites are potentially phytotoxic and strongly enhanced levels of selenate or selenite in the root environment may negatively affect production of crop plants. Selenium is an essential micronutrient for humans and animals, which rely on plants as source for their diet (Hawkesford and Zhao, 2007; Zhu et al., 2009). A controlled uptake of selenium is essential in the biofortiﬁcation of crops, though may strongly be affected by the sulfur nutritional status (Hawkesford and Zhao, 2007; Zhu et al., 2009; Shinmachi et al., 2010; Stroud et al., 2010). Targets for crop breeding Sulfur use efﬁciency has seldom been an important target for crop improvement; however, with the widespread appearance of sulfur deﬁciency symptoms in Europe and elsewhere at the end of the 20th century, the importance of sulfur nutrition has been rerecognized. Legislation controlling pollution has led to substantial reductions in the aerial deposition of sulfur, which was previously at levels to fulﬁll many crop requirements. Together with the use of high analysis fertilizer containing no sulfur, this led to deﬁciencies being apparent in crops as evidenced by yield penalties and quality issues (Zhao et al., 1999). Targets for crop improvement either focus on acquisition efﬁciency and/or conversion into protein, or nutritional issues related to speciﬁc sulfur-containing end products. While readily available and relatively cheap fertilizer application can effectively

SULFUR NUTRITION IN CROP PLANTS

Fig. 14.2.

305

Are there targets to improve sulfur use efﬁciency in crop plants: the central role of the sulfate

transporters.

correct for sulfur deﬁciencies, solutions aimed at optimizing acquisition also have merits. Not least, as already noted (Shinmachi et al., 2010), the interaction between sulfate and selenate (and molybdate) can either result in overaccumulation of potentially toxic levels of selenate under deﬁcient conditions, or conversely, sulfur fertilization can result in nutritionally suboptimal levels. The central role of the transporters is evident from Figure 14.2 (see also Hawkesford, 2000). One target may be to select for transporters with altered discrimination between the anions. For improving sulfate acquisition, logical targets may be enhanced by root architectural characteristics and the spatial expression of the high-afﬁnity transporters involved in uptake. The repression of expression of these transporters due to high availability or low demand might be target to encourage luxury uptake. Subsequent to uptake, storage and remobilization patterns will depend on transporter expression. Both the temporary storage and the ﬁnal sinks may need to be manipulated (see Chapter 1). In the latter cases particularly this may have

advantageous consequences for either resistance to stress or nutritional value. The breeding for low glucosinolate Brassica species was to speciﬁcally enhance nutritional quality (Schnug, 1990). Acknowledgments Rothamsted Research is an institute of the Biotechnology and Biological Sciences Research Council of the United Kingdom. Research (MJH) is also supported by the Biotechnology and Biological Sciences Research Council (BB/G022437/1 and BB/ C514066/1) and the Department of Environment, Food and Rural Affairs (WGIN project IF0146). The authors thank Dick Visser for drawing Figure 14.1. References Anderson, J.W. (1993) Selenium interactions in sulfur metabolism. In: Sulfur Nutrition and Assimilation in Higher Plants; Regulatory, Agricultural and Environmental Aspects (eds. L.J. De Kok, I. Stulen, H. Rennenberg, et al.), pp. 49–60. SPB Academic Publishing, The Hague.

306

NUTRIENT USE EFFICIENCY IN CROPS

Anderson, J.W. (2005) Regulation of sulfur distribution and redistribution in grain plants. In: Sulfur Transport and Assimilation in Plants in the Post Genomic Era (eds. K. Saito, L.J. De Kok, I. Stulen, et al.), pp. 23–31. Backhuys Publishers, Leiden. Baxter, I., Muthukumar, B., Park, H.C., et al. (2008) Variation in molybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter (MOT1). PLoS Genetics 4, e1000004. doi:10.1371/journal.pgen.1000004. Benning, C. (1998) Biosynthesis and function of the sulfolipid sulphoquinovosyl diacylglycerol. Annual Review of Plant Physiology and Plant Molecular Biology 49, 53–77. Benning, C., Garavito, R.M., & Shimojima, M. (2008) Sulfolipid biosynthesis and function in plants. In: Sulfur Metabolism in Phototrophic Organisms (eds. R. Hell, C. Dahl, D.B. Knaff & T. Leustek), pp. 185–200. Springer, Dordrecht. Bloem, E., Haneklaus, S., Salac, I., et al. (2007) Facts and ﬁction about sulfur metabolism in relation to plant-pathogen interactions. Plant Biology 9, 596–607. Brunold, C., Von Ballmoos, P., Hesse, H., et al. (2003) Interactions between sulfur, nitrogen and carbon metabolism. In: Sulfur Transport and Assimilation in Plants: Regulation, Interaction and Signaling (eds. J.-C. Davidian, D. Grill, L.J. De Kok, et al.), pp. 45–56. Backhuys Publishers, Leiden. Buchner, P., Stuiver, C.E.E., Westerman, S., et al. (2004a) Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H2S and pedospheric sulfate nutrition. Plant Physiology 136, 3396–3408. Buchner, P., Takahashi, H., & Hawkesford, M.J. (2004b) Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport. Journal of Experimental Botany 55, 1765–1773. Buchner, P., Parmar, S., Kriegel, A., et al. (2010) The sulfate transporter family in wheat: tissue-speciﬁc gene expression in relation to nutrition. Molecular Plant 3, 374–389. Clarkson, D.T., Hawkesford, M.J., & Davidian, J.-C. (1993) Membrane and long-distance transport of sulfate. In: Sulfur Nutrition and Assimilation in Higher Plants: Regulatory, Agricultural and Environmental Aspects (eds. L.J. De Kok, I. Stulen, H. Rennenberg, et al.), pp. 3–19. SPB Academic Publishing, The Hague. Clement, C.R., Hopper, M.J., & Jones, J.H.P. (1978) The uptake of nitrate by Lolium perenne from ﬂowing nutrient solution. I. Effect of concentration. Journal of Experimental Botany 29, 453–464.

Cram, W.J. (1990) Uptake and transport of sulfate. In: Sulfur Nutrition and Sulfur Assimilation in Higher Plants (eds. H. Rennenberg, C. Brunold, L.J. De Kok & I. Stulen), pp. 3–11. SPB Academic Publishing, The Hague. Davidian, J.-C., Hatzfeld, Y., Cathala, N., et al. (2000) Sulfate uptake and transport in plants. In: Sulfur Nutrition and Sulfur Assimilation in Higher Plants: Molecular, Biochemical and Physiological Aspects (eds. C. Brunold, H. Rennenberg, L.J. De Kok, et al.), pp. 19–40. Paul Haupt, Bern. De Kok, L.J. & Stulen, I. (1993) Functions of glutathione in plants under oxidative stress. In: Sulfur Nutrition and Assimilation in Higher Plants; Regulatory, Agricultural and Environmental Aspects (eds. L.J. De Kok, I. Stulen, H. Rennenberg, et al.), pp. 125–138. SPB Academic Publishing, The Hague. De Kok, L.J. & Tausz, M. (2001) The role of glutathione in plant reaction and adaptation to air pollutants. In: Signiﬁcance of Glutathione to Plant Adaptation to the Environment (eds. D. Grill, M. Tausz & L.J. De Kok), pp. 185–201. Kluwer Academic Publishers, Dordrecht. De Kok, L.J., Stuiver, C.E.E., & Stulen, I. (1998) The impact of elevated levels of atmospheric H2S on plants. In: Responses of Plant Metabolism to Air Pollution and Global Change (eds. L.J. De Kok & I. Stulen), pp. 51–63. Backhuys Publishers, Leiden. De Kok, L.J., Castro, A., Durenkamp, M., et al. (2002a) Sulphur in plant physiology. In: Proceedings 500, pp. 1–26. International Fertilizer Society, York. De Kok, L.J., Stuiver, C.E.E., Westerman, S., & Stulen, I. (2002b) Elevated levels of hydrogen sulﬁde in the plant environment: nutrient or toxin. In: Air Pollution and Biotechnology in Plants (eds. K. Omasa, H. Saji, S. Yousseﬁan & N. Kondo), pp. 201–213. Springer-Verlag, Tokyo. De Kok, L.J., Castro, A., Durenkamp, M., et al. (2005) Pathways of plant sulfur uptake and metabolism— an overview. Landbauforschung Völkenrode, Germany Special Issue 283, 5–13. De Kok, L.J., Durenkamp, M., Yang, L., & Stulen, I. (2007) Atmospheric sulfur. In: Sulfur in Plants—An Ecological Perspective (eds. M.J. Hawkesford & L.J. De Kok), pp. 91–106. Springer, Dordrecht. De Kok, L.J., Yang, L., Stuiver, C.E.E., & Stulen, I. (2009) Negative versus positive functional plant responses to air pollution: a study establishing cause-effect relationships of SO2 and H2S. In: Air Quality and Ecological Impacts: Relating Sources to Effects (ed. A.H. Legge), Developments in Environmental Sciences 9, 121–134, Elsevier, Amsterdam.

SULFUR NUTRITION IN CROP PLANTS

Durenkamp, M., De Kok, L.J., & Kopriva, S. (2007) Adenosine 5’ phosphosulphate reductase is regulated differently in Allium cepa L. and Brassica oleracea L. upon exposure to H2S. Journal of Experimental Botany 58, 1571–1579. El Kassis, E., Cathala, N., Rouached, H., et al. (2007) Characterization of a selenate-resistant Arabidopsis mutant. Root growth as a potential target for selenate toxicity. Plant Physiology 143, 1231–1241. Eriksen, J. (2002) Organic manures as sources of fertilizer sulphur. In: Proceedings 505, pp. 1–19. International Fertilizer Society, York. Fourcroy, F., Rouached, H., Berthomieu, P., et al. (2005) Involvement of the STAS domain in the regulation of the Arabidopsis thaliana sulfate transporter Sultr1;2. In: Sulfur Transport and Assimilation in Plants in the Post Genomic Era (eds. K. Saito, L.J. De Kok, I. Stulen, et al.), pp. 39–42. Backhuys Publishers, Leiden. Haneklaus, S., Bloem, E., & Schnug, E. (2003) The global sulphur cycle and its links to plant environment. In: Sulphur in Plants (eds. Y.P. Abrol & A. Ahmad), pp. 1–28. Kluwer Academic Publishers, Dordrecht. Haneklaus, S., Bloem, E., & Schnug, E. (2007a) Sulfur interactions in crop ecosystems. In: Sulfur in Plants—An Ecological Perspective (eds. M.J. Hawkesford & L.J. De Kok), pp. 17–58. Springer, Dordrecht. Haneklaus, S., Bloem, E., Schnug, E., et al. (2007b) Sulfur. In: Handbook of Plant Nutrition (eds. A.V. Baker & D.J. Pilbeam), pp. 183–238. Taylor and Francis, Boca Raton, FL. Hawkesford, M.J. (2000) Plant responses to sulphur deﬁciency and the genetic manipulation of sulphate transporters to improve S-utilisation efﬁciency. Journal of Experimental Botany 51, 131–138. Hawkesford, M.J. (2003) Transporter gene families in plants: the sulfate transporter gene family— redundancy or specialization? Physiologia Plantarum 117, 155–163. Hawkesford, M.J. (2007) Sulfur and plant ecology: a central role of sulfate transporters for responding to sulfur availability. In: Sulfur in Plants—An Ecological Perspective (eds. M.J. Hawkesford & L.J. De Kok), pp. 1–15. Springer, Dordrecht. Hawkesford, M.J. (2008) Uptake, distribution and subcellular transport of sulfate. In: Sulfur Metabolism in Phototrophic Organisms (eds. R. Hell, C. Dahl, D.B. Knaff & T. Leustek), pp. 15–30. Springer, Dordrecht. Hawkesford, M.J. & De Kok, L.J. (2006) Managing sulphur metabolism in plants. Plant, Cell & Environment 29, 382–395.

307

Hawkesford, M.J. & Smith, F.W. (1997) Molecular biology of higher plant sulphate transporters. In: Sulphur Metabolism in Higher Plants—Molecular, Ecophysiological and Nutritional Aspects (eds. W.J. Cram, L.J. De Kok, I. Stulen, et al.), pp. 13–25. Backhuys Publishers, Leiden. Hawkesford, M.J. & Wray, J.L. (2000) Molecular genetics of sulphur assimilation. Advances in Botanical Research 33, 159–223. Hawkesford, M.J. & Zhao, F.J. (2007) Selenium— strategies for increasing selenium content of wheat. Journal of Cereal Science 46, 282–292. Hawkesford, M.J., Buchner, P., Hopkins, L., & Howarth, J.R. (2003a) Sulphate uptake and transport. In: Sulphur in Plants (eds. Y.P. Abrol & A. Ahmad), pp. 71–86. Kluwer Academic Publishers, Dordrecht. Hawkesford, M.J., Buchner, P., Hopkins, L., & Howarth, J.R. (2003b) The plant sulfate transporter family: specialized functions and integration with whole plant nutrition. In: Sulfur Transport and Assimilation in Plants: Regulation, Interaction and Signaling (eds. J.-C. Davidian, D. Grill, L.J. De Kok, et al.), pp. 1–10. Backhuys Publishers, Leiden. Hell, R. (1997) Molecular physiology of plant sulfur metabolism. Planta 202, 138–148. Hell, R. & Kruse, C. (2007) Sulfur in biotic interactions of plants. In: Sulfur in Plants—An Ecological Perspective (eds. M.J. Hawkesford & L.J. De Kok), pp. 197–224. Springer, Dordrecht. Hoefgen, T. & Hesse, H. (2007) Sulfur in plants as part of a metabolic network. In: Sulfur in Plants—An Ecological Perspective (eds. M.J. Hawkesford & L.J. De Kok), pp. 107–142. Springer, Dordrecht. Kataoka, T., Hayashi, N., Yamaya, T., & Takahashi, H. (2004a) Root-to-shoot transport of sulfate in Arabidopsis. Evidence for the role of SULTR3;5 as a component of low-afﬁnity sulfate transport system in the root vasculature. Plant Physiology 136, 4198–4204. Kataoka, T., Watanabe-Takahashi, A., Hayashi, N., et al. (2004b) Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis. The Plant Cell 16, 2693–2704. Kopriva, S. & Koprivova, A. (2003) Sulphate assimilation: a pathway which likes to surprise. In: Sulphur in Higher Plants (eds. Y.P. Abrol & A. Ahmad), pp. 87–112. Kluwer Academic Publishers, Dordrecht. Kopriva, S. & Koprivova, A. (2004) Plant adenosine 5’-phosphosulphate reductase: the past, the present, and the future. Journal of Experimental Botany 55, 1775–1783. Kopriva, S., Patron, N.J., Keeling, P., & Leustek, T. (2008) Phylogenetic analysis of sulfate assimilation

308

NUTRIENT USE EFFICIENCY IN CROPS

and cysteine biosynthesis in phototrophic organisms. In: Sulfur Metabolism in Phototrophic Organisms (eds. R. Hell, C. Dahl, D.B. Knaff & T. Leustek), pp. 31–58. Springer, Dordrecht. Koralewska, A., Posthumus, F.S., Stuiver, C.E.E., et al. (2007) The characteristic high sulfate content in Brassica oleracea is controlled by the expression and activity of sulfate transporters. Plant Biology 9, 654–661. Koralewska, A., Stuiver, C.E.E., Posthumus, F.S., et al. (2008) Regulation of sulfate uptake, expression of the sulfate transporters Sultr1;1 and Sultr1;2, and APS reductase in Chinese cabbage (Brassica pekinensis) as affected by atmospheric H2S nutrition and sulfate deprivation. Functional Plant Biology 35, 318–327. Koralewska, A., Buchner, P., Stuiver, C.E.E., et al. (2009a) Expression and activity of sulfate transporters and APS reductase in curly kale in response to sulfate deprivation and re-supply. Journal of Plant Physiology 166, 168–179. Koralewska, A., Stuiver, C.E.E., Posthumus, F.S., et al. (2009b) The up-regulated sulfate uptake capacity, but not the expression of sulfate transporters is strictly controlled by the shoot sink capacity for sulfur in curly kale. In: Sulfur Metabolism in Plants: Regulatory Aspects, Signiﬁcance of Sulfur in the Food Chain, Agriculture and the Environment (eds. A. Sirko, L.J. De Kok, S. Haneklaus, et al.), pp. 61–68. Margraf Publishers, Weikersheim. Kredich, N.M. (1993) Gene regulation of sulfur assimilation. In: Sulfur Nutrition and Assimilation in Higher Plants; Regulatory, Agricultural and Environmental Aspects (eds. L.J. De Kok, I. Stulen, H. Rennenberg, et al.), pp. 37–47. SPB Academic Publishing, The Hague. Leustek, T. & Saito, K. (1999) Sulfate transport and assimilation in plants. Plant Physiology 120, 637–643. Migge, A., Bork, C., Hell, R., & Becker, T.W. (2000) Negative regulation of nitrate reductase gene expression by glutamine or asparagine accumulating in leaves of sulfur-deprived tobacco. Planta 211, 587–595. North, K.A. & Kopriva, S. (2007) Sulfur in resistance to environmental stresses. In: Sulfur in Plants—An Ecological Perspective (eds. M.J. Hawkesford & L.J. De Kok), pp. 143–168. Springer, Dordrecht. Parmar, S., Buchner, P., & Hawkesford, M.J. (2007) Leaf developmental stage affects sulfate depletion and speciﬁc sulfate transporter expression during sulfur deprivation in Brassica napus L. Plant Biology 9, 647–653.

Roberts, S.K. (2006) Plasma membrane anion channels in higher plants and their putative functions in roots. The New Phytologist 169, 647–666. Saito, K. (2004) Sulfur assimilatory metabolism. The long and smelling road. Plant Physiology 136, 2443–2450. Schnug, E. (1990) Glucosinolates—fundamental, environmental and agricultural aspects. In: Sulfur Nutrition and Sulfur Assimilation in Higher Plants; Fundamental, Environmental and Agricultural Aspects (eds. H. Rennenberg, C. Brunold, L.J. De Kok & I. Stulen), p. 97. SPB Academic Publishing, The Hague. Schröder, P. (1993) Plants as source of atmospheric sulfur. In: Sulfur Nutrition and Assimilation in Higher Plants; Regulatory, Agricultural and Environmental Aspects (eds. L.J. De Kok, I. Stulen, H. Rennenberg, et al.), pp. 253–270. SPB Academic Publishing, The Hague. Shahbaz, M., Tseng, H.W., Stuiver, C.E.E., et al. (2010) Copper exposure interferes with the regulation of the uptake, distribution and metabolism of sulfate in Chinese cabbage. Journal of Plant Physiology 167, 438–446. Shibagaki, N., Rose, A., McDermott, J.P., et al. (2002) Selenate-resistant mutants of Arabidopsis thaliana identify Sultr1;2, a sulfate transporter required for efﬁcient transport of sulfate into roots. The Plant Journal 29, 475–486. Shinmachi, F., Buchner, P., Stroud, J.L., et al. (2010) Inﬂuence of sulfur deﬁciency on the expression of speciﬁc sulfate transporters and the distribution of sulfur, selenium and molybdenum in wheat. Plant Physiology 153, 327–336. Sirko, A. & Gotor, C. (2007) Molecular links between metals in the environment and plant sulfur metabolism. In: Sulfur in Plants—An Ecological Perspective (eds. M.J. Hawkesford & L.J. De Kok), pp. 169–195. Springer, Dordrecht. Smith, F.W. & Diatloff, E. (2005) Sulfate transport processes in plants. In: Sulfur Transport and Assimilation in Plants in the Post Genomic Era (eds. K. Saito, L.J. De Kok, I. Stulen, et al.), pp. 3–11. Backhuys Publishers, Leiden. Smith, F.W., Ealing, P.M., Hawkesford, M.J., & Clarkson, D.T. (1995) Plant members of a family of sulfate transporters reveal functional subtypes. Proceedings of the National Academy of Sciences of the United States of America 92, 9373–9377. Smith, F.W., Hawkesford, M.J., Ealing, P.M., et al. (1997) Regulation of expression of a cDNA from barley roots encoding a high afﬁnity sulphate transporter. The Plant Journal 12, 875–884.

SULFUR NUTRITION IN CROP PLANTS

Stroud, J.L., Zhao, F.J., Buchner, P., et al. (2010) Impacts of sulphur nutrition on micronutrient (Se and Mo) concentrations in wheat grain. Journal of Cereal Science 52, 111–113. Stuiver, C.E.E., De Kok, L.J., & Westerman, S. (1997) Sulfur deﬁciency in Brassica oleracea L.: development, biochemical characterization, and sulfur/ nitrogen interactions. Russian Journal of Plant Physiology 44, 505–513. Stuiver, C.E.E., Koralewska, A., Posthumus, F.S., & De Kok, L.J. (2009) The impact of sulfur deprivation on root formation, and activity and expression of sulfate transporters in Chinese cabbage. In: Sulfur Metabolism in Plants: Regulatory Aspects, Signiﬁcance of Sulfur in the Food Chain, Agriculture and the Environment (eds. A. Sirko, L.J. De Kok, S. Haneklaus, et al.), pp. 61–68. Margraf Publishers, Weikersheim. Stulen, I. & De Kok, L.J. (1993) Whole plant regulation of sulfate uptake and metabolism—a theoretical approach and comparison with current ideas on regulation of nitrogen metabolism. In: Sulfur Nutrition and Assimilation in Higher Plants; Regulatory, Agricultural and Environmental Aspects (eds. L.J. De Kok, I. Stulen, H. Rennenberg, et al.), pp. 77–91. SPB Academic Publishing, The Hague. Takahashi, H. (2005) Functions and regulation of plant sulfate transporters. In: Sulfur Transport and Assimilation in Plants in the Post Genomic Era (eds. K. Saito, L.J. De Kok, I. Stulen, et al.), pp. 13–21. Backhuys Publishers, Leiden. Takahashi, H., Watanabe-Takahashi, A., Smith, F.W., et al. (2000) The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana. The Plant Journal 23, 171–182. Ter Steege, M.W., Stulen, I., Wiersema, P.K., et al. (1998) Growth requirement for N as a criterion to assess the effects of physical manipulation on nitrate uptake ﬂuxes in spinach. Physiologia Plantarum 103, 181–192. Ter Steege, M.W., Stulen, I., Wiersema, P.K., et al. (1999) Efﬁciency of nitrate uptake in spinach: impact of external nitrate concentration and relative growth rate on nitrate inﬂux and efﬂux. Plant and Soil 208, 125–134. Tomatsu, H., Takano, J., Takahashi, H., et al. (2007) An Arabidopsis thaliana high-afﬁnity molybdate transporter required for efﬁcient uptake of molybdate from soil. Proceedings of the National Academy of Sciences of the United States of America 104, 18807–18812.

309

Walker, K. (2002) Sulphur fertilizer recommendations in Europe. In: Proceedings 506, pp. 1–22. International Fertilizer Society, York. Westerman, S., De Kok, L.J., & Stulen, I. (2000) Interaction between metabolism of atmospheric H2S in the shoot and sulfate uptake by the roots of curly kale (Brassica oleracea L.). Physiologia Plantarum 109, 443–449. Westerman, S., Blake-Kalff, M.M.A., De Kok, L.J., & Stulen, I. (2001a) Sulfate uptake and utilization by two varieties of Brassica oleracea with different sulfur need as affected by atmospheric H2S. Phyton 41, 49–62. Westerman, S., Stulen, I., Suter, M., et al. (2001b) Atmospheric H2S as sulfur source for Brassica oleracea: consequences for the activity of the enzymes of the assimilatory sulfate reduction pathway. Plant Physiology and Biochemistry 39, 425–432. Yang, L., Stulen, I., & De Kok, L.J. (2006a) Sulfur dioxide: relevance of toxic and nutritional effects for Chinese cabbage. Environmental and Experimental Botany 57, 236–245. Yang, L., Stulen, I., & De Kok, L.J. (2006b) Impact of sulfate nutrition on the utilization of atmospheric SO2 as sulfur source for Chinese cabbage. Journal of Plant Nutrition and Soil Science 169, 529–534. Yoshimoto, N., Takahashi, H., Smith, F.W., et al. (2002) Two distinct high-afﬁnity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots. The Plant Journal 29, 465–473. Yoshimoto, N., Inoue, E., Saito, K., et al. (2003) Phloem-localizing sulfate transporter, Sultr1;3, mediates re-distribution of sulfur from source to sink organs in Arabidopsis. Plant Physiology 131, 1511–1517. Yoshimoto, N., Inoue, E., Watanabe-Takahashi, A., et al. (2007) Posttranscriptional regulation of highafﬁnity sulfate transporters in Arabidopsis by sulfur nutrition. Plant Physiology 145, 378–388. Zhao, F.J., Hawkesford, M.J., & McGrath, S.P. (1999) Sulphur assimilation and effects on yield and quality of wheat. Journal of Cereal Science 30, 1–17. Zhao, F., Tausz, M., & De Kok, L.J. (2008) Uptake, distribution and subcellular transport of sulfate. In: Sulfur Metabolism in Phototrophic Organisms (eds. R. Hell, C. Dahl, D.B. Knaff & T. Leustek), pp. 15–30. Springer, Dordrecht. Zhu, Y.G., Pilon-Smits, E.A.H., Zhao, F.J., et al. (2009) Selenium in higher plants: understanding mechanisms for biofortiﬁcation and phytoremediation. Trends in Plant Science 14, 436–442.

Chapter 15

Iron Nutrition and Implications for Biomass Production and the Nutritional Quality of Plant Products Jean-François Briat

Abstract Iron homeostasis is required to avoid deficiency or toxicity of this metal, which would be deleterious for the physiology and the growth of the plant. Basic knowledge of the molecular and cellular mechanisms establishing iron homeostasis concerns iron uptake from the soil, long-distance trafficking, and subcellular compartmentation and storage. These various aspects and the integration of these mechanisms at the wholeplant level have been studied in detail in the last decade. A direct output of this knowledge will be the development of new breeding strategies and new biotechnological approaches to improve (1) the plant resistance to iron deficiency, and as a consequence, biomass production, and (2) the nutritional quality of plant products, in particular the increase of iron content and bioavailability.

Introduction Agriculture will have to face tremendous changes in the near future. The major chal-

lenges will be increasing biomass productivity, while simultaneously improving plant product quality, and achieving this in a sustainable way within the perspective of a global climate modification involving CO2 concentration and temperature increases (Pretty, 2008). Mineral nutrients are key components of these challenges as they are essential for both plant productivity and quality, and they can, through fertilization, impact the environment. Among minerals, metals such as magnesium, manganese, and iron are essential because of their role in CO2 fixation by the photosynthesis process, iron being a key element in ensuring the electron flow through the Photosystem (PS) II-b6f/Rieske–PSI complex. It is well documented that iron is a limiting factor for biomass production since phytoplankton primary productivity in 30–40% of the world’s oceans is limited by availability of the micronutrient iron (Martin and Fitzwater, 1988). More recently, it was shown that iron was a limiting factor for biomass production by the model higher plant Arabidopsis thaliana (Ravet et al., 2009a). In both cases, the iron storage

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 311

312

NUTRIENT USE EFFICIENCY IN CROPS

IRON UTILIZATION IRON STORAGE

BIOMASS PRODUCTION QUALITY OF PRODUCTS

IRON ALLOCATION

IRON UPTAKE

Sensing

Nongrass plants Grass plants

GENOTYPE Iron efficient Iron inefficient

IRON STATUS

AIR : Light, Temperature, CO2

ENVIRONMENT SOIL : pH, Water, N/P, Organic matter

Fig. 15.1. Biomass production and plant product quality as an output of iron homeostasis. The iron status of a plant is not only defined by the quantity of the metal at a given moment. It also includes the redox state of the metal, its speciation with chelating molecules, and its tissular, cellular, and subcellular compartmentalization. It is dependent of the interactions between a given genotype and the various parameters of the environment. This iron status is sensed by the plant and transduced to regulate iron uptake, its distribution throughout the plant, and ultimately its utilization for physiological purposes, or its storage. The output of this iron homeostasis process will directly impact plant biomass and the quality of plant products.

protein ferritin was shown to be necessary to buffer transiently the iron in a safe form (Marchetti et al., 2009; Ravet et al., 2009a), revealing that iron-dependent biomass production requires the control of iron homeostasis. It is also well established that plant product quality in the future must include an improvement of nutritional content and availability. Among the micronutrients, iron is of primary importance because it is the most commonly deficient micronutrient in the human diet, and iron deficiency affects an estimated 2 billion people. In this context, it was proposed that feeding humans safely with enough iron directly within their diet could become possible by using iron-fortified transgenic plants overexpressing ferritin (Newell-McGloughlin, 2008). Reaching

such a goal requires an integrated knowledge of the establishment and control of iron homeostasis in plants, which will be reviewed in the first part of this chapter. The second part of this chapter will address the relationship between iron homeostasis and plant productivity and plant product quality (Fig. 15.1). Iron uptake by the roots Molecular components involved in iron uptake Iron enters the plant via the roots from where it is distributed inside the plant. According to the plant family considered—that is, graminacea plants versus other plants—two

IRON NUTRITION

mechanisms prevail for mining iron from the soil solution. They involve respectively chelation of the ferric iron (Fe3+) by small organic molecules, or its reduction in its ferrous form (Fe2+) prior to transport across the plasmalemma of root epidermal cells. These processes were recently reviewed in detail (Morrissey and Guerinot, 2009), and they will be briefly summarized here prior to considering lesser known aspects of iron fertilization and apoplasmic occurrence of iron. The iron chelation mechanism occurring in graminacea plants relies on the synthesis of methionine derivatives known as phytosiderophores and belonging to the mugineic acid family (Mori and Nishizawa, 1987). Methionine is converted to Sadenosylmethionine (SAM) by SAM synthetase (Shojima et al., 1989), and this enzyme activity shows no difference in irondeficient and iron-sufficient barley roots (Takizawa et al., 1996). Therefore, constitutive SAM synthase activity is likely to be sufficient for mugineic acid (MA) synthesis. Then, nicotianamine synthase (NAS) combines three molecules of SAM to form one molecule of nicotianamine (NA) (Shojima et al., 1989). Barley, rice, or corn NASs are induced in roots at a transcriptional level in response to low iron supply (Herbik et al., 1999; Higuchi et al., 1999, 2001; Inoue et al., 2003; Mizuno et al., 2003). Although NA is produced by both monocotyledons and dicotyledons, the subsequent steps leading to mugineic acid synthesis are specific to grasses. The critical enzymes in this specific pathway are nicotianamine aminotransferase (NAAT) (Takahashi et al., 2001) followed by deoxymugineic acid synthase (DMAS) (Bashir et al., 2006), which catalyze the removal of an amino residue from NA, resulting in the production of 2′-deoxymugineic acid, the precursor of all other mugineic acids. NAAT and DMAS activity are strongly induced by

313

iron deficiency in roots and represent a limiting step in the production of mugineic acids. Subsequent hydroxylation of 2′deoxymugineic acid results in the formation of other members of the mugineic acid family. Two barley cDNAs specifically expressed in iron-deficient roots, HvIDS2 and HvIDS3 (IRON DEFICIENCY SPECIFIC), were shown to encode dioxygenases involved in the production of 3epihydroxy-2′-deoxymugineic acid and 3epihydroxy–mugineic acid (Nakanishi et al., 2000; Kobayashi et al., 2001). In contrast to the biosynthetic pathway of mugineic acids, the molecular mechanisms of phytosiderophore secretion in the rhizosphere remain poorly understood. It has been suggested that vesicular transport may be involved since the appearance of swollen vesicles in iron-deficient barley roots correlates with phytosiderophore release. The production of phytosiderophores is increased in response to iron deficiency, and tolerance to iron deficiency is correlated to the quantity and the type of phytosiderophores secreted (Negishi et al., 2002). The yellow stripe 3 (ys3) maize mutant shows interveinal chlorosis characteristic of iron deficiency, due to a defect in MA secretion, and can be rescued by exogenous application of MAs or cocultivation with wild-type plants (Lanfranchi et al., 2002). The uptake of Fe(III)–PS complexes in grasses occurs through a specialized transporter. The gene encoding this transporter was discovered by investigating the yellow stripe 1 (ys1) mutant of maize, which is unable to respond to iron deficiency due to a defect in the uptake of Fe(III)–PS complexes. The ZmYS1 gene encodes a plasma membrane protein belonging to the oligopeptide transporter (OPT) family (Curie et al., 2001). ZmYS1 mRNA and protein are upregulated by iron deficiency in roots and shoots, where ZmYS1 functions as a proton-coupled symporter to transport Fe(III)-PS and Fe(III)-NA (Curie

NUTRIENT USE EFFICIENCY IN CROPS

SHOOT

YSLs Fe(III)-ITP Fe(II)-NA

FRD3 FPN1

? FPN2

I Iron

AHA2 FRO2 IRT1

PHLOEM

FRO3 FRO6 IRTs Fe(III)-citrate

et al., 2001; Roberts et al., 2004; Schaaf et al., 2004). Nongrass plants respond to iron deficiency with both morphological and physiological changes. Adaptative morphogenesis of roots in response to iron deficiency includes root hair formation, swelling of root tips, as well as enhanced lateral root development and reduced lateral root growth (Schmidt, 1999). +H-ATPases are involved in these processes, and it was recently reported that, in A. thaliana, two out of 12 + H-ATPase isoforms participate to establish the root responses to iron deficiency. The + H-ATPase encoded by the AHA2 gene is required to acidify the rhizosphere, whereas the one encoded by the AHA7 gene promotes root hair development (Santi and Schmidt, 2009). In addition to acidification, the second physiological response to iron deficiency in nongrass plants is an enhanced Fe3+ reduction capacity of the roots (Yi and Guerinot, 1996). Ferric reduction takes place at the plasma membrane of root epidermal cells. It catalyzes transmembrane (TM) electron transport from cytosolic reduced pyridine nucleotides to extracellular iron compounds serving as electron acceptors. The characterization of Arabidopsis mutants (frd1 mutants) lacking induction of Fe3+-chelate reductase under iron-deficient conditions confirms that iron must be reduced prior to its transport and that Fe3+ reduction can be uncoupled from proton release (Yi and Guerinot, 1996). The Arabidopsis Fe3+chelate reductase gene FRO2 has been cloned, based on sequence similarity to the yeast FRE genes (Robinson et al., 1999). FRO2-like genes were identified from other plant species such as pea and tomato (Waters et al., 2002; Li et al., 2004). After its reduction, iron is then transported across the root plasma membrane as the ferrous (Fe2+) form via a divalent metal transporter (Fig. 15.2). In Arabidopsis, the IRT1 gene encodes the founding member of this class of eukary-

XYLEM

314

Va

IRT2 Ve

ROOT

Fig. 15.2. Schematic representation of iron uptake by

Arabidospsis roots and of its long-distance trafficking throughout the plant. Iron from the soil is solubilized by acidification of the rhizosphere, which is mediated by the AHA2 H+-ATPase. Fe(III)-chelates are then reduced by the FRO2 ferric-chelate reductase (belonging to the NADPH oxidase family). The resulting Fe(II) is then transported across the plasmalemma of root epidermal cells by the IRT1 transporter. This rapid intake of iron within cortex root cells being potentially toxic, the metal can be transiently stored (1) in vacuoles (Va), where it is uploaded by the FPN2 transporter belonging to the ferroportin family, or (2) in uncharacterized vesicles (Ve) via the activity of the IRT2 transporter. Iron unloading within the xylem sap requires citrate, which chelates Fe(III) for long-distance circulation; the FRD3 transporter, belonging to the MATE family, has been characterized as a citrate effluxer important for iron translocation from root to shoot by the xylem sap. The ferroportin FPN1 is also involved in iron transport into the xylem vessels. Reduction of Fe(III)-chelates and transport within the shoots requires reductases, likely encoded by the FRO3 and/or FRO6 genes, and transporters of the IRT and YSL families. These latter transporters are important for long-distance transport of iron chelated to nicotianamine, a S-adenosylmethionine derivative, in the phloem sap, where Fe(III) can also be found complexed with small peptides, such as the ITP protein. YSLs are also important for iron cycling between the xylem and the phloem streams.

IRON NUTRITION

otic metal ion transporters (Eide et al., 1996), referred to as the ZIP (ZRT, IRT-like transporters) gene family (Guerinot, 2000), with related sequences in plants, yeast, animals, and humans. IRT1 is a protein with eight TM domains; IRT1 mediates uptake of manganese, zinc, and cadmium in yeast cells (Korshunova et al., 1999). In plants, IRT1 mediates transport of manganese, zinc, cadmium, and cobalt (Vert et al., 2002). Determinants for this broad substrate specificity of IRT1 have been investigated by sitedirected mutagenesis (Rogers et al., 2000). Mutant plants of irt1 are chlorotic and have a severe growth defect in soil, which can be rescued by foliar application of iron (Henriques et al., 2002; Varotto et al., 2002; Vert et al., 2002). IRT1 homologs have also been characterized in pea, tomato, and other plants (Cohen et al., 1998; Eckhardt et al., 2001). Rice has a unique behavior since in addition to the Fe3+-chelate uptake shared by all grass plants, it is also able to acquire iron through the uptake of Fe2+ through an IRT1 ortholog, as observed for nongrass plants (Ishimaru et al., 2006). Moreover, yeast ferric chelate reductase, when expressed in transgenic rice plants, increases the iron efficiency of these plants under iron-limiting conditions, compared with untransformed plants (Ishimaru et al., 2007). Additional factors should be considered in addition to this classical view of the molecular actors of iron uptake by roots. The first concerns the biology of the plant. In addition to the already characterized components of iron uptake, the roles of many other genes are likely to be discovered. This claim is documented by a recent report showing that in A. thaliana, up to 85% of the genes expressed in a particular region of the root are differentially regulated (Dinneny et al., 2008). Indeed, the ability to study iron metabolism in the various cell layers of a root is necessary and will be

315

required to improve our knowledge of these processes. From an agronomical point of view, upstream of the plant processes described above, it is important to consider that iron uptake by crops in the field will depend on the iron status, which can be defined by the availability of this metal being itself dependent of the interaction between a given genotype and the soil conditions (for review see Robin et al., 2008, Fig. 15.1). The solubility of soil iron is in particular considerably affected by the complexation or chelation of Fe3+ by organic ligands. Some of these organic ligands are produced by soil microorganisms (e.g., bacterial siderophores) or secreted by plant roots (organic acids such as citrate, or phenolic compounds). The amount and composition of humic acids of a soil also influence the iron status. The role of humic acids on the availability of micronutrients, including iron, is still a matter of debate that can be explained by their complex action. In the case of iron, humic substances not only contribute to increasing iron bioavailability through their chelating properties, but also have redox properties (Weber et al., 2006). These properties are related to phenolic groups contributing to Fe(III) reduction (Szilâgyi, 1971; Deiana et al., 1995; Chen et al., 2003). The chemical reduction of Fe(III) by humic substances is strongly pH dependent, the highest reduction capacities occurring at pH = 3 (Chen et al., 2003). As pH increases, humic substances are more frequently bound to metal cations and therefore have a decreased reducing ability (Chen et al., 2003). In addition to organic matter, nitrogen fertilization could contribute to determining the fate of iron availability since it has been reported that NO3 nutrition can promote iron deficiency chlorosis of sunflower leaves by inhibiting iron acquisition by roots due to high pH at the root surface (Nikolic and Römheld, 2003).

316

NUTRIENT USE EFFICIENCY IN CROPS

Regulation of iron uptake The iron uptake systems described above are upregulated transcriptionally in response to iron deficiency, both in grass and in nongrass plants (Walker and Connolly, 2008; Fig. 15.3). The orthologous genes of the Arabidopsis AtIRT1 and AtFRO2 genes are not expressed in response to iron deficiency in the tomato fer mutant, suggesting that this gene controls the expression of the uptake system, directly or indirectly (Bereczky et al.,

(A)

(B) Barley/Rice

Arabidopsis

XXX

2003; Li et al., 2004). The corresponding gene was cloned, revealing that it encodes a transcription factor belonging to the basic helix–loop–helix (bHLH) family (Ling et al., 2002). A FER gene ortholog is present in the Arabidopsis genome (Jakoby et al., 2004), able to complement the fer mutation when expressed in this tomato genetic background (Yuan et al., 2005). This gene was independently characterized by Colangelo and Guerinot (2004) and named FIT1 for iron deficiencyinduced transcription factor 1 (Fig. 15.3A).

FRO2 IDE2

IDE1

IDS2

IRT1

IRT1 mRNA RNA

K146

bHLH38

IDEF2

IDEF1

IRO2 RegTFs

IRO2

bHLH39 K171

FIT1

IRT1 protein XXXXXX

XXX

CACGTG

X

FeDRG

FeDRG

Degradation

Transcription factor networks for the regulation of iron uptake and homeostasis in grass and nongrass plants. (A) In Arabidopsis, FIT1 belongs to the bHLH family of transcription factors. It interacts with two other bHLHs (bHLH38 and 39) to regulate the increase in transcript abundance of the FRO2 reductase, and to control the expression of an uncharacterized gene (X) involved in the repression of the IRT1 protein degradation. The ciselement(s) recognized by FIT1 (XXX) are so far uncharacterized. The posttranslational regulation of the IRT1 protein stability requires two lysine residues (K146 and K171), suggesting that ubiquitination and proteasome-mediated degradation could participate in the turnover of this metal transporter. (B) In grass plants, the IDE1 and IDE2 ciselements were initially described within the promoter sequence of the barley IDS2 gene, which encodes a 2-oxoglutarate-dependent dioxygenase required for deoxymugineic acid synthesis. The trans-acting factors IDEF1 and IDEF2 able to bind these regulatory elements have been characterized. IDEF1 also regulates the expression of another transcription factor (IRO2) in response to iron deficiency. The IRO2 transcription factor is either (1) involved in the direct transactivation of some FeDRG (Fe-deficiency-response-genes) by binding to a CACGTG core sequence within FeDRG promoters, or (2) by controlling the expression of IRO2-regulated-transcription-factors (IRO2RegTFs) that will recognize as yet uncharacterized cis-elements (XXXX) within the promoter sequence of some FeDRG. Fig. 15.3.

IRON NUTRITION

FIT1 is required for regulating the FRO2/IRT1 iron uptake system at two different levels. It transcriptionally controls Fe(III)-chelate reductase FRO2 mRNA abundance, and posttranscriptionally controls the amount of the Fe(II) transporter protein IRT1. However, real-time polymerase chain reaction (RT–PCR) indicated that IRT1 and FRO2 transcript levels were reduced in fit-1 mutants grown in sufficient iron. The IRT1 protein, which was reported to be drastically less abundant in –Fe treated fit-1 (Colangelo and Guerinot, 2004), was also accumulated several fold less in fit-1 mutants grown in iron-replete conditions (Séguéla et al., 2008). Therefore, IRT1 and FRO2 are less expressed in fit-1 than in wild type, but they are still regulated by iron availability. Although, FIT1 is important to control the iron uptake machinery, it seems to do so by contributing to the general level of gene expression rather than by controlling the iron starvationinduced response. The specificity of the iron regulation of the uptake system in nongrass plants could therefore be provided by the interaction of FIT with other transcription factors. Indeed, the FIT gene is itself controlled by the iron status of the plant (Colangelo and Guerinot, 2004; Brumbarova and Bauer, 2005), consistent with the hypothesis that regulators could act upstream or interact with FIT to regulate the plant response to iron deficiency. Such a hypothesis is reinforced by the observation that four other bHLH transcription factors participate in the regulation of the iron deficiency response of nongrass plants (Yuan et al., 2005; Wang et al., 2007; Yuan et al., 2008); Two (bHLH38 and bHLH39) physically interact with FIT (Fig. 15.3A), and transgenic plants that constitutively coexpress either bHLH38 or bHLH39 with FIT show iron-independent high-level expression of FRO2 and IRT1 (Yuan et al., 2008).

317

In addition to the transcriptional control of the iron uptake system of roots of nongrass plants described above, a posttranscriptional control of IRT1 and FRO2 has been proposed (Connolly et al., 2002; Connolly et al., 2003). It is based on the observation that transgenic Arabidopsis plants transformed with a p35SCAMV::IRT1 or a p35SCAMV::FRO2 construct overexpress IRT1 and FRO2 transcripts regardless of the iron nutrition status of the plant, whereas the corresponding proteins are accumulated only under iron deficiency conditions. An iron-dependent control of the IRT1 and FRO2 protein stability was suggested, which recently received experimental support (Kerkeb et al., 2008). IRT1 contains an intracellular loop composed of a histidine, which might bind metals, and of two lysine residues (K146 or K171) that could serve as ubiquitination sites, known to be involved in the degradation of protein through the proteasome pathway. Mutation of the histidine or lysine residues did not eliminate the ability of IRT1 to transport iron or zinc. Expression of these variants in transgenic A. thaliana plants revealed that either K146 or K171 is required for ironinduced protein turnover. It was therefore hypothesized by these authors, that iron may signal modification and turnover of the A. thaliana IRT1 protein through K146 or K171 ubiquitination, resulting in the targeting of the protein to proteasome degradation (Kerkeb et al., 2008; Fig. 15.3A). In grass plants, the regulation of the iron uptake system has been studied at the transcriptional level in barley and rice (Fig. 15.3B). The barley IDS2 gene encodes a 2-oxoglutarate-dependent dioxygenase required for deoxymugineic acid synthesis, and is upregulated in response to iron deficiency (Nakanishi et al., 2000). Two cisacting elements of its promoter, named iron deficiency-responsive elements 1 and 2 (IDE1 and IDE2), act synergistically to

318

NUTRIENT USE EFFICIENCY IN CROPS

induce iron deficiency-specific expression of a reporter gene in transgenic plants (Kobayashi et al., 2003). Bioinformatic analysis indicated that many promoters of genes regulated in response to iron deficiency harbor sequences homologous to IDE1 and IDE2. Trans-acting factors interacting with the IDE1 and IDE2 cis-elements have been characterized (Kobayashi et al., 2007; Ogo et al., 2008). The rice IDEF1 protein belongs to the ABI3/VP1 family of transcription factors; in rice, IDEF1 knockdown plants grown under iron deficiency conditions are susceptible to early-stage iron deficiency. This is consistent with the observation that the IDEF1 expression level is positively correlated to the level of induction of known iron-regulated genes, just after the onset of iron starvation. However, this overall IDEF1 trans-activation became less evident in subsequent, later developmental stages. In fact, genes positively regulated by IDEF1 at this early stage all contain the IDE1 cis-element (CATGC) within their proximal promoter regions. These regions are also enriched in RY elements (CATGCA), known to regulate gene expression during seed maturation (Kobayashi et al., 2009). Indeed, expression of several iron deficiency-induced genes encoding late embryogenesis abundant proteins is increasingly regulated by IDEF1 at subsequent, later stages. A dual function of IDEF1 in the iron deficiency response in grass plants was therefore proposed (Kobayashi et al., 2010). This transcription factor would transactivate iron-regulated genes by interacting with IDE1 cis-elements at early stages. Transcriptional transactivation of seed maturation-related genes occurs later on, during the subsequent stages of iron deficiency, via RY elements. In rice, IDEF1 overexpression causes upregulation of OsIRO2, which encodes a bHLH transcription factor (Ogo et al., 2006; Fig. 15.3B). OsIRO2 is strongly expressed in both roots and shoots, specifically in response to iron

deficiency stress, and it has numerous homologs among graminaceous plants. The cisacting sequence bound by IRO2 was determined, and is found upstream of several genes involved in iron acquisition by grasses. The IDEF1 regulation of IRO2 reveals a potential network of transcription factors for the regulation of the iron responses in grass plants. Less information has been reported concerning the IDEF2 protein, which binds to the IDE2 cis-element; it is, however, known that it belongs to the NAC family of transcription factors (Kobayashi et al., 2007; Ogo et al., 2008). Iron distribution and compartmentation throughout the plant Long-distance trafficking of iron Once taken up by root epidermal cells, iron has to move through the symplast of cortical cells prior to reaching the pericycle and being loaded within the xylem vasculature to be transported to the aerial part of the plant via the sap (Fig. 15.2). Due to its reactivity with oxygen, iron will not diffuse freely in the cytoplasm. In A. thaliana, it could be safely stored within the vacuoles of root periphery cells as a consequence of the activity of one of the two ferroportin (FPN2) effluxers (Morrissey et al., 2009), or may reach the pericycle through various mechanisms; one of these mechanisms would require chelation of iron by still uncharacterized chaperones. Alternatively, iron compartmentalization within intracellular vesicles loaded by the IRT2 transporter (a close homolog of IRT1), and acting as a shuttle, has recently been proposed (Vert et al., 2009; Fig. 15.2). Organic acids, and especially citrate, have been suggested to be the main metal chelators in the xylem (Cataldo et al., 1988). It is, however, only very recently that it has

IRON NUTRITION

been unambiguously demonstrated that a triFe(III), tri-citrate complex (Fe(3)Cit(3)) was found in the xylem sap of iron-deficient tomato resupplied with iron. A second complex, a di-Fe(III), di-citrate complex was also detected along with Fe(3)Cit(3), with the allocation of iron between the two complexes depending on the iron to citrate ratio (Rellan-Alvarez et al., 2010). These reports suggest that active root transporters must load iron from the root cortex cells to the xylem and that citrate could play a role in this process. Such efflux iron transporters have recently been characterized at the molecular level in A. thaliana. The FRD3 gene is involved in citrate efflux into the xylem (Rogers and Guerinot, 2002; Fig. 15.2). It is a member of the multidrug and toxic compound extrusion (MATE) family, expressed in the root vasculature, and upregulated twofold in response to iron starvation. Genes involved in the iron deficiency response are constitutively expressed in an frd3 null mutant, consistent with its chlorotic phenotype. However, citrate (at 60% of wild type levels) is still measured in the xylem sap of this mutant, suggesting a role for other effluxers. Indeed, it has been recently proposed that the ferroportin FPN1 effluxer could also participate in the loading of iron into the root vasculature (Fig. 15.2). Yet fpn1 plants show no change in the iron deficiency response (Morrissey et al., 2009). Once in the leaves, Fe(III)-citrate is likely to be the substrate of leaf ferric chelate reductase since such an enzymatic activity has been described in leaf mesophyll cells (Bruggemann et al., 1993). In A. thaliana, some of the FRO genes could be involved in this process (Fig. 15.2), the best candidate being FRO3, and perhaps FRO2, which is also expressed in leaves in addition to roots, although to a much lower level (Mukherjee et al., 2006). Although presenting a low reductase activity when expressed in yeast, FRO6 has expression restricted to green

319

tissues and is regulated by light in a tissueor cell differentiation-specific manner (Feng et al., 2006). In pea, the fact that FRO1 is also expressed in leaves (Waters et al., 2002) makes it a good candidate for the function of leaf ferric reduction. Iron is not only trafficking through the xylem sap; mobility of iron from source to sink tissues via the phloem sap has also been reported (Stephan et al., 1994; Grusak, 1995). One of the molecules identified as a potential phloem metal transporter is NA (Stephan and Scholz, 1993). It is a ubiquitous molecule in the plant kingdom, used specifically in grass plants as a precursor for the synthesis of the siderophores of the mugineic acid family (see the “Molecular components involved in iron uptake” section). The three amino and three carboxy groups in the molecule enable the formation of hexadentate coordination, driving the formation of very stable octahedral chelates with a central metal ion. In vitro, NA is able to form stable complexes with manganese (Mn), Fe(II), and Fe(III), cobalt (Co), zinc (Zn), nickel (Ni), and copper (Cu) (reviewed by Curie et al., 2009). Long-distance transport of the Fe–NA complex, especially in the phloem, requires at least some of the yellow stripe-like (YSL) transporters (Fig. 15.2), a subclass of the OPT family, first identified by the characterization of the maize YS1 transporter of deoxymugineic acid (Curie et al., 2001; Curie et al., 2009). A common feature of the YSL genes is that their expression is limited to the vascular system. The precise localization of their expression to one type of vessel has not been systematically reported, except for A. thaliana AtYSL1 (xylem parenchyma) and Oryza sativa OsYSL2 (phloem companion cells). In other cases, the expression seems to be spread around or between the phloem and xylem, for example, Thlaspi caerulescens TcYSL3, and A. thaliana AtYSL3 or AtYSL5 (for review see Curie et al., 2009). Various

320

NUTRIENT USE EFFICIENCY IN CROPS

physiological roles of YSL proteins have been hypothesized (Curie et al., 2009). They could participate in the unloading of metals from the xylem sap through the uptake by xylem parenchyma cells, and in the loading of phloem sieve tubes via uptake by phloemassociated cells. In addition, their function in the xylem–phloem exchange (Fig. 15.2) could be important in all supporting tissues such as petioles and stems for the fine balance of NA–metal nutrition, and also in young organs or, where xylem is either not as yet differentiated or is interrupted, as for the seed coat and embryo. In A. thaliana seeds, AtYSL1 and AtYSL3 genes are required to maintain the amount of iron, zinc, and copper as well as NA (Le Jean et al., 2005; Waters et al., 2006) and could play important roles in circulating metals from the xylem to the phloem in this organ. In addition to NA, small peptides could also participate in the travel of iron in the phloem (Maas et al., 1988). Indeed, iron transport protein (ITP), a phloem protein of Ricinus communis, has been purified and shown to complex Fe(III) in vivo, but not Fe(II). ITP is a 96-amino acid protein belonging to the late embryogenesis abundant (LEA) family (Kruger et al., 2002). The preference of ITP for ferric iron is in agreement with the observation that only 4% of the total iron in the phloem exudate of R. communis seedlings is in the ferrous form (Schmidke et al., 1999). Although the stability constant (log K = [Fe-NA]/[Fe][NA]) of nicotianamine (NA) for Fe(III) is 20.6, and only 12.8 for Fe(II), the Fe(II)–NA complex possesses an unusual kinetic stability, explaining why NA is found complexed to Fe(II), and not to Fe(III), in the phloem sap (von Wiren et al., 1999). Since there is a low but significant steady-state concentration of ferrous iron in the phloem (Maas et al., 1988), and since the bulk of iron in the phloem is chelated in the Fe(III) form by ITP (Krueger et al., 2002), it is speculated that NA could

shuttle iron by chelating Fe(II) from ITPbound Fe(III) during loading and unloading of the phloem. Such a hypothesis would imply the existence of an as yet uncharacterized redox system within the phloem for ensuring Fe(III)/Fe(II) cycling. Intracellular compartmentation and cellular iron homeostasis Until recently, very little information was available concerning intracellular iron movement in plant cells. However, although still incomplete, our knowledge of the molecules involved in subcellular iron compartmentation has tremendously improved (for review see Morrissey and Guerinot, 2009; Fig. 15.4). For a long time is has been hypothesized that vacuoles were likely to play a major role in iron storage and homeostasis. Indeed, in Arabidopsis, two elegant pieces of work have demonstrated that three transporters were necessary for the efflux and influx of iron in and out seed vacuoles. Vacuolar iron transporter (VIT) 1 is required for delivery of iron into storage vacuoles of vascular cells in the embryo, whereas NRAMP3/ NRAMP4 divalent metal transporters remobilize this vacuolar iron upon germination (Lanquar et al., 2005; Kim et al., 2006; Fig. 15.4). Mutant plants altered in these transporters exhibit severe defects during germination. Mitochondria contain iron proteins important for respiration, and they host a universal iron–sulfur cluster biogenesis machinery (Briat et al., 2007); thus, iron needs to enter this organelle. So far no data have been reported on potential mechanisms. With respect to iron efflux from mitochondria, it has been reported that the STA1 gene from Arabidopsis encodes a homolog of the yeast ATM1p (Kushnir et al., 2001), an adenosine triphosphate binding cassette (ABC) transporter located at the mitochon-

IRON NUTRITION

YSL ?

N Fe(III)

321

VIT1

V

Fe-NA? Fe(II) PIC1?

FRO7

[Fe-S]

Fer Fe(III)

C

? ?

[Fe-S]

Fer Fe(III)

Frataxin

M

NRAMP3/4

ATM3 (STA1)

[Fe-S]

Fig. 15.4. Subcellular distribution of iron within plant cells. The distribution of iron between the various subcellular compartments is an important aspect of iron homeostasis. A reduction step of Fe(III) by the FRO7 reductase is required prior to Fe(II) uptake by the chloroplast (C). It has been suggested that PIC1 could be responsible for this transport activity. Inside the chloroplast, iron can be buffered within the iron storage protein ferritin (Fer) to avoid iron-mediated oxidative stress, or used for [Fe-S] cluster biogenesis, since these organelles are autonomous for this activity. The mitochondria (M) are also autonomous for their iron–sulfur cluster biosynthesis by a universal mitochondrial system conserved in all eukaryote cells, and the STA1/ATM3 ABC transporter is required for mitochondrial iron efflux. Such an iron efflux by the chloroplast has been hypothesized, but so far such a function has not been attributed to any transporter. Iron uptake by plant mitochondria is so far uncharacterized at the molecular level. Although ferritin could also be present in plant mitochondria of some specific cell types, the main form of iron storage in this organelle is achieved by the frataxin protein, as observed in yeast or animal and human mitochondria. The vacuole (V) also plays a major role in iron storage and homeostasis. Iron loading of vacuoles is performed by the VIT1 transporter and likely by some members of the YSL family of transporters. Iron efflux from the vacuole, in particular during seed germination, requires two members of the NRAMP family of transporters, namely NRAMP3 and NRAMP4. NA, nicotianamine; N, nucleus.

drial inner membrane (Fig. 15.4) and involved in the export of iron–sulfur clusters from the mitochondrial matrix to the cytoplasm (Kispal et al., 1999; Lill and Kispal, 2000). A subfamily of three A. thaliana halfmolecule adenosine triphosphate (ATP)binding cassette transporters, ATM1, 2 and 3, have been localized to the mitochondria when expressed in yeast; however, only ATM3 was able to rescue the Δatm1 yeast mutant (Chen et al., 2007). Consistent with this finding, the activity of cytosolic aconitase, an iron–sulfur-dependent enzyme, was strongly decreased across a range of atm3 alleles, whereas mitochondrial and plastid iron–sulfur enzymes were unaffected (Bernard et al., 2009). However, in contrast

to mutants in the yeast and mammalian orthologs, Arabidopsis atm3 mutants did not display a dramatic iron homeostasis defect and did not accumulate iron in mitochondria. The bulk of iron in leaves is found within the chloroplasts, where it is engaged in the photosynthetic process. Iron transport into the plastids is therefore of primary importance in plant physiology, and paradoxically this subcellular iron transport activity is poorly documented. Light was shown to be necessary for efficient iron transport from the leaf veins to the mesophyll cells. Iron uptake studies with isolated barley chloroplasts indicated that this process is also light dependent and requires a Fe(III)-chelate

322

NUTRIENT USE EFFICIENCY IN CROPS

reductase activity (Bughio et al., 1997). In agreement with this result, an inwarddirected Fe(II) transport across the chloroplast inner membrane occurs by a potential-stimulated uniport mechanism, as shown by stopped flow spectrofluorometry using inner membrane vesicles (Shingles et al., 2002). At the molecular level, the localization of ferric reductase oxidase 7 (FRO7), one of the eight members of the A. thaliana FRO family, to the chloroplast, reinforces the previous physiological data evidencing the necessity of a reduction step in the iron uptake process by this organelle (Fig. 15.4). Chloroplasts from fro7 loss-offunction mutants have 75% less Fe(III)chelate reductase activity, contain 33% less iron per microgram of chlorophyll than wild-type chloroplasts, and are altered in photosynthetic electron transport. Hence, fro7 seedlings show severe chlorosis and die without setting seeds when germinated in alkaline soil, unless watered with high levels of soluble iron (Jeong et al., 2008). A potential chloroplast iron transporter could be encoded by the A. thaliana PIC1 gene (Duy et al., 2007), which displays homology with cyanobacterial permease-like proteins. Yeast complementation with PIC1 and studies of a pic-1 knockout allele suggest that PIC1 functions in iron transport across the inner envelope of chloroplasts (Fig. 15.4). Cellular iron homeostasis is not only dependent on transporter activities in charge of an adequate iron allocation within various subcellular compartments. At least two soluble proteins, the ferritins (for review see Briat et al., 2010) and frataxin (for review see Ramirez et al., 2010), play a key role in cellular iron metabolism, and in particular within the plastid and mitochondria compartments (Fig. 15.4). Both these proteins are involved in iron storage and buffering. Ferritins are mainly found within plastids, but they can also be detected in mitochondria, whereas frataxin is exclusively a mitochondria protein. The structure of ferritins

is highly conserved between plants and animals, but contol of ferritin gene expression in response to iron excess occurs at the transcriptional level in plants, in contrast to animals, which regulate ferritin expression at the translational level. Reverse genetic and physiological approaches revealed strong links between plant ferritins and protection against oxidative stress. In contrast, their putative iron-storage function to furnish iron during various developmental processes is unlikely to be essential. Ferritins, by buffering iron, exert a finetuning of the quantity of metal required for metabolic purposes and help plants to cope with adverse situations, the deleterious effects of which would be amplified if no system had evolved to take care of free reactive iron. Frataxin has been recently identified in plants where it plays an important role in mitochondria biogenesis and in maintaining mitochondrial iron homeostasis, likely through a role in iron–sulfur cluster biosynthesis. From an integrative point of view, these various subcellular compartments are likely to cooperate in order to establish iron homeostasis at the cell level. Such integration has been recently supported by studying iron homeostasis in Arabidopsis seeds (Ravet et al., 2009b). Analysis of the expression of the seed-specific AtFer2 ferritin gene in different genetic backgrounds modified in iron homeostasis of plastid or vacuolar compartments (fer, nramp, and vit knockout mutants, and NRAMP and VIT overexpressors) revealed that ferritin stability in seeds depends on a proper allocation of iron from vacuoles to plastids. It highlights a potential crosstalk between the vacuolar and plastidial seed compartments for iron store allocation. Iron and plant productivity: iron interactions with light and CO2 The best evidence that iron is a limiting factor for biomass production comes from

IRON NUTRITION

mesoscale iron addition experiments launched more than 20 years ago, and which have unequivocally demonstrated that iron supply limits production in one third of the world’s ocean by controlling the dynamics of plankton blooms (Boyd et al., 2007). Iron, light, and photosynthesis Until recently, the physiological mechanisms driving biomass increase in oceanic phytoplankton in response to iron fertilization were not clearly identified. In one of these experiments, it was reported that iron supply was leading to a ninefold increase in chlorophyll concentration and that the maximum quantum yield of photosynthesis was doubled (Hiscock et al., 2008). The impact of iron on phytoplankton biomass was therefore attributed to its effect on lightlimited photosynthesis rates and not on light-saturated photosynthesis rates. This observation is consistent with the fact that iron deficiency decreases light-limited photosynthesis of phytoplankton by decreasing the synthesis of functional proteins for biogenesis or repair of reaction centers. The fact that photosynthetic reaction center core and electron transport chain proteins requiring iron are not synthesized or repaired under iron deficiency conditions is consistent with the observation of a reduced ability to process absorbed light energy into chemical energy. Furthermore, decrease in variable fluorescence, indicative of damaged photosystem II, and in reaction center turnover rate, revealing defects in the photosynthetic electron chain, are characteristics of iron limitation effects on photosynthesis by oceanic phytoplankton. From a molecular point of view, the more advanced characterization of the alterations of the structure and function of the photosynthetic apparatus in response to iron was obtained with the green unicellular algae Chamydomonas reinhardtii (Moseley et al., 2002; Busch et al., 2008; Petroutsos

323

et al., 2009). The antenna protein complexes are differentially affected by iron deficiency. It leads to a pronounced degradation of PSI. The induction of ferritin synthesis (a chloroplast located iron storage protein) correlated with the degree of PSI degradation during iron deficiency, and with the observation that the PSI level can be restored to normal within 24 h after iron repletion at the expense of the accumulated ferritin (Busch et al., 2008). This indicates that the iron– ferritin is likely to participate in the fast adjustment of the photosynthetic apparatus with respect to iron availability. RNAi strains with a reduced ferritin amount exhibit a delay in the degradation of PSI under iron deficiency, and these Chlamydomonas strains are more sensitive to photo-oxidative stress under high-light conditions. In addition to iron-deficiency-mediated degradation of PSI, a remodeling of the PSI-associated light-harvesting antenna (LHCI) also occurs. This adaptation is a sequential process; it starts with uncoupling the antenna from the PSI core and is followed by specific degradation of LHCs and induction of new LHCs, prior to ending with assembly of new antenna complexes in Fe-deficient cells (Moseley et al., 2002). Quantitative proteomic analysis has shown that the proton gradient regulation L1 (PGRL1) protein has an increased abundance in C. reinhardtii cells deprived of iron, compared with unstarved cells. This protein is one of 90 members of a family absolutely conserved across all the organisms containing a photosynthetic plastid. PGRL1 participates in cyclic electron transfer (CEF), as do the two A. thaliana orthologs, and is likely to be involved in the switch between cyclic (CEF) and linear photosynthetic electron transfer (LEF) (DalCorso et al., 2008; Petroutsos et al., 2009). In C. reinhardtii, PGRL1 also plays a role in modulating acclimation to iron likely by binding, sensing, or distributing iron, by changing reaction center stoichiometries leading to remodeling

324

NUTRIENT USE EFFICIENCY IN CROPS

photosynthetic electron transfer, and/or by partitioning the electron flow between LEF, CEF, and respiration in order to adapt ATP synthesis capacity to the overall cellular demand (Petroutsos et al., 2009). Chlamydomonas cell responses to iron deficiency are comparable to those reported for vascular plants. A proteolytically induced loss of photosynthetic components including both photosystems and the cyt b6/f complex make the cells chlorotic. Iron deficiency therefore alters both structure and function of chloroplasts from higher plants (for review see Briat, 2008). It has been reported recently that iron nutrition was also a growth-limiting factor for the model plant A. thaliana (Ravet et al., 2009a). Under regular tap water irrigation, the biomass of the wild-type Col0 ecotype plants was found lower than under Fe(III)-ethylenediamineN-N′-bis(2-hydroxyphenylacetic acid) (EDDHA) fertilization. However, this irondependent gain of biomass requires the presence of ferritins in vegetative organs since “ferrirrigation” of the fer1-3-4 triple ferritin mutant line is reduced when compared to wild-type plants. The reduced growth of fer1-3-4 is likely due to a decrease in CO2 fixation. In mature leaves, photosynthesis is not affected by the absence of ferritin. The observation that the electron flux through PSII is not different in the mutant and in Col0 under iron or water irrigation suggests that the absence of ferritins in the triple mutant does not have a severe impact on the photosynthetic electron transfer machinery. However, the decrease in CO2 fixation observed suggests that the photosynthetic electron transfer chain is less efficiently used by the Calvin cycle enzymes in the absence of ferritins (Ravet et al., 2009a). Iron and CO2 interactions Global warming is in part correlated to substantial increases in carbon dioxide (CO2),

which is one of the most important greenhouse gases in the atmosphere. It is likely to have a major impact on plant growth since it is known that elevated CO2 increases net photosynthesis rate in C3 plants by suppressing ribulose-1,5-bisphosphate oxygenase activity, decreasing photorespiration, and increasing carbon assimilates for plant growth and development (Lawlor and Mitchell, 2000). Consequently, the biomass of C3 plants increases under elevated CO2 concentration (Dijkstra et al., 2002). As a consequence of such an enhancement of plant growth, the demand for nutrients also increases, and macronutrient limitation under elevated CO2 has generally been found to suppress the CO2-mediated gain of biomass. In this context, it is of particular interest to extend this knowledge to micronutriments, and in particular to iron because of its major role in the photosynthetic process. Iron content in soil regularly exceeds plant requirements, but its bioavailability to plants is often limited (Robin et al., 2008), particularly in calcareous soils, which represent one third of cultivated lands. Therefore, plant iron nutrition is likely to be affected by the continued elevation of atmospheric CO2, which, in turn, will affect crop production. Indeed, two recent studies have addressed this question in grass (barley) and nongrass (tomato) plants (Haase et al., 2008; Jin et al., 2009). In tomato, a relative increase in biomass at elevated CO2 was observed both under iron-limited or iron-sufficient conditions. However, the biomass increase was greater under iron deficiency, compared with the measurement performed with ironreplete plants (Jin et al., 2009). The increase in tomato plant biomass under elevated CO2 and restricted iron supply therefore cannot be attributed to increased photosynthesis alone; raising CO2 concentration also results in the improvement of iron nutrition of the plants. The same observation was reported with barley, a grass plant using chelation

IRON NUTRITION

rather than reduction to take up iron from the soil. The stimulated biomass production in iron-sufficient and iron-deficient barley plants under elevated atmospheric CO2 treatments was observed both in hydroponics and in soil cultures (Haase et al., 2008). Iron biofortification and plant product quality General considerations: relationships between iron deficiency, sustainable agriculture, and public health Iron deficiency is one of the top 10 health challenges in modern society and is particularly prevalent in women of child-bearing age. It is the major cause of anemia, which affects at least 2 billion people worldwide. Symptoms associated with iron deficiency anemia can be severe, including increased susceptibility for infections and retardation of mental and psychomotor development, and of growth. More than half of the cases of iron deficiency anemia could be overcome by increasing the amount of iron in the diet. However, this goal is difficult to attain in developing countries where the population relies on plant products, including cereal grains, which contain very low levels of iron and antinutritional compounds such as phytate (Gomez-Galera et al., 2010). In Europe, cereals and their products provide 44% of the daily intake of iron, and there is increasing concern among public health authorities that the dietary supply of iron is below the lower recommended limit for some people. The “Green Revolution,” with the breeding of semi-dwarf, high-yielding crop cultivars that respond more to increased inputs of fertilizers, has markedly increased grain yield these last 50 years. This orientation of modern agriculture toward higher agronomic yield rather than the nutritional quality has raised the question of whether

325

increased grain yield may have resulted in a lower density of minerals in grain. Measurements, over a period of 160 years, were made with archived wheat grain and soil samples from the Broadbalk Wheat Experiment, established in 1843 at Rothamsted, United Kingdom, in order to evaluate changes in the mineral concentration of wheat grain, including iron, and to establish whether trends are due to plant factors (e.g., cultivar, yield) or changes in soil nutrient concentration (Fan et al., 2008). Among the various analyses performed, it was observed that the iron concentrations of grain remained stable between 1845 and the mid-1960s, but subsequently has decreased significantly, which coincided with the introduction of semi-dwarf, high-yielding cultivars. In comparison, the concentrations in soil have either increased or remained stable. In conclusion, multiple regression analysis showed that both increasing yield and harvest index were highly significant factors that explained the downward trend in grain mineral concentration, including iron. Iron improvement of human diets can be achieved in different ways (Gomez-Galera et al., 2010). The Flour Fortification Initiative has created a network of governmental and private sectors in several developing countries in Latin America to promote the fortification of wheat flour. It enabled the increase of the use of iron-fortified wheat flour in these countries from 18% in 2004 to 27% in 2007, helping 540 million people avoid iron deficiency (Centers for Disease Control and Prevention, 2008). However, food fortification programs are not easy to implement, especially in developing countries, and biofortification has been proposed as an alternative long-term approach for improving mineral nutrition. It aims to improve the mineral nutritional qualities of crops, both by increasing the mineral content of the edible part of plants, seeds being a prime target, and by improving

326

NUTRIENT USE EFFICIENCY IN CROPS

mineral bioavailability. Agronomical practices, plant breeding, and genetic engineering are used separately or in combination to reach these goals (Gomez-Galera et al., 2010). Iron biofortification and agronomical practices For iron, agronomical solutions to correct plant iron deficiency consist of using synthetic Fe(III)-chelates as fertilizers. Fe(III)chelates are generally derivatives from the family of ethylenediamine carboxylic acids. They are expensive and their use is therefore restricted to soil-less horticulture as well as to high added-value field-grown crops. Furthermore, these xenobiotics strongly impact metal availability and mobility in the soil because of their high persistence in the environment, raising questions about their sustainability in modern agriculture (Álvarez-Fernández et al., 2007). Alternatively to fertilization with iron chelates, it is well established that intercropping of grass and nongrass plants, especially on calcareous soils, where iron is poorly available, helps nongrass plants to resist iron deficiency (Zuo et al., 2000; Inal et al., 2007; Zuo and Zhang, 2009). Consequently, this agronomical practice can lead to 1.5 to 2.5 increases in iron content of shoots and seeds of the nongrass plant of such intercropping system. However, no advantage has been reported for the grass plant in this dual system. A simple explanation would be that phytosiderophores secreted by the grass plant help to solubilize iron, which is then actively reduced and taken up by the irondeficient nongrass partner. Molecular evidence to support this hypothesis has been recently reported for the peanut/maize couple (Ding et al., 2009). The Fe(III)chelate reductase activity of peanut and its transcript levels were higher in the maizeintercropped than in the monocropped

peanuts, and the maize roots secreted more phytosiderophore when intercropped with peanut. Iron biofortification and plant breeding Plant breeding offers the attractive possibility of increasing the iron content of crops. Indeed, natural variation for iron content of most plant species is well established. For example, the amount of iron in edible tissues varies between 6 and 22 mg kg−1 in rice, between 10 and 160 mg kg−1 in maize, and between 15 and 360 mg kg−1 in wheat (White and Broadley, 2005). Furthermore, including iron-enriched rice in nutrition trials of women in developing countries, where iron deficiency anemia is widespread, revealed that it has a positive impact on their health (Haas et al., 2005), validating the concept of biofortification. However, despite such genetic variation, cultivated varieties still have low iron levels, failing to provide the reference daily intake of this metal. Nevertheless, promising programs are still being developed. For example, the recent success in producing interspecific F1 hybrids between different iron-rich accessions of Aegilops longissima, and elite durum and bread wheat cultivars with low iron content have opened new possibilities for improved germplasm (Tiwari et al., 2008). As well as cereals, legume seeds are also of major importance for human diet, and they are therefore key targets for breeding with the aim of mineral biofortification, including iron. The model legume plants Lotus japonicus and Medicago truncatula were used to identify quantitative trait loci (QTLs), which determine the genetic basis for seed nutrient density, with the objective of facilitating the identification of synthenic regions in the legume genome, which will be of interest for the human diet, and to characterize beneficial alleles to assist

IRON NUTRITION

legume breeding programs (Klein and Grusak, 2009; Sankaran et al., 2009). Fiveseed iron QTLs were mapped on chromosome 1, 4, and 6 of L. japonicus (Klein and Grusak, 2009) and three-seed iron concentration QTLs, but no iron content QTL, were characterized in M. truncatula (Sankaran et al., 2009). Iron biofortification and transgenic approaches As an alternative to classical plant breeding methods, transgenic approaches for improving iron accumulation and bioavailability have been intensively developed in recent years. The most promising results with such a biotechnological strategy have been obtained by overexpressing the iron storage protein ferritin in transgenic plants, leading, on average, to a threefold increase in iron content. At the same time, it was reported that oral administration of plant ferritin (Beard et al., 1996), or a meal based on rice expressing high levels of ferritin (MurrayKolb et al., 2002), was suppressing rat anemia, demonstrating that plant ferritin constitutes an efficient iron source in the diet. Among the various significant trials performed, soybean ferritin has been expressed in several cereal crops under the control of an endosperm-specific promoter (Goto et al., 1999; Vasconcelos et al., 2003; Drakakaki et al., 2005; Qu et al., 2005), and pea ferritin has been constitutively expressed in rice (Hong-Xia et al., 2008). The impossibility of obtaining an improvement in iron content greater than threefold, although the amount of recombinant protein observed in western blots was often much higher, can be explained by bottlenecks existing upstream of iron sequestration within ferritins. Two such areas deserve attention: (1) iron unloading within the seeds is a critical control step, and NA and some of the YSL transporters play key roles in this process (Le Jean et al.,

327

2005), and (2) iron loading of seed plastids is an important parameter for the posttranscriptional stabilization of the ferritin within these organelles (Ravet et al., 2009b). Consistent with these remarks, it has been recently reported that coexpression of NAS and of ferritin in rice endosperm led to a more than sixfold increase in seed iron content (Wirth et al., 2009), doubling what was currently obtained with single ferritin gene transformation. As well as the importance of an overall increase in iron content, the bioavailability of this iron is also a major point to be addressed because of the presence of antinutrients in plants, among which phytate is well known. To address this, the combined expression of ferritin and phytase has been achieved in rice and maize, resulting in an increase in iron levels and availability as assayed in simulated digestion/absorption trials (Lucca et al., 2002; Drakakaki et al., 2005). Although encouraging, these biotechnological approaches need to be integrated with physiological and agronomical knowledge, keeping in mind that metal benefit and metal toxicity can be “two sides of the same coin” (Guerinot and Salt, 2001; Zhao and McGrath, 2009). It is well known that iron uptake by plant roots results from complex interactions between plant and soil within the rhizosphere, and not only from the plant genotype. Solid phases controlling iron solubility in soils, chemical speciation of iron in solution, importance of redox in the solubilization of iron, and the role of synthetic and natural chelates in transport processes that occur near roots are among soil-dependent factors determining iron bioavailability (Lindsay, 1995). In addition, plant iron uptake mechanisms are intimately linked with loading processes of other metals, some of which are potentially toxic for humans. Ferritin overaccumulation in transgenic tobacco leaves leads to an unnatural iron

328

NUTRIENT USE EFFICIENCY IN CROPS

sequestration. As a consequence, these transgenic plants behave as if iron deficient, and activate iron transport systems as revealed by an increase in root ferric reductase activity, explaining why these plants have an increased iron content (Van Wuytswinkel et al., 1999). This iron deficiency situation has been reported to be responsible for cadmium loading of plants, through activation of the IRT1 ferrous iron transporter (Vert et al., 2002). In grasses, phytosiderophores of the mugineic acid family are involved in root iron(III) uptake; this system, activated under iron deficiency conditions, is also able to transport zinc, copper, nickel, manganese, and cadmium (Schaaf et al., 2004). Indeed, the influence of various soil conditions on the increase in leaf iron content of various tobacco plant genotypes has been tested (Vansuyt et al., 2000). One control transgenic tobacco and two transgenic tobaccos overexpressing ferritin in the plastids or in the cytoplasm, respectively, were grown on five different soils, two of them being sewage sludge amended. Although a significant increase in leaf iron concentration was measured in transgenics overexpressing ferritin grown on three out of the five soils, this increase was not a general rule. On some soils, leaf iron concentration of control plants was as high as in transgenics grown on other soils. In addition, an increased phosphorus concentration in the two sewage sludge-amended soils correlated with a high leaf iron concentration in control plants, similar to the one measured in ferritin-transformed plants. Growing plants in vitro with various increasing phosphate concentrations revealed a direct phosphorous involvement in iron loading of control plants, at a similar level to the overexpressing ferritin plants. In addition, with one of the soils tested, not only iron but also manganese, zinc, and cadmium, and to a much lesser extent copper, nickel and lead, were found to be more abundant

in ferritin-transformed plants than in the control plants. These data indicate that the iron fortification of leaves, based on ferritin overexpression, could be limited in its biotechnological application because of its high soil dependence. Conclusion The molecular understanding of iron nutrition has tremendously increased in the last 10 years. Transporters and chelating molecules responsible for iron uptake, translocation, subcellular compartmentation, and storage have been described. More recently, the physiological integration of these molecular partners at the cell, organ, and even whole-plant levels has started to give promising results. A major challenge in the future concerns the regulation of these various targets, primarily driven by the iron status of the plant and its sensing. A major output of this molecular studies is to offer molecular markers for plant breeding or to propose alternative biotechnological transgenic approaches with aims of (1) remediating iron deficiency of plants on calcareous soils, and (2) increasing the iron content and bioavailability of edible parts of crops in order to improve the diet and to fight human iron deficiency anemia, a worldwide public health challenge. However, in order to be successful in the future, these applied science outputs will need to benefit of multidisciplinary programs promoting collaboration between molecular plant physiologists, soil scientists, agronomists, and plant, animal, and human nutritionists. References Álvarez-Fernández, A., Orera, I., Abadía, J., et al. (2007) Determination of synthetic ferric chelates used as fertilizers by liquid chromatographyelectrospray/mass spectrometry in agricultural matrices. Journal of the American Society of Mass Spectrometry 18, 37–47.

IRON NUTRITION

Bashir, K., Inoue, H., Nagasaka, S., et al. (2006) Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants. The Journal of Biological Chemistry 281, 32395–32402. Beard, J.L., Burton, J.W., & Theil, E.C. (1996) Purified ferritin and soybean meal can be sources of iron for treating iron deficiency in rats. Journal of Nutrition 126, 154–160. Bereczky, Z., Wang, H.Y., Schubert, V., et al. (2003) Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato. The Journal of Biological Chemistry 278, 24697–24704. Bernard, D.G., Cheng, Y., Zhao, Y., et al. (2009) An allelic mutant series of ATM3 reveals its key role in the biogenesis of cytosolic iron sulfur proteins in Arabidopsis. Plant Physiology 151, 590–602. Boyd, P.W., Jickells, T., Law, C.S., et al. (2007) Mesoscale iron enrichment experiments 1993– 2005: synthesis and future directions. Science 315, 612–617. Briat, J.F. (2008) Iron dynamics in plants. Advances in Botanical Research 46, 137–180. Briat, J.F., Gaymard, F., & Curie, C. (2007) Iron utilization and metabolism in plants. Current Opinion in Plant Biology 10, 276–282. Briat, J.F., Duc, C., Ravet, K., et al. (2010) Ferritins and iron storage in plants. Biochimica Biophysica Acta 1800, 806–814. Bruggemann, W., Maas-Kantel, K., & Moog, P.R. (1993) Iron uptake by leaf mesophyll cells: the role of the plasma membrane-bound ferric-chelate reductase. Planta 190, 151–155. Brumbarova, T. & Bauer, P. (2005) Iron-mediated control of basic helix-loop-helix protein FER, a regulator of iron uptake in tomato. Plant Physiology 137, 1018–1026. Bughio, N., Takahashi, M., Yoshimura, E., et al. (1997) Light-dependent iron transport into isolated barley chloroplasts. Plant Cell Physiology 38, 101–105. Busch, A., Rimbauld, B., Naumann, B., et al. (2008) Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photooxidative stress in response to iron availability in Chlamydomonas reinhardtii. The Plant Journal 55, 201–211. Cataldo, D.A., McFadden, K.M., Garland, T.R., et al. (1988) Organic constituents and complexation of nickel (II), iron (III), cadmium (II), and plutonium (IV) in soybean xylem exudates. Plant Physiology 86, 734–739. Centers for Disease Control and Prevention (2008) Trends in wheat-flour fortification with folic acid, iron. Worldwide, 2004 and 2007. Morbidity and Mortality Weekly Report 57, 8–10.

329

Chen, J., Gu, B.H., Royer, R.A., et al. (2003) The roles of natural organic matter in chemical and microbial reduction of ferric iron. Science of The Total Environment 307, 167–178. Chen, S., Sanchez-Fernandez, R., Lyver, E.R., et al. (2007) Functional characterization of AtATM1, AtATM2, and AtATM3, a subfamily of Arabidopsis half-molecule ATP-binding cassette transporters implicated in iron homeostasis. The Journal of Biological Chemistry 282, 21581–21571. Cohen, C.K., Fox, T.C., Garvin, D.F., et al. (1998) The role of iron-deficiency stress responses in stimulating heavy-metal transport in plants. Plant Physiology 116, 1063–1072. Colangelo, E.P. & Guerinot, M.L. (2004) The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response. The Plant Cell 16, 3400–3412. Connolly, E.L., Fett, J.P., & Guerinot, M.L. (2002) Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. The Plant Cell 14, 1347–1357. Connolly, E.L., Campbell, N.H., Grotz, N., et al. (2003) Overexpression of the FRO2 ferric chelate reductase confers tolerance to growth on low iron and uncovers post-transcriptional control. Plant Physiology 133, 1102–1110. Curie, C., Panaviene, Z., Loulergue, C., et al. (2001) Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409, 346–349. Curie, C., Cassin, G., Couch, D., et al. (2009) Metal movement within the plant: contribution of nicotianamine and yellow stripe 1-like transporters. Annals of Botany 103, 1–11. DalCorso, G., Pesaresi, P., Masiero, S., et al. (2008) A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis. Cell 132, 273–285. Deiana, S., Gessa, C., Marchetti, M., et al. (1995) Phenolic acid redox properties: pH influence on iron(III) reduction by caffeic acid. Soil Science Society of America Journal 59, 1301–1307. Dijkstra, P., Hymus, G., Colavito, D., et al. (2002) Elevate atmospheric CO2 stimulates aboveground biomass in a fire-regenerated scrub-oak system. Global Change Biology 8, 90–103. Ding, H., Duan, L., Wu, H., et al. (2009) Regulation of AhFRO1, an Fe(III)-chelate reductase of peanut, during iron deficiency stress and intercropping with maize. Physiologia Plantarum 136, 274–283. Dinneny, J.R., Long, T.A., Wang, J.Y., et al. (2008) Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320, 942–945.

330

NUTRIENT USE EFFICIENCY IN CROPS

Drakakaki, G., Marcel, S., Glahn, R.P., et al. (2005) Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Molecular Biology 59, 869–880. Duy, D., Wanner, G., Meda, A.R., et al. (2007) PIC1, an ancient permease in Arabidopsis chloroplasts, mediates iron transport. The Plant Cell 19, 986–1006. Eckhardt, U., Marques, A.M., & Buckhout, T.J. (2001) Two iron-regulated cation transporters from tomato complement metal uptake-deficient yeast mutants. Plant Molecular Biology 45, 437–448. Eide, D., Broderius, M., Fett, J., et al. (1996) A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proceedings of the National Academy of Sciences of the United States of America 93, 5624–5628. Fan, M.S., Zhao, F.J., Fairweather-Tait, S.J., et al. (2008) Evidence of decreasing mineral density in wheat grain over the last 160 years. Journal of Trace Elements in Medicine and Biology 22, 315–324. Feng, H., An, F., Zhang, S., et al. (2006) Light-regulated, tissue-specific, and cell differentiation-specific expression of the Arabidopsis Fe(III)-chelate reductase gene AtFRO6. Plant Physiology 140, 1345–1344. Gomez-Galera, S., Rojas, E., Sudhakar, D., et al. (2010) Critical evaluation of strategies for mineral fortification of staple food crops. Transgenic Research 19, 165–180. Goto, F., Yoshihara, T., Shigemoto, N., et al. (1999) Iron fortification of rice seeds by the soybean ferritin gene. Nature Biotechnology 17, 282–286. Grusak, M.A. (1995) Whole-root iron(III)-reductase activity throughout the life cycle of iron-grown Pisum sativum L. (Fabaceae): relevance to the iron nutrition of developing seeds. Planta 197, 111–117. Guerinot, M.L. (2000) The ZIP family of metal transporters. Biochimica Biophysica Acta-Biomembranes 146, 190–198. Guerinot, M.L. & Salt, D.E. (2001) Fortified foods and phytoremediation. Two sides of the same coin. Plant Physiology 125, 164–167. Haas, J.D., Beard, J.L., Murray-Kolb, L.E., et al. (2005) Iron-biofortified rice improves the iron stores of nonanemic Filipino women. Journal of Nutrition 135, 2823–2830. Haase, S., Rothe, A., Kania, A., et al. (2008) Responses to iron limitation in Hordeum vulgare L. as affected by the atmospheric CO2 concentration. Journal of Environmental Quality 37, 1254–1262.

Henriques, R., Jásik, J., Klein, M., et al. (2002) Knockout of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects. Plant Molecular Biology 50, 587–597. Herbik, A., Koch, G., Mock, H.P., et al. (1999) Isolation, characterization and cDNA cloning of nicotianamine synthase from barley—a key enzyme for iron homeostasis in plants. European Journal of Biochemistry 265, 231–239. Higuchi, K., Suzuki, K., Nakanishi, H., et al. (1999) Cloning of nicotianamine synthase genes, novel genes involved in the biosynthesis of phytosiderophores. Plant Physiology 119, 471–479. Higuchi, K., Watanabe, S., Takahashi, M., et al. (2001) Nicotianamine synthase gene expression differs in barley and rice under Fe-deficient conditions. The Plant Journal 25, 159–167. Hiscock, M.R., Lance, V.P., Apprill, A.M., et al. (2008) Photosynthetic maximum quantum yield increases are an essential component of the Southern Ocean phytoplankton response to iron. Proceedings of the National Academy of Sciences of the United States of America 105, 4775–4780. Hong-Xia, Y., Mei, L., Ze-Jian, G., et al. (2008) Evaluation and application of two high-iron transgenic rice lines expressing a pea ferritin gene. Rice Science 15, 51–56. Inal, A., Gunes, A., Zhang, F., et al. (2007) Peanut/ maize intercropping induced changes in rhizosphere and nutrient concentrations in shoots. Plant Physiology and Biochemistry 45, 350–356. Inoue, H., Higuchi, K., Takahashi, M., et al. (2003) Three rice nicotianamine synthase genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long-distance transport of iron and differentially regulated by iron. The Plant Journal 36, 366–381. Ishimaru, Y., Kim, S., Tsukamoto, T., et al. (2007) From the cover: mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil. Proceedings of the National Academy of Sciences of the United States of America 104, 7373–7378. Ishimaru, Y., Suzuki, M., Tsukamoto, T., et al. (2006) Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+. The Plant Journal 45, 335–346. Jakoby, M., Wang, H.Y., Reidt, W., et al. (2004) FRU (bHLH029) is required for induction of iron mobilization genes in Arabidopsis thaliana. FEBS Letters 577, 528–534. Jeong, J., Cohu, C., Kerkeb, L., et al. (2008) Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions. Proceedings of the National Academy of Sciences

IRON NUTRITION

of the United States of America 105, 10619–10624. Jin, C.W., Du, S.T., Chen, W.W., et al. (2009) Elevated carbon dioxide improves plant iron nutrition through enhancing the iron-deficiency-induced responses under iron-limited conditions in tomato. Plant Physiology 150, 272–280. Kerkeb, L., Mukherjee, I., Chatterjee, I., et al. (2008) Iron-induced turnover of the Arabidopsis IRONREGULATED TRANSPORTER 1 metal transporter requires lysine residues. Plant Physiology 146, 1964–1973. Kim, S.A., Punshon, T., Lanzirotti, A., et al. (2006) Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1. Science 314, 1295–1298. Kispal, G., Csere, P., Prohl, C., et al. (1999) The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins. The EMBO Journal 18, 3981–3989. Klein, M.A. & Grusak, M.A. (2009) Identification of nutrient and physical seed trait QTL in the model legume Lotus japonicus. Genome 52, 677–691. Kobayashi, T., Nakanishi, H., Takahashi, M., et al. (2001) In vivo evidence that Ids3 from Hordeum vulgare encodes a dioxygenase that converts 2’-deoxymugineic acid to mugineic acid in transgenic rice. Planta 212, 864–871. Kobayashi, T., Nakayama, Y., Itai, R.N., et al. (2003) Identification of novel cis-acting elements, IDE1 and IDE2, of the barley IDS2 gene promoter conferring iron-deficiency-inducible, root-specific expression in heterogeneous tobacco plants. The Plant Journal 36, 780–793. Kobayashi, T., Ogo, Y., Itai, R.N., et al. (2007) The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants. Proceedings of the National Academy of Sciences of the United States of America 104, 19150–19155. Kobayashi, T., Itai, R.N., Ogo, Y., et al. (2009) The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes. The Plant Journal 60, 948–961. Kobayashi, T., Nakanishi, H., & Nishizawa, N.K. (2010) Dual regulation of iron deficiency response mediated by the transcription factor IDEF1. Plant Signaling & Behavior 5, 157–159. Korshunova, Y.O., Eide, D., Clark, W.G., et al. (1999) The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range. Plant Molecular Biology 40, 7–44. Kruger, C., Berkowitz, O., Stephan, U.W., et al. (2002) A metal-binding member of the late embryogenesis

331

abundant protein family transports iron in the phloem of Ricinus communis L. The Journal of Biological Chemistry 277, 25062–25069. Kushnir, S., Babiychuk, E., Storozhenko, S., et al. (2001) A mutation of the mitochondrial ABC transporter Sta1 leads to dwarfism and chlorosis in the Arabidopsis mutant starik. The Plant Cell 13, 89–100. Lanfranchi, S., Basso, B., & Soave, C. (2002) The yellow stripe3 mutant of maize is defective in phytosiderophore secretion. Maydica 47, 81–184. Lanquar, V., Lelievre, F., Bolte, S., et al. (2005) Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron. The EMBO Journal 24, 4041–4051. Lawlor, D.W. & Mitchell, R.A.C. (2000) Crop ecosystem responses to climatic change: wheat. In: Climate Change and Global Crop Productivity (eds. K.R. Reddy & H.F. Hodges), p. 57. CAB International, Wallingford, UK. Le Jean, M., Schikora, A., Mari, S., et al. (2005) A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading. The Plant Journal 44, 769–782. Li, L., Cheng, X., & Ling, H.Q. (2004) Isolation and characterization of Fe(III)-chelate reductase gene LeFRO1 in tomato. Plant Molecular Biology 54, 125–136. Lill, R. & Kispal, G. (2000) Maturation of cellular Fe-S proteins: an essential function of mitochondria. Trends in Biochemical Sciences 25, 352–356. Lindsay, W.L. (1995) Chemical reactions in soils that affect iron availability to plants. A quantitative approach. In: Iron in Plants and Soils (ed. J. Abadia), pp. 7–14. Kluwer Academic Publishers, Amsterdam. Ling, H.Q., Bauer, P., Bereczky, Z., et al. (2002) The tomato fer gene encoding a bHLH protein controls iron-uptake responses in roots. Proceedings of the National Academy of Sciences of the United States of America 99, 13938–13943. Lucca, P., Hurrell, R., & Potrykus, I. (2002) Fighting iron deficiency anemia with iron-rich rice. Journal of the American College of Nutrition 21, 184S–190S. Maas, F.M., Wetering, D.A., Beusichem, M.L., et al. (1988) Characterization of phloem iron and its possible role in the regulation of Fe-efficiency reactions. Plant Physiology 87, 167–171. Marchetti, A., Parker, M.S., Moccia, L.P., et al. (2009) Ferritin is used for iron storage in bloom-forming marine pennate diatoms. Nature 457, 467–470. Martin, J.H. & Fitzwater, S. (1988) Iron deficiency limits phytoplankton growth in the north-east Pacific subarctic. Nature 331, 341–343.

332

NUTRIENT USE EFFICIENCY IN CROPS

Mizuno, D., Higuchi, K., Sakamoto, T., et al. (2003) Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status. Plant Physiology 132, 1989–1997. Mori, S. & Nishizawa, N.K. (1987) Methionine as a dominant precursor of phytosiderophores in Gramineae plants. Plant & Cell Physiology 28, 1081–1092. Morrissey, J. & Guerinot, M.L. (2009) Iron uptake and transport in plants: the good, the bad, and the ionome. Chemical Review 109, 4553–4567. Morrissey, J., Baxter, I.R., Lee, J., et al. (2009) The ferroportin metal efflux proteins function in iron and cobalt homeostasis in Arabidopsis. The Plant Cell 21, 3326–3338. Moseley, J.L., Allinger, T., Herzog, S., et al. (2002) Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus. The EMBO Journal 21, 6709–6720. Mukherjee, I., Campbell, N.H., Ash, J.S., et al. (2006) Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper. Planta 223, 1178–1190. Murray-Kolb, L.E., Takaiwa, F., Goto, F., et al. (2002) Transgenic rice is a source of iron for iron-depleted rats. Journal of Nutrition 132, 957–960. Nakanishi, H., Yamaguchi, H., Sasakuma, T., et al. (2000) Two dioxygenase genes, Ids3 and Ids2, from Hordeum vulgare are involved in the biosynthesis of mugineic acid family phytosiderophores. Plant Molecular Biology 44, 199–207. Negishi, T., Nakanishi, H., Yazaki, J., et al. (2002) cDNA microarray analysis of gene expression during Fe-deficiency stress in barley suggests that polar transport of vesicles is implicated in phytosiderophore secretion in Fe-deficient barley roots. The Plant Journal 30, 83–94. Newell-McGloughlin, M. (2008) Nutritionally improved agricultural crops. Plant Physiology 147, 939–953. Nikolic, M. & Römheld, V. (2003) Nitrate does not result in iron inactivation in the apoplast of sunflower leaves. Plant Physiology 132, 1303–1314. Ogo, Y., Itai, R.N., Nakanishi, H., et al. (2006) Isolation and characterization of IRO2, a novel iron-regulated bHLH transcription factor in graminaceous plants. Journal of Experimental Botany 57, 2867–2878. Ogo, Y., Kobayashi, T., Itai, R.N., et al. (2008) A novel NAC transcription factor IDEF2 that recognizes the iron deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants. Journal of Biological Chemistry 283, 13407–13417.

Petroutsos, D., Terauchi, A.M., Busch, A., et al. (2009) PGRL1 participates in iron-induced remodeling of the photosynthetic apparatus and in energy metabolism in Chlamydomonas reinhardtii. The Journal of Biological Chemistry 284, 32770–32781. Pretty, J. (2008) Agricultural sustainability: concepts, principles and evidence. Philosophical Transactions of the Royal Society B 363, 447–465. Qu, L.Q., Yoshihara, T., Ooyama, A., et al. (2005) Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds. Planta 222, 225–233. Ramirez, L., Zabaleta, E.J., & Lamattina, L. (2010) Nitric oxide and frataxin: two players contributing to maintain cellular iron homeostasis. Annals of Botany 105, 801–810. Ravet, K., Touraine, B., Boucherez, J., et al. (2009a) Ferritins control interaction between iron homeostasis and oxidative stress in Arabidopsis. The Plant Journal 57, 400–412. Ravet, K., Touraine, B., Kim, S.A., et al. (2009b) Posttranslational regulation of AtFER2 ferritin in response to intracellular iron trafficking during fruit development in Arabidopsis. Molecular Plant 2, 1095–1106. Rellan-Alvarez, R., Giner-Martinez-Sierra, J., Orduna, J., et al. (2010) Identification of a tri-iron(III), tricitrate complex in the xylem sap of iron-deficient tomato resupplied with iron: new insights into plant iron long-distance transport. Plant Cell Physiology 51, 91–102. Roberts, L.A., Pierson, A.J., Panaviene, Z., et al. (2004) Yellow Stripe1, expanded roles for the maize ironphytosiderophore transporter. Plant Physiology 135, 112–120. Robin, A., Vansuyt, G., Hinsinger, P., et al. (2008) Iron dynamics in the rhizosphere: consequences for plant health and nutrition. Advances in Agronomy 99, 183–225. Robinson, N.J., Proctor, C.M., Connolly, E.L., et al. (1999) A ferric-chelate reductase for iron uptake from soils. Nature 397, 694–697. Rogers, E.E. & Guerinot, M.L. (2002) FRD3, a member of the multidrug and toxin efflux family, controls iron deficiency responses in Arabidopsis. The Plant Cell 14, 1787–1799. Rogers, E.E., Eide, D.J., & Guerinot, M.L. (2000) Altered selectivity in an Arabidopsis metal transporter. Proceedings of the National Academy of Sciences of the United States of America 97, 12356–12360. Sankaran, R.P., Huguet, T., & Grusak, M.A. (2009) Identication of QTL affecting seed mineral concentrations and content in the model legume Medicago truncatula. Theoritical and Applied Genetics 119, 241–253.

IRON NUTRITION

Santi, S. & Schmidt, W. (2009) Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots. The New Phytologist 183, 1072–1084. Schaaf, G., Ludewig, U., Erenoglu, B.E., et al. (2004) ZmYS1 functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals. The Journal of Biological Chemistry 279, 9091–9096. Schmidke, I., Kruger, C., Frommichen, R., et al. (1999) Phloem loading and transport characteristics of iron in interaction with plant-endogenous ligands in castor bean seedlings. Physiologia Plantarum 106, 82–89. Schmidt, W. (1999) Mechanisms and regulation of reduction-based iron uptake in plants. The New Phytologist 141, 1–26. Séguéla, M., Briat, J.F., Vert, G., et al. (2008) Cytokinins negatively regulate the root iron uptake machinery in Arabidopsis through a growth-dependent pathway. The Plant Journal 55, 289–300. Shingles, R., North, M., & McCarty, R.E. (2002) Ferrous ion transport across chloroplast inner envelope membranes. Plant Physiology 128, 1022–1030. Shojima, S., Nishizawa, N.K., & Mori, S. (1989) Establishment of a cell free system for the biosynthesis of nicotianamine. Plant & Cell Physiology 30, 673–677. Stephan, U.W., Schmidke, I., & Pich, A. (1994) Phloem translocation of Fe, Cu, Mn and Zn in Ricinus seedlings in relation to the concentration of nicotianamine, an endogenous chelator of divalent metal ions, in different seedling parts. Plant and Soil 165, 181–188. Stephan, U.W. & Scholz, G. (1993) Nicotianamine: mediator of transport of iron and heavy metals in the phloem. Physiologia Plantarum 88, 522–529. Szilâgyi, M. (1971) Reduction of Fe3+ ion by humic acid preparations. Soil Science 111, 233–235. Takahashi, M., Nakanishi, H., Kawasaki, S., et al. (2001) Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes. Nature Biotechnology 19, 466–469. Takizawa, R., Nishizawa, N.K., Nakanishi, H., et al. (1996) The effect of an iron deficiency on S-adenosylmethionine synthetase in barley roots. Journal of Plant Nutrition 19, 1189–1200. Tiwari, V.K., Rawat, N., Neelam, K., et al. (2008) Development of Triticum turgidum subsp. durum— Aegilops longissima amphiploids with high iron and zinc content through unreduced gamete formation in F1 hybrids. Genome 51, 757–766.

333

Van Wuytswinkel, O., Vansuyt, G., Grignon, N., et al. (1999) Iron homeostasis alteration by ferritin overexpression in transgenic tobacco. The Plant Journal 17, 93–98. Vansuyt, G., Mench, M., & Briat, J.F. (2000) Soildependent variability of leaf iron accumulation in transgenic tobacco overexpressing ferritin. Plant Physiology and Biochemistry 38, 499–506. Varotto, C., Maiwald, D., Pesaresi, P., et al. (2002) The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana. The Plant Journal 31, 589–599. Vasconcelos, M., Datta, K., Oliva, N., et al. (2003) Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene. Plant Science 164, 371–378. Vert, G., Grotz, N., Dedaldechamp, F., et al. (2002) IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. The Plant Cell 14, 1223–1233. Vert, G., Barberon, M., Zelazny, E., et al. (2009) Arabidopsis IRT2 cooperates with the highaffinity iron uptake system to maintain iron homeostasis in root epidermal cells. Planta 229, 1171–1179. von Wiren, N., Klair, S., Bansal, S., et al. (1999) Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants. Plant Physiology 119, 1107–1114. Walker, E.L. & Connolly, E.L. (2008) Time to pump iron: iron-deficiency-signaling mechanisms of higher plants. Current Opinion in Plant Biology 11, 530–535. Wang, H.Y., Klatte, M., Jakoby, M., Baumlein, H., et al. (2007) Iron deficiency-mediated stress regulation of four subgroup Ib bHLH genes in Arabidopsis thaliana. Planta 226, 897–908. Waters, B.M., Blevins, D.G., & Eide, D.J. (2002) Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition. Plant Physiology 129, 85–94. Waters, B.M., Chu, H.H., Didonato, R.J., et al. (2006) Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds. Plant Physiology 141, 1446–1448. Weber, T., Allard, T., Tipping, E., et al. (2006) Modelling iron binding to organic matter. Environmental Science & Technology 40, 7488–7493. White, P.J. & Broadley, M.R. (2005) Biofortifying crops with essential mineral elements. Trends in Plant Sciences 10, 586–593. Wirth, J., Poletti, S., Aeschlimann, B., et al. (2009) Rice endosperm iron biofortification by targeted and

334

NUTRIENT USE EFFICIENCY IN CROPS

synergistic action of nicotianamine synthase and ferritin. Plant Biotechnology Journal 7, 631–644. Yi, Y. & Guerinot, M.L. (1996) Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency. The Plant Journal 10, 835–844. Yuan, Y.X., Zhang, J., Wang, D.W., et al. (2005) AtbHLH29 of Arabidopsis thaliana is a functional ortholog of tomato FER involved in controlling iron acquisition in Strategy I plants. Cell Research 15, 613–621. Yuan, Y.X., Wu, H., Wang, N., et al. (2008) FIT interacts with AtbHLH38 and AtbHLH39 in regulating

iron uptake gene expression for iron homeostasis in Arabidopsis. Cell Research 18, 385–397. Zhao, F.J. & McGrath, S.P. (2009) Biofortification and phytoremediation. Current Opinion in Plant Biology 12, 373–380. Zuo, Y. & Zhang, F. (2009) Iron and zinc biofortification strategies in dicot plants by intercropping with graminaceous species. A review. Agronomy for Sustainable Development 29, 63–71. Zuo, Y., Zhang, F., Li, X., et al. (2000) Studies on the improvement in iron nutrition of peanuts by intercropping with maize on a calcareous soil. Plant and Soil 220, 13–25.

Chapter 16

Zinc in Soils and Crop Nutrition Behzad Sadeghzadeh and Zed Rengel

Abstract As a micronutrient, zinc (Zn) is involved in enzyme activation (about 300 enzymes), gene expression, phytohormone activity, protein synthesis, photosynthesis and carbohydrate metabolism, fertility and seed production, and disease resistance. Zinc is taken up by root-cell membrane transporters of the zinc iron premeases (ZIP) family in all plant species, as well as by yellow stripe-like (YSL) proteins (Zn–phytosiderophore complexes) in grasses, and is loaded into the stele via heavy metal ATPases (HMA). About half of the arable soils in the world have low zinc availability, resulting in hampered crop growth, poor yields, and low zinc density in edible crop parts. There are commercially available cultivars with increased efﬁciency of zinc acquisition from soil as well as zinc utilization in tissues. Mechanisms underlying zinc efﬁciency are not completely understood. Given that 2–3 billion people have diets deﬁcient in zinc, biofortiﬁcation of staple crops is one of the global challenges being tackled at present. Soil and foliar fertilization with zinc (agronomic biofortiﬁcation) as well as breeding genotypes

for increased zinc density in edible parts (genetic biofortiﬁcation) are the options currently pursued for improving zinc intake in susceptible human populations.

Introduction Interest in plant micronutrients has risen in the last decades because poor availability of micronutrients in soil is widespread in agricultural lands and is becoming one of the major limiting factors for crop production. Not only crop yields, but also the quality of crop products, is impaired when the supply of plant-available micronutrients is insufﬁcient. The essentiality of zinc for plant growth as a micronutrient was recognized only about 70 years ago. In some parts of the world, the existence of zinc deﬁciency in crops has only been established during the last 10 or 20 years. Zinc deﬁciency can be exacerbated by a change from traditional subsistence agriculture to growing modern varieties for commercial purposes using relatively large amounts of macronutrient fertilizers.

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 335

336

NUTRIENT USE EFFICIENCY IN CROPS

Many of the high-yielding new varieties in intensively managed farming systems remove large amounts of zinc from soil at every harvest, thus depleting the soil and increasing the likelihood of zinc deﬁciency. For example, a harvest of 6.5 t grain ha−1 per year removed 416 g Zn ha−1 per year in soybean–wheat cropping system (Takkar, 1996). Furthermore, in intensively managed farming systems, increased use of macronutrient fertilizers, especially phosphorus as well as fertilizers containing little zinc impurities, can exacerbate zinc deﬁciency (Loneragan and Webb, 1993). Zinc deﬁciency causes yield losses in large areas under crop production. In developing countries, the need to maximize food production is crucial, and hence improving land productivity is necessary. Therefore, any factor (such as zinc deﬁciency) that prevents crops from reaching their potential yield needs to be addressed in these countries. However, zinc deﬁciency also occurs in many other technologically advanced countries such as Australia, most of the states in the United States, and in parts of Europe. In addition to staple crops such as wheat, maize, barley, and rice, the productivity of many other crops such as cotton, tea, fruit trees, grapes, and many vegetables can be signiﬁcantly reduced by zinc deﬁciency. Fortunately, the occurrence and reason behind zinc deﬁciency in many crops growing in most farmlands on different soil types of the world are commonly known. Therefore, zinc deﬁciency problems can be solved if farmers are made aware of the condition and how to treat it. Zinc is essential for the normal healthy growth and reproduction of plants, animals, and humans. It is required as a structural component or regulatory cofactor of a large number of proteins and enzymes (such as metalloenzymes and transcription factors) involved in many important biochemical pathways (carbohydrate metabolism, protein

biosynthesis, growth regulator metabolism) and functions (e.g., maintenance and integrity of cell membranes, resistance to infection) (Vallee and Falchuk, 1993; Berg and Shi, 1996). If the available amount of zinc is insufﬁcient (= Zn deﬁciency), plants suffer from physiological stresses caused by a failure of metabolic processes in which zinc plays a critical role. Among the essential trace elements for plants, zinc deﬁciency is the most widespread and most frequently encountered deﬁciency problem that has recently received global attention (Hotz and Brown, 2004). In cases of marginal or moderate deﬁciency, yield losses of 40% or more (without obvious visible symptoms on crops) can have a severe economic effect on farmers due to reduced income (Alloway, 2004). In addition to the yield losses, zinc deﬁciency can cause large reductions in crop quality by lowering zinc concentration in edible parts. Hence, increasing the zinc content of staple food crops is expected to enhance dietary intake of bioavailable zinc and improve zinc status of human populations (House et al., 2002). Applying zinc fertilizers does not always correct zinc deﬁciency due to factors such as subsoil constraints, topsoil drying, or disease interactions (Graham and Rengel, 1993). Where fertilizers are applied to correct zinc deﬁciency, the added zinc is likely to remain near the surface, especially in no-till farming systems, thus limiting its use by crops. In the semi-arid areas, applying the liquid form of nitrogen, phosphatic, and zinc fertilizers to the subsoil (up to 0.4 m deep) can increase zinc uptake and grain yield by crops more than applying granular fertilizers to the surface. However, the cost of the liquid form of fertilizers is relatively high in areas with extensive production (Holloway, 1996). Moreover, zinc fertilizers may be unavailable or unaffordable in developing countries.

ZINC IN SOILS AND CROP NUTRITION

Due to widespread zinc deﬁciency and difﬁculties in alleviating it by the use of fertilizers, development of crops that are efﬁcient zinc accumulators (especially under low plant-available soil zinc) is an important approach for improving tolerance to zinc deﬁciency (= Zn efﬁciency) and consequently grain productivity and micronutrient quality of food. In addition, zinc-efﬁcient genotypes could reduce land degradation by limiting the use of machinery and minimizing fertilizer inputs on agricultural lands. There are commercially available zincefﬁcient cultivars of wheat, barley, and rice (Gregorio et al., 2000; Genc and McDonald, 2004; Sadeghzadeh et al., 2009a), which are grown quite widely in soils with a low plantavailable concentration of zinc. Tolerance of plant genotypes to zinc deﬁciency, as a genetic trait, is usually called zinc efﬁciency and deﬁned as the ability of a cultivar to grow and yield well in soils too deﬁcient in zinc for a standard cultivar (Graham, 1984). While physiological and molecular mechanisms of tolerance to zinc deﬁciency are partially known (Hacisalihoglu and Kochian, 2003), a better understanding of the physiological, morphological, and genetic bases of zinc efﬁciency is needed for developing fast, simple, and reliable screening procedures to be used in identifying and breeding genotypes for zinc efﬁciency. This chapter gives a broad review of the relevant aspects of soil–plant relationships: from the origins of zinc in soils, zinc deﬁciency distribution and the factors affecting zinc availability, to the interactions between zinc and other soil nutrients. Soil testing is then reviewed along with the lower critical concentrations of zinc in soil test extractions. The chapter also covers the importance of zinc in optimal growth and yield, followed by zinc uptake and transport in plants as well as physiological aspects and symptoms of zinc deﬁciency in cereals and the role of zinc in alleviating drought stress. Types of zinc

337

compounds used as fertilizers and their application methods and rates are dealt with. Zinc concentration (density) in staple foods in relation to zinc deﬁciency problem in humans and improving this density by plant breeding are discussed, which leads to consideration of genotypic variation for seed zinc density, and mechanisms, screening, and genetics of zinc efﬁciency. Zinc in Soils Total zinc concentration of soils is related to the composition of the parent rock material and the nature and extent of weathering processes (Chesworth, 1991). The sedimentary rocks typically comprise 10–120 mg Zn kg−1, whereas the lithosphere contains 70–80 mg Zn kg−1 (Barak and Helmke, 1993; Alloway, 1995). Mean total zinc concentration in most agricultural soils is 10–300 mg Zn kg−1(Barber, 1995). Soils containing high amounts of zinc originated from parent rocks containing weathered zinc minerals (e.g., zinc sulfate, zinc carbonate, zinc oxide) (Barak and Helmke, 1993; Cappuyns et al., 2006). The range of total zinc concentrations in Australian soils that include many ancient, heavily weathered areas is <2 to 180, with a mean zinc concentration (34 mg kg−1) that is lower than for world soils (55 mg Zn kg−1) (Tiller, 1983). Zinc in soils can be separated into fractions based on particle size distribution and/ or chemical analysis. There are three primary zinc fractions in soil: (1) water-soluble zinc (present in the soil solution and soluble organic fractions); (2) adsorbed and exchangeable zinc in the colloidal fraction (associated with clay particles, iron, and aluminum hydroxides and humic compounds); and (3) insoluble zinc complexes in the solid phase (Alloway, 1995; Barber, 1995). Zinc distribution among soil fractions is determined by soil-speciﬁc (1) precipitation and dissolution, (2) complexation and

338

NUTRIENT USE EFFICIENCY IN CROPS

decomplexation, and (3) adsorption and desorption reactions (Kiekens, 1995). Soil pH is the dominant factor determining soil zinc distribution. At higher pH, zinc is readily adsorbed on cation exchange sites, whereas adsorbed zinc is displaced by CaCl2 at lower pH. In soils with low soluble organic matter, soluble zinc and the ratio of ionic Zn2+ to organically complexed zinc increases at low pH. At soil pH below 7.7, Zn2+ is the main species, but ZnOH+ predominates above pH 7.7, and the neutral species Zn(OH)2 is dominant above pH 9. At pH 8, the activity of Zn2+ is 10−10 M, but it increases to 10−4 M (6.5 mg L−1) at pH 5 (Kiekens, 1995). At soil pH below 5.8, the exchangeable zinc fraction may increase at the expense of organically complexed zinc (Yang et al., 2010). Distribution of Soils with Low Plant-Available Zinc The availability of zinc to plants depends on several soil factors, such as the concentration of zinc in solution, ion speciation, and the interaction of zinc with other macro- and micronutrients (Halvorson and Lindsay, 1977; Foth and Ellis, 1997; Zhu et al., 2001; Li et al., 2003). Zinc deﬁciency in crops occurs on a wide range of soil types in many parts of the world, but tropical regions with highly weathered soils, semi-arid areas with calcareous high pH soils, sandy soils, and acid-leached soils with low total zinc content tend to be most seriously affected (Lucas and Davis, 1961; Takkar and Walker, 1993). The results of 190 ﬁeld trials in 15 countries around the world showed that zinc deﬁciency was the most frequently occurring micronutrient deﬁciency problem. Zinc deﬁciency was recorded in 49% of the trials, with 25% of these being acute forms with visible symptoms, and 24% with hidden deﬁciencies conﬁrmed by yield responses to zinc amendments (Sillanpää, 1990).

Soils with low plant-available zinc have been reported in the arid and semi-arid regions of India (Nayyar et al., 1990; White and Zasoski, 1999), paddy ﬁelds in Pakistan (Rashid and Raﬁque, 1998), poorly drained calcareous paddy soils in China (Sillanpää, 1982), and highly alkaline soils with low organic matter in Central Anatolia of Turkey (Eyüpoglu et al., 1994). In China, about 40% of land is estimated to be zinc-deﬁcient according to plant-response studies. Around 50% of soil has less than 1 mg kg−1 diethyltriamine pentaacetic acid (DTPA)-extractable zinc (Liu, 1994; Yang et al., 2007). In Australia, soils with low plant-available zinc are found in South Australia, Victoria, Queensland, New South Wales, and Western Australia (Graham and Rengel, 1993). The south-west of Western Australia is considered the largest continuous zinc-deﬁcient area in the world (Donald and Prescott, 1975). Such large-scale deﬁciencies have made Australia a world center of research on micronutrient problems. Zinc Uptake from Soil Zinc enters the plant primarily via absorption of Zn2+ from the soil solution by the roots, which is a dynamic and complex process. Uptake depends on ion concentrations at the root surface, plant demand, and root absorption capacity (Fageria et al., 1991). Zinc reaches the root surface via mass ﬂow, diffusion, and root interception (Mengel and Kirkby, 1987; Salisbury and Ross, 1992). Mass ﬂow is the passive nutrient transport from soil to roots, driven by transpiration. When the solution moving through the soil to the root contains a relatively large concentration of zinc, mass ﬂow becomes the dominant mechanism bringing zinc to the root surface (Barber, 1984). When the concentration of zinc is low, particularly in soils with low plant-available

ZINC IN SOILS AND CROP NUTRITION

zinc, diffusion plays an important role in the transport of zinc and other nutrients such as phosphorus, potassium, copper, iron, and manganese to the root surface (Barber and Silberbush, 1984). Diffusion, in contrast to mass ﬂow, operates only in the immediate volume of soil surrounding a root. The interception of nutrients by roots may be an important uptake mechanism for soil immobile nutrients like zinc (Marschner, 1993). Goos et al. (2000) reported that poor root interception limits zinc uptake by maize if granules of ZnSO4 are banded in the soil, particularly if a low rate of ZnSO4 is used. Factors Affecting Availability of Soil Zinc to Plants The term “availability” is commonly used to describe the ability of plants to take up nutrients from soil (Tiller, 1983). Several authors have extensively reviewed the factors affecting zinc solubility in soils and availability to plants (Marschner, 1993; Catlett et al., 2002). Zinc availability to plants can be affected by factors such as total soil zinc content, soil pH, organic matter, soil temperature and moisture regimes, root distribution, and rhizosphere effects. Soils with a low total zinc concentration (e.g., sandy soils) often produce zincdeﬁcient crops. Such cases of zinc deﬁciency are related to absolute zinc deﬁciency rather than problems with zinc availability. Studies on crops have shown a negative correlation between soil pH and metal uptake (Narwal et al., 1983; Castilho and Chardon, 1995; Wang et al., 2006a). Micronutrient availability generally decreases as the soil pH increases, with the exception of molybdenum. Increasing soil pH stimulates zinc adsorption to the surfaces of various soil constituents, such as metal oxides and clay minerals; this results in decreased solubility and availability of zinc to plants (Brümmer et al., 1988; Barrow,

339

1993). High pH decreases desorption of zinc from soil surfaces, which also reduces availability of zinc to plants (Dang et al., 1993). Zinc can precipitate in the form of Zn(OH)2, ZnCO3, and Zn2SiO4 (Marschner, 1993) at high pH. In soil solution, zinc concentration is largely dependent on pH; for example, the concentration of zinc in soil solution is about 10−4 M at pH 5.0 and 10−10 M at pH 8.0 (Lindsay, 1991). An increase in soil pH from 5.2 to 6.8 by liming resulted in about a 10fold decrease in zinc concentration in plants (Parker and Walker, 1986). Conversely, the availability of natural and supplied zinc doubled when soil pH was reduced from 7 to 5 by using ammonium sulfate (Viets, 1966). At low pH, poor stability of metal– organic complexes increases zinc availability (Norvell, 1991). Total soil zinc concentration in calcareous and noncalcareous soils is often similar, but zinc deﬁciency is frequently reported for calcareous soils (Cakmak et al., 1996c; Singh et al., 2005). In a study of 1511 Turkish soil samples, DTPA-extractable zinc concentrations were inversely related to soil pH and soil organic matter (Eyüpoglu et al., 1994). Reddy and Dunn (1987) reported signiﬁcant differences in seed yield among the soybean genotypes under different soil pH conditions. Calcareous soils (pH > 7) with moderate to high organic matter content (>15 g organic C per kg soil) are likely zincdeﬁcient due to high HCO3− in the soil solution (Singh et al., 2005). In alkaline soil with low zinc supply, increasing zinc application increased zinc concentration in plants and reduced the deﬁciency symptoms, but only slightly improved plant growth. It is concluded that plant growth on alkaline soils was repressed more by soil alkalinity than zinc deﬁciency (Liu and Tang, 1999). Soil organic matter has a critical role in solubility and transport of zinc to roots (Marschner, 1993; Obrador et al., 2003) due

340

NUTRIENT USE EFFICIENCY IN CROPS

to adsorption of zinc by organic ligands (Lindsay, 1972). Similarly, Catlett et al. (2002) reported a strong inverse relationship between the soil organic matter content and soluble zinc concentrations in 18 Colorado soils. However, an adequate level of organic matter increases solubility and zinc diffusion rate in soils (Sharma and Deb, 1988). The wheat experiment showed zinc content to be positively correlated with the soil organic matter content (Sillanpää, 1982; Hamilton et al., 1993). In the United States, one of the most frequent causes of zinc deﬁciency problems is removal of surface soil by land leveling (Alloway, 2004). The subsoil has lower organic matter and, in many cases, also a higher pH than the topsoil. Both the DTPA-extractable zinc and organic matter content decrease with depth in the soil proﬁle (Alloway, 2004). Other factors that contribute to zinc deﬁciency are low soil moisture and low temperature (Moraghan and Mascagni, 1991; Marschner, 1993). Soil moisture affects nutrient supply via impaired diffusion to the root surface (Marschner, 1986). Given that zinc diffusion in soils is highly dependent on soil moisture, zinc nutrition of plants may be at risk in semi-arid and arid areas, where soils are usually deﬁcient in water for long periods during the growing season. Accordingly, in zinc-deﬁcient calcareous soils, wheat yield reductions are more severe under rain-fed than irrigated conditions (Ekiz et al., 1998). On the other hand, waterlogging alters zinc chemistry in the soil, whereby submerging the soil decreases water-soluble zinc concentration compared with well-drained soils (Amer et al., 1980). In addition, a decrease in zinc solubility and low zinc uptake in poorly drained soils may be due to the coprecipitation of zinc with soluble iron and aluminum (Singh and Abrol, 1986). Early in the growing season, zinc deﬁciency may occur when soil temperature is

still relatively low, and then diminishes as temperature rises (Brennan et al., 1993). Both incidence and severity of zinc deﬁciency symptoms are exacerbated by low soil temperature (Moraghan and Mascagni, 1991). It was suggested that colder root zone temperature decreases root colonization with arbuscular mycorrhizae, root growth, zinc uptake, and its translocation into the shoots (Schwartz et al., 1987; Moraghan and Mascagni, 1991). In barley, shoot zinc uptake was 82% higher in plants grown in solution at 20°C compared with 10°C (Schwartz et al., 1987). Finally, the interaction of zinc with other elements decreases zinc availability to plants, inﬂuencing its adsorption, distribution, and utilization in plants (Loneragan and Webb, 1993). Zinc interactions with phosphorus (P) and nitrogen (N) are most widespread in crop production on soils with limiting supplies of zinc and phosphorus or nitrogen. Interaction between Zinc and Other Soil Nutrients High amounts of applied nitrogen in the absence of zinc can cause zinc deﬁciency through a dilution effect brought about by promotion of plant growth, and to a lesser extent by changing soil pH (Table 16.1; Loneragan and Webb, 1993). Also, nitrogen application increases shoot-to-root ratio due to a stimulation of shoot growth to a greater extent than root growth. Promotion of plant growth without increasing root absorption rate or root size results in a decrease in zinc concentration in plants (Loneragan and Webb, 1993). High levels of phosphorus in soil can sometimes increase the symptoms of zinc deﬁciency (Foth and Ellis, 1997). Zinc and phosphorus coprecipitation in the soil may cause formation of insoluble ZnO3(PO4)2 that would decrease the soil solution zinc

ZINC IN SOILS AND CROP NUTRITION

Table 16.1.

341

Interactions of zinc with other elements

Element

Zinc × Element Interactions

Reference

Nitrogen (N)

Increasing N fertilization may increase Zn requirement due to increased growth and/ or changing rhizosphere pH Higher level of P decreased Zn availability

Loneragan and Webb (1993); Hamlin et al. (2003)

Phosphorus (P)

High P may increase Zn deﬁciency symptoms P application depressed Zn concentration

Potassium (K)

Accumulation of Zn in plants may lead to a suppression of K accumulation

Calcium (Ca)

Ca decreased Zn availability

Manganese (Mn)

Increasing level of Zn inhibited uptake of Mn and vice versa in several plant species

Magnesium (Mg)

Cell Zn content was not inﬂuenced by Mn nutrition Toxic level of Zn suppressed accumulation of magnesium

Boron (B)

Nontoxic levels of Zn did not affect magnesium concentration Increasing Zn supplies may either increase, decrease, or have no effect on the Fe status High concentrations of Zn can induce iron chlorosis through reducing Fe availability in soil Zn absorption was suppressed by high concentration of Fe in nutrient solution culture Zinc can alleviate boron toxicity

Copper (Cu)

High level of Cu inhibited Zn uptake

Cesium (Cs)

Cesium application increased Zn concentration

Iron (Fe)

concentration to a deﬁciency level (Barrow, 1993). Moreover, phosphorus fertilization reduced the zinc absorption rate (Safaya, 1976). Under limiting or marginal supplies of zinc and phosphorus, application of phosphorus decreased zinc concentration to deﬁcient levels due to the dilution of zinc concentration in the plant by growth stimulation (Sharma et al., 1968; Singh et al.,

Barrow (1993); Das et al. (2005) Foth and Ellis (1997) Wagar et al. (1986); Imtiaz et al. (2006); Agbim (2009) Kalyanaraman and Sivagurunathan (1994); Thalooth et al. (2006) Chaney (1993); Loneragan and Webb (1993) Galvez et al. (1989); de Varennes et al. (2001); Kaya et al. (2001); Xu et al. (2007b) Allen et al. (2007) Kalyanaraman and Sivagurunathan (1994); Bonnet et al. (2000) Gunes et al. (1998); Fontes et al. (1999) Loneragan and Webb (1993) Romheld and Marschner (1986) Alloway (2008)

Güne et al. (1999); Hosseini et al. (2007) Loneragan and Webb (1993); Arredondo et al. (2006) Isaure et al. (2006)

1988). Some studies, however, showed that a drop in shoot zinc concentration could be greater than what could be explained just by a dilution effect. In these cases, it was concluded that phosphorus-induced zinc deﬁciency depressed zinc uptake, ﬁrst by curtailing its translocation from root to shoot, and second by lowering its rate of absorption (Sharma et al., 1968).

342

NUTRIENT USE EFFICIENCY IN CROPS

High phosphorus supply can induce zinc deﬁciency in crops, for example, in wheat (Wagar et al., 1986). At a low zinc supply, zinc concentration in wheat shoots decreased with increased phosphorus applications, and severe zinc deﬁciency and lower yields were observed at 250 mg P kg−1 soil. Also, zinc deﬁciency was either induced or made more severe with increasing phosphorus concentration in the leaves (Cakmak and Marschner, 1987). Interestingly, increased application of phosphorus affected zinc concentration in the zinc-inefﬁcient but not the zinc-efﬁcient cultivars (Imtiaz et al., 2006). The zinc-efﬁcient cultivars maintained phosphorus concentrations at a lower level, absorbed more zinc, and grew better. In zinc-inefﬁcient cultivars, phosphorus may reduce the physiological activity of zinc (Leece, 1978), leading to higher zinc requirements (Boawn and Brown, 1968). Macronutrient cations such as calcium, potassium, and magnesium decrease the rate of zinc absorption by plants. Potassium, NH4, and magnesium cations strongly inhibit the rate of zinc absorption from solutions of low calcium concentration (Loneragan and Webb, 1993). In a study with wheat seedlings, tissue zinc concentration decreased as Ca(NO3) concentration was increased from 0 to 40 mM (Chaudhry and Loneragan, 1972). In addition, micronutrient cations, especially Cu2+, may diminish zinc uptake (Loneragan and Webb, 1993) due to competition between Cu2+ and Zn2+ for absorption sites on the root plasma membranes (Chaudhry et al., 1973). The iron and zinc interaction is complex, with conﬂicting results reported. With increasing iron supply, shoot zinc concentration was either decreased (Zhang et al., 1991), unchanged (Chaudhry and Loneragan, 1972), or increased (Giordano et al., 1974). Predicting Zinc Deﬁciency in Soil Soil analysis can be used as a tool for predicting nutrient deﬁciency in existing crops.

The results obtained can be compared with calculated critical zinc values in the soil types for the speciﬁc crop. Soil testing can be carried out at any time; therefore, it has an advantage over plant tissue analysis. Soil tests enable prediction of possible deﬁciencies in advance of growing the crop, so that appropriate fertilization or other treatments can be applied to avoid the yield and/or quality loss due to zinc deﬁciency. Several soil analytical procedures are available for predicting zinc availability in soils, including extractions with the chelating agents like DTPA, ethylenediamine tetraacetic acid (EDTA), hydrochloric acid and ammonium bicarbonate–DTPA, and Mehlich 1 test. The DTPA method is widely used for predicting plant-available zinc in soils (Lindsay and Norvell, 1978). Based on greenhouse and ﬁeld experiments, about 0.6 mg kg−1 DTPA-extractable zinc has been suggested as a critical concentration for wheat grown in calcareous soils of arid regions in India (Bansal et al., 1990). In contrast, adding zinc fertilizer to soils containing 0.5 mg kg−1 soil of DTPA-extractable zinc had no signiﬁcant effect on grain yield of barley and wheat grown in Saskatchewan (Singh et al., 1987). In Australia, the critical DTPA-extractable zinc concentration in acidic soils for wheat is 0.25 mg kg−1, and an application of zinc to soils containing more than 0.25 mg extractable Zn kg−1 is not effective in increasing yields (Brennan, 1996). Zinc in Plants The Functions of Zinc in Plants Zinc is required in small amounts to allow normal function of several key plant physiological pathways as well as in the structural and functional integrity of membranes. These functions have important roles in growth regulation, enzyme activation, gene expression and regulation, phytohormone activity, protein synthesis, photosynthesis

ZINC IN SOILS AND CROP NUTRITION

and carbohydrate metabolism, fertility and seed production, and defense against disease (Marschner, 1995). Zinc deﬁciency impairs these physiological functions, leading to severe reduction in growth, lower yields (or even crop failure), and poor quality crop products (Brown et al., 1993). Zinc is the only metal represented in all six enzyme classes (oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases) (Webb, 1992). The zinc requirement in a wide range of enzymes means that protein, carbohydrate, and auxin metabolism as well as reproductive processes are depressed under zinc deﬁciency (Römheld and Marschner, 1991; Brown et al., 1993). There are three primary Zn2+-ligand binding sites underpinning structural, catalytic, and cocatalytic function of zinc in enzymes (Auld, 2001; Maret, 2005). Structural zinc sites comprise four ligands for proper protein folding such as in protein kinases. At catalytic sites (e.g., carbonic anhydrase), Zn2+ is directly involved in the enzyme function by complexing with water and sulfur, nitrogen, or oxygen donors. At cocatalytic sites where two or three Zn2+ are bridged by amino acid residues (e.g., superoxide dismutases), Zn2+ is involved in catalytic, structural, and regulatory functions. Zinc is involved in carbohydrate metabolism through its effects on photosynthesis and sugar transformation (Salama et al., 2002). A decrease in photosynthesis by zinc deﬁciency can result from a decrease in (1) carbonic anhydrase (CA) activity (Sharma et al., 1982; Ohki, 2006), (2) the photochemical activity of chloroplasts (Tkachuk et al., 1994), (3) chlorophyll content, and (4) chloroplast structure. Low CA may inhibit photosynthetic electron transfer and consequently limit chlorophyll content (Römheld and Marschner, 1991). The activity of enzyme CA can be an indicator of the concentration of physiologically active zinc in leaves as well as the amount of zinc accumulation in leaves (Lazova et al., 2004).

343

Zinc is essential in protein metabolism, being involved in the stability and functioning of genetic material. Zinc is essential in chromatin structure, DNA and RNA metabolism, and gene expression (Vallee and Falchuk, 1993; Ho, 2004). Zinc deﬁciency decreases protein synthesis (Marschner, 1995; Obata et al., 1999) due to RNA degradation (Ciais et al., 2004) and lower activity of RNA polymerase (Marschner, 1995) as well as deformation and reduction of ribosomes (Kitagishi et al., 1987; Brown et al., 1993). In zinc-deﬁcient bean leaves compared with control plants, free amino acids concentration was increased by a factor of 6.5; following zinc resupply, this increase factor was 5.1 after 24 h, 2.7 after 48 h, and 1.4 after 72 h (Cakmak et al., 1989). Zinc has an important physiological effect in maintaining the integrity and function of cellular membranes (Pinton et al., 1994) by controlling generation and detoxiﬁcation of reactive oxygen species (ROS; Cakmak and Marschner, 1988b; Cakmak, 2000). ROS can damage membrane lipids (Bettger and O’Dell, 1981) and sulfhydryl groups (Rengel, 1995b). When these compounds are damaged by oxidative stress, there is increased leakage of organic compounds (such as carbohydrates and amino acids) from zinc-deﬁcient root cells (Cakmak and Marschner, 1988b). Due to the increased leakage of carbon-containing compounds into the rhizosphere, zinc-deﬁcient plants may be susceptible to root diseases such as Fusarium graminearum (Sparrow and Graham, 1988; Grewal et al., 1996). Zinc Uptake and Movement in Plants Roots take up zinc from the soil solution as a divalent cation (Zn2+), and at high pH, zinc is absorbed as a monovalent ZnOH+ (Marschner, 1995). Zinc accumulation in roots has biphasic kinetics, with an initial rapid binding in the root cell walls, followed by a slower linear transport phase across the

344

NUTRIENT USE EFFICIENCY IN CROPS

plasma membrane (Santa Maria and Cogliatti, 1988). The process of Zn2+ movement from the external solution to the root cell wall free space is via diffusion followed by zinc transport across the plasma membrane, via ion transport proteins such as zinc iron premeases (ZIP) family transport proteins (Guerinot, 2000). ZIP family genes appear to be the primary proteins responsible for zinc uptake from the rhizosphere (Grotz et al., 1998). The cytoplasm is negatively charged, and there is an electrical gradient drawing cations (including Zn2+) into the cell. Reid et al. (1996) reported that for zinc transport across the plasma membrane, it appears unnecessary to invoke an active transport system because zinc inﬂux will be thermodynamically favored. Root zinc uptake is mediated by the high-velocity, low-afﬁnity (Km = 2–5 μM) and low-velocity, highafﬁnity transport systems (Km = 0.6–2 nM), with the latter dominating under low soil zinc conditions (Hacisalihoglu et al., 2001; Hacisalihoglu, 2002). Zinc-inefﬁcient plants have lower maximum rates of uptake and lower afﬁnity values than zinc-efﬁcient plants (Rengel and Wheal, 1997b; Hacisalihoglu et al., 2001). From the root surface, nutrients are transported into the root xylem through epidermis, cortex, and endodermis (Marschner, 1995). Zinc may pass through the root to the xylem either through the extracellular spaces between root cells (apoplast) or through the cytoplasmic continuum of root cells linked by plasmodesmata (symplast) (White et al., 2002; Broadley et al., 2007). The apoplastic ﬂux is largely determined by the cation exchange properties of the cell wall, water ﬂows, and the presence of the Casparian band, and contributes increasingly to zinc uptake and inﬂux to the xylem as external zinc concentration increases (White et al., 2002). In Thlaspi caerulescens and Thlaspi arvense, the entry point for zinc accumula-

tion is across the plasma membrane of root cells, and zinc reaches the xylem via the symplastic pathway in both species (Lasat et al., 1996; Lasat and Kochian, 2000). Zinc can be taken up across the root-cell plasma membranes as Zn2+ or as a zinc– phytosiderophore (PS) complex (Grotz and Guerinot, 2006; Suzuki et al., 2006; Broadley et al., 2007). Most Zn2+ inﬂux to the cytoplasm is mediated by ZIP family transport proteins such as ZIP1, ZIP3, and ZIP4 (Guerinot, 2000; Pence et al., 2000; Broadley et al., 2007), and the uptake of zinc–PS complexes is catalyzed by yellow stripe-like (YSL) proteins in Strategy II plants (the grasses) (Suzuki et al., 2006; Haydon and Cobbett, 2007b). The heavy metal ATPase (HMA) family appears to be the most likely candidate for loading zinc into the xylem for root-to-shoot transport (Eren and Arguello, 2004; Hussain et al., 2004). In xylem, zinc may be transferred as Zn2+ or complexed with the organic acids, histidine, or nicotianamine (Welch, 1995; Von Wirén et al., 1999; Broadley et al., 2007). Zinc in xylem has been measured in soluble form bound to small proteins and as insoluble complexes such as zinc–phytate (Tinker et al., 1981) or anionic complexes (White et al., 1981). ZIP family members appear to mediate Zn2+ inﬂux to the phloem and leaf cells (Ishimaru et al., 2005). YSL proteins may also load zinc into the phloem (Haydon and Cobbett, 2007b; Waters and Grusak, 2008). For a large body of research on zinc transporters and the cellular trafﬁcking of zinc, readers are referred to Eide (2006). Zinc Deﬁciency in Plants When the supply of zinc to plants is inadequate, physiological functions of zinc are impaired, and the growth is adversely affected. Zinc deﬁciency is a severe micronutrient disorder that threatens world food

ZINC IN SOILS AND CROP NUTRITION

production (Alloway, 2001; Welch and Graham, 2004). The main causes of zinc deﬁciency in crops are: low zinc availability (high pH, calcareous, and sodic soils), low total soil zinc concentration (especially in sandy, sodic, and calcareous soils), high levels of nitrogen and phosphate, and restricted root exploration due to soil compaction or high water table, particularly in soils of marginal zinc status (Alloway, 2004). Wheat and barley show a signiﬁcant decrease in growth and grain yield under zinc-deﬁcient conditions in the ﬁeld (Graham et al., 1992; Cakmak et al., 1996c; McDonald et al., 2001). Zinc deﬁciency in soils also reduces zinc concentration and content in edible portions of staple food crops and diminishes their nutritional quality (Welch and Graham, 2004). About 40% of world’s human population suffers from micronutrient deﬁciencies, including zinc deﬁciency (the so-called hidden hunger) (Bouis, 1996; Graham and Welch, 1996). A high proportion of cereal-based foods with low zinc content in the diet is considered one of the major reasons for the widespread occurrence of zinc deﬁciency in humans, especially in developing countries (Welch and Graham, 1999; Graham et al., 2001).

345

Diagnosing Zinc Deﬁciency in Plants Visible symptoms of zinc deﬁciency, such as small and distorted leaves, interveinal chlorosis in young leaves, and shortened internodes (Marschner, 1995), can be a quick and simple diagnostic tool for severe zinc deﬁciency in many crops. However, in some cases, visible symptoms are not easy to recognize. Climatic conditions can also affect symptom development. In some instances, visual diagnosis can be hampered by the simultaneous phoshphorus toxicity (Webb and Loneragan, 1988) and chlorosis induced by intense light (Cakmak et al., 1995). Perhaps most importantly, mild or even moderate cases of deﬁciency often do not give rise to clear diagnostic symptoms. Such hidden zinc deﬁciency can sometimes cause 20% yield loss or more. In barley, visual zinc deﬁciency symptoms are stunted plants and leaves, chlorotic areas on leaves, necrosis, and leaves collapsing around the middle (Fig. 16.1) (Genc et al., 2003; Lombnaes and Singh, 2003; Sadeghzadeh et al., 2009a). In wheat, symptoms of zinc deﬁciency are usually observed in young seedlings, later resulting in whitishbrown patches and then necrotic lesions on the leaf blades, with leaves collapsing in the

Zinc deﬁciency symptoms on barley leaves: development of necrotic lesions and collapsing leaves in the center (Sadeghzadeh, 2008).

Fig. 16.1.

346

NUTRIENT USE EFFICIENCY IN CROPS

middle (Rengel and Graham, 1995c; Cakmak and Braun, 2001). The appearance of these symptoms can vary with environmental conditions, plant age, deﬁciency stage and severity, as well as the supply of other nutrients (Brennan et al., 1993). In addition, the visual symptoms of zinc deﬁciency vary considerably among crop genotypes and may be signiﬁcantly correlated with zinc efﬁciency and grain yield (Genc et al., 2002b; Sadeghzadeh, 2008). Given that individual varieties (or cultivars) can often vary considerably in their susceptibility to zinc deﬁciency, it is important to screen crop varieties and identify tolerant (or zinc-efﬁcient) varieties to be grown on soils of low plant-available zinc. Genotypes can be screened for zinc efﬁciency based on expression and severity of visual zinc deﬁciency symptoms on leaves. By using a visual score of deﬁciency symptoms in barley, it was found that the greater tolerance to zinc deﬁciency in a zinc-efﬁcient genotype compared with a zinc-inefﬁcient genotype at the seedling stage was controlled by a single gene. Hence, visual zinc deﬁciency scores are useful for genetic analysis of tolerance to zinc deﬁciency (Genc et al., 2003). Critical (or threshold) concentration (usually deﬁned as 90% of maximum yield) in tissues vary with plant species, cultivar, plant age, plant parts, and the environment. Whole shoot, root, young leaves, and grain are used for diagnosing zinc status, although leaves have been considered the most appropriate plant part to sample for determining nutrient status (Dang et al., 1993; Huang et al., 1996). The critical zinc concentrations in leaves vary between 20 mg Zn kg−1 in wheat, 15 mg Zn kg−1 in rice, and 22 mg Zn kg−1 in maize and groundnut. For the whole young plant, values reported include 8 mg Zn kg−1 for sorghum, 22 mg Zn kg−1 in rice and 25 mg Zn kg−1 in wheat and chickpea. However, differences can also occur between different varieties of these crops (Alloway, 2001).

It has been suggested that biochemical analysis can be an excellent indicator of nutrient status, particularly when plant tissues have a large amount of physiologically inactive nutrients (Table 16.2; Gibson and Leece, 1981). To diagnose zinc deﬁciency, the activity of CA was used in wheat (Rengel, 1995a), mustard (Chatterjee and Khurana, 2007), and maize (Gibson and Leece, 1981). Similarly, the activity of aldolase and ribonuclease appear to be reliable biological indicators of the zinc status in mustard (Chatterjee and Khurana, 2007). Correcting Zinc Deﬁciency in Plants Zinc deﬁciency in crop production can be ameliorated through agronomy and/or genetic improvement. Substantial crop responses to zinc fertilization were reported in Australia, India, and especially in Central Anatolia (Turkey), where wheat grain yields have increased by over 600% since the mid1990s (Cakmak, 2004). Fertilization is used to provide crops with the macronutrients nitrogen, phosphorus, and potassium (Constant and Sheldrick, 1991). These nutrients, by promoting root and shoot development, can increase the uptake of all nutrients, including zinc, required by the crop. In addition, the widespread use of macroelement fertilizers makes them a convenient vehicle, by adding zinc fertilizers during the manufacturing process, to supply crops with zinc as well. Alternatively, zinc-containing impurities in fertilizers may make a signiﬁcant contribution to zinc supply to crops (Bolland et al., 1993). Fertilization could correct zinc deﬁciency and ensure optimum yields as well as increased zinc concentration in grain. However, fertilizer studies focusing speciﬁcally on increasing zinc concentration in edible crop parts (such as grain) have received much less attention compared with those on the role of soil and foliar zinc fertilization to correct zinc deﬁciency and

ZINC IN SOILS AND CROP NUTRITION

347

Table 16.2. Enzyme activities as indicators of zinc nutrient stress used in screening plant genotypes for Zn

efﬁciency Enzyme Type

Plant Species

Reference

Carbonic anhydrase (CA) activity

Maize Cotton Mustard Rice Wheat

Alcohol dehydrogenase (ADH) activity Fructose 1,6 biphosphate aldolase (FBPAse) activity Total superoxide dismutase (SOD)

Rice

Gibson and Leece (1981) Ohki (2006) Chatterjee and Khurana (2007) Sasaki et al. (1998) Rengel (1995a); Hacisalihoglu et al. (2003a) Moore and Patrick (1988); Reddy (2006) Pandey et al. (2002)

Cu/Zn superoxide dismutase activity

Acid phosphatase (APase) activity Ribonuclease (RNAse) activity

Ascorbate peroxidase (APx) activity Production of superoxidegenerating NADPH-oxidases Nicotianamine synthase (NAS) activity Peroxidase (POD) activity

Black gram (Vigna mungo L.) Narrow-leaf lupin Tobacco Wheat, rye Maize Narrow-leaf lupin Rice Rye Wheat

Tobacco

Yu et al. (1998); Yu and Rengel (1999) Yu et al. (1998) Cakmak et al. (1998) Wang and Jin (2005) Yu and Rengel (1999) Obata et al. (1999) Cakmak et al. (1997c) Cakmak et al. (1998); Yu et al. (1999); Hacisalihoglu et al. (2003a) Chatterjee and Khurana (2007) Hayes et al. (1999) Kaya et al. (2000) Pandey et al. (2002) Brown et al. (1993); Azhar et al. (2005) Yu et al. (1998)

Bean, tomato

Cakmak and Marschner (1988a)

Wheat

Singh et al. (2002)

Mustard Wheat

Chatterjee and Khurana (2007) Salama and Fouly (2008)

Mustard Pasture species Tomato Black gram Wheat

increase plant growth and yield (Martens and Westermann, 1991; Mortvedt and Gilkes, 1993; Rengel et al., 1999). Zinc deﬁciency can be treated by applying fertilizers to soils and/or foliage, treating seeds with zinc (a technique called seed priming) as well as sowing zinc-dense seeds. Several different zinc sources, including ZnSO4, ZnCO3, ZnO, Zn(NO3)2, and ZnCl2, are being used as fertilizers (Shuman, 1998). Application rates of zinc fertilizers vary

depending on the crop, the zinc form, soil conditions, and the application method. Zinc can be applied to soil as organic and/ or inorganic zinc fertilizers. Zinc sulfate (ZnSO4) is the most commonly used inorganic source of zinc due to its high solubility in water, existence in both crystalline and granular, and low cost compared with synthetic zinc chelates such as zinc−ethylenedi aminetetraacetate (ZnEDTA; Martens and Westermann, 1991; Mortvedt and Gilkes,

348

NUTRIENT USE EFFICIENCY IN CROPS

1993). For soil applications, the rates can range from 2.5–22 kg Zn ha−1 for inorganic forms such as ZnSO4 and 0.3–6 kg Zn ha−1 for chelated forms (Alloway, 2001). One application of zinc sulfate between 20 and 30 kg ha−1 can improve zinc status of the soil, which will last for around 5 years before another application is required (Alloway, 2004). However, the rates of zinc application can be affected by variations in soil DTPA-extractable zinc and the soil type; for example, the application rates of zinc fertilizer may have to be higher and more frequent in soils with a high content of calcium carbonate. Ekiz et al. (1998) reported that 7 kg broadcast applications of zinc sulfate per hectare on severely zinc-deﬁcient calcareous soils signiﬁcantly enhanced wheat yield, but that increasing the zinc rate from 7 to 21 kg did not result in an additional increase in grain yield. The agronomic effectiveness (e.g., magnitude of the crop response per unit of applied micronutrient) is higher with ZnEDTA than for inorganic zinc fertilizers. Inorganic zinc fertilizers (such as zinc sulfate) may become ineffective rapidly as the dissolved zinc reacts with soil minerals and organic matter, whereas synthetic zinc chelates have the advantage of keeping the applied zinc in solution in a less reactive form (Martens and Westermann, 1991; Mortvedt and Gilkes, 1993). In maize, it was reported that zinc sulfate was generally less effective than ZnEDTA in increasing zinc uptake on calcareous soils (Maftoun and Karimian, 1989). However, due to its high cost, the use of ZnEDTA in cereal farming is limited. There is also a leaching risk associated with zinc chelates application because the more mobile the chelate, or the less biodegradable the carrier, the greater the leaching risk (Gonzalez et al., 2007). Foliar application of zinc can be effective in alleviating zinc deﬁciency in plants, particularly in fruit and vegetable production.

Application of 0.5 to 1.0 kg Zn ha−1 as zinc sulfate or 0.2 kg Zn ha−1 as ZnEDTA can often correct zinc deﬁciency in plants (Martens and Westermann, 1991). The advantage of foliar fertilization is that the fertilizer is applied directly to the plant rather than to the soil. Thus, the absorption of zinc is not interfered by the soil–root transfer processes. Foliar application of zinc was more effective than soil application for increasing grain zinc density but not grain yield (Paterson et al., 1991). When high grain yield and grain zinc concentration are desired, a combined soil and leaf application of zinc was the most effective method. However, in the long term, soil application alone could be the most economical method because zinc applied to soil has a good residual effect and does not need to be reapplied every year (Martens and Westermann, 1991; Yilmaz et al., 1997). During early seed development, zinc transfer from shoots into wheat grains is particularly high, which suggests that the timing of foliar zinc application would be a critical factor for an effective increase in grain zinc density (= biofortiﬁcation) (Ozturk et al., 2006). Depending on the growth stage of ﬁeld-grown wheat, foliar applications of ZnEDTA and zinc sulfate at the rate of 400– 450 g Zn ha−1 were either equally effective or ZnEDTA was superior to zinc sulfate in correcting zinc deﬁciency (Brennan, 1991). Under zinc-deﬁcient conditions, sowing seeds containing higher amounts of zinc can be a practical solution to increasing crop yield. In greenhouse experiments, wheat plants derived from seed low in zinc (250 ng Zn seed−1) had worse seedling vigor and grain yield compared with those arising from seed high in zinc (700 ng Zn seed−1) (Rengel and Graham, 1995b). Seed priming (applying zinc to seeds) is a simple and inexpensive technique, especially for resource-poor farmers. Improving seed zinc content by pretreating seeds in fer-

ZINC IN SOILS AND CROP NUTRITION

tilizer solutions can have high agronomic beneﬁts due to accelerated seedling emergence and improved seedling vigor in soils with low zinc availability. However, seed priming alone was not found to increase the seed zinc density of the progeny in wheat (Yilmaz et al., (1997). Similarly, it was reported that zinc-primed rice grains produced larger seedlings, but seed priming had no effect on the zinc content of progeny seeds in rice, lentil, chickpea, and wheat (Johnson et al., 2005). Although seed priming may not substitute for soil or foliar fertilization in biofortifying staple food crops with zinc for human nutrition, it is obvious that seed with high zinc content is advantageous from the perspective of yield and thus food security. Furthermore, seed priming could provide a practical solution in soils with low zinc availability when farmers are not aware of zinc deﬁciency, and zinc applications are not practiced. There is increasing evidence that enhanced grain nutrient concentration may allow good crop establishment when such seeds were sown in soils with low plantavailable nutrients. In zinc-dense seeds, from an agronomic point of view, there may be enough seed reserves to last until a large root system is developed to compensate for the low nutrient supply in zinc-poor soils (Welch, 1999). Sowing seeds with high zinc concentration may be a practical solution for increasing yield under zinc deﬁciency (Cakmak and Braun, 2001). In glasshouse experiments, wheat plants grown from high zinc seed had better growth and grain yield than plants derived from the seed with low zinc content (Rengel and Graham, 1995a,b). In the zinc-deﬁcient ﬁeld, wheat plants derived from seed with high and medium zinc content had a signiﬁcantly greater grain yield than plants grown from seed with low zinc content (Cakmak and Braun, 2001). In barley, Genc et al. (2000) reported that high seed zinc content reduced visual zinc deﬁ-

349

ciency symptoms and improved vegetative growth, especially when zinc supply from the rooting medium was deﬁcient for plant growth. The use of seed with high zinc content could provide a practical approach to alleviating the zinc deﬁciency problem, especially where farmers are not aware of zinc deﬁciency, and zinc fertilization is not practical. An alternative approach to increasing crop yields on soils with low plant-available zinc is by exploiting genotypic differences in zinc uptake and tissue use efﬁciency that exist within crop species (Rengel, 2001; Cakmak, 2002; Hacisalihoglu and Kochian, 2003; Alloway, 2004). There are zincefﬁcient cultivars of rice, wheat, and other crops that are grown widely in soils with low plant-available zinc. This approach is one of matching the plant to the soil rather than modifying the soil to suit the plant. Zinc Deﬁciency and Drought Tolerance Much of world cereal-growing areas suffer from drought stress and irregular distribution of precipitation during the growing season. Tolerance to environmental stresses is usually associated with a high requirement for zinc to regulate and maintain the expression of genes involved in protecting cells from the harmful effects of stress. Drought stress results in substantial yield losses, and a yield decrease becomes more severe in combination with zinc deﬁciency (Ekiz et al., 1998; Bagci et al., 2007). Low water availability under drought generally results in reduced total nutrient uptake and frequently causes reduced concentrations of zinc in plant tissues (Gunes et al., 2006). Under greenhouse conditions, drought stress reduced the accumulation of micronutrients, including zinc, in shoots of wheat and chickpea (Gunes et al., 2007). Limited water supply may reduce root

350

NUTRIENT USE EFFICIENCY IN CROPS

growth and nutrient mobility in soil and uptake by plants (Fageria et al., 2002; Samarah et al., 2004). Decreased absorption of nutrients like zinc under drought results from an interference in nutrient uptake and unloading mechanisms, and in reduced transpiration ﬂow (Marschner, 1995; Baligar et al., 2001). However, there is genotypic variation among species and genotypes within species in capacity to take up zinc under water stress (Abo-Hegazi et al., 1996; Zubaidi et al., 1999; Garg, 2003; Gunes et al., 2006; Bagci et al., 2007). Under drought stress, nutrient uptake in plants may have an important role in the tolerance (Samarah et al., 2004; Bagci et al., 2007). Water deﬁcit causes oxidative stress by enhancing production of ROS, especially during photosynthesis (Sairam and Saxena, 2001; Li et al., 2004; Selote et al., 2004; Goodman and Newton, 2005). The ROS, including hydrogen peroxide (H2O2), superoxide (O2−), hydroxyl radicals (OH), and singlet oxygen (1O2), are unavoidable byproducts of cell metabolism. Under optimal physiological conditions, the production and destruction of ROS is successfully regulated by cell metabolism. However, under stress conditions, the formation of these radicals might be in excess of the amount present under optimal conditions, thus causing oxidative stress. ROS constitute a major threat to plants due to DNA strand breakage (Brawn and Fridovich, 1981), peroxidation of membrane lipids (Moran et al., 1994), enzyme inactivation (Hernandez-Ruiz et al., 2001), protein denaturation (Bowler et al., 1992), and inhibition of cell cycle progression (Reichheld et al., 1999). Such drought stress-induced production of ROS and increased sensitivity of plants to photoxidative damage in chloroplasts are exacerbated when plants are simultaneously under zinc-deﬁciency stress. Zinc deﬁciency can modulate the activities of antioxidative enzymes (Cakmak and

Marschner, 1988a; Wenzel and Mehlhorn, 1995); therefore, oxidative stress is a component of zinc deﬁciency stress. Cells have antioxidative systems that scavenge superoxide radicals and convert them to H2O2, while the resulting H2O2 can be detoxiﬁed in the ascorbate–glutathione cycle, which includes ascorbate peroxidase (APx) and glutathione reductase (Nakano and Asada, 1981). Adequate zinc nutrition may alleviate drought stress in different ways such as inhibition of photoxidative damage caused by ROS in chloroplasts (Cakmak, 2000; Wang and Jin, 2005). Zinc is involved in diminished production as well as detoxiﬁcation of ROS by anti-oxidative enzymes such as copper/zinc superoxide dismutase (SOD) (Cakmak and Marschner, 1988b; Cakmak, 2000). Copper/zinc SOD is located mainly in the cytosol and/or chloroplasts (Fridovich, 1986). Many transcription factors are involved in tolerance to changes in environmental conditions (Chen et al., 2002), such as zinc ﬁnger proteins. More than 600 zinc ﬁnger proteins were found in Arabidopsis (Eulgem et al., 2000). However, only a few of these factors respond in a similar way to a variety of different stresses. One representative of the small group of genes that responds similarly to many stresses (e.g., cold, drought, and heat) is the zinc ﬁnger protein Zat12 (Fowler and Thomashow, 2002; Kreps et al., 2002; Rizhsky et al., 2004; Davletova et al., 2005). Zat12 was originally isolated as a light-stress-response cDNA (rhl41) by Iida et al. (2000), and was later identiﬁed by transcriptome analyses of plants subjected to different biotic and abiotic stress conditions. Zinc in Human Nutrition An emphasis on producing enough calories (and neglecting food quality) has resulted in unforeseen nutritional problems for nearly

ZINC IN SOILS AND CROP NUTRITION

50% of the world’s people, especially among the poor populations (UNACCSN, 1992). Around 30% of the world’s population has zinc-deﬁcient diets (Alloway, 2008), emphasizing the importance of zinc content in staple foods, such as rice, wheat, and maize. Zinc deﬁciency in humans affects physical growth, the functioning of the immune system, reproductive health, and neurobehavioral development. Worldwide, zinc deﬁciency caused nearly half million deaths in children under 5 years of age in 2004 (Black et al., 2008). In humans, zinc deﬁciency can be caused by inadequate intake, increased requirements, malabsorption, increased losses, and impaired utilization. In most situations, inadequate dietary intake of absorbable zinc (low total dietary intake, heavy reliance on foods with low zinc content, and/or poorly absorbable zinc) is likely to be the primary cause of zinc malnutrition (Solomons and Cousins, 1984). Poor absorption of zinc may occur as a result of a number of different conditions, such as malabsorption syndromes and inﬂammatory diseases of the bowel (Bremner and Beattie, 2007; Cummings and Kovacic, 2007), whereas certain drugs that chelate zinc may cause impaired utilization of zinc in tissues (Moretti and Caprara, 2010). Enhancing nutrient content and nutritional quality of crops for human nutrition is currently a global challenge because it was mostly ignored in breeding in the past. Recently, there has been a growing research interest in increasing the micronutrient density in edible portions of crops (Rengel et al., 1999; Schachtman and Barker, 1999; Welch and Graham, 2004; Uauy et al., 2006; Distelfeld et al., 2007; Cakmak, 2008; Sadeghzadeh et al., 2009b), which will result in improved human health as well as better crop production. Micronutrient concentration in grains destined for human food is a more important

351

parameter than the micronutrient content. Nutrient concentration in seed is dependent on parent plant growth conditions such as soil type, nutrient availability, crop species, and genotypes (Ascher et al., 1994; Rengel et al., 1999). There is a rapidly developing ﬁeld of research on the biofortiﬁcation of plant foods with zinc. This involves both the breeding of new varieties of cereal crops with the genetic potential to accumulate high concentration of zinc in grains (genetic biofortiﬁcation) and the use of zinc fertilizers to increase grain zinc concentration (agronomic biofortiﬁcation). Although the plant breeding route is likely to be the most costefﬁcient approach in the long run, for the time being, the use of fertilizers is necessary to improve the zinc density in diets while the plant breeding programs are being carried out. However, it will be necessary to monitor both the zinc concentrations in the cereal grains and also the soil to ensure that the enrichment of the grains occurs without the accumulation of zinc in soils to possibly harmful levels. Breeding for Zinc Efﬁciency The primary objective of plant breeding has been to enhance farm productivity, usually by developing crops with higher yields. In contrast, improving micronutrient efﬁciencies and increasing nutrient concentrations in plants has rarely been a breeding objective. In fact, crop nutritional problems have been mostly ignored in breeding. Some nutritional problems cannot be easily resolved by altering soil fertility or chemistry, and application of modern breeding techniques to breed crops adapted to soils of poor nutritional status is required. With micronutrient deﬁciencies induced by high pH (in case of iron and maganese), agronomic solutions (fertilizers) are not always successful, and a genetic solution is

352

NUTRIENT USE EFFICIENCY IN CROPS

necessary (Cakmak and Braun, 2001). Furthermore, correction of zinc deﬁciency induced by subsoil constraints, topsoil drying, and diseases is not effective via fertilization (Graham and Rengel, 1993). Hence, breeding and use of zinc-efﬁcient plant genotypes that can more effectively function under zinc deﬁciency is an effective and sustainable solution to zinc deﬁciency limitations in crop production (Lynch and Steponkus, 1987). Some progress has been made relative to the ﬁrst stage of breeding, which consists of screening for genetic variability in tissue concentration of trace minerals such as zinc. Most plants contain between 30 and 100 mg Zn kg−1 dry matter, and concentrations above 300 mg Zn/kg are generally toxic (Marschner, 1995). For example, in a glasshouse- and ﬁeld-grown doubled-haploid barley population (150 genotypes), the range of shoot zinc concentrations were 22–61 mg kg−1 (Sadeghzadeh, 2008). In general, zincefﬁcient genotypes have higher zinc concentration in the shoot than zinc-inefﬁcient ones (Genc et al., 2002b; Sadeghzadeh, 2008). However, some studies do not recommend using zinc concentration as criteria to select for zinc efﬁciency due to the effect of dilution by growth (Cakmak and Braun, 2001). With the existence of large genotypic variations in zinc efﬁciency among crops (Cakmak et al., 1998), there is a need for targeted selection and/or breeding of plants with higher efﬁciency both in terms of higher grain yield and grain zinc concentration. In the past, a lack of a suitable screening procedure to allow screening of large numbers of genotypes in a short time hampered breeding for zinc efﬁciency. However, screening in the ﬁeld at nutrient-responsive sites and comparing yields at limiting and nonlimiting rates of zinc has been used extensively to assess efﬁciency (Takkar et al., 1983; Graham et al., 1992). The results of such screening can be variable because

the severity of the nutrient deﬁciency varies between sites and years due to the effects of other growth-limiting factors like drought and diseases. Hence, reliable alternative methods are required. The use of controlled environments for screening is a common practice. Considerations of funding and time mean that the screening of large populations for development of molecular markers requires a pot culture screening system. Soil-based pot assays under controlled conditions allowed the relative efﬁciency of genotypes to be assessed (Cakmak et al., 1997b; Genc and McDonald, 2004), but these are generally based on seedling growth rather than grain yield. Finally, a major challenge will be to demonstrate the relevance of these screening methods under ﬁeld conditions. Lombnaes and Singh (2003) chose four approaches to characterize tolerance to zinc deﬁciency in barley and wheat: (1) relative shoot weight at low compared with high zinc supply (zinc efﬁciency index), (2) relative shoot-to-root ratio at low compared with high zinc supply, (3) total shoot uptake of zinc under deﬁcient conditions, and (4) shoot dry weight under deﬁcient conditions. Evaluating severity of zinc deﬁciency symptoms on leaves together with the zinc efﬁciency ratio (yield at –Zn/yield at +Zn) appears to be a reasonable approach to reliably and quickly screen large numbers of genotypes for zinc efﬁciency in a short time (Cakmak and Braun, 2001; Genc et al., 2003). Mechanisms of Zinc Efﬁciency Various mechanisms have been proposed to explain zinc efﬁciency at the molecular, physiological, structural, and developmental levels, but the actual mechanisms involved are not clear. In general, zinc-efﬁcient genotypes show increased zinc uptake efﬁciency by roots and/or more efﬁcient utilization of

ZINC IN SOILS AND CROP NUTRITION

zinc within the cells. It was suggested that zinc-efﬁcient plant species have the ability to mine zinc from soil by enhancing availability of zinc in the rhizosphere (Dong et al., 1995; Cakmak et al., 1996b; Rengel et al., 1998; Gao et al., 2005). Genc et al. (2006) reported that there are a number of different mechanisms contributing to zinc efﬁciency in barley, but uptake is the major one, and its effect is modiﬁed by the physiological efﬁciency within shoot. Increased zinc uptake might be dependent on root surface area, root colonization by mycorrhizae, pH decline in rhizosphere, release of zinc-chelating PS from roots, and induction of polypeptides involved in zinc uptake and transport across the plasma membranes (Rengel and Graham, 1995c; Cakmak and Braun, 2001). Zinc efﬁciency can be inﬂuenced by root size and morphology, which vary among plant species (Dong et al., 1995; Genc et al., 2007; Chen et al., 2009b) (see also Chapter 2). Longer and thinner roots with increased root surface area may inﬂuence availability of zinc and other nutrients such as copper, manganese, iron (Rengel and Graham, 1995c). Furthermore, soil biological activity such as root colonization by mycorrhizal fungi increases the uptake of diffusionlimited nutrients, including zinc, phosphorus, and copper (see Chapters 3, 12, and 17). Arbuscular mycorrhizae (AM) enables plants to increase zinc uptake by expanding the volume of soil explored by the root system (Gao et al., 2007; Subramanian et al., 2009). The fungal hyphae extend from the roots into the soil, and explore greater distance than root hairs. In maize, AM increased zinc uptake and shoot zinc content in soils with low plant-available zinc concentration (Faber et al., 1990). Roots alter rhizosphere chemistry by changing the rhizosphere pH (Fageria and Stone, 2006; Wang et al., 2006b) and/or releasing PS that could chelate soil zinc and

353

increase its availability (Cakmak et al., 1994; Suzuki et al., 2006; Masuda et al., 2008). Root-mediated decline in pH increases zinc availability by solubilizing zinc from inorganic and organic soil complexes (Hacisalihoglu and Kochian, 2003). A number of studies have focused on the role of root exudates in zinc efﬁciency. PS are nonproteinogenic amino acids released from roots of graminaceous species under deﬁciency of iron (Marschner, 1995) or zinc (Zhang et al., 1991; Kochian, 1993). In the rhizosphere, root exudation plays an important role in increasing the mass and activity of soil microbes that can enhance decomposition of soil organic matter and nutrient cycling (Baudoin et al., 2003; Butler et al., 2003). PS released from roots can also change the solubility, adsorption/desorption, fractionation, and migration of metals in soils through dissolution, chelation, and oxidation/reduction (Clemens et al., 2002; Kuang et al., 2003; Xu et al., 2007a; RasouliSadaghiani et al., 2010). They are involved in mobilizing zinc from the apoplast of wheat roots (Zhang et al., 1991) and, possibly, in translocation and solubility of zinc within the plant (Welch, 1995). Root exudation is closely linked to plant nutritional status, with enhanced rates of exudation in response to nutrient deﬁciency (Ström et al., 2001; Dakora and Phillips, 2002; Rengel, 2002; Ryan et al., 2003; Shen et al., 2003; Xu et al., 2007a). Under zinc deﬁciency, release of PS is about 6–8 times lower in zinc-inefﬁcient durum wheat than zincefﬁcient bread wheat (Cakmak et al., 1996c, 1998). In rice grown in nutrient solution, zinc uptake efﬁciency correlated with exudation rates of low-molecular-weight organic anions (Hofﬂand et al., 2006). Under low soil zinc, the total concentration of organic acid anions in root exudates of barley genotypes was twofold and that of total amino acids 2.5-fold greater in Sahara

354

NUTRIENT USE EFFICIENCY IN CROPS

(zinc-efﬁcient) than Clipper (zinc-inefﬁcient) (Rasouli-Sadaghiani et al., 2010). The results on the role of root PS release in zinc efﬁciency are both contradictory and somewhat controversial. Erenoglu et al. (1996) found that PS release under zinc deﬁciency did not correlate with differences in zinc efﬁciency of efﬁcient and inefﬁcient wheat genotypes. Zinc deﬁciency did not induce PS release in barley or in wheat cultivars that had previously been reported to exude more PS under zinc deﬁciency than zinc-inefﬁcient genotypes (Gries et al., 1995; Pedler et al., 2000). Although root exudation of PS does not appear to have a major importance in zinc efﬁciency, it cannot be ruled out, and the ﬁnal determination awaits the results of further studies (Rengel, 2001). In comparison, with zinc-inefﬁcient genotypes, zinc-efﬁcient ones may differentially affect the microbe populations in the rhizosphere (Rengel, 1997; Bais et al., 2006; Farinati et al., 2009; Xin-Xian et al., 2009). The rhizosphere is a biologically active zone of the soil surrounding the plant roots (Chapter 3). This zone is an important component of biogeochemical cycling of nitrogen, carbon, phosphorus, and other nutrients (Singh et al., 2004), which is essential for promoting plant growth (Morrissey et al., 2004). The number of pseudomonads increased signiﬁcantly in the rhizosphere of zinc-efﬁcient wheat genotypes under zinc deﬁciency (Rengel et al., 1996; Rengel, 1997). However, further research is needed to investigate a possible causal relationship between zinc efﬁciency and increased microbial populations in the rhizosphere. Zn-efﬁcient genotypes can deliver more zinc from roots to shoots than zinc-inefﬁcient genotypes under low but not under zincsufﬁcient supply (Rengel and Graham, 1995c; Cakmak et al., 1996b). The high zinc efﬁciency of rye is mainly related to its capacity to take up and translocate zinc to

shoots at much higher rates than other cereals (Cakmak et al., 1997a). Khan et al. (1998) concluded that an efﬁcient zinc uptake coupled with a better root-to-shoot transport could be important for zinc efﬁciency in chickpea genotypes. There are some reports suggesting that the zinc transport from the soil into the root and then to the shoot is not an important factor in differential zinc efﬁciency. There are no signiﬁcant differences in shoot zinc concentration between inefﬁcient and efﬁcient genotypes grown in soils with low plant-available zinc, even when large differences in visual zinc deﬁciency symptoms can be observed in contrasting genotypes (Cakmak et al., 1999; Hacisalihoglu et al., 2003a). From these studies, the implication is that zinc efﬁciency might be a shootmediated trait. Most likely possibilities for a shoot-mediated mechanism for zinc efﬁciency would be: (1) changes in subcellular zinc compartmentation and homeostasis such that the efﬁcient genotypes accumulate higher levels of zinc in the cytoplasm of leaf cells; and (2) more efﬁcient biochemical use of cellular zinc such that zinc-requiring macromolecules can efﬁciently incorporate zinc as a cofactor under low zinc conditions (Hacisalihoglu and Kochian, 2003). However, both the efﬁcient and inefﬁcient cultivars had similar zinc content in the cytoplasm (9–11%) and vacuole (83–85%) (Hacisalihoglu et al., 2003a), suggesting that subcellular zinc compartmentation may not be involved in zinc efﬁciency. More studies are needed in this area. For zinc homeostasis, metal-responsive elements (MRE) may play a role by controlling gene expression in relation to changes in plant metal status. The metal response element-binding transcription factor-1 (MTF1) mediates the regulation of genes involved in zinc homeostasis, including responses to both zinc deﬁciency and toxicity. MTF1 appears to be involved in the

ZINC IN SOILS AND CROP NUTRITION

regulation of the free zinc concentration in the cell by allowing zinc to bind to MRE and initiate metallothionein gene transcription (Andrews, 2001). There is a positive correlation between biochemical utilization involving zincrequiring enzymes and zinc efﬁciency in wheat and bean (Hacisalihoglu et al., 2003a,b). Zinc-efﬁcient genotypes might contain a higher amount of zinc that readily participates in metabolic reactions and binds to zinc-requiring enzymes. Zinc-efﬁcient wheat genotypes showed higher activity of zinc-requiring enzymes (CA and copper/ zinc SOD) than zinc-inefﬁcient genotypes under zinc-deﬁcient conditions and at similar zinc concentrations in leaves (Rengel, 1995a; Cakmak et al., 1997c; Hacisalihoglu and Kochian, 2003). Internal utilization of zinc is considered an important potential zinc efﬁciency mechanism when zinc-efﬁcient and zincinefﬁcient plants have similar leaf zinc concentrations, and only zinc-inefﬁcient plants show severe zinc deﬁciency symptoms (Rengel and Graham, 1995c). Genc et al. (2002a) indicated that the greater efﬁciency of barley genotypes may be attributed to more efﬁcient utilization at the cellular level. Genotypic Variation for Seed Zinc Accumulation Some crops genotypes have a large capacity to take up trace minerals and accumulate them in grain even when grown in soils with low plant-available micronutrients (Graham and Welch, 1996); however, the physiological processes controlling micronutrient accumulation in seeds are not well understood (Welch and Graham, 2002). Nevertheless, micronutrient uptake and accumulation traits in plants are heritable and could therefore be improved by selective breeding. Screening for genetic variabil-

355

ity in these traits is the ﬁrst step in plant breeding (Ruel and Bouis, 1998). Similar to vegetative tissues, there is a signiﬁcant genotypic variation for seed zinc accumulation in several staple crops including rice, wheat, maize, and bean (Graham et al., 1999; Moraghan and Grafton, 1999; Gregorio et al., 2000; Genc et al., 2002b; Mantovi et al., 2003). For example, the seed zinc concentration of wheat cultivars ranged from 25 to 64 mg kg−1 (Frossard et al., 2000), and ﬁeld-grown barley seed zinc content varied from 0.7 to 2.9 μg seed−1 (Sadeghzadeh, 2008). It is desirable to combine the ability to load zinc into the grain with a high yield potential. The traits of zinc uptake efﬁciency from soil and loading into seed are achievable together with breeding for high yield. Farmers can be expected to adopt higher yielding genotypes with zinc-dense seeds to achieve higher proﬁts. The grain zinc concentration of a genotype reﬂects its ability to take up zinc from the soil, mobilize zinc from different plant parts, and load it into the grain (Pearson et al., 1995; Genc et al., 2006). However, to obtain reliable information on genetic variation in grain nutrient concentrations, any possible dilution and concentration effects associated with differences in yield potential need to be taken into account (McDonald et al., 2008). Screening for Zinc Efﬁciency It is important to clearly deﬁne zinc efﬁciency before describing the screening methods. The deﬁnition proposed by Graham (1984) is useful in looking at genetic differences in zinc efﬁciency among plants. He deﬁned zinc efﬁciency as the ability of a cultivar to grow and yield well in soils too deﬁcient in zinc for a standard cultivar. Zinc efﬁciency can be calculated as the ratio of yield (shoot or grain dry matter) produced under zinc deﬁciency (–Zn) to yield

356

NUTRIENT USE EFFICIENCY IN CROPS

Fig. 16.2. Genotypic variation in the expression of zinc deﬁciency symptoms and growth in barley genotypes Sahara (zinc-efﬁcient) and Clipper (zinc-inefﬁcient) grown for 50 days in Lancelin soil fertilized with 0.0 and 0.8 mg Zn kg−1 soil (Sadeghzadeh, 2008).

produced with supplied zinc fertilizer (+Zn). Zinc efﬁciency can be divided into two components: uptake, or the plant’s ability to acquire the nutrient from the soil, and utilization efﬁciency, or the ability to convert the absorbed nutrient into grain yield (see Chapter 1). Plant genotypes are widely different in their tolerance to zinc deﬁciency, both in zinc uptake and in zinc utilization. A wide range of wheat, barley, rice, bean, chickpea, and maize germplasm has been studied, indicating there is enough genotypic variation to allow breeding for nutritional improvement (Fig. 16.2, Table 16.3) (Khan et al., 1998; Genc et al., 1999; Banziger and Long, 2000; Beebe et al., 2000; Gregorio et al., 2000; Ortiz-Monasterio and Graham, 2000; Wissuwa et al., 2006; Sadeghzadeh et al., 2009a). Nutritional traits are generally stable across environments, despite some reported genotype × environment interactions; it is possible to combine high micronutrient traits with high yield (Gregorio, 2002). Rengel and Wheal (1997b) indicated that differences in zinc efﬁciency between wheat

cultivars are closely related to differences in zinc uptake capacity. Similarly, higher zinc efﬁciency of rye over wheat cultivars is accompanied by an increase in zinc concentration of shoots; rye has a higher genetic ability to absorb zinc from soils with low plant-available zinc (Cakmak et al., 1997a). In ﬁeld experiments on zinc-deﬁcient calcareous soils, zinc efﬁciency is positively correlated with total amount (uptake) of zinc in shoots (Graham et al., 1992; Cakmak et al., 1997b). Although zinc-efﬁcient genotypes have greater zinc uptake ability, they do not necessarily have a higher zinc concentration (amount of zinc per unit of dry weight) in leaf or shoot tissue, or grain (Graham et al., 1992). Indeed, zinc-inefﬁcient wheat genotypes may even have greater zinc concentrations in leaves or grains than zincefﬁcient genotypes (Rengel and Graham, 1996; Cakmak et al., 1997b; Cakmak et al., 1998). Under zinc deﬁciency, increased zinc uptake by efﬁcient genotypes improves dry matter production and results in corresponding decreases in zinc concentration similar to those present in zinc-inefﬁcient

Table 16.3.

Plant traits used in screening plant genotypes for zinc efﬁciency

Plant Trait

Plant Type

Reference

Phytosiderophore release

Barley

Gries et al. (1995); Suzuki et al. (2006); RasouliSadaghiani et al. (2010) Cakmak et al. (1996a) Masuda et al. (2008) Hopkins et al. (1998) Cakmak et al. (1994); Walter et al. (1994) Cayton et al. (1985) Cakmak et al. (1996b) Grewal and Williams (1999) Norvell and Welch (1993) Fageria (2002) Yu et al. (1999) Rengel and Graham (1995c; Cakmak et al. (1996b); Rengel and Romheld (2000) Genc et al. (2002a); Sadeghzadeh et al. (2009a) Khan et al. (1998) Pearson and Rengel (1997) Genc et al. (2003); Sadeghzadeh et al. (2009a) Hacisalihoglu and Kochian (2003) Cakmak et al. (1996a); Yu et al. (1999) Jolley and Brown (1991) Cakmak et al. (1998) Cakmak et al. (1997b) Erenoglu et al. (1996) Genc et al. (2007) Gao et al. (2005) Cakmak et al. (1997b) Dong et al. (1995); Rengel and Wheal (1997a) Wu et al. (2003) Cakmak et al. (1996b) Chen et al. (2003) Gao et al. (2005) Pearson and Rengel (1997) Genc et al. (2002a); Sadeghzadeh (2008); Lonergan et al. (2009); Rasouli-Sadaghiani et al. (2010) Khan et al. (1998) Hopkins et al. (1998) Cakmak et al. (1996b) Cakmak et al. (1997b) Erenoglu et al. (1996) Norvell and Welch (1993); Sadeghzadeh et al. (2009a) Cakmak et al. (1996b) Rengel and Graham (1995d) Sadeghzadeh (2008); Birsin (2010) Ranjbar and Bahmaniar (2007) Harris and Taylor (2004) Cakmak et al. (1997c) Lasat et al. (1998) Kothari et al. (1991) Lynch and Whipps (1990)

Shoot/root ratio of Zn (translocation factor) Shoot/root dry weight

Root/shoot dry weight

Scoring Zn deﬁciency visual symptoms

Fine roots and root surface area

Zn translocation into shoot

Shoot Zn concentration and content

Root Zn concentration and content Flag leaf Zn concentration 65 Zn translocation into shoot Microbial growth in the rhizosphere

Durum Rice Sorghum Wheat Rice Wheat Lucerne Barley Bean Durum Wheat Barley Chickpea Wheat Barley Common bean Durum Navy bean Oat Triticale Wheat Barley Rice Rye Wheat Barley Durum Red clover Rice Wheat Barley Chickpea Maize Durum Rye Wheat Barley Durum Wheat Barley Wheat Durum Wheat Thlaspi Maize Wheat

357

358

NUTRIENT USE EFFICIENCY IN CROPS

genotypes (dilution by growth) (cf. Marschner, 1995). A lack of a suitable screening procedure to allow screening of a large number of genotypes (e.g., doubled-haploid populations) in short time has hampered breeding for zinc efﬁciency. Different screening methods have been used to evaluate zinc efﬁciency: nutrient solution culture (Rengel and Graham, 1995c; Sadeghzadeh et al., 2009a), ﬁeld evaluations (Bagci et al., 2007; Sadeghzadeh, 2008), and greenhouse soil bioassays (Genc et al., 2002b; Sadeghzadeh, 2008). Soil-free systems such as chelatorbuffered hydroponic system have been used commonly to study plant mineral nutrition at realistically low zinc concentrations (Parker, 1997; Rengel, 1999). Chelatorbuffered nutrition solutions simulate soil solution conditions with accurate control of micronutrient activity (Parker et al., 1995), so that zinc deﬁciency could be induced in a predictable and reproducible manner. Moreover, this system with appropriately low activities of zinc may be useful in ranking genotypes for differential tolerance to zinc deﬁciency that can be conﬁrmed in the ﬁeld (Rengel and Graham, 1995c; Rengel, 1999). In a study on differential zinc efﬁciency of two barley genotypes (Sahara = zinc-efﬁcient; Clipper = zincinefﬁcient) grown in soil and chelatorbuffered nutrient solution, Sadeghzadeh et al. (2009a) reported that the responses of the genotypes to zinc fertilization in chelatorbuffered nutrient solution were consistent with their response in soils in terms of visual zinc deﬁciency symptoms and shoot and root zinc concentration and content. Although zinc-efﬁcient genotypes possess extensive root systems (Cakmak and Marschner, 1988b; Cakmak et al., 1989), they do not necessarily have bigger roots than inefﬁcient genotypes when grown in nutrient solution. For example, Sahara had less root dry matter than Clipper in nutrient

solution with deﬁcient zinc supply (Sadeghzadeh et al., 2009a). Similar results were reported by Rengel and Graham (1995c), where root growth of zincinefﬁcient wheat genotypes increased in chelator-buffered nutrient solution with deﬁcient zinc supply. In contrast, in chelatorbuffered nutrient solution, Rengel and Romheld (2000) observed a more severe decline in root growth of zinc-inefﬁcient wheat Songlen than zinc-efﬁcient Aroona as a consequence of zinc deﬁciency, which is consistent with other studies on wheat (Webb and Loneragan, 1990), as well as reports on unchanged root growth of barley grown in chelator-buffered nutrient solution at low zinc activity (Norvell and Welch, 1993; Welch and Norvell, 1993). Therefore, using root growth as a criterion to select for zinc-efﬁcient genotypes when grown in nutrient solution is not recommended. Field-based techniques are more laborious than pot or solution culture ones. The results can be variable because the severity of the nutrient deﬁciency varies among sites and years due to the effects of other growthlimiting factors (such as drought and disease) as well as the irregular spatial distribution of zinc in soil. Screening under glasshouse condition in pots is generally easier than under ﬁeld conditions because it is fast, cost-effective, and can overcome problems of soil heterogeneity. The growth containers should be big enough to eliminate root restriction, reduced sink strength at the whole plant level, and reduced photosynthetic enhancement (Arp, 1991). However, pot screening is less realistic than ﬁeld conditions, especially for grain yield production. Nevertheless, using 150 barley doubled-haploid lines grown to maturity, a strong correlation between zinc efﬁciency in ﬁeld and glasshouse conditions was shown (Sadeghzadeh, 2008). However, the suitability of pot screening can be affected when additional nutrient stress

ZINC IN SOILS AND CROP NUTRITION

exists. For example, the correlation between zinc efﬁciency in the ﬁeld and glasshouse was weak in soils with both boron toxicity and zinc deﬁciency (Cakmak and Braun, 2001).

Genetics of Zinc Efﬁciency Most research related to zinc efﬁciency in crops has concentrated on the physiological aspects of zinc uptake, or has compared genotypes for their relative efﬁciency for growing in soils with low plant-available zinc. Although some physiological mechanisms involved in zinc efﬁciency have been documented, limited information is available on the genetic control of these mechanisms and the genes responsible for zinc efﬁciency. Genetic variability for zinc efﬁciency and its possibly simple inheritance should allow progress toward improving zinc efﬁciency in crops. A limited number of studies have yielded some preliminary evidence regarding the genetics of zinc efﬁciency in several crop species. Studies of chromosome addition lines in rye have shown that copper, zinc, and manganese efﬁciencies were independent traits located on different chromosomes (Graham, 1984). Results from a diallel analysis in rice suggested that the genes controlling zinc efﬁciency are additive and, to a lesser degree, dominant (Majumder et al., 1990). In maize, four additive genes were reported to affect zinc concentration in the ear leaf (El-Bendary et al., 1993). Several loci on chromosomes 1R, 2R, and 7R enhance zinc efﬁciency in rye, with genes on short arms of 1R and 7R being the most effective (Schlegel and Cakmak, 1997; Cakmak et al., 1997a). The distribution of F3 populations from the cross between zincefﬁcient and zinc-inefﬁcient genotypes showed that only a few genes control zinc efﬁciency in soybean based on measure-

359

ments of foliar zinc concentrations (Hartwing et al., 1991). The results from a diallel experiment comparing seven wheat cultivars differing in zinc efﬁciency with their F1 derivatives showed that genes controlling zinc efﬁciency are dominant (Cakmak and Braun, 2001). The overexpression of zinc transporters in cereals may affect plant growth, seed mineral content, and zinc transport rates (Table 16.4). Lasat et al. (1996) found that enhanced expression of genes encoding zinc transporters can increase zinc uptake in T. caerulescens and T. arvense. Further studies in this system found a zinc transporter ZNT1 gene that was highly expressed in T. caerulescens, allowing enhanced zinc uptake (Lasat et al., 2000; Pence et al., 2000; Assunção et al., 2001). ZNT1 gene is a member of the ZIP family of metal transporters (Guerinot, 2000) that are known to transport a variety of divalent cations. Transgenic Arabidopsis that overexpressed a plasma membrane ion transporter, ZAT, exhibited enhanced zinc content in the roots of plants grown in a high-zinc environment (Van der Zaal et al., 1999). In barley, overexpression of an Arabidopsis zinc transporter increased short-term zinc uptake and seed cation content (Ramesh et al., 2004). Uauy et al. (2006) reported that a reduction in RNA levels of the T. aestivum, No Apical Meristem (TaNAM) gene, is associated with a decrease in wheat grain zinc and iron concentrations and an increase in residual nitrogen, zinc, and iron in the ﬂag leaf. These results suggest that the reduced grain zinc and iron concentrations were the result of reduced translocation from leaves, rather than a dilution effect caused by larger grains. By using visual zinc deﬁciency symptoms, it was reported that a single dominant gene controls tolerance to zinc deﬁciency in common bean (Singh and Westermann, 2002). Similarly, using a visual score of deﬁciency symptoms in barley, Genc et al.

360

NUTRIENT USE EFFICIENCY IN CROPS

Examples of up-regulated expression of genes encoding proteins responsible for Zn uptake, sequestration and redistribution within the plant during Zn deﬁciency

Table 16.4.

Gene

Encoded Protein

Plant Type

Reference

ZIPs

Zinc iron-like proteins (Zinc iron permeases)

Arabidopsis

HMAs

Heavy-metal ATPase family

Grotz et al. (1998); Wintz et al. (2003); Talke et al. (2006); Van De Mortel et al. (2006); Krämer et al. (2007) Ishimaru et al. (2005) Wintz et al. (2003); Papoyan and Kochian (2004); Verret et al. (2004); Van De Mortel et al. (2006) Suzuki et al. (2006) Wintz et al. (2003); Schaaf et al. (2005); Grotz and Guerinot (2006); Waters et al. (2006) Arrivault et al. (2006); Rout and Das (2009) Chen et al. (2009a)

Rice Arabidopsis

YSLs

Yellow stripe-like proteins Metal tolerance proteins

Barley Arabidopsis

MTPs

Zn-induced facilitator

Arabidopsis

Ferric reductase defective 3 Nicotianamine synthase

Medicago

ZIF

Thlaspi Arabidopsis

FRD3

Arabidopsis

NAS

Arabidopsis

Tobacco

Assunção et al. (2001) Hussain et al. (2004); Haydon and Cobbett (2007a) Delhaize (1996); Rogers and Guerinot (2002); Van De Mortel et al. (2006) Wintz et al. (2003); Becher et al. (2004); Weber et al. (2004); Talke et al. (2006) Takahashi et al. (2003)

FRD, ferric reductase defective.

(2003) reported that tolerance to zinc deﬁciency at the seedling stage is controlled by a single gene with no dominance. Concluding Remarks Low plant availability of soil zinc is a critical problem for crop production, causing severe reduction in yield and nutritional quality of the edible portion of ﬁeld crops. High zinc efﬁciency in crops appears to be related to various morphological and physiological traits such as root surface area, zinc-mobilizing root exudates, and better utilization of zinc at the cellular level. Combining improved tolerance to zinc deﬁciency in soils and increased zinc concentra-

tion and content in seed is a highpriority research topic. This topic, however, requires a comprehensive exploration of potential genetic resources and an indepth understanding of zinc accumulation mechanisms. The processes and factors that control zinc transfer from soil to roots and shoots and then to the grain are far from being understood. More work is also needed on the effects of cultivation methods on phytate concentrations in food crops (phytate limits zinc absorption in the human digestive system) and the implications for human health. Research is also needed on the role of soil biota in zinc accumulation by plants.

ZINC IN SOILS AND CROP NUTRITION

With the growing awareness of the large numbers of people suffering from zinc deﬁciency, there is enough nutritional evidence to persuade plant breeders to consider zinc density traits as an important objective in the breeding programs targeted to the developing world. Currently, the application of zinc fertilizers appears to be a cheap, effective, and short-term means of increasing zinc concentration in grains. This increased zinc density in grain would have to be achieved without concomitant yield depression. However, increased application of zinc fertilization beyond a critical level can cause a vegetative and grain yield decrease because of zinc toxicity. The long-term solution is to develop new genotypes with higher concentration of zinc as well as nutritional enhancers that promote zinc bioavailability. Such biofortiﬁcation would improve the health of people (especially susceptible populations) consuming these crops. There is signiﬁcant genetic variation for zinc efﬁciency in cereals, suggesting that selection for improved zinc efﬁciency is possible. Evaluating the severity of zinc deﬁciency symptoms on leaves as well as monitoring shoot and seed zinc concentrations and content appear to be useful approaches for screening large populations for zinc efﬁciency. In view of the difﬁculty in developing fast and reliable methods of screening for efﬁciency (such as seedling selection in pots) or their limited applicability to ﬁeld conditions, development of molecular markers for zinc efﬁciency is considered important for success in cereal breeding programs. The recent identiﬁcation of DNA markers diagnostic of zinc efﬁciency should accelerate production of cultivars yielding well on soils with low plant-available zinc (Lonergan et al., 2009; Sadeghzadeh et al., 2009b), and may be the starting point for identifying the speciﬁc genes responsible for differences in the response of crops to zinc deﬁciency.

361

Agronomic biofortiﬁcation of staple crops with zinc using suitable cultivation methods is an achievable objective. This is a complementary approach to the improvement of genotypes by breeding and genetic engineering, as well as to improvements in the processing of crops into food products. References Abo-Hegazi, A.M.T., Rofail, N.B., Eissa, E.A., & Hassan, A.M. (1996) Effect of zinc on grain characteristics of draught-resistant rice mutants. Journal of Radioanalytical and Nuclear Chemistry 206, 349–357. Agbim, N.N. (2009) Interactions of phosphorus, magnesium and zinc on the yield and nutrient content of maize. The Journal of Agricultural Science 96, 509–514. Allen, M.D., Kropat, J., Tottey, S., Del Campo, J.A., & Merchant, S.S. (2007) Manganese deﬁciency in Chlamydomonas results in loss of photosystem II and MnSOD function, sensitivity to peroxides, and secondary phosphorus and iron deﬁciency. Plant Physiology 143, 263–277. Alloway, B.J. (1995) Heavy Metals in Soils. Blackie Academic & Professional, London, UK. Alloway, B.J. (2001) Zinc—The Vital Micronutrient for Healthy, High-Value Crops. International Zinc Association, Brussels. Alloway, B.J. (2004) Zinc in Soils and Crop Nutrition. International Zinc Association Communications. IZA publications, Brussels, Belgium. Alloway, B.J. (2008) Zinc in Soils and Crop Nutrition, 2nd ed. published by IZA and IFA, Brussels, Belgium and Paris, France. Amer, F., Rezk, A.I., & Khalid, H.M. (1980) Fertilizer zinc efﬁciency in ﬂooded calcareous soils. Soil Science Society of America Journal 44, 1025–1030. Andrews, G.K. (2001) Cellular zinc sensors: MTF-1 regulation of gene expression. Biometals 14, 223–237. Arp, W.J. (1991) Effects of source-sink relations on photosynthetic acclimation to elevated CO2. Plant, Cell & Environment 14, 869–875. Arredondo, M., Martínez, R., Núñez, M.T., Ruz, M., & Olivares, M. (2006) Inhibition of iron and copper uptake by iron, copper and zinc. Biological Research 39, 95–102. Arrivault, S., Senger, T., & Krämer, U. (2006) The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deﬁciency and Zn oversupply. The Plant Journal 46, 861–879.

362

NUTRIENT USE EFFICIENCY IN CROPS

Ascher, J.S., Graham, R.D., Elliott, D.E., Scott, J.M., & Jessop, R.S. (1994) Agronomic value of seed with high nutrient content. In: Wheat in HeatStressed Environments: Irrigated, Dry Areas and Rice-Wheat Farming Systems (eds. D.A. Saunders & G.P. Hettel), pp. 297–308. CIMMYT, Mexico. Assunção, A.G.L., Martins, P.D.C., De Folter, S., Vooijs, R., Schat, H., & Aarts, M.G.M. (2001) Elevated expression of metal transporter genes in three accessions of the metal hyperaccumulator Thlaspi caerulescens. Plant, Cell & Environment 24, 217–226. Auld, D.S. (2001) Zinc coordination sphere in biochemical zinc sites. Biometals 14, 271–313. Azhar, K.F., Nawaz, S., & Ali, M.S. (2005) Effect of some trace elements on the nucleic acid metabolising enzymes level of germinating wheat seeds. Natural Product Research 19, 337–346. Bagci, S.A., Ekiz, H., Yilmaz, A., & Cakmak, I. (2007) Effects of zinc deﬁciency and drought on grain yield of ﬁeld-grown wheat cultivars in Central Anatolia. Journal of Agronomy and Crop Science 193, 198–206. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S., & Vivanco, J.M. (2006) The role of root exudates in rhizosphere interactions with plants and other organisms. Annual Review of Plant Biology 57, 233–266. Baligar, V.C., Fageria, N.K., & He, Z.L. (2001) Nutrient use efﬁciency in plants. Communications in Soil Science and Plant Analysis 32, 921–950. Bansal, R.L., Singh, S.P., & Nayyar, V.K. (1990) The critical zinc deﬁciency level and response to zinc application of wheat on Typic Ustochrepts. Experimental Agriculture 26, 303–306. Banziger, M. & Long, J. (2000) The potential for increasing the iron and zinc density of maize through plant breeding. Food and Nutrition Bulletin 21, 397–400. Barak, P. & Helmke, P.A. (1993) The chemistry of zinc. In: Zinc in Soils and Plants (ed. A.D. Robson), Kluwer Academic Publishers, Dordrecht, The Netherlands. Barber, S.A. (1984) Soil Nutrient Bioavailability: A Mechanistic Approach, 2nd ed. John Wiley, New York. Barber, S.A. (1995) Soil Nutrient Bioavailability. John Wiley & Sons, Inc, New York, NY. Barber, S.A. & Silberbush, M. (1984) Plant root morphology and nutrient uptake. In: Roots, Nutrients and Water Inﬂux and Plant Growth, vol. 49 (eds. S.A. Barber & R. Bouldin), pp. 65–88. American Society of Agronomy Special Publication, Madison, WI. Barrow, N.J. (1993) Mechanisms of reaction of zinc with soil and soil components. In: Zinc in Soils and

Plants (ed. A.D. Robson), pp. 15–31. Kluwer Academic Publishers, Dordrecht, The Netherlands. Baudoin, E., Benizri, E., & Guckert, A. (2003) Impact of artiﬁcial root exudates on the bacterial community structure in bulk soil and maize rhizosphere. Soil Biology and Biochemistry 35, 1183–1192. Becher, M., Talke, I.N., Krall, L., & Krämer, U. (2004) Cross-species microarray transcript proﬁling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. The Plant Journal 37, 251–268. Beebe, S., Gonzalez, A., & Rengifo, J. (2000) Research on trace minerals in the common bean. Food and Nutrition Bulletin 21, 387–391. Berg, J.M. & Shi, Y. (1996) The galvanization of biology: a growing appreciation for the roles of zinc. Science 271, 1081–1085. Bettger, W.J. & O’Dell, B.L. (1981) A critical physiological role of zinc in the structure and function of biomembranes. Life Science 28, 1425–1438. Birsin, M.A. (2010) Mineral element distribution and accumulation patterns within two barley cultivars. Journal of Plant Nutrition 33, 267–284. Black, R.E., Allen, L.H., Bhutta, Z.A., et al. (2008) Maternal and child undernutrition: global and regional exposures and health consequences. The Lancet 371, 243–260. Boawn, L.C. & Brown, J.C. (1968) Further evidence of P-Zn imbalance in plants. Proceedings of the Soil Science Society of America 32, 94–97. Bolland, M.D.A., Jarvis, R.J., Coates, P., & Harris, D.J. (1993) Effect of phosphate fertilizers on the elemental composition of seed of wheat, lupin, and triticale. Communication in Soil Science and Plant Analysis 24, 1991–2014. Bonnet, M., Camares, O., & Veisseire, P. (2000) Effects of zinc and inﬂuence of Acremonium lolii on growth parameters, chlorophyll a ﬂuorescence and antioxidant enzyme activities of ryegrass (Lolium perenne L. cv Apollo). Journal of Experimental Botany 51, 945–953. Bouis, H. (1996) Enrichment of food staples through plant breeding: a new strategy for ﬁghting micronutrient malnutrition. Nutrition Reviews 54, 131–137. Bowler, C., Montagu, M.V., & Inze, D. (1992) Superoxide dismutase and stress tolerance. Annual Review of Plant Biology 43, 83–116. Brawn, K. & Fridovich, I. (1981) DNA strand scission by enzymatically generated oxygen radicals. Archives of Biochemistry and Biophysics 206, 414–419. Bremner, I. & Beattie, J.H. (2007) Copper and zinc metabolism in health and disease: speciation and

ZINC IN SOILS AND CROP NUTRITION

interactions. The Proceedings of the Nutrition Society 54, 489–499. Brennan, R.F. (1991) Effectiveness of zinc sulfate and zinc chelate as foliar sprays in alleviating zinc deﬁciency of wheat grown on zinc-deﬁcient soils in Western Australia. Australian Journal of Experimental Agriculture 31, 831–834. Brennan, R.F. (1996) Availability of previous and current applications of zinc fertilizer using single superphosphate for the grain production of wheat on soils of South Western Australia. Journal of Plant Nutrition 19, 1099–1115. Brennan, R.F., Armour, J.D., & Reuter, D.J. (1993) Diagnosis of zinc deﬁciency. In: Zinc in Soils and Plants (ed. A.D. Robson), pp. 168–181. Kluwer Academic, Dordrecht. Broadley, M.R., White, P.J., Hammond, J.P., Zelko, I., & Lux, A. (2007) Zinc in plants. The New Phytologist 173, 677–702. Brown, P.H., Cakmak, I., & Zhang, Q. (1993) Form and function of zinc in plants. In: Zinc in Soils and Plants (ed. A.D. Robson), pp. 93–106. Kluwer Academic Publishers, Dordrecht, The Netherlands. Brümmer, G.W., Gerth, J., & Tiller, K.G. (1988) Reaction kinetics of the adsorption and desorption of nickel, zinc and cadmium by goethite. I. Adsorption and diffusion of metals. Journal of Soil Science 39, 37–52. Butler, J.L., Williams, M.A., Bottomley, P.J., & Myrold, D.D. (2003) Microbial community dynamics associated with rhizosphere carbon ﬂow. Applied and Environmental Microbiology 69, 6793–6800. Cakmak, I. (2000) Possible roles of zinc in protecting plant cells from damage by reactive oxygen species. The New Phytologist 146, 185–205. Cakmak, I. (2002) Plant nutrition research: priorities to meet human needs for food in sustainable ways. Plant and Soil 247, 3–24. Cakmak, I. (2004) Identiﬁcation and correction of widespread zinc deﬁciency in Turkey—a success story (a NATO-Science for Stability Project). Proceedings of the International Fertiliser Society 552, 1–26. Cakmak, I. (2008) Enrichment of cereal grains with zinc: agronomic or genetic biofortiﬁcation? Plant and Soil 302, 1–17. Cakmak, I. & Marschner, H. (1987) Mechanism of phosphorus-induced zinc deﬁciency in cotton. III: changes in physiological availability of zinc in plants. Physiologia Plantarum 70, 13–20. Cakmak, I. & Marschner, H. (1988a) Enhanced superoxide radical production in roots of zinc-deﬁcient plants. Journal of Experimental Botany 39, 1449–1460.

363

Cakmak, I. & Marschner, H. (1988b) Increase in membrane permeability and exudation in roots of zinc deﬁcient plants. Journal of Plant Physiology 132, 356–361. Cakmak, I. & Braun, H.J. (2001) Genotypic variation for zinc efﬁciency. In: Application of Physiology in Wheat Breeding (eds. M.P. Reynolds, J.I. OrtizMonasterio & A. McNab), pp. 183–199. D.F. CIMMYT, Mexico. Cakmak, I., Marschner, H., & Bangerth, F. (1989) Effect of zinc nutritional status on growth, protein metabolism and levels of indole-3-acetic acid and other phytohormones in bean (Phaseolus vulgaris L.). Journal of Experimental Botany 40, 405–412. Cakmak, I., Gulut, K.Y., Marschner, H., & Graham, R.D. (1994) Effect of zinc and iron deﬁciency on phytosiderophore release in wheat genotypes differing in zinc efﬁciency. Journal of Plant Nutrition 17, 1–17. Cakmak, I., Atli, M., Kaya, R., Evliya, H., & Marschner, H. (1995) Association of high light and zinc deﬁciency in cold-induced leaf chlorosis in grapefruit and mandarin trees. Journal of Plant Physiology 146, 355–360. Cakmak, I., Sari, N., Marschner, H., et al. (1996a) Phytosiderophore release in bread and durum wheat genotypes differing in zinc efﬁciency. Plant and soil 180, 183–189. Cakmak, I., Sari, N., Marschner, H., et al. (1996b) Dry matter production and distribution of zinc in bread and durum wheat genotypes differing in Zn efﬁciency. Plant and Soil 180, 173–181. Cakmak, I., Yilmaz, A., Kalayci, M., et al. (1996c) Zinc deﬁciency as a critical problem in wheat production in central Anatolia. Plant and Soil 180, 165–172. Cakmak, I., Derici, R., Torun, B., Tolay, I., Braun, H.J., & Schlegel, R. (1997a) Role of rye chromosome in improvement of zinc efﬁciency in wheat and triticale. Plant and Soil 196, 249–253. Cakmak, I., Ekiz, H., Yilmaz, A., et al. (1997b) Differential response of rye, triticale, bread and durum wheats to zinc deﬁciency in calcareous soils. Plant and Soil 188, 1–10. Cakmak, I., Oztürk, L., Eker, S., Torun, B., Kalfa, H.I., & Yılmaz, A. (1997c) Concentration of zinc and activity of copper/zinc-superoxide dismutase in leaves of rye and wheat cultivars differing in sensitivity to zinc eﬁciency. Journal of Plant Physiology 151, 91–95. Cakmak, I., Torun, B., Erenoglu, B., et al. (1998) Morphological and physiological differences in the response of cereals to zinc deﬁciency. Euphytica 100, 349–357. Cakmak, I., Kalayci, M., Ekiz, H., Braun, H.J., Kilinc, Y., & Yilmaz, A. (1999) Zn deﬁciency as a practical

364

NUTRIENT USE EFFICIENCY IN CROPS

problem in plant and human nutrition in Turkey: a NATO-Science for stability project. Field Crops Research 60, 175–188. Cappuyns, V., Swennen, R., Vandamme, A., & Niclaes, M. (2006) Environmental impact of the former Pb-Zn mining and smelting in East Belgium. Journal of Geochemical Exploration 88, 6–9. Castilho, P.D. & Chardon, W.J. (1995) Uptake of soil cadmium by three ﬁeld crops and its prediction by a pH-dependent Freundlich sorption model. Plant and Soil 171, 263–266. Catlett, K.M., Heil, D.M., Lindsay, W.L., & Ebinger, M.H. (2002) Soil chemical properties controlling zinc2+ activity in 18 Colorado soils. Soil Science Society of America Journal 66, 1182–1189. Cayton, M.T.C., Reyes, E.D., & Neue, H.U. (1985) Effect of zinc fertilization on the mineral nutrition of rices differing in tolerance to zinc deﬁciency. Plant and Soil 87, 319–327. Chaney, R.L. (1993) Zinc phytotoxicity. In: Zinc in Soils and Plants (ed. A.D. Robson), Kluwer Academic Publishers, Dordrecht. Chatterjee, C. & Khurana, N. (2007) Zinc stressinduced changes in biochemical parameters and oil content of mustard. Communications in Soil Science and Plant Analysis 38, 751–761. Chaudhry, F.M. & Loneragan, J.F. (1972) Zinc absorption by wheat seedlings. II. Inhibitation by hydrogen ions and by micronutrient cations. Soil Science Society of America, Proceedings 36, 327–331. Chaudhry, F.M., Sharif, M., Latif, A., & Qureshi, R.H. (1973) Zinc-copper antagonism in the nutrition of rice (Oryza sativa L.). Plant and soil 38, 573–580. Chen, W.R., He, Z.L., Yang, X.E., & Feng, Y. (2009b) Zinc efﬁciency is correlated with root morphology, ultrastructure, and antioxidative enzymes in rice. Journal of Plant Nutrition 32, 287–305. Chen, B.D., Li, X.L., Tao, H.Q., Christie, P., & Wong, M.H. (2003) The role of arbuscular mycorrhiza in zinc uptake by red clover growing in a calcareous soil spiked with various quantities of zinc. Chemosphere 50, 839–846. Chen, W., Provart, N.J., Glazebrook, J., et al. (2002) Expression proﬁle matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. The Plant Cell Online 14, 559–574. Chen, M., Shen, X., Li, D., Ma, L., Dong, J., & Wang, T. (2009a) Identiﬁcation and characterization of MtMTP1, a Zn transporter of CDF family, in the Medicago truncatula. Plant Physiology and Biochemistry 47, 1089–1094. Chesworth, W. (1991) Geochemistry of micronutrients. In: Micronutrients in Agriculture, 2nd ed. (eds.

J.J. Mortvedt, F.R. Cox, L.M. Shuman & R.M. Welch), pp. 1–30. Soil Science Society of America Inc., Madison, WI. Ciais, D., Cherradi, N., Bailly, S., et al. (2004) Destabilization of vascular endothelial growth factor mRNA by the zinc-ﬁnger protein TIS11b. Oncogene 23, 8673–8680. Clemens, S., Palmgrenb, M.G., & Krämer, U. (2002) A long way ahead: understanding and engineering plant metal accumulation. Trends in plant science 7, 309–315. Constant, K.M. & Sheldrick, W.F. (1991) An outlook for fertilizer demand, supply, and trade. World Bank Technical Paper No. 137, Asia Technical Department Series. The World Bank, Washington, DC. Cummings, J.E. & Kovacic, J.P. (2007) The ubiquitous role of zinc in health and disease. Journal of Veterinary Emergency and Critical Care 19, 215–240. Dakora, F.D. & Phillips, D.A. (2002) Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant and Soil 245, 35–47. Dang, Y.P., Edwards, D.G., Dalal, R.C., & Tiller, K.G. (1993) Identiﬁcation of an index tissue to predict zinc status of wheat. Plant and Soil 154, 161–167. Das, K., Dang, R., Shivananda, T.N., & Sur, P. (2005) Interaction effect between phosphorus and zinc on their availability in soil in relation to their contents in stevia (Stevia rebaudiana). The Scientiﬁc World Journal 5, 490–495. Davletova, S., Schlauch, K., Coutu, J., & Mittler, R. (2005) The zinc-ﬁnger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis. Plant Physiology 139, 847–856. de Varennes, A., Carneiro, J.P., & Goss, M.J. (2001) Characterization of manganese toxicity in two species of annual medics. Journal of Plant Nutrition 24, 1947–1955. Delhaize, E. (1996) A metal-accumulator mutant of Arabidopsis thaliana. Plant Physiology 111, 849–855. Distelfeld, A., Cakmak, I., Peleg, Z., et al. (2007) Multiple QTL-effects of wheat Gpc-B1 locus on grain protein and micronutrient concentrations. Physiologia Plantarum 129, 635–643. Donald, C.M. & Prescott, J.A. (1975) Trace elements in Australian crop and pasture production1924–74. In: Trace Elements in Soil-Plant-Animal Systems (eds. D.J.D. Nicholas & A.R. Egan), pp. 7–37. Academic Press, Inc., New York, NY. Dong, B., Rengel, Z., & Graham, R.D. (1995) Root morphology of wheat genotypes differing in zinc efﬁciency. Journal of Plant Nutrition 18, 2761–2773.

ZINC IN SOILS AND CROP NUTRITION

Eide, D.J. (2006) Zinc transporters and the cellular trafﬁcking of zinc. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1763, 711–722. Ekiz, H., Bagcý, S.A., Kýral, A., et al. (1998) Effects of zinc fertilization and irrigation on grain yield and zinc concentration of various cereals grown in zincdeﬁcient calcareous soils. Journal of plant nutrition 21, 2245–2256. El-Bendary, A.A., El-Fouly, M.M., Raksa, F.A., Omar, A.A., & Abou-Youssef, A.Y. (1993) Mode of inheritance of zinc accumulation in maize. Journal of Plant Nutrition 16, 2043–2053. Eren, E. & Arguello, J.M. (2004) Arabidopsis HMA2, a divalent heavy metal-transporting PIB-type ATPase, is involved in cytoplasmic Zn2+ homeostasis. Plant Physiology 136, 3712–3723. Erenoglu, B., Cakmak, I., Marschner, H., et al. (1996) Phytosiderophore release does not relate well with zinc efﬁciency in different bread wheat genotypes. Journal of Plant Nutrition 19, 1569–1580. Eulgem, T., Rushton, P.J., Robatzek, S., & Somssich, I.E. (2000) The WRKY superfamily of plant transcription factors. Trends in Plant Science 5, 199–205. Eyüpoglu, F., Kurucu, N., & Sanısag, U. (1994) Status of plant available micronutrients in Turkish soils. In: Soil and Fertilizer. Research Institute Annual Report No. R-118. Ankara, Turkey, pp. 25–32. Faber, B.A., Zasoski, R.J., Burau, R.G., & Uriu, K. (1990) Zinc uptake by corn as affected by vesiculararbuscular mycorrhizae. Plant and Soil 129, 121–130. Fageria, N.K. (2002) Micronutrients’ inﬂuence on root growth of upland rice, common bean, corn, wheat, and soybean. Journal of Plant Nutrition 25, 613–622. Fageria, N.K. & Stone, L.F. (2006) Physical, chemical, and biological changes in the rhizosphere and nutrient availability. Journal of Plant Nutrition 29, 1327–1356. Fageria, N.K., Baligar, V.C., & Jones, C.A. (1991) Growth and Mineral Nutrition of Field Crops. Marcel Dekker, New York, NY. Fageria, N.K., Baligar, V.C., & Clark, R.B. (2002) Micronutrients in crop production. Advances in Agronomy 77, 185–167. Farinati, S., DalCorso, G., Bona, E., et al. (2009) Proteomic analysis of Arabidopsis halleri shoots in response to the heavy metals cadmium and zinc and rhizosphere microorganisms. Proteomics 9, 4837–4850. Fontes, P.C.R., Moreira, M.A., Fontes, R.L.F., & Cardoso, A.A. (1999) Effects of zinc fungicides and different zinc fertilizer application methods on soluble and total zinc in potato plant shoots.

365

Communications in Soil Science and Plant Analysis 30, 1847–1859. Foth, H.D. & Ellis, B.G. (1997) Soil Fertility. CRC Press, Boca Raton, FL. Fowler, S. & Thomashow, M.F. (2002) Arabidopsis transcriptome proﬁling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. The Plant Cell Online 14, 1675–1690. Fridovich, I. (1986) Superoxide dismutases. Advances in Enzymology and Related Areas of Molecular Biology 58, 61–97. Frossard, E., Bucher, M., Mächler, F., Mozafar, A., & Hurrell, R. (2000) Potential for increasing the content and bioavailability of Fe, Zn and Ca in plants for human nutrition. Journal of the Science of Food and Agriculture 80, 861–879. Galvez, L., Clark, R.B., Gourley, L.M., & Maranville, J.W. (1989) Effect of silicon on mineral composition of sorghum growth with excess manganese. Journal of Plant Nutrition 12, 547–561. Gao, X., Zou, C., Zhang, F., van der Zee, S.E., & Hofﬂand, E. (2005) Tolerance to zinc deﬁciency in rice correlates with zinc uptake and translocation. Plant and Soil 278, 253–261. Gao, X., Kuyper, T.W., Zou, C., Zhang, F., & Hofﬂand, E. (2007) Mycorrhizal responsiveness of aerobic rice genotypes is negatively correlated with their zinc uptake when nonmycorrhizal. Plant and Soil 290, 283–291. Garg, B.K. (2003) Nutrient uptake and management under drought: nutrient-moisture interaction. Current Agriculture 27, 1–8. Genc, Y. & McDonald, G.K. (2004) The potential of synthetic hexaploid wheats to improve zinc efﬁciency in modern bread wheat. Plant and Soil 262, 23–32. Genc, Y., McDonald, G.K., Rengel, Z., & Graham, R.D. (1999) Genotypic variation in the response of barley to zinc deﬁciency. In: Plant Nutrition-Molecular Biology and Genetics. Proceeding of the Sixth International Symposium on Genetics and Molecular Biology of Plant Nutrition (eds. G. Gissel-Nielsen & A. Jensen), pp. 205–221. Kluwer Academic Publishers, Dordrecht, Netherlands. Genc, Y., McDonald, G.K., & Graham, R.D. (2000) Effect of seed zinc content on early growth of barley (Hordeum vulgare L.) under low and adequate soil zinc supply. Australian Journal of Agricultural Research 51, 37–45. Genc, Y., McDonald, G.K., & Graham, R.D. (2002a) Critical deﬁciency concentration of zinc in barley genotypes differing in zinc efﬁciency and its relation to growth responses. Journal of Plant Nutrition 25, 545–560.

366

NUTRIENT USE EFFICIENCY IN CROPS

Genc, Y., McDonald, G.K., & Graham, R.D. (2002b) A soil-based method to screen for zinc efﬁciency in seedlings and its ability to predict yield responses to zinc efﬁciency in mature plants. Australian Journal of Agricultural Research 53, 409–421. Genc, Y., Shepherd, K.W., McDonald, G.K., & Graham, R.D. (2003) Inheritance of tolerance to zinc deﬁciency in barley. Plant Breeding 122, 283–284. Genc, Y., McDonald, G.K., & Graham, R.D. (2006) Contribution of different mechanisms to zinc efﬁciency in bread wheat during early vegetative stage. Plant and Soil 281, 353–367. Genc, Y., Huang, C.Y., & Langridge, P. (2007) A study of the role of root morphological traits in growth of barley in zinc-deﬁcient soil. Journal of Experimental Botany 58, 4017–4018. Gibson, T.S. & Leece, D.R. (1981) Estimation of physiologically active zinc in maize by biochemical assay. Plant and Soil 63, 395–406. Giordano, P.M., Noggle, J.C., & Mortvedt, J.J. (1974) Zinc uptake by rice, as affected by metabolic inhibitors and competing cations. Plant and Soil 41, 637–646. Gonzalez, D., Obrador, A., & Alvarez, J.M. (2007) Behavior of zinc from six organic fertilizers applied to a navy bean crop grown in a calcareous soil. Journal of Agricultural and Food Chemistry 55, 7084–7092. Goodman, B.A. & Newton, A.C. (2005) Effects of drought stress and its sudden relief on free radical processes in barley. Journal of the Science of Food and Agriculture 85, 47–53. Goos, R.J., Johnson, B.E., & Thiollet, M. (2000) A comparison of the availability of three zinc sources to maize (Zea mays L.) under greenhouse conditions. Biology and Fertility of Soils 31, 343–347. Graham, R.D. (1984) Breeding for nutritional characteristics in cereals. Advances in Plant Nutrition 1, 57–102. Graham, R.D. & Rengel, Z. (1993) Genotypic variation in Zn uptake and utilization by plants. In: Zinc in Soils and Plants (ed. A.D. Robson), pp. 107–114. Kluwer Academic Publishers, Dordrecht, The Netherlands. Graham, R.D. & Welch, R.M. (1996) Breeding for staple-food crops with high micronutrient density. Working Papers on Agricultural Strategies for Micronutrients, No. 3. Washington, D.C, International Food Policy Research Institute. Graham, R.D., Ascher, J.S., & Hynes, S.C. (1992) Selecting zinc-efﬁcient cereal genotypes for soils of low zinc status. Plant and Soil 146, 241–250. Graham, R.D., Senadhira, D., Beebe, S., Iglesias, C., & Monesterio, I. (1999) Breeding for micronutrient density in edible portions of staple food crops: con-

ventional approaches. Field Crops Research 60, 57–80. Graham, R.D., Welch, R.M., & Bouis, H.E. (2001) Addressing micronutrient malnutrition through enhancing the nutritional quality of staple foods: principles, perspectives and knowledge gaps. Advances in Agronomy 70, 77–142. Gregorio, G.B. (2002) Progress in breeding for trace minerals in staple crops. Journal of Nutrition 132, 500S–502S. Gregorio, G.B., Senadhira, D., Htut, T., & Graham, R.D. (2000) Breeding for trace mineral density in rice. Food and Nutrition Bulletin 21, 382–386. Grewal, H.S. & Williams, R. (1999) Alfalfa genotypes differ in their ability to tolerate zinc deﬁciency. Plant and Soil 214, 39–48. Grewal, H.S., Graham, R.D., & Rengel, Z. (1996) Genotypic variation in zinc efﬁciency and resistance to crown rot disease (Fusarium graminearum Schw. Group 1) in wheat. Plant and Soil 186, 219–226. Gries, D., Brunn, S., Crowley, D.E., & Parker, D.R. (1995) Phytosiderophore release in relation to micronutrient metal deﬁciencies in barley. Plant and Soil 172, 299–308. Grotz, N. & Guerinot, M.L. (2006) Molecular aspects of Cu, Fe and Zn homeostasis in plants. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1763, 595–608. Grotz, N., Fox, T., Connolly, E., Park, W., Guerinot, M.L., & Eide, D. (1998) Identiﬁcation of a family of zinc transporter genes from Arabidopsis that respond to zinc deﬁciency. Proceedings of the National Academy of Sciences of the United States of America 95, 7220–7224. Guerinot, M.L. (2000) The ZIP family of metal transporters. Biochimica et Biophysica Acta 1465, 190–198. Gunes, A., Alpaslan, M., & Inal, A. (1998) Critical nutrient concentrations and antagonistic and synergistic relationships among the nutrients of NFTgrown young tomato plants. Journal of Plant Nutrition 21, 2035–2047. Günes, A., Alpaslan, M., Çikili, Y., & Özcan, H. (1999) Effect of zinc on the alleviation of boron toxicity in tomato. Journal of Plant Nutrition 22, 1061–1068. Gunes, A., Cicek, N., Inal, A., et al. (2006) Genotypic response of chickpea (Cicer arietinum L.) cultivars to drought stress implemented at pre-and postanthesis stages and its relations with nutrient uptake and efﬁciency. Plant, Soil & Environment 52, 368–376. Gunes, A., Inal, A., Adak, M.S., et al. (2007) Mineral nutrition of wheat, chickpea and lentil as affected

ZINC IN SOILS AND CROP NUTRITION

by mixed cropping and soil moisture. Nutrient Cycling in Agroecosystems 78, 83–96. Hacisalihoglu, G. (2002) Physiological and biochemical mechanisms underlying zinc efﬁciency in monocot and dicot crop plants. PhD thesis. New York, USA, Cornell University, Ithaca. Hacisalihoglu, G. & Kochian, L.V. (2003) How do some plants tolerate low levels of soil zinc? Mechanisms of zinc efﬁciency in crop plants. New Phytologist 159, 341–350. Hacisalihoglu, G., Hart, J.J., & Kochian, L.V. (2001) High- and low-afﬁnity zinc transport systems and their possible role in zinc efﬁciency in bread wheat. Plant Physiology 125, 456–463. Hacisalihoglu, G., Hart, J.J., Wang, Y., Cakmak, I., & Kochian, L.V. (2003a) Zinc efﬁciency is correlated with enhanced expression and activity of Cu/Zn superoxide dismutase and carbonic anhydrase in wheat. Plant Physiology 131, 595–602. Hacisalihoglu, G., Ozturk, L., Cakmak, I., Welch, R.M., & Kochian, L.V. (2003b) Genotypic variation in common bean in response to zinc deﬁciency in calcareous soil. Plant and Soil 259, 71–83. Halvorson, A.D. & Lindsay, W.L. (1977) The critical Zn2+ concentration for corn and the nonabsorption of chelated zinc. Soil Science Society of America Journal 41, 531–534. Hamilton, M.A., Westermann, D.T., & James, D.W. (1993) Factors affecting zinc uptake in cropping systems. Soil Science Society of America Journal 57, 1310–1315. Hamlin, R.L., Schatz, C., & Barker, A.V. (2003) Zinc accumulation in Brassica juncea as inﬂuenced by nitrogen and phosphorus nutrition. Journal of Plant Nutrition 26, 177–190. Harris, N.S. & Taylor, G.J. (2004) Cadmium uptake and translocation in seedlings of near isogenic lines of durum wheat that differ in grain cadmium accumulation. BMC Plant Biology 4, 4. Hartwing, E.E., Jones, W.F., & Kilen, T.C. (1991) Identiﬁcation and inheritance of inefﬁcient zinc absorption in soybean. Crop Science 31, 61–63. Haydon, M.J. & Cobbett, C.S. (2007a) A novel major facilitator superfamily protein at the tonoplast inﬂuences zinc tolerance and accumulation in Arabidopsis. Plant Physiology 143, 1705–1719. Haydon, M.J. & Cobbett, C.S. (2007b) Transporters of ligands for essential metal ions in plants. The New Phytologist 174, 499–506. Hayes, J.E., Richardson, A.E., & Simpson, R.J. (1999) Phytase and acid phosphatase activities in extracts from roots of temperate pasture grass and legume seedlings. Australian Journal of Plant Physiology 26, 801–810.

367

Hernandez-Ruiz, J., Arnao, M.B., Hiner, A.N., GarciaCanovas, F., & Acosta, M. (2001) Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2. Biochemical Journal 354, 107–114. Ho, E. (2004) Zinc deﬁciency, DNA damage and cancer risk. The Journal of Nutritional Biochemistry 15, 572–578. Hofﬂand, E., Wei, C., & Wissuwa, M. (2006) Organic anion exudation by lowland rice (Oryza sativa L.) at zinc and phosphorus deﬁciency. Plant and Soil 283, 155–162. Holloway, R. (1996) Zinc as a subsoil nutrient for cereals. PhD thesis. University of Adelaide. Hopkins, B.G., Whitney, D.A., Lamond, R.E., & Jolley, V.D. (1998) Phytosiderophore release by sorghum, wheat, and corn under zinc deﬁciency. Journal of Plant Nutrition 21, 2623–2637. Hosseini, S.M., Maftoun, M., Karimian, N., Ronaghi, A., & Emam, Y. (2007) Effect of zinc× boron interaction on plant growth and tissue nutrient concentration of corn. Journal of Plant Nutrition 30, 773–781. Hotz, C. & Brown, K.H. (2004) Assessment of the risk of zinc deﬁciency in populations and options for its control. Food and Nutrition Bulletin 25, 94–204. House, W.A., Welch, R.M., Beebe, S., & Cheng, Z. (2002) Potential for increasing the amounts of bioavailable zinc in dry beans (Phaseolus vulgaris L.) through plant breeding. Journal of the Science of Food and Agriculture 82, 1452–1457. Huang, L., Ye, Z., & Bell, R.W. (1996) The importance of sampling immature leaves for the diagnosis of boron deﬁciency in oilseed rape (Brassica napus cv. Eureka). Plant and Soil 183, 187–198. Hussain, D., Haydon, M.J., Wang, Y., et al. (2004) Ptype ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis. The Plant Cell 16, 1327–1339. Iida, A., Kazuoka, T., Torikai, S., Kikuchi, H., & Oeda, K. (2000) A zinc ﬁnger protein RHL41 mediates the light acclimatization response in Arabidopsis. The Plant Journal 24, 191–203. Imtiaz, M., Alloway, B.J., Memon, M.Y., et al. (2006) Zinc tolerance in wheat cultivars as affected by varying levels of phosphorus. Communications in Soil Science and Plant Analysis 37, 1689–1702. Isaure, M.P., Fraysse, A., Deves, G., et al. (2006) Micro-chemical imaging of cesium distribution in Arabidopsis thaliana plant and its interaction with potassium and essential trace elements. Biochimie 88, 1583–1590. Ishimaru, Y., Suzuki, M., Kobayashi, T., et al. (2005) OsZIP4, a novel zinc-regulated zinc transporter in

368

NUTRIENT USE EFFICIENCY IN CROPS

rice. Journal of Experimental Botany 56, 3207–3214. Johnson, S.E., Lauren, J.G., Welch, R.M., & Duxbury, J.M. (2005) A comparison of the effects of micronutrient seed priming and soil fertilization on the mineral nutrition of chickpea (Cicer arietinum), lentil (Lens culinaris), rice (Oryza sativa) and wheat (Triticum aestivum) in Nepal. Experimental Agriculture 41, 427–448. Jolley, V.D. & Brown, J.C. (1991) Factors in iron-stress response mechanism enhanced by Zn-deﬁciency stress in Sanilac, but not Saginaw navy bean. Journal of Plant Nutrition 14, 257–265. Kalyanaraman, S.B. & Sivagurunathan, P. (1994) Effect of zinc on some important macro-and microelements in blackgram leaves. Communications in Soil Science and Plant Analysis 25, 2247–2259. Kaya, C., Higgs, D., & Burton, A. (2000) Plant growth, phosphorus nutrition, and acid phosphatase enzyme activity in three tomato cultivars grown hydroponically at different zinc concentrations. Journal of Plant Nutrition 23, 569–579. Kaya, C., Burton, M.A.S., & Higgs, D.E.B. (2001) Responses of tomato CVs growth to fruit-harvest stage under zinc stress in glasshouse conditions. Journal of Plant Nutrition 24, 369–382. Khan, H.R., McDonald, G.K., & Rengel, Z. (1998) Chickpea genotypes differ in their sensitivity to Zn deﬁciency. Plant and Soil 198, 11–18. Kiekens, L. (1995) Zinc. In: Heavy Metals in Soils, 2nd ed. (ed. B.J. Alloway), Blackie Academic and Professional, London. Kitagishi, K., Obata, H., & Kondo, T. (1987) Effect of zinc deﬁciency on 80S ribosome content of meristematic tissues of rice plant. Soil Science and Plant Nutrition 33, 423–429. Kochian, L.V. (1993) Zinc absorption from hydroponic solutions by plant roots. In: Zinc in Soils and Plants (ed. A.D. Robson), pp. 45–57. Kluwer Academic Publishers, Dordrecht, The Netherlands. Kothari, S.K., Marschner, H., & Romheld, V. (1991) Contribution of VA mycorrhizal hyphae in acquisition of phosphorus and zinc by maize grown calcareous soil. Plant and Soil 131, 177–185. Krämer, U., Talke, I.N., & Hanikenne, M. (2007) Transition metal transport. FEBS Letters 581, 2263–2272. Kreps, J.A., Wu, Y., Chang, H.S., Zhu, T., Wang, X., & Harper, J.F. (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiology 130, 2129–2141. Kuang, Y.W., Wen, D.Z., Zhong, C.W., & Zhou, G.Y. (2003) Root exudates and their roles in phytoremediation. Acta Phyloecologica Sinica (in Chinese) 27, 709–717.

Lasat, M.M. & Kochian, L.V. (2000) Physiology of Zn hyperaccumulation in Thlaspi caerulescens. In: Phytoremediation of Contaminated Soil and Water (eds. N. Terry & G. Banuelos), pp. 159–169. CRC Press LLC, Boca Raton, FL. Lasat, M.M., Baker, A.J.M., & Kochian, L.V. (1996) Physiological characterization of root Zn2+ absorption and translocation to shoots in Zn hyperaccumulator and nonaccumulator species of Thlaspi. Plant Physiology 112, 1715–1722. Lasat, M.M., Baker, A.J.M., & Kochian, L.V. (1998) Altered Zn compartmentation in the root symplasm and stimulated Zn absorption into the leaf as mechanisms involved in Zn hyperaccumulation in Thlaspi caerulescens. Plant Physiology 118, 875–883. Lasat, M.M., Pence, N.S., Garvin, D.F., Ebbs, S.D., & Kochian, L.V. (2000) Molecular physiology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens. Journal of Experimental Botany 51, 71–79. Lazova, G.N., Naidenova, T., & Velinova, K. (2004) Carbonic anhydrase activity and photosynthetic rate in the tree species Paulownia tomentosa Steud. Effect of dimethylsulfoxide treatment and zinc accumulation in leaves. Journal of Plant Physiology 161, 295–301. Leece, D.R. (1978) Distribution of physiologically inactive zinc in maize growing in black earth soil. Australian Journal of Agricultural Research 29, 749–758. Li, H.Y., Zhu, Y.G., Smith, S.E., & Smith, F.A. (2003) Phosphorus-zinc interactions in two barley cultivars differing in phosphorus and zinc efﬁciencies. Journal of Plant Nutrition 26, 1085– 1099. Li, C.Z., Jiao, J., & Wang, G.X. (2004) The important roles of reactive oxygen species in the relationship between ethylene and polyamines in leaves of spring wheat seedlings under root osmotic stress. Plant Science 166, 303–315. Lindsay, W.L. (1972) Zinc in soils and plant nutrition. Advances in Agronomy 24, 147–186. Lindsay, W.L. (1991) Inorganic equilibria affecting micronutrients in soils. In: Micronutrients in Agriculture (eds. J.J. Mordvedt, F.R. Cox, L.M. Shuman & R.M. Welch), pp. 89–112. SSSA Book Series No. 4, Soil Science Society of America, Madison, WI. Lindsay, W.L. & Norvell, W.A. (1978) Development of a DTPA soil test for zinc, iron, manganese and copper. Soil Science Society of America Journal 42, 421–428. Liu, Z. (1994) The soil zinc distribution in China. Chinese Agricultural Science 27, 30–37.

ZINC IN SOILS AND CROP NUTRITION

Liu, A. & Tang, C. (1999) Responses of two genotypes of Lupinus albus L. to zinc application on an alkaline soil. Journal of Plant Nutrition 22, 467–477. Lombnaes, P. & Singh, B.R. (2003) Varietal tolerance to zinc deﬁciency in wheat and barley grown in chelator-buffured nutrient solution and its effect on uptake of Cu, Fe, and Mn. Journal of Plant Nutrition and Soil Science 166, 76–83. Loneragan, J.F. & Webb, M.J. (1993) Interactions between zinc and other nutrients affecting the growth of plants. In: Zinc in Soils and Plants (ed. A.D. Robson), pp. 119–134. Kluwer Academic Publishers, Dordrecht, The Netherlands. Lonergan, P.F., Pallotta, M.A., Lorimer, M., Paull, J.G., Barker, S.J., & Graham, R.D. (2009) Multiple genetic loci for zinc uptake and distribution in barley (Hordeum vulgare). The New Phytologist 184, 168–179. Lucas, R.E. & Davis, J.F. (1961) Relationships between pH values of organic soils and availabilities of 12 plant nutrients. Soil Science 92, 177–182. Lynch, D.V. & Steponkus, P.L. (1987) Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L. cv Puma). Plant Physiology 83, 761–767. Lynch, J.M. & Whipps, J.M. (1990) Substrate ﬂow in the rhizosphere. Plant and Soil 129, 1–10. Maftoun, M. & Karimian, N. (1989) Relative efﬁciency of two zinc sources for maize (Zea mays L.) in two calcareous soils from an arid area of Iran. Agronomie 9, 771–775. Majumder, N.D., Rakshit, S.C., & Borthakur, D.N. (1990) Genetic effects on uptake of selected nutrients in some rice (Oryza sativa L.) varieties in phosphorus deﬁcient soil. Plant and Soil 123, 117–120. Mantovi, P., Bonazzi, G., Maestri, E., & Marmiroli, N. (2003) Accumulation of copper and zinc from liquid manure in agricultural soils and crop plants. Plant and Soil 250, 249–257. Maret, W. (2005) Zinc coordination environments in proteins determine zinc functions. Journal of Trace Elements in Medicine and Biology 19, 7–12. Marschner, H. (1986) Mineral Nutrition of Higher Plants, 1st ed. Academic Press, San Diego, CA. Marschner, H. (1993) Zinc uptake from soils. In: Zinc in Soils and Plants (ed. A.D. Robson), pp. 59–77. Kluwer Academic Publishers, Dordrecht, The Netherlands. Marschner, H. (1995) Mineral Nutrition of Higher Plants. Academic Press, London. Martens, D.C. & Westermann, D.T. (1991) Fertilizer application for correcting micronutrient deﬁciencies. In: Micronutrients in Agriculture (eds. J.J. Mortvedt, F.R. Cox, L.M. Shuman & R.M. Welch), SSSA Book Series No. 4. SSSA Book, Madison, WI.

369

Masuda, H., Suzuki, M., Morikawa, K.C., et al. (2008) Increase in iron and zinc concentrations in rice grains via the introduction of barley genes involved in phytosiderophore synthesis. Rice 1, 100–108. McDonald, G.K., Graham, R.D., Lloyd, J., Lewis, J., Lonergan, P., & Khabaz-Saberi, H. (2001) Breeding for improved zinc and manganese efﬁciency in wheat and barley. Proceeding of the 10th Australian Agronomy Conference. Hobart, Department of Plant Science, Waite Institute, Glen Osmond, SA. McDonald, G.K., Genc, Y., & Graham, R.D. (2008) A simple method to evaluate genetic variation in grain zinc concentration by correcting for differences in grain yield. Plant and Soil 306, 49–55. Mengel, K. & Kirkby, E.A. (1987) Principles of Plant Nutrition. International Potash Institute, Bern, Switzerland. Moore, P.A. Jr & Patrick, J.W.H. (1988) Effect of zinc deﬁciency on alcohol dehydrogenase activity and nutrient uptake in rice. Agronomy Journal 80, 882–885. Moraghan, J.T. & Grafton, K. (1999) Seed-zinc concentration and the zinc-efﬁciency trait in navy bean. Soil Science Society of America Journal 63, 918–922. Moraghan, J.T. & Mascagni, H.J. Jr (1991) Environmental and soil factors affecting micronutrient deﬁciencies and toxicities. In: Micronutrients in Agriculture (eds. J.J. Mordvedt, F.R. Cox, L.M. Shuman & R.M. Welch), pp. 371–425. Soil Science Society of America, Madison, WI. Moran, J.F., Becana, M., Iturbe-Ormaetxe, I., Frechilla, S., Klucas, R.V., & Aparicio-Tejo, P. (1994) Drought induces oxidative stress in pea plants. Planta 194, 346–352. Moretti, M.E. & Caprara, D.L. (2010) Drug–Nutrient Interaction Considerations in Pregnancy and Lactation. In: Handbook of Drug-Nutrient Interactions (eds. I.B. Joseph & T.A. Vincent), Humana Press, Totowa, NJ. Morrissey, J.P., Dow, J.M., Mark, G.L., & O’Gara, F. (2004) Are microbes at the root of a solution to world food production? EMBO Reports 5, 922–926. Mortvedt, J.J. & Gilkes, R.J. (1993) Zinc fertilisers. In: Zinc in Soils and Plants (ed. A.D. Robson), pp. 33–44. Kluwer Academic Publishers, Dordrecht, The Netherlands. Nakano, Y. & Asada, K. (1981) Hydrogen peroxide is scavenged by ascorbate-speciﬁc peroxidase in spinach chloroplasts. Plant and Cell Physiology 22, 867–880. Narwal, R.P., Singh, B.R., & Panhwar, A.R. (1983) Plant availability of heavy metals in a sludgetreated soil: I. Effect of sewage sludge and soil pH

370

NUTRIENT USE EFFICIENCY IN CROPS

on the yield and chemical composition of rape. Journal of Environmental Quality 12, 358–365. Nayyar, V.K., Takkar, P.N., Bansal, R.L., Singh, S.P., Kaur, N.P., & Sadana, U.S. (1990) Micro-nutrients in soils and crops of Punjab. Ludhiana, India, Research Bulletin. Department of Soils, Punjab Agricultural University. Norvell, W.A. (1991) Reactions of metal chelates in soils and nutrient solutions. In: Micronutrients in Agriculture (eds. J.J. Mortvedt, F.R. Cox, L.M. Shuman & R.M. Welch), pp. 187–228. Soil Science Society of America, Inc., Madison, WI. Norvell, W.A. & Welch, R.M. (1993) Growth and nutrient uptake by barley (Hordeum vulgare L. cv Herta): studies using an N-(2-hydroxyethyl) ethylenedinitrilotriacetic acid-buffered nutrient solution technique. I. Zinc ion requirements. Plant Physiology 101, 619–625. Obata, H., Kawamura, S., Senoo, K., & Tanaka, A. (1999) Changes in the level of protein and activity of Cu/Zn-superoxide dismutase in zinc deﬁcient rice plant, Oryza sativa L. Soil Science and Plant Nutrition 45, 891–896. Obrador, A., Novillo, J., & Alvarez, J.M. (2003) Mobility and availability to plants of two zinc sources applied to a calcareous soil. Soil Science Society of America Journal 67, 564–572. Ohki, K. (2006) Effect of zinc nutrition on photosynthesis and carbonic anhydrase activity in cotton. Physiologia Plantarum 38, 300–304. Ortiz-Monasterio, J.I. & Graham, R.D. (2000) Breeding for trace minerals in wheat. Food and Nutrition Bulletin 21, 392–396. Ozturk, L., Yazici, M.A., Yucel, C., et al. (2006) Concentration and localization of zinc during seed development and germination in wheat. Physiologia Plantarum 128, 144–152. Pandey, N., Pathak, G.C., Singh, A.K., & Sharma, C.P. (2002) Enzymic changes in response to zinc nutrition. Journal of Plant Physiology 159, 1151–1153. Papoyan, A. & Kochian, L.V. (2004) Identiﬁcation of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase. Plant Physiology 136, 3814–3823. Parker, D.R. (1997) Responses of six crop species to solution zinc2+ activities buffered with HEDTA. Soil Science Society of America Journal 61, 167–176. Parker, M.B. & Walker, M.E. (1986) Soil pH and manganese effects on manganese nutrition of peanut. Agronomy Journal 78, 614–620. Parker, D.R., Chaney, R.L., & Norvell, W.A. (1995) Chemical equilibrium models: applications to plant nutrition research. In: Chemical Equilibrium and Reaction Models (eds. R.H. Loeppert, P. Schwab &

S. Goldberg), pp. 163–200. Soil Science Society of America, Madison, WI. Special Publication No. 42. Paterson, J.E., Berndt, G.F., Cameron, D., & Rowbottom, W. (1991) Investigation into the response of barley to applied zinc. Journal of the Science of Food and Agriculture 54, 387–392. Pearson, J.N. & Rengel, Z. (1997) Genotypic differences in the production and partitioning of carbohydrates between roots and shoots of wheat grown under zinc or manganese deﬁciency. Annals of Botany (London) 80, 803–808. Pearson, J.N., Rengel, Z., Jenner, C.F., & Graham, R.D. (1995) Transport of zinc and manganese to developing wheat grains. Physiologia Plantarum 95, 449–455. Pedler, J.F., Parker, D.R., & Crowley, D.E. (2000) Zinc deﬁciency-induced phytosiderophore release by the Triticaceae is not consistently expressed in solution culture. Planta 211, 120–126. Pence, N.S., Larsen, P.B., Ebbs, S.D., et al. (2000) The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proceedings of the National Academy of Sciences of the United States of America 97, 4956–4960. Pinton, R., Cakmak, I., & Marschner, H. (1994) Zinc deﬁciency enhanced NAD(P)H-dependent superoxide radical production in plasma membrane vesicles isolated from roots of bean plants. Journal of Experimental Botany 45, 45–50. Ramesh, S.A., Choimes, S., & Schachtman, D.P. (2004) Over-expression of an Arabidopsis zinc transporter in Hordeum vulgare increases short-term zinc uptake after zinc deprivation and seed zinc content. Plant Molecular Biology 54, 373–385. Ranjbar, G.A. & Bahmaniar, M.A. (2007) Effects of soil and foliar application of Zn fertilizer on yield and growth characteristics of bread wheat (Triticum aestivum L.) cultivars. Asian Journal of Plant Science 6, 1000–1005. Rashid, A. & Raﬁque, E. (1998) Micronutrients in Pakistani Agriculture: Signiﬁcance and Use. Pakistan Agricultural Research Council, Islamabad, Pakistan. Rasouli-Sadaghiani, M.H., Sadeghzadeh, B., Sepehr, E., & Rengel, Z. (2011) Root exudation and Zn uptake by barley genotypes differing in Zn efﬁciency. Journal of Plant Nutrition 34, 1120–1132. Reddy, K.J. (2006) Nutrient stress. In: Physiology and Molecular Biology of Stress Tolerance in Plants (eds. K.W. Madhava Rao, A.S. Raghavendra & K.J. Reddy), Springer, Heidelberg, Germany. Reddy, M.R. & Dunn, S.J. (1987) Differential response of soybean genotypes to soil pH and manganese application. Plant and Soil 101, 123–126. Reichheld, J.P., Vernoux, T., Lardon, F., Van Montagu, M., & Inzé, D. (1999) Speciﬁc checkpoints regulate

ZINC IN SOILS AND CROP NUTRITION

plant cell cycle progression in response to oxidative stress. The Plant Journal 17, 647–656. Reid, R.J., Brookes, J.D., Tester, M.A., & Smith, F.A. (1996) The mechanism of zinc uptake in plants. Planta 198, 39–45. Rengel, Z. (1995a) Carbonic anhydrase activity in leaves of wheat genotypes differing in Zn efﬁciency. Journal of Plant Physiology 147, 251–256. Rengel, Z. (1995b) Sulfhydryl groups in root-cell plasma membranes of wheat genotypes differing in Zn efﬁciency. Physiologia Plantarum 95, 604–612. Rengel, Z. (1997) Root exudation and microﬂora populations in rhizosphere of crop genotypes differing in tolerance to micronutrient deﬁciency. Plant and Soil 196, 255–260. Rengel, Z. (1999) Zinc deﬁciency in wheat genotypes grown in conventional and chelator-buffered nutrient solutions. Plant Science (Shannon) 143, 221–230. Rengel, Z. (2001) Genotypic differences in micronutrient use efﬁciency in crops. Communications in Soil Science and Plant Analysis 32, 1163–1186. Rengel, Z. (2002) Genetic control of root exudation. Plant and Soil 245, 59–70. Rengel, Z. & Graham, R.D. (1995a) Importance of seed Zn content for wheat grown on Zn-deﬁcient soil. I. Vegetative growth. Plant and Soil 173, 259–266. Rengel, Z. & Graham, R.D. (1995b) Importance of seed Zn content for wheat growth on Zn-deﬁcient soil. II. Grain yield. Plant and Soil 173, 267–274. Rengel, Z. & Graham, R.D. (1995c) Wheat genotypes differ in zinc efﬁciency when grown in the chelatebuffered nutrient solution. I. Growth. Plant and Soil 176, 307–316. Rengel, Z. & Graham, R.D. (1995d) Wheat genotypes differ in Zn efﬁciency when grown in chelatebuffered nutrient solution: II. Nutrient uptake. Plant and Soil 176, 317–324. Rengel, Z. & Graham, R. (1996) Uptake of zinc from chelate-buffered nutrient solutions by wheat genotypes differing in zinc efﬁciency. Journal of Experimental Botany 47, 217–226. Rengel, Z. & Romheld, V. (2000) Differential tolerance to Fe and Zn deﬁciencies in wheat germplasm. Euphytica 113, 219–225. Rengel, Z. & Wheal, M.S. (1997a) Herbicide chlorsulfuron decreases growth of ﬁne roots and micronutrient uptake in wheat genotypes. Journal of Experimental Botany 48, 927–934. Rengel, Z. & Wheal, M.S. (1997b) Kinetic parameters of zinc uptake by wheat are affected by the herbicide chlorsulfuron. Journal of Experimental Botany 48, 935–941.

371

Rengel, Z., Gutteridge, R., Hirsch, P., & Hornby, D. (1996) Plant genotype, micronutrient fertilization and take-all infection inﬂuence bacterial populations in the rhizosphere of wheat. Plant and Soil 183, 269–277. Rengel, Z., Romheld, V., & Marschner, H. (1998) Uptake of zinc and iron by wheat genotypes differing in tolerance to zinc deﬁciency. Journal of Plant Physiology 152, 433–438. Rengel, Z., Batten, G.D., & Crowley, D.E. (1999) Agronomic approaches for improving the micronutrient density in edible portions of ﬁeld crops. Field Crops Research 60, 27–40. Rizhsky, L., Liang, H., Shuman, J., Shulaev, V., Davletova, S., & Mittler, R. (2004) When defense pathways collide: the response of Arabidopsis to a combination of drought and heat stress. Plant Physiology 134, 1683–1696. Rogers, E.E. & Guerinot, M.L. (2002) FRD3, a member of the multidrug and toxin efﬂux family, controls iron deﬁciency responses in Arabidopsis. The Plant Cell Online 14, 1787–1799. Römheld, V. & Marschner, H. (1986) Evidence for a speciﬁc uptake system for iron phytosiderophores in roots of grasses. Plant Physiology 80, 175–180. Römheld, V. & Marschner, H. (1991) Function of micronutrients in plants. In: Micronutrients in Agriculture (eds. J.J. Mortvedt, F.R. Cox, L.M. Shuman & R.M. Welch), pp. 297–328. Soil Science Society of America, Madison, WI. Book Series No. 4. Rout, G.R. & Das, P. (2009) Effect of metal toxicity on plant growth and metabolism: I. Zinc. In: Sustainable Agriculture (eds. E. Lichtfouse, M. Navarrete, P. Debaeke, S. Veronique, & C. Alberola), Springer, Dordrecht, pp. 873–884. Ruel, M.T. & Bouis, H.E. (1998) Plant breeding: a long-term strategy for the control of zinc deﬁciency in vulnerable populations. The American Journal of Clinical Nutrition 68, 488S–494S. Ryan, P.R., Delhaize, E., & Jones, D.L. (2003) Function and mechanism of organic anion exudation from plant roots. Annual Reviews in Plant Physiology and Plant Molecular Biology 52, 527–560. Sadeghzadeh, B. (2008) Mapping of chromosome regions associated with seed Zn accumulation in barley, PhD thesis. Faculty of Natural and Agricultural Sciences. Perth, The University of Western Australia. Sadeghzadeh, B., Rengel, Z., & Li, C. (2009a) Differential zinc efﬁciency of barley genotypes grown in soil and chelator-buffered nutrient solution. Journal of Plant Nutrition 32, 1744–1767. Sadeghzadeh, B., Rengel, Z., Li, C., & Yang, H. (2009b) Molecular marker linked to a chromosome region

372

NUTRIENT USE EFFICIENCY IN CROPS

regulating seed Zn accumulation in barley. Molecular Breeding 25, 167–177. Safaya, N.M. (1976) Phosphorus-zinc interaction in relation to absorption rate of phosphorus, zinc, copper, manganese and iron in corn. Soil Science Society of America Journal 40, 719–722. Sairam, R.K. & Saxena, D.C. (2001) Oxidative stress and antioxidants in wheat genotypes: possible mechanism of water stress tolerance. Journal of Agronomy and Crop Science 184, 55–61. Salama, Z.A. & Fouly, M.E. (2008) Evaluation of the efﬁciency of some egyptian wheat (Triticum aestivum L.) cultivars to Zn deﬁciency through peroxidase activity and protein proﬁle techniques. Notulae Botanicae, Horti Agrobotanici, Cluj-Napoca 36, 42–46. Salama, Z.A., Lazova, G.N., Stoinova, Z.G., Popova, L.P., & El-Fouly, M.M. (2002) Effect of zinc defﬁciecny on photosynthesis in chick-pea and maize plants. Comptes rendus de l’Académie bulgare des sciences: sciences mathématiques et naturelles: 65. Salisbury, F.B. & Ross, C.W. (1992) Plant Physiology. Wadsworth Publishing Company, Belmont, CA. Samarah, N., Mullen, R., & Cianzio, S. (2004) Size distribution and mineral nutrients of soybean seeds in response to drought stress. Journal of Plant Nutrition 27, 815–835. Santa Maria, G.E. & Cogliatti, D.H. (1988) Bidirectional Zn-ﬂuxes and compartmentation in wheat seedling roots. Journal of Plant Physiology 132, 312–315. Sasaki, H., Hirose, T., Watanabe, Y., & Ohsugi, R. (1998) Carbonic anhydrase activity and CO2transfer resistance in Zn-deﬁcient rice leaves. Plant Physiology 118, 929–934. Schaaf, G., Schikora, A., Haberle, J., et al. (2005) A putative function for the Arabidopsis Fephytosiderophore transporter homolog AtYSL2 in Fe and Zn homeostasis. Plant and Cell Physiology 46, 762–774. Schachtman, D.P. & Barker, S.J. (1999) Molecular approaches for increasing the micronutrient density in edible portions of food crops. Field Crops Research 60, 81–92. Schlegel, R. & Cakmak, I. (1997) Physical mapping of rye genes determining micronutritional efﬁciency in wheat. In: Plant Nutrition for Sustainable Food Production and Environment (eds. T. Ando, K. Fujita, T. Mae, H. Matsumoto, S. Mori & J. Sekiya), pp. 287–288. Kluwer Academic Publishers, Dordrecht, The Netherlands. Schwartz, S.M., Welch, R.M., Grunes, D.L., et al. (1987) Effect of zinc, phosphorus and root-zone temperature on nutrient uptake by barley. Soil Science Society of America Journal 51, 371–375. Selote, D.S., Bharti, S., & Khanna-Chopra, R. (2004) Drought acclimation reduces O2*-accumulation

and lipid peroxidation in wheat seedlings. Biochemical and Biophysical Research Communications 314, 724–729. Sharma, K.N. & Deb, D.L. (1988) Effect of organic manuring on zinc diffusion in soils of varying texture. Journal of the Indian Society of Soil Science 36, 219–224. Sharma, K.C., Krantz, B.A., Brown, A.L., & Quick, J. (1968) Interaction of Zn and P in top and root of corn and tomato. Agronomy Journal 60, 453–456. Sharma, C.P., Sharma, P.N., Bisht, S.S., & Nautiyal, B.D. (1982) Zinc deﬁciency induces changes in cabbage (Brassica oleracea var. Capitat). In: Proceedings of the Ninth international Plant Nutrition Colloquium (ed. A. Scaife), pp. 601–606. Commonwealth Agricultural Bureau, Warwick, England. Shen, J., Rengel, Z., Tang, C., & Zhang, F. (2003) Role of phosphorus nutrition in development of cluster roots and release of carboxylates in soil-grown Lupinus albus. Plant and Soil 248, 199–206. Shuman, L.M. (1998) Micronutrient fertilizers. In: Nutrient Use in Crop Production (ed. Z. Rengel), The Haworth Press, New York. Sillanpää, M. (1982) Micronutrients and the nutrient status of soils: a global study. FAO Soils Bulletin No. 48. Rome, Food and Agriculture Organization of the United Nations, pp. 75–82. Sillanpää, M. (1990) Micronutrient assessment at the country level: an international study. FAO Soils Bulletin No. 63. Rome, Food and Agriculture Organisation of the United Nations. Singh, M.V. & Abrol, I.P. (1986) Transformation and movement of zinc in an alkali soil and their inﬂuence on the yield and uptake of zinc by rice and wheat crops. Plant and Soil 94, 445–449. Singh, S.P. & Westermann, D.T. (2002) A single dominant gene controlling resistance to soil zinc deﬁciency in common bean. Crop Science 42, 1071–1074. Singh, B., Erenoglu, B., Neumann, G., Römheld, V., & vonWiren, N. (2002) Role of phytosiderophores in zinc efﬁciency of wheat. In: Durchwurzelung, Rhizodeposition und Pﬂanzenverfügbarkeit von Nahrstoffen und Schwemetallen: Eco-Physiology of Rhizosphere (eds. W. Merbach, B.W. Hutsch, L. Wittenmayer & J. Augustin), pp. 52–60. Teubner, Stuttgart, Germany. Singh, J.P., Karamanos, R.E., & Stewart, J.W.B. (1987) The zinc fertility of Saskatchewan soils. Canadian Journal of Soil Science 67, 103–116. Singh, J.P., Karmanos, R.E., & Stewart, J.W.B. (1988) The mechanism of phosphorus-induced zinc deﬁciency in beans (Phaseolus vulgaris L.). Canadian Journal of Soil Science 68, 345–358. Singh, B.K., Millard, P., Whiteley, A.S., & Murrell, J.C. (2004) Unravelling rhizosphere-microbial interac-

ZINC IN SOILS AND CROP NUTRITION

tions: opportunities and limitations. Trends in Microbiology 12, 386–393. Singh, B., Natesan, S.K.A., Singh, B.K., & Usha, K. (2005) Improving zinc efﬁciency of cereals under zinc deﬁciency. Current Science 88, 36–44. Solomons, N.W. & Cousins, R.J. (1984) Zinc. (eds. N.W. Solomons & I.H. Rosenberg), Absorption and Malabsorption of Mineral Nutrients. Alan R. Liss, New York, NY. Sparrow, D.H. & Graham, R.D. (1988) Susceptibility of zinc-deﬁcient wheat plants to colonization by Fusarium graminearum Schw. Group I. Plant and Soil 112, 261–266. Ström, L., Owen, A.G., Godbold, D.L., & Jones, D.L. (2001) Organic acid behaviour in a calcareous soil: sorption reactions and biodegradation rates. Soil Biology and Biochemistry 33, 2125–2133. Subramanian, K.S., Tenshia, V., Jayalakshmi, K., & Ramachandran, V. (2009) Role of arbuscular mycorrhizal fungus (Glomus intraradices)–(fungus aided) in zinc nutrition of maize. Journal of Agricultural Biotechnology and Sustainable Development 1, 29–38. Suzuki, M., Takahashi, M., Tsukamoto, T., et al. (2006) Biosynthesis and secretion of mugineic acid family phytosiderophores in zinc-deﬁcient barley. The Plant Journal 48, 85–97. Takahashi, M., Terada, Y., Nakai, I., et al. (2003) Role of nicotianamine in the intracellular delivery of metals and plant reproductive development. The Plant Cell Online 15, 1263–1280. Takkar, P.N. (1996) Micronutrient research and sustainable agricultural productivity in India. Journal of the Indian Society of Soil Science 44, 562–581. Takkar, P.N. & Walker, C.D. (1993) The distribution and correction of zinc deﬁciency. In: Zinc in Soils and Plants (ed. A.D. Robson), pp. 151–165. Kluwer Academic Publishers, Dordrecht, The Netherlands. Takkar, P.N., Singh, S.P., Bansal, R.L., & Nayyar, V.K. (1983) Tolerance of barley varieties to Zn deﬁciency. Indian Journal Agricultural Science 53, 971–979. Talke, I.N., Hanikenne, M., & Kramer, U. (2006) Zincdependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant Physiology 142, 148–167. Thalooth, A.T., Tawﬁk, M.M., & Mohamed, H.M. (2006) A comparative study on the effect of foliar application of zinc, potassium and magnesium on growth, yield and some chemical constituents of mungbean plants grown under water stress conditions. World Journal of Agricultural Sciences 2, 37–46.

373

Tiller, K.G. (1983) Micronutrients. In: Soils: An Australian Viewpoint (eds. J. Lenaghan & G. Katsntoni), pp. 365–387. Division of Soils, CSIRO and Academic Press, Melbourne and London. Tinker, P.B., MacPherson, A., & West, T.S. (1981) Levels, distribution and chemical forms of trace elements in food plants. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 294, 41–55. Tkachuk, K.S., Savchenko, N.P., & Pidryads’kii, V.G. (1994) Effect of Mg2+ and Zn2+ on the membrane potential of root cells and the photochemical activity of chloroplasts of winter wheat leaves. Fiziologiya I Biokhimiya Kul’turnykh Rastenii 26, 147–150. Uauy, C., Distelfeld, A., Fahima, T., Blechl, A., & Dubcovsky, J. (2006) A NAC gene regulating senescence, improves grain protein, zinc, and iron content in wheat. Science 314, 1298–1301. UNACCSN (1992) Second Report on the World Nutrition Situation. Volume I. Global and Regional Results. Geneva, United Nations Administrative Committee on Coordination, Subcommittee on Nutrition. Vallee, B.L. & Falchuk, K.H. (1993) The biochemical basis of zinc physiology. Physiological Reviews 73, 79–118. Van De Mortel, J.E., Almar Villanueva, L., Schat, H., et al. (2006) Large expression differences in genes for iron and zinc homeostasis, stress response, and lignin biosynthesis distinguish roots of Arabidopsis thaliana and the related metal hyperaccumulator Thlaspi caerulescens. Plant Physiology 142, 1127–1147. Van der Zaal, B.J., Neuteboom, L.W., Pinas, J.E., et al. (1999) Overexpression of a novel Arabidopsis gene related to putative zinc transporter genes from animals can lead to enhanced zinc resistance and accumulation. Plant Physiology 119, 1047–1055. Verret, F., Gravot, A., Auroy, P., et al. (2004) Overexpression of AtHMA4 enhances root-to-shoot translocation of zinc and cadmium and plant metal tolerance. FEBS Letters 576, 306–312. Viets, F.G. (1966) Zinc deﬁciency in the soil-plant system. In: Zn Metabolism (ed. A.S. Prasad), pp. 90–128. Charles C. Thomas, Illinois, USA. Von Wirén, N., Klair, S., Bansal, S., et al. (1999) Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants. Plant Physiology 119, 1107–1114. Wagar, B.I., Stewart, J.W.B., & Henry, J.L. (1986) Comparison of single large broadcast and small annual seed-placed phosphorus treatments on yield and phosphorus and zinc contents of wheat on Chernozemic soils. Canadian Journal of Soil Science 66, 237–248.

374

NUTRIENT USE EFFICIENCY IN CROPS

Walter, A., Römheld, V., Marschner, H., & Mori, S. (1994) Is the release of phytosiderophores in zincdeﬁcient wheat plants a response to impaired iron utilization? Physiologia Plantarum 92, 493–500. Wang, H. & Jin, J.Y. (2005) Photosynthetic rate, chlorophyll ﬂuorescence parameters, and lipid peroxidation of maize leaves as affected by zinc deﬁciency. Photosynthetica 43, 591–596. Wang, A.S., Angle, J.S., Chaney, R.L., Delorme, T.A., & Reeves, R.D. (2006a) Soil pH effects on uptake of Cd and Zn by Thlaspi caerulescens. Plant and Soil 281, 325–337. Wang, P., Bi, S., Ma, L., & Han, W. (2006b) Aluminum tolerance of two wheat cultivars (Brevor and Atlas66) in relation to their rhizosphere pH and organic acids exuded from roots. Journal of Agricultural and Food Chemistry 54, 10033–10039. Waters, B.M. & Grusak, M.A. (2008) Whole-plant mineral partitioning throughout the life cycle in Arabidopsis thaliana ecotypes Columbia, Landsberg erecta, Cape Verde Islands, and the mutant line ysl1ysl3. New Phytologist 177, 389–405. Waters, B.M., Chu, H.H., DiDonato, R.J., et al. (2006) Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds. Plant Physiology 141, 1446–1458. Webb, E.C. (1992) Enzyme Nomenclature: Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Academic Press, New York. Webb, M.J. & Loneragan, J.F. (1988) Effect of zinc deﬁciency on growth, phosphorus concentration, and phosphorus toxicity of wheat plants. Soil Science Society of America Journal 52, 1676–1680. Webb, M.J. & Loneragan, J.F. (1990) Zinc translocation to wheat roots and its implications for a phosphorus/ zinc interaction in wheat plants. Journal of Plant Nutrition 13, 1499–1512. Weber, M., Harada, E., Vess, C., Roepenack-Lahaye, E., & Clemens, S. (2004) Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identiﬁes nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors. The Plant Journal 37, 269–281. Welch, R.M. (1995) Micronutrient nutrition of plants. Critical Reviews in Plant Sciences 14, 49–82. Welch, R.M. (1999) Importance of seed mineral nutrient reserves in crop growth and development. In: Mineral Nutrition of Crops: Fundamental Mechanisms and Implications (ed. Z. Rengel), pp. 205–226. Food Products Press, New York, NY.

Welch, R.M. & Graham, R.D. (1999) A new paradigm for world agriculture: meeting human needs productive, sustainable, nutritious. Field Crops Research 60, 1–10. Welch, R.M. & Graham, R.D. (2002) Breeding crops for enhanced micronutrien content. Plant and Soil 245, 205–214. Welch, R.M. & Graham, R.D. (2004) Breeding for micronutrients in staple food crops from a human nutrition perspective. Journal of Experimental Botany 55, 353–364. Welch, R.M. & Norvell, W.A. (1993) Growth and nutrient uptake by barley (Hordeum vulgare L. cv Herta): studies using an N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid-buffered nutrient solution technique. I. Role of zinc in the uptake and root leakage of mineral nutrients. Plant Physiology 101, 627–631. Wenzel, A.A. & Mehlhorn, H. (1995) Zinc deﬁciency enhances ozone toxicity in bush beans (Phaseolus vulgaris L. cv. Saxa). Journal of Experimental Botany 46, 867–872. White, J.G. & Zasoski, R.J. (1999) Mapping soil micronutrients. Field Crop Research 60, 11–26. White, M.C., Decker, A.M., & Chaney, R.L. (1981) Metal complexation in xylem ﬂuid. I. Chemical composition of tomato and soybean stem exudate. Plant Physiology 67, 292–300. White, P.J., Whiting, S.N., Baker, A.J.M., & Broadley, M.R. (2002) Does zinc move apoplastically to the xylem in roots of Thlaspi caerulescens? New Phytologist 153, 201–207. Wintz, H., Fox, T., Wu, Y.Y., et al. (2003) Expression proﬁles of Arabidopsis thaliana in mineral deﬁciencies reveal novel transporters involved in metal homeostasis. Journal of Biological Chemistry 278, 47644–47653. Wissuwa, M., Ismail, A.M., & Yanagihara, S. (2006) Effects of zinc deﬁciency on rice growth and genetic factors contributing to tolerance. Plant Physiology 142, 731–741. Wu, F., Zhang, G., & Yu, J. (2003) Interaction of cadmium and four microelements for uptake and translocation in different barley genotypes. Communications in Soil Science and Plant Analysis 34, 2003–2020. Xin-Xian, L., Yu-Gang, Z., Dai, J., & Qixing, Z. (2009) Zinc, cadmium and lead accumulation and characteristics of rhizosphere microbial population associated with hyperaccumulator sedum alfredii hance under natural conditions. Bulletin of Environmental Contamination and Toxicology 82, 460–467. Xu, W.H., Liu, H., Ma, Q.F., & Xiong, Z.T. (2007a) Root exudates, rhizosphere Zn fractions, and Zn accumulation of ryegrass at different soil Zn levels. Pedosphere 17, 389–396.

ZINC IN SOILS AND CROP NUTRITION

Xu, X., Chen, X., Shi, J., Chen, Y., Wu, W., & Perera, A. (2007b) Effects of manganese on uptake and translocation of nutrients in a hyperaccumulator. Journal of Plant Nutrition 30, 1737–1751. Yang, X.E., Chen, W.R., & Feng, Y. (2007) Improving human micronutrient nutrition through biofortiﬁcation in the soil–plant system: China as a case study. Environmental Geochemistry and Health 29, 413–428. Yang, W., Jingshuang, L.I.U., & Na, Z. (2010) Effects of pH on the fraction transformations of Zn in black soil at the condition of freeze/thaw cycles. Journal of Arid Land Resources and Environment. DOI: CNKI:SUN:GHZH.0.2010-01-033. Yilmaz, A., Ekiz, H., Torun, B., et al. (1997) Effect of different zinc application methods on grain yield and zinc concentration in wheat cultivars grown on zinc-deﬁcient calcareous soils. Journal of Plant Nutrition 20, 461–471. Yu, Q. & Rengel, Z. (1999) Micronutrient deﬁciency inﬂuences plant growth and activities of superoxide dismutases in narrow-leafed lupins. Annals of Botany 83, 175–182.

375

Yu, Q., Osborne, L., & Rengel, Z. (1998) Micronutrient deﬁciency changes activities of superoxide dismutase and ascorbate peroxidase in tobacco plants. Journal of Plant Nutrition 21, 1427–1437. Yu, Q., Worth, C., & Rengel, Z. (1999) Using capillary electrophoresis to measure Cu/Zn superoxide dismutase concentration in leaves of wheat genotypes differing in tolerance to zinc deﬁciency. Plant Science (Shannon) 143, 231–239. Zhang, F.S., Römheld, V., & Marschner, H. (1991) Diurnal rhythm of release of phytosiderophores and uptake rate of zinc in iron-deﬁcient wheat. Soil Science and Plant Nutrition 37, 671–678. Zhu, Y.G., Smith, S.E., & Smith, F.A. (2001) Zinc (Zn)-phosphorus (P) interactions in two cultivars of spring wheat (Triticum aestivum L.) differing in P uptake efﬁciency. Annals of Botany 88, 941–945. Zubaidi, A., McDonald, G.K., & Hollamby, G.J. (1999) Nutrient uptake and distribution by bread and durum wheat under drought conditions in South Australia. Australian Journal of Experimental Agriculture 39, 721–732.

Chapter 17

Overview of the Acquisition and Utilization of Boron, Chlorine, Copper, Manganese, Molybdenum, and Nickel by Plants and Prospects for Improvement of Micronutrient Use Efﬁciency Patrick H. Brown and Elias Bassil

Abstract The development of crops with improved micronutrient use efﬁciency requires an integrated consideration of the soil, plant, and environmental processes that determine nutrient acquisition and utilization. In the following, an overview of the extent of global deﬁciency, the chemistry of element behavior in soil, and the biological mechanisms of acquisition and utilization of boron, chlorine, copper, manganese, molybdenum, and nickel by plants is provided with emphasis on the molecular and physiological processes that hold most promise for optimizing the efﬁciency of use of these elements.

Introduction Agronomically important deﬁciencies of the essential micronutrients boron (B), chlorine (Cl), copper (Cu), manganese (Mn), molybdenum (Mo), and nickel (Ni) occur worldwide (Alloway, 2008). The scale and importance of these deﬁciencies, however,

varies greatly, boron deﬁciency occurs globally across a wide range of climates, cropping systems and soils; copper, manganese, and molybdenum deﬁciencies are of great importance in particular soil types and cropping systems, while nickel and chlorine deﬁciencies have been demonstrated in only isolated environments with limited crop species. The development of crops with improved micronutrient use efﬁciency requires consideration of the soil, plant, and environmental processes that determine nutrient acquisition and utilization. This chapter provides an overview of these processes with an emphasis on the physiological and molecular basis of micronutrient acquisition and an analysis of the prospects for improvement of micronutrient use efﬁciency. The micronutrients iron and zinc are dealt with in Chapters 15 and 16, respectively. Among the techniques used to predict crop response to micronutrients is the integration of data drawn from soil maps, tissue testing, and on-farm and off-farm testing.

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 377

378

NUTRIENT USE EFFICIENCY IN CROPS

Sillanpää (1982, 1990) utilized ﬁeld and laboratory trials, tissue sampling, and soil analysis and mapping to provide the ﬁrst detailed maps of micronutrient-responsive soils for Africa, Europe, and parts of Asia. This approach has now been replicated in many countries and regions (see summaries in Alloway, 2008; Brown, 2008; Zou et al., 2008; Singh, 2008). The utility of these maps for micronutrient management is limited, however, since they generally lack sufﬁcient resolution for farm-scale management and do not adequately integrate local environmental conditions, cropping system, and management practices. The occurrence of visual symptoms can also be used to identify micronutrient deﬁciencies but requires considerable expertise and is complicated by plant age, disease, and herbicide damage, and often varies with environment. Visual symptoms also do not appear until after plant growth has been reduced and yield has been substantially impacted. Conclusive diagnosis of micronutrient deﬁciency generally requires tissue analysis, which is expensive and difﬁcult to perform effectively. Neither the visual analysis of symptoms nor tissue analysis provides any information on the cause of the deﬁciency or the approach required to correct the problem. The difﬁculties in assessments of micronutrient needs are exacerbated by the frequent ineffectiveness and high cost of micronutrient fertilizers and the oftenvariable crop response. Together these factors confound experimentation, make crop management decisions difﬁcult, and add great complexity to the development of micronutrient-efﬁcient cultivars. For the micronutrients discussed in this chapter, our understanding of the processes involved in soil acquisition and utilization of micronutrients is inadequate, and as a consequence most micronutrient management strategies are not based on well-established chemical or biological principles but are the result of

ﬁeld experimentation and grower experience. Practices not based upon a clear physical or physiological understanding are frequently difﬁcult to transfer to other locations or adapt to changing production practices. Micronutrient fertilization is often viewed as an uncertain, high-risk, and costprohibitive undertaking in low-input systems, while risk aversion in high-input systems has resulted in widespread application of micronutrients even in the absence of a predictable response (Brown, 2008). To achieve the goal of optimizing the efﬁciency of micronutrient use and maximizing crop productivity and quality, a better understanding of the processes involved in acquisition, and utilization of micronutrients by plants is required. Optimization of nutrient use efﬁciency cannot be achieved through improvements in understanding of fundamental processes alone but will require a sound understanding of soil supply processes, the environment, and agronomy of the cropping system of interest. Graham (1984) deﬁned nutrient efﬁciency as the ability to produce a high plant yield in a soil, or other media, that would otherwise limit plant growth. While this deﬁnition is highly relevant in a crop improvement context for a given environment, it does not provide a mechanistic understanding of the processes involved and hence may not be transferable to new environments. This deﬁnition of efﬁciency, which was developed before the advent of low-cost molecular genetic techniques, may also fail to identify germplasm with unique efﬁciency traits if those traits are present in an otherwise unproductive background. Approaches for identifying and manipulating nutrient efﬁciency that focus solely on isolated mechanistic processes are equally constrained and can lead to the identiﬁcation of germplasm of little agronomic relevance (for discussion see Gourley et al., 1994). Thus, a very highly nutrient-efﬁcient

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

germplasm may be of little agronomic value if maximal potential yield is low. Additionally, a cultivar that is inefﬁcient at very low nutrient availability may have high nutrient utilization efﬁciency (yield per unit available nutrient) when nutrient availability is high. Nutrient use efﬁciency is ultimately determined by a complex of factors of varying importance including: (1) the amount, solubility, and distribution of the element in the soil, (2) the characteristics and regulation of nutrient acquisition from the soil by plant roots, (3) the function and demand for the element in the plant life cycle, and the (4) the mobility of the element within the cell and the plant. The relative contribution of each of these factors to overall nutrient use efﬁciency and the prospects for improvement in NUE differs widely among the nutrients. Boron, chlorine, copper, manganese, molybdenum, and nickel are extremely diverse in their chemical properties, which in turn inﬂuences soil reactions, the form of nutrient absorbed by roots and utilized in metabolism, and the function and mobility of the nutrient in plants. Available information on the molecular and physiological mechanisms of acquisition and utilization of boron, chlorine, copper, manganese, molybdenum, and nickel varies greatly in both quality and quantity. For the two most recently deﬁned plant essential elements, chlorine (Broyer et al., 1954) and nickel (Brown et al., 1987), very little molecular and physiological information exists, while in contrast, the past decade has seen a tremendous increase in understanding of boron, copper, and manganese. The great diversity in chemistry, biology, and information available for the elements boron, chlorine, copper, manganese, molybdenum, and nickel precludes a uniﬁed discussion of the acquisition, utilization, and prospects for improvement of nutrient use

379

efﬁciency of these elements. Collectively, boron, chlorine, copper, manganese, molybdenum, and nickel, however, do provide illustrative examples of each of the varied factors that determine nutrient efﬁciency and the diverse approaches that have been used to improve micronutrient use efﬁciency in crop plants. Optimized micronutrient status is essential not only for maximal productivity but also to ensure optimal crop quality, disease resistance, and the efﬁcient use of both water and nitrogen by the crop. Boron, chlorine, copper, manganese, molybdenum, and nickel are beneﬁcial for human health; however, there is very little data to suggest these elements are deﬁcient in human populations and little rationale for explicit attempts to enhance the content of these nutrients in foods. In the following, an overview of the extent of global deﬁciency, the chemistry of element behavior in soil, and the biological mechanisms of acquisition and utilization of boron, chlorine, copper, manganese, molybdenum, and nickel by plants is provided with emphasis on the molecular and physiological processes that hold most promise for optimizing the efﬁciency of use of these elements. Boron Boron deﬁciency occurs widely and is considered the second most important micronutrient deﬁciency globally. It is a major constraint in cereal and brassica crops throughout south Asia and in forest and horticultural production in high rainfall zones worldwide. Reproductive growth is especially sensitive to boron deﬁciency and substantial crop losses can occur when no clear vegetative signs of deﬁciency are observed. Deﬁciencies are exacerbated by climate, temperature, and water stress, and are frequently erratic and transient in occurrence and hence difﬁcult to detect or predict. Boron

380

NUTRIENT USE EFFICIENCY IN CROPS

is among the least understood of all plant micronutrients and has only a single known essential function, as a component of the cell wall. While much progress has been made toward understanding the mechanisms of boron acquisition from soils and transport within the plant, our understanding is still far from adequate and many observed responses to boron deﬁciency cannot be explained. Species and genotypes vary signiﬁcantly in their ability to grow in boron-deﬁcient and toxic environments and success has been achieved in identifying and developing superior germplasm with greater efﬁciency of boron use and tolerance to toxicity. Boron in soils and plants and occurrence of global deﬁciencies Among the essential plant micronutrients, boron deﬁciency is the most widespread in agriculture (Loomis and Durst, 1992; Shorrocks, 1997) and impacts plant productivity by reducing not only quantity but also the quality of yield. One signiﬁcant cause of boron deﬁciency that distinguishes it from other micronutrient deﬁciencies is that boron deﬁciency speciﬁcally inhibits growing tissues and especially reproductive structures, which represent more than 80% of the world’s agricultural product (Brown et al., 2002). Boron deﬁciency occurs in diverse cropping systems throughout the world and across a wide range of climates and is not restricted to speciﬁc soil types or crops. While boron deﬁciency is more prevalent on leached sandy, alkaline, and heavily limed soils, boron is easily leached from most soils and deﬁciencies often occur in areas with high rainfall (South East Asia, Japan, and Brazil) or in irrigated zones utilizing water with low boron content (<0.3 μg mL−1). Boron availability is signiﬁcantly affected by soil water content and becomes limiting in dry conditions where mass ﬂow to roots is reduced (Shorrocks, 1997). The main soil factors that affect boron availability include

pH, soil texture, organic matter, and clay mineralogy, which mostly dictate the extent of adsorption of boron to soil surfaces (Goldberg, 1997). In arid regions of the world where parent material is high in boron, boron toxicity can be a problem. These areas are commonly found around the Eastern Mediterranean, parts of California, Southern Australia, and Chile. For example, in Southern Australia, 5 million ha (corresponding to 30%) contain 15 mg kg−1 boron or more, causing signiﬁcant losses to barley yield (Cartwright et al., 1986). High concentrations of boron are typically toxic to most organisms, and for this reason boron is used as a potent biocide in industry. The discovery of boron functions in biology would not have been possible without an a priori understanding of the physical and chemical properties of boron (Loomis and Durst, 1992; Goldbach, 1997; Blevins and Lukaszewski, 1998; Brown et al., 2002). At cytoplasmic pH, more than 98% of boron exists as unionized free boric acid B(OH)3 and less than 2% exists as the borate ion, B(OH)4− (Woods, 1996). Boron acts as a weak Lewis acid, with a pKa of 9.2, and accepts a hydroxyl ion rather than donate a proton. Borate (ionized boron) forms diester bonds with molecules containing cis-diols (i.e., sortibol, apiose). Because borate contains two pairs of hydroxyl moieties, it allows borate to form diester complexes with two molecules on each side of the borate ion, thus serving a cross-linking or bridging function. Diester complexes are believed to be energetically more favorable than monoester complexes as evidenced by the fact that all known boron complexes in nature are diester cross-linked (Brown et al., 2002; Bolanos et al., 2004; Goldbach and Wimmer, 2007). Function and deﬁciency symptoms The function of boron in plants is poorly understood and remains a great challenge in

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

plant nutrition. Although boron has been accepted as a necessary micronutrient for plant growth and development for over 85 years (Warington, 1923), it has not been until recent years that some of its roles in biology have become evident. Numerous difﬁculties unique to boron have hindered progress in understanding boron function in plants and include technical limitations to measuring the low cellular concentrations present in cells, boron’s labile nature, lack of radioisotopes, and the ability to bind to diverse molecules. Classical studies aimed at identifying boron function in plants have relied on the withdrawal of boron from growing media, which resulted in an almost immediate arrest of growth and the subsequent manifestation of numerous secondary effects, making the identiﬁcation of primary effects difﬁcult to observe. In fact, the literature is clouded with many purported functions of boron, making meaningful understanding of the true role of boron in biology difﬁcult (Brown et al., 2002). For example, it has long been held that boron is required for phenol metabolism and carbohydrate transport, but this is most likely a secondary cause of boron deﬁciency and not likely to be a primary role of the nutrient. Such conclusions are drawn from studies that typically involved many hours to days (long term) of boron deﬁciency. Primary effects on plant processes are likely to occur much sooner given that boron affects cell wall dynamics, the cytoskeleton, and plasma membrane associated processes, all within 10–15 min (Findeklee and Goldbach, 1996; Goldbach et al., 2001; Yu et al., 2003). Secondary effects including oxidative stress responses could occur within 30 min (Lukaszewski and Blevins, 1996; Kobayashi et al., 2004; Koshiba et al., 2009). Visual symptoms of boron deﬁciency generally become evident at tissue concentrations of less than 10 mg kg−1 dry weight in monocots and 20–30 mg kg−1 dry weight in dicots (Brown and Hu, 1997). In fruit and

381

nut trees, boron deﬁciency often results in decreased fruit set even when vegetative symptoms are not apparent (Nyomora et al., 1997). Foliar application of boron prior to anthesis often results in improved yield in tree crops even when soil and subsequent plant tissue analyses appear “adequate” (Brown et al., 2002). This is suggestive of a transient, but critical, reproductive deﬁciency. Boron deﬁciency causes a wide range of anatomical, physiological, as well as biochemical symptoms. These include the inhibition of apical growth, reduction in leaf expansion, necrosis of terminal buds, breaking of tissues due to brittleness and fragility, abortion of ﬂower initials, and shedding of fruits (Goldbach, 1997; Brown et al., 2002). Most anatomical deﬁciency symptoms have been associated with cell wall abnormalities (Loomis and Durst, 1992; Brown et al., 2002) and the numerous biochemical and physiological effects often observed under boron deﬁciency have been interpreted by most experts to be secondary effects of cell wall damage (Goldbach, 1997; Blevins and Lukaszewski, 1998; Brown et al., 2002; Bolanos et al., 2004). In plants, elevated (mM) concentrations of boron become toxic. Boron toxicity reduces shoot growth, primarily in expanding tissues, followed by chlorosis, beginning at the older leaf tips and margins, before ﬁnally causing necrosis (Nable et al., 1997; Reid et al., 2004; Reid and Fitzpatrick, 2009). The mechanism of boron toxicity remains unknown. The most prominent symptoms of boron deﬁciency are often associated with primary cell walls and include abnormally formed walls that are often thick, brittle, have altered mechanical properties, and do not expand normally (see Brown et al., 2002). A signiﬁcant amount of total plant boron is associated with cell wall pectins (Hu and Brown, 1994) and is often correlated with the whole-plant boron requirement (Hu et al., 1996). Isolation of a boron–polysaccharide complex, (Matoh et al., 1993), later identiﬁed as

382

NUTRIENT USE EFFICIENCY IN CROPS

RGII (Kobayashi et al., 1996; O’Neill et al., 1996), demonstrated that boron in the cell wall predominantly cross-links the apiosyl residue in the A side chain of each of two neighboring monomeric RGII molecules to form a dimeric B-dRGII pectin complex (Ishii and Matsunaga, 1996; Kobayashi et al., 1996; O’Neill et al., 1996; Pellerin et al., 1996; Ishii et al., 1999). Indisputable evidence demonstrating this role of boron in cell walls and its subsequent importance to plant growth and development came from the Arabidopsis mur1 mutant. In mur1, shoot RGII, which has a substituted sugar residue, forms B-dRGII less rapidly, which once formed is less stable than RGII from wild-type plants (O’Neill et al., 2001). mur1 plants are dwarfed with brittle stems, but the phenotype is rescued with added boron. Interestingly, a role of boron cross-linked RGII in intercellular attachment of tissues was shown in another RGII biosynthesis mutant (Iwai et al., 2002). Boron is also required for legume– Rhizobium symbiotic interactions. Boron plays a role in the maintenance of nodule cell wall and membrane structure (Bolanos et al., 1994; Bonilla et al., 1997) for rhizobial infection and nodule cell invasion processes (Bolanos et al., 1996; Redondo-Nieto et al., 2001) as well as for symbiosome development and bacteroid maturation (Bolanos et al., 2001). More recent studies have shown the participation of boron in nodule organogenesis and in plant–bacteria interactions, suggesting that boron has a wide range of functions beyond its role in cell wall structure (Redondo-Nieto et al., 2001; Reguera et al., 2009; Reguera et al., 2010). Evidence supporting the essentiality of boron in other organisms, including yeast, bacteria, and animal embryos that lack a cell wall, suggests functions of boron beyond RGII. Additional but unexplained effects of boron deﬁciency include swelling of lipo-

somes, increased ﬂuidity of microsomes, and the disruption of membrane transport processes. The discovery that boron is required in bacterial quorum sensing (Chen et al., 2002), in vibrioferrin, a boroncontaining siderophore (Harris et al., 2007), as well as the identiﬁcation of a boron transporter in animal cells (Park et al., 2004), highlights novel roles of boron. In plant cells, Bassil et al. (2004) proposed that boron was necessary for cell to wall adhesion and the organization of the architectural integrity of the cell while Brown et al. (2002) suggested that boron functions to stabilize membrane raft formation and maintain membrane function. Uptake and transport The past few years have witnessed tremendous advances in the understanding of boron uptake mechanisms and transport into cells. Recent identiﬁcation of membrane transporters, their tissue, and cellular localization as well as regulation provides clues about how plants control their boron status. A summary of known and putative wholeplant and cellular boron transporters is provided in Figures 17.1 and 17.2. Under conditions of moderate or excess boron, boron uptake is the result of passive absorption of boric acid and is primarily determined by the concentration of boron outside the root (Brown and Hu, 1994), membrane permeability (Raven, 1980), B-complexation inside root or outside the root, translocation within the plant, and transpiration (Brown et al., 2002). Raven (Raven, 1980) calculated a theoretical lipid permeability coefﬁcient in the order of 10−6 cm s−1, which is adequate to satisfy plant boron requirements under almost all naturally occurring soil conditions. Experimental data using artiﬁcial liposomes veriﬁed this theoretical permeability and also showed that permeability was inﬂuenced by mem-

Cortex

MoO42MOT1

B(OH)4-

Endodermis

Passive diffusion

Pericycle

MoO42-

BOR 1

Mesophyll Shoot

H3BO3

B(OH)4-

H3BO3

BOR 1

CLCe

Sultr? Xylem

Cl-

NIP6;1

Phloem

H3BO3

Cl-?

B-Polyol (B-sucrose?)

CCC?

Companion cell

NIP5;1?

Guard Cell

Cl-?

Reproductive Organs

TIP5;1?

Fig. 17.1.

Intercellular metal transport in dicots. Boron is taken up into the root as H3BO3 through the NIP5:1 channel then loaded into the xylem through the boron transporter BOR1. Mechanisms of xylem unloading and subsequent distribution to leaves and reproductive tissues are not well described but may involve homologs of NIP and BOR. BOR4 is an export transporter responsible for export of B(OH)4 into the rhizosphere under high boron conditions. H3BO3 is membrane permeable and is present in many soils at concentrations great enough to supply most plant boron requirements by passive diffusion. Boron transport to reproductive structures and meristematic tissues is facilitated by polyol-B complex formation in polyol-producing species and by B-CHO complexation (to a much lesser extent) in other species. The mechanisms of boron loading into the phloem are unknown. Chloride is present in abundant amounts in most environments. Uptake is likely mediated by the chloride channels (CLC) family (CLC a-g) and by the cation chloride cotransporters. The CLC channels have afﬁnity for diverse anions (including nitrate), and the details of the roles of these transporters in chlorine uptake and homeostasis are not well understood. Molybdenum is transported across plasma membranes as MoO42– via members of the Sultr family (MOT1/Sultr5;2 and Sultr 1;1/SHST1). The localization and speciﬁcity of molybdate transporters in plant tissues and the mechanisms of subcellular transport are unclear. Modiﬁed with permission from Palmer and Guerinot, 2009.

Epidermis

NIP5;1

H3BO3 Root

CLCb

Cl- ?

MOT1/Sultr 5;2

BO OR 4

MoO42-

H3BO3

B(OH)4-

MoO42-

Sultr 1;1 1 ShST1

383

384

NUTRIENT USE EFFICIENCY IN CROPS

B-RGII BOR 1? BOR 4?

ClCL

B(OH)4-

Ce

?

Cl-

CLCa,c,g

Cl-

GmCLC1

Cl-

OsCLC1

Cl-

AtTIP5;1

H3BO3

Sultr4;1

MoO42-

PSII

Chloroplast

Cl-

CLCd,f

Golgi/ER

Mitochondrion MoO4

2-

PVC

MOT1 MoCo

Vacuole

Intracellular transport of H3BO3/B(OH)4–, Cl−, MoO42–. Information on the subcellular transport and regulation of boron, chlorine, and molybdenum is very poor. The mechanisms of boron uptake and delivery to the cell wall, the only known essential role for boron in plants, are unknown. Seven members of the chloride channel family (CLCa-g) have been identiﬁed in plants and their localization has been demonstrated. To date, the speciﬁcity of these channels for chlorine and the essentiality for plant function have not been determined. Molybdenum is transported as MoO4− in plants by members of the SULTR family: MOT1 (SULTR5;2) appears to be essential for molybdenum acquisition though its localization to plasma membranes or mitochondrial membranes is currently uncertain. Sultr Sultr4;1 (tonoplast) is upregulated under molybdenum deﬁciency and has structural characteristics suitable for MoO4− transport. Modiﬁed with permission from Palmer and Guerinot, 2009.

Fig. 17.2.

brane lipid composition (Dordas and Brown, 2000). In other studies using plant membranes (Dordas and Brown, 2001a) and the charophyte alga (Stangoulis et al., 2001b), a boron permeability coefﬁcient of 20–300fold slower than that predicted by Raven (1980) or determined experimentally in artiﬁcial liposomes (Dordas et al., 2000) was found. Although passive diffusion can theoretically account for boron uptake in the major-

ity of agronomic conditions, it is not adequate to explain boron uptake under conditions of low boron supply and does not provide a mechanistic understanding of differential species response and observed patterns of tissue boron distribution. Boron transporters are likely to be required for regulated delivery of boron for cell wall synthesis and for delivery of boron to reproductive tissues for which passive diffusion will often be inadequate. In some species and genotypes, boron

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

transporters also function to mitigate boron toxicity. Regardless of the demonstrated existence and function of boron transporters in plants, it is apparent that boron uptake is under very poor control since deﬁciencies and toxicities can be rapidly induced and plant boron concentrations generally reﬂect the product of transpirational water loss and the boron concentration in that water. Channel-mediated transport The observation that boron uptake is passively mediated is at odds with other observed differences in uptake between cultivars that can be as large as sevenfold even when grown under identical conditions (Nable et al., 1997; Dordas and Brown, 2001a,b) and which cannot be explained by differences in water use efﬁciency. Possible involvement of channel proteins in boron uptake was provided by Dordas et al (Dordas and Brown, 2001a,b) and Nuttall (2000). The permeation of boric acid across vesicles of squash and Arabidopsis root plasma membranes was partially inhibited by mercury and reversed by 2-mercaptoethanol, suggesting that boron may be facilitated through protein channels. In addition, heterologous expression of major intrinsic proteins (MIPS, also known as aquaporins), such as PIP1 in Xenopus oocytes, resulted in a 30% increase in boron permeability and was inhibited by the presence of other noncharged compounds of similar size (Dordas and Brown, 2001a,b). NOD26-like intrinsic protein (NIP5;1), an MIP subfamily member, was identiﬁed from a transcriptional analysis in which AtNIP5;1 was signiﬁcantly upregulated in low-boron-treated Arabidopsis (Takano et al., 2006). In roots, NIP5;1 localizes to the plasma membrane of epidermal, cortical, and endodermal cells of the elongations zone. Heterologous expression in Xenopus oocytes increased transport of boron into

385

cells compared with water-injected controls. Loss of function mutants of NIP5;1 had severe shoot and root growth retardation and low boron content when grown under low boron. These results suggest that NIP5;1 acts as a boric acid importer required for boron uptake under low boron. Some NIP members are multifunctional channels that also transport other uncharged molecules such as glycerol, urea, silicon, arsenite (Ma et al., 2006; Ma et al., 2008) lactic acid, and water (see (Miwa et al., 2010). A close homolog to AtNIP5;1 is AtNIP6;1, which facilitates boron transport across membranes but not water (Tanaka et al., 2008). AtNIP6;1 transcript is detected in shoots but not roots, and promoter-β-glucuronidase (GUS) analysis revealed speciﬁc expression in nodes, especially near the phloem. When grown in low boron, AtNIP6;1 knockout lines have reduced leaf expansion and decreased concentration of boron in the young leaves. Tanaka et al. suggested that NIP6;1 plays a role in the xylem to phloem transfer of boron in nodes of shoot tissue. Recently, it was observed that overexpression of AtTIP5;1, a tonoplast and reproductive organ localized aquaporin, resulted in increased tolerance of Arabidopsis to moderately high levels of boron in the growing medium (Pang et al., 2010). There was a modest increase in tissue boron in plants overexpressing AtTIP5;1 grown at high boron but not under control boron concentrations; veriﬁcation of these early results is required. The “active” transport of boron (i.e., against a concentration gradient) was ﬁrst suggested in studies of low-boron-treated sunﬂower in which the boron concentration in the xylem was found to be higher than in the growth medium. The boron concentrating mechanism could be inhibited by low temperature and metabolic inhibitors and followed Michaelis–Menten kinetics. Boron concentrations in xylem exudates did not

386

NUTRIENT USE EFFICIENCY IN CROPS

differ from that of growing medium when plants were grown in adequate solution boron (Dannel et al., 2000). High-afﬁnity uptake under low boron was also reported in Chara (Stangoulis et al., 2001a). These data suggested that in addition to passive uptake, a high-afﬁnity boron uptake system may be operating at low boron. The ﬁrst boron transporter identiﬁed in any organism was AtBOR1. BOR1 is an efﬂux transporter, which belongs to the SLC4 bicarbonate family of transporters (Frommer & von Wiren, 2002). Heterologous expression of AtBOR1 in yeast reduced cellular boron concentration, suggesting that BOR1 is an efﬂux-type transporter (Takano et al., 2002). In Arabidopsis, AtBOR1 is expressed in the pericycle of the root stele, and is required for xylem loading. Under low boron, the bor1 mutant exhibits reduced growth and poor seed set but otherwise resembles wild type at adequate boron supply (Noguchi et al., 1997). Overexpression of BOR1 enhanced root-to-shoot translocation of boron and improved seed set under boron-limiting conditions under which wildtype plants had reduced seed set (Miwa et al., 2006). BOR1 activity is downregulated by boron through a process of endocytotic degradation of the transport protein when boron in growth medium is adequate. In barley (Hordeum vulgare L.), BOR4, one of the six paralogs of BOR1, is not responsive to boron changes, and accumulates in the presence of high boron. BOR4 overexpressing transgenic lines of Arabidopsis exhibit enhanced tolerance of high boron (Miwa et al., 2007). The difference in regulation of BOR1 and BOR4 suggests that complex mechanisms for the perception and control of boron homeostasis must exist. BOR1 homologs have also been characterized in yeast (Frommer and von Wiren, 2002) and in animals (Park et al., 2004). Our current understanding of how boron is transported across tissues is explained as

follows. At adequate concentrations of boron, most plant demand is met by passive uptake. At low boron, boric acid enters the symplasm of root cells through NIP5;1 channels that increase membrane permeability to boron. Symplastically boron travels to the pericycle, where it is exported by BOR1 into the xylem against its concentration gradient, for long-distance transport to shoots. The mechanism of unloading, delivery to cell wall synthetic processes, and translocation to reproductive tissues are not understood. Within-plant transport The long-distance transport of boron is largely dependent on transpiration and boron accumulates at the margins of mature leaves (Brown and Shelp, 1997; Brown et al., 2002). Symptoms of both boron deﬁciency in young tissues, and toxicity in mature tissues (Brown and Shelp, 1997; Nable et al., 1997), suggests that boron is highly immobile in most species (Brown and Shelp, 1997; Brown and Hu, 1998). It was later determined that boron mobility was species speciﬁc and that boron was highly phloem mobile in species that transport sugar alcohols (polyols) within the phloem as the main photosynthate. Hu et al. (1997) subsequently identiﬁed the existence of boronpolyol complexes in phloem sap of celery and peach as boron cross-linked mixtures of mannitol, fructose, and sorbitol, and provided a mechanistic explanation for the observed phloem boron mobility in polyolproducing species (Hu et al., 1997). More recent evidence suggests that nonpolyolproducing plants, such as wheat, canola, and sunﬂower can also transport boron in the phloem (Matoh and Ochiai, 2005; Stangoulis et al., 2010). The relevance of these ﬁndings to whole-plant boron transport are uncertain since wheat and canola are known to be very sensitive to boron deﬁciencies, especially

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

during reproductive growth when dependence on phloem-translocated boron would be greatest (Xue et al., 1998; Rerkasem and Jamjod, 2004). The processes that control boron distribution in shoots are poorly understood, and implications for diagnosis and management of boron deﬁciency, especially during reproductive development when boron requirements are greatest, remain challenging. Prospects for optimizing boron use efﬁciency Genotypes and cultivars differ widely in their response to either high or low boron and have been grouped into distinct boron efﬁciency classes (Jamjod et al., 2004). Below a critical boron level, inefﬁcient genotypes typically display sterility and set few or no grains, while efﬁcient genotypes set grain normally (Rerkasem and Jamjod, 2004). Rerkasem and Jamjod (1997) listed 24 species in which signiﬁcant genotypic variations in boron efﬁciency have been observed, particularly in wheat (Rerkasem and Jamjod, 1997; Xu et al., 2001; Jamjod et al., 2004; Rerkasem and Jamjod, 2004). In many cases, differences in boron efﬁciency can be quite large. When grown in low-boron soil, the boron-efﬁcient wheat cultivar Fang 60, yields 97% of controls grown in sufﬁcient boron, while the inefﬁcient cultivar SW41 yields only 11% of controls (Rerkasem and Jamjod, 1997). Improved yield of Fang 60 when grown under low boron, correlated with a higher partitioning of boron to ears (nearly double that of SW41), (Nachiangmai et al., 2004). The authors suggested that boron efﬁciency in Fang 60 was associated with improved xylem and long-distance transport as well as retranslocation of boron from vegetative tissues. Given the recent identiﬁcation of bis-sucrose borate complexes in wheat phloem (Stangoulis et al., 2010), it will

387

interesting to determine if cultivars such as Fang 60 are more efﬁcient in utilizing available boron pools within the plant because they are capable of higher boron transport in the phloem due to favorable complex formation. Other known genotypic variation in boron include canola (Xu et al., 2001; Zeng et al., 2007) and Arabidopsis (Zeng et al., 2007). In canola, using the application of boron isotopes to the mature leaves, Stangoulis et al., (2001a) found that one efﬁcient cultivar, Huashuang-2, retranslocated more boron to younger leaves, and concluded that multiple mechanisms for boron efﬁciency must exist. It is now clear that the nutrient-efﬁcient characteristics are usually genetically controlled and a number of these genes have been mapped using restriction fragment length polymorphism (RFLP) and ampliﬁed fragment length polymorphism (AFLP) markers (Xu et al., 2001). The ratio of high boron efﬁciency to low boron efﬁciency individuals ﬁtted the expected ratio of 3:1, indicating a major gene controlling the boron efﬁciency trait. The major gene was mapped in the ninth linkage group of Brassica napus. The boron efﬁciency of wheat is controlled by two genes (Jamjod et al., 2004). The efﬁciency analysis indicated that genetic control varied from recessive to additive, to completely dominant with different cross combinations and boron levels. Collectively, these reports suggest that perhaps enhanced partition into the grain and/or enhanced nutrient transport may be key factors that determine differences in boron efﬁciency and which have important implications for plant breeding. Since genotypic differences in nutrient efﬁciency are genetically controlled, genetic and molecular tools can be used to improve cultivars considered to be boron inefﬁcient but which otherwise have good crop characteristics.

388

NUTRIENT USE EFFICIENCY IN CROPS

The molecular characterization of boron transporters as well as studies in transgenic Arabidopsis and tobacco raise interesting questions about the potential for engineering crops for improve tolerance to low boron. The work of Fujiwara et al. has provided the initial context to examine transgenic approaches for improve yield and quality in conditions of low boron (Miwa et al., 2006; Tanaka and Fujiwara, 2008; Miwa et al., 2010). The overexpression of AtBOR1 increased boron transport to shoots and improved shoot growth and fertility when plants were grown under low boron and without obvious consequences to growth if the same plants are grown under high boron. Added tolerance to low boron was observed in the overexpression of AtBOR1 in NIP5;1 activation tag lines plants (Kato et al., 2009), while overexpression of BOR4 generated plants with improved tolerance to high boron (Miwa et al., 2006). The possibilities of using additional transgenic approaches to improve plant growth in conditions of limited boron supply also exist. Engineering of sorbitol synthesis facilitated improved boron uptake, efﬁcient remobilization of boron, and improved growth in expanding tissues and seed yield under low boron, compared to wild-type tobacco (Bellaloui et al., 1999; Brown et al., 1999) and rice (Bellaloui et al., 2003). Despite such encouraging laboratory results, it remains to be determined if such results also persist in plants grown under ﬁeld conditions. Chlorine Chloride (Cl) is abundant in most soils, water, and the atmosphere, and rainfall, dust deposition, and irrigation generally supply adequate chlorine to support crop growth. Chlorine functions in electrical charge balance, as a cofactor for oxygen evolution in photosystem II (PSII) and in osmoregula-

tion and seismonasty of speciﬁc cell types. Chlorine deﬁciencies are rare, generally occurring only in areas a great distance from oceans. The chloride ion (Cl−) is highly soluble, forms only weak complexes in most soils, and is readily mobile within plants. Comparatively little is known about the mechanisms of Cl− acquisition, although recent molecular analyses have validated earlier kinetic and electrophysical evidence for the presence of Cl−/anion channels and cation-Cl− symporters. Species and cultivars differ in both the requirement for Cl− and the efﬁciency of Cl− use, although the mechanism underlying these differences is not known. Chlorine in soils and plants and occurrence of global deﬁciencies Chlorine is abundant in the lithosphere and atmosphere. Deposition from rain can provide 0.5–1000 kg ha−1 year−1 (Fixen, 1993; Christensen and Hayes, 2009), and irrigation, even with waters low in Cl−, provides in excess of 1000 kg ha−1 year−1 and generally much higher. Soils do not adsorb or release Cl− in signiﬁcant amounts, although newly deposited organic matter from leaf fall can provide substantial crop inputs. Highly leached, permeable, sandy textured soils such as Arenosols, Ferrasols, and Acrisols in areas with little atmospheric or irrigation input are most likely to give rise to Cl− deﬁciency in sensitive crops (Heckman, 2007). Chloride is present as a secondary ion in many potassium, magnesium, and calcium fertilizers (KCl, MgCl2, CaCl2). The use of KCl as a potassium source easily provides all required Cl−. Chloride removal in crops is low, ranging from 1–8 kg ha−1 in grain crops to 5–80 kg ha−1 in forage crops, and in almost all cropping systems removal in crops is substantially less than deposition (Fixen, 1993). Minimum plant requirement for Cl− ranges from 0.1 to

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

4.5 g kg−1 dry matter (DM) and the majority of plant species are satisﬁed with 1.0 g kg−1 DM. Species with high Cl− requirement include barley (H. vulgare L.) and wheat (Triticum aestivum L.) at heading (4.0 g kg−1 DM), coconut (Cocos nucifera L.), kiwifruit (Actinidia deliciosa A. Chev), oil-palm, and sugar beet (Beta vulgaris L.) (>2.0 g kg−1 DM) (Heckman, 2007). The best documented example of agricultural chlorine deﬁciency is in the wheatgrowing regions of the Great Plains of the United States (Fixen, 1993; Heckman, 2007). These rain-fed regions are typiﬁed by very low chlorine deposition in rain (<0.5 kg ha−1; (Xu et al., 2000), leached soils with very low chlorine content, and highproduction and high-demand species (wheat, barley) (Fixen, 1993). Equivalent conditions to the Great Plains of the United States exist in central Asia and Russia, although there are no unequivocal reports of chlorine deﬁciencies from these regions. Field deﬁciencies and responses have also been observed in kiwifruit, coconut, and oil-palm grown under rain-fed conditions at signiﬁcant distances from the ocean and in isolated vegetable and kiwifruit production areas in China and east of the Cascade mountains in the Paciﬁc Northwest of the United States (Xu et al., 2000).

Function and deﬁciency symptoms The growth of many plants is reduced substantially in Cl-free media (White and Broadley, 2001). Deﬁciency causes reduced leaf growth and wilting, followed by chlorosis, bronzing, and ﬁnally, necrosis. Roots become stunted and the development of laterals is suppressed. Fruits are decreased in numbers and size. While more than 130 Clcontaining organic compounds in plants have been identiﬁed (Engvild, 1986), the only established requirement is as a cofactor

389

for the fast turnover of the oxygen-evolving water oxidation complex in PSII (Guskov et al., 2010; Kusunoki, 2007; Kusunoki, 2008). The chlorine atom in PSII has been assigned close to the entrance of putative proton transfer pathways, and its participation in proton release from the Mn4OxCa-cluster is very likely. One or more Cl− ions are required for the water oxidation cycle to proceed, and a depletion of Cl− has been shown to inhibit the S2 → S3 and S3 → S0 transitions. Proposed roles of Cl− in the water oxidation complex include ligation to manganese or Ca atoms, regulation of the redox potential of the Mn4OxCa cluster, maintaining a hydrogen bond network, and activation of the substrate water (Kawakami et al., 2009). There remains some controversy as to the number of chloride-binding sites (1 or 2) in the Mn4OxCa cluster (Kawakami et al., 2009; Guskov et al., 2010). Chloride acts as a counter anion to stabilize membrane potential and is involved in turgor and pH regulation, and at concentrations present in most environments, chloride is the most abundant inorganic anion in plant cells. Average plant Cl− concentrations range from 1 to 20 g kg−1 DM, and the majority of plant species are satisﬁed with 0.2 to 0.5 g kg−1 DM, that is, about 10 to 100 times lower. Thus, while chloride plays a quantitatively important role in ion balance when chloride is abundant, other anions (nitrate, malate) can fulﬁll this role when chloride supply is reduced. Chlorine is also required for the osmoregulation of leaf motor cells in Mimosa pudica (Moran, 2007), and the proton-pumping V-type ATPase on endomembranes is speciﬁcally stimulated by chloride and other anions (Sze, 1985). Fluorescent-labeled AtClC-d colocalizes with V-type ATPase in the trans-Golgi network, and mutations in either the AtCLCd or V-ATPase result in similar phenotypes (von der Fecht-Bartenbach et al., 2007). In addition, Cl− or permeant anions dissipate

390

NUTRIENT USE EFFICIENCY IN CROPS

the electrical potential (+ inside) generated by the electrogenic H+-V-ATPase in vesicles, and this is accompanied by an increase in delta pH (acid inside). These results suggest the presence of an anion conductance on the same membrane (Sze, 1985) and support a functional linkage between AtCLC-d and V-ATPase. Interestingly, a strong interdependence between chloride channel (CLC) activity and acidiﬁcation of intracellular compartments has also been established in mammals. A functional linkage between AtCLC-d and V-ATPase may also explain the striking similarities between the chloride-stimulated V-ATPase and root elongation. Plants harboring a loss of function mutation of AtCLCd had impaired root growth and cell elongation, an effect that was also reported following H+-ATPase knockdown by RNAi (Padmanaban et al., 2004). Severe inhibition of root elongation in chlorine-deﬁcient plants might be related to the function of chloride in stimulating V-ATPase-mediated compartmental acidiﬁcation and plant growth. Chloride is an important anion in opening and closing of stomatal guard cells (Roelfsema and Hedrich, 2005). Opening and closure of stomata is mediated by ﬂuxes of potassium and accompanying anions such as malate and chloride, and it has long been hypothesized that tonoplast Cl−/H+ antiporters mediate stomatal opening (Pierce and Higinbotham, 1970). Recently, the chloride transporters AtCLC-c and SLAC1 have been localized to the guard cell vacuole, and endomembrane compartments though their function in chloride transport and stomatal opening has not been resolved. Field evidence suggests that chlorine status has a signiﬁcant impact on disease resistance in wheat (Fixen, 1993; Heckman, 2007). An interaction between disease and chlorine have been observed for 15 foliar diseases in 11 crops, and the presence of disease pressure can result in an apparent

increase in the tissue chlorine required for optimal yield (Fixen, 1993). Uptake, transport, and homeostasis The discovery of the essentiality of chlorine coincided with a period of intense interest in the characterisitics and mechanisms of ion uptake into plants. This research has been summarized in two excellent reviews (White and Broadley, 2001; Teakle and Tyerman, 2010). A summary of known and putative whole plant and cellular Cl− transporters is provided in Figures 17.1 and 17.2. On the basis of premolecular research, White and Broadley (2001) concluded that under nonsaline conditions (1) active Cl− inﬂux across the plasma membrane occurs though a H+/C1− symport; (2) plasma membrane Cl− efﬂux is mediated by anion channels; (3) tonoplast Cl− ﬂuxes are passive and mediated by anion channels; and (4) xylem loading of Cl− is down its electrochemical gradient. Chloride inﬂux across the plasma membrane becomes passive at high soil concentrations (White and Broadley, 2001). This conclusion has been partly supported by subsequent cloning, molecular, and positional analyses of transporters involved in Cl− homeostasis. Known transporters include the CLC protein family, which includes both Cl− channels and the (Cl−/NO3−) /H+ antiporters, secondary active transporters (Lv et al., 2009; Zifarelli and Pusch, 2010), and cation chlorine transporter (CCC) (Colmenero-Flores et al., 2007). Although Cl− does clearly stimulate H+ ﬂuxes and there is clear evidence of tight coupling between H+ and anion transport (De Angeli et al., 2007), there is no evidence to date of a H+/C1− symporter in plants. Indeed, there is growing evidence that CLCs function primarily as (Cl−/NO3−)/H+ antiporters (Zifarelli and Pusch, 2010). Currently, none of the known chlorine transporters have been shown to be essential for plant chlorine

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

transport, the speciﬁcity of the transporters for chlorine has not been well characterized, and no chlorine uptake knockout mutants are known. Additional Cl− transporters have been identiﬁed in a number of nonplant species, and homologous genes are present in plants, including chloride intracellular channel (CLIL), (nucleotide-sensitive chloride conductance regulator protein (ICln), and ATPBinding Cassette (ABC) transporter (Tejada-Jiménez et al., 2009). A function of the CLIL, ICln, and ABC transporters in Cl− transport in plants has not been shown. Seven CLC members have been identiﬁed in the Arabidopsis genome (Marmagne et al., 2007; Lv et al., 2009; Isayenkov et al., 2010; Zifarelli and Pusch, 2010). CLC members are localized in different organs (Isayenkov et al., 2010); AtCLCe is localized in thylakoid membranes, while AtCLCf and CLCd are targeted to Golgi vesicles (Marmagne et al., 2007; von der FechtBartenbach et al., 2007). AtCLCc displays signiﬁcant expression in guard cells. AtCLCb is strongly expressed in roots, while AtCLCe displayed an almost complementary expression pattern and was predominantly expressed in leaves with very low levels in roots and stems (Lv et al., 2009). Lv et al. (2009) also found that all the AtCLC members were expressed in vascular tissues in both roots and shoots, implying a possible role in long-distance ion transport within the plant. Collectively, results suggest that all CLC proteins are associated with endosomal membranes. In addition to differential expression patterns, the CLC family of transporters show differential selectivity for Cl− and NO3−. AtClCb is selective for NO3−, whereas AtClCc and -AtClCg are selective for Cl−, and AtClCc exhibits highest expression in guard cells (Lv et al., 2009) and may be critical in osmotically regulated guard cell opening. This observation is consistent with

391

early physiological observations that predicted the presence of guard cell tonoplast Cl−/H− antiporters (Pierce and Higinbotham, 1970). Colmenero-Flores et al. (2007) cloned an Arabidopsis cDNA encoding a member of the CCC family. Expression in Xenopus laevis oocytes and knockout analysis suggests the AtCCC functions as a Na+:K+:Cl− cotransporter. Simultaneous presence of Na+:K+:Cl− was required for activity. Preferential expression was observed in root and shoot vasculature, suggesting involvement in Cl− homeostasis and perhaps plant development. Further functional characterization of CCCs in plants under nonsalt stress conditions is required to better deﬁne the role of these transporters in Cl− homeostasis. Chloride is relatively mobile within the phloem (Marschner, 1995) and the recirculation of Cl− (deﬁned as the ratio of phloem/ xylem nutrient ﬂuxes) is about 20% in a number of plants (White and Broadley, 2001). Analysis of phloem sap also indicates that the phloem Cl− concentration varies directly with the Cl− solution in which plants are grown. Prospects for optimizing Cl− use efﬁciency Given the limited occurrence of Cl− deﬁciencies globally, and the ease with which deﬁciencies can be corrected by soil or foliar Cl− application, there has been no effort to develop more efﬁcient cultivars. Clear and heritable differences between cultivars and species in their tolerance to deﬁciencies (Fixen, 1993) and toxicity (reviewed by (White and Broadley, 2001), however, have been observed in a number of species. With recent advances in the molecular understanding of Cl− homeostasis and evidence that these traits are heritable (White and Broadley, 2001), the potential exists for

392

NUTRIENT USE EFFICIENCY IN CROPS

selection of cultivars with enhanced tolerance to low-Cl− and high-Cl− soils. Breeding for low Cl− tolerance and enhanced efﬁciency of Cl− is unlikely to be relevant in developed countries where chlorine fertilization is a trivial expense but may be important in less developed countries where inputs are limited. Much greater potential exists for selection, breeding, and molecular manipulation of Cl− transporters to enhance tolerance of saline conditions. As discussed by Teakle and Tyerman (2010) and demonstrated by Brumos et al. (2009), Cl− likely plays a signiﬁcant but much overlooked role in plant tolerance of saline soils. Copper Agricultural copper (Cu) deﬁciency is relatively rare, although signiﬁcant agronomic deﬁciencies do occur in cereal crops in many regions of Europe, as well as in southern and western Australia, and in isolated crops globally. The primary source of copper for plant uptake is Cu(II) complexed with dissolved organic matter (Cu(II)-DOM) and to a lesser extent free Cu2+ in the soil solution. The behavior of copper in soils is poorly understood and copper deﬁciency is not easily predicted from soil type, mineralogy or pH and existing soil and plant tests are generally inadequate. Reproductive growth is sensitive to copper deﬁciency and under moderate copper deﬁciencies crops may exhibit signiﬁcant yield loss with no apparent vegetative symptoms. While our understanding of soil and crop copper response is poor our understanding of copper transport and homeostasis is perhaps the best of all micronutrients. To maintain the essential functions of copper while protecting cellular metabolism from the negative effects of copper ions, plants have developed an array of transport, complexation and localization mechanisms. Copper is transported into the root sym-

plasm as Cu+ through the copper transporter family COPT and requires an as yet unidentiﬁed root plasma membrane reduction step. Transmembrane transport may also occur through zinc iron permeases (ZIP) and other undeﬁned processes. Speciﬁc copper transporters have been identiﬁed for copper delivery to chloroplasts, plasma membranebound ethylene receptors, mitochondria, and endoplasmic reticulum (ER). Copper chaperones are low-molecular-weight metalreceptor proteins involved in the intracellular trafﬁcking of metal ions and insertion of copper into the active sites of Cu-dependent enzymes (O’Halloran and Culotta, 2000). Chaperones for Cu-dependent plastocyanin, cytochrome oxidase, and CuZn superoxide dismutase (SOD) have been identiﬁed. Copper metabolism is tightly regulated, and copper shortage results in a microRNAfacilitated reduction in copper allocation to nonessential copper enzymes, which allows maintenance of critical Cu-plastocyanin function. Improvements in the efﬁciency of copper use will require an improved understanding of copper behavior in soils and of the mechanisms of copper transport to reproductive tissues. The limited extent of copper deﬁciency and the difﬁculty in predicting or measuring response, coupled with the low relative cost of copper fertilizers, suggests that routine prophylactic copper fertilization should be practiced in areas of suspected response. In areas or crops for which fertilization is not possible, management of soil organic matter or selection of cultivars with higher copper levels in reproductive tissues may be viable. Copper in soils and plants and occurrence of global deﬁciencies The behavior of copper in soils is complex, and our ability to predict crop responses is inadequate. In nonﬂooded agricultural soils,

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

copper in solution is present mainly as Cu(II), either as the free ion Cu2+ (usually only a minor fraction)or as Cu(II) complexed with dissolved organic matter (Cu(II)-DOM) (Hodgson et al., 1966; Loneragan, 1981; Amery et al., 2007). Barber (1984) predicted that soluble complexed copper must be involved in copper uptake since free Cu2+ was unlikely to be present in adequate concentrations to support growth. Evidence that Cu(II)-DOM is a major source for plant copper is supported by the observation that Cu2+ is rarely present in adequate concentrations and by the ﬁnding that >98% of soluble copper in soils is complexed as Cu(II)-DOM (Sauve et al., 1997). It is estimated that a solution Cu2+ concentration of >5 × 10−9 M is required to support plant growth (Degryse et al., 2006; Bravin et al., 2010); soil solution Cu2+, however, is rarely present in noncontaminated soils in excess of 1 × 10−12 M, suggesting that free Cu2+ is not the sole source of copper for plants (Sauve et al., 1997; Nolan et al., 2005; Nolan et al., 2010). This suggestion is further supported by the observation that normal plant growth can also be attained in nutrientbuffered solutions in which free Cu2+ concentrations of <10−12 M are maintained (Bell et al., 1991; Parker and Norvell, 1999) and demonstrates that complexed (buffered) Cu(II) is essential for plant copper supply. While Cu(II)-DOM complexes are the most important determinants of soil copper availability, much of the available research on the behavior of copper in agricultural soils has focused on the activity of soil solution Cu2+ (Barber, 1984; Bravin et al., 2010). The relative lack of experimentation on the kinetics of Cu+ and Cu(II)-DOM in soils and their role in copper availability in noncontaminated soils is undoubtedly a consequence of the substantial technical difﬁculties in obtaining soil solution to carry out the speciation analysis, the cofounding effects of extraction procedures on labile complexes, and

393

the analytical challenges of metal speciation at low concentration with highly varied organic ligands (Bravin et al., 2010). Given the complexity of soil copper reactions and the recent ﬁnding that copper must ﬁrst be reduced to Cu+ prior to uptake by the main copper transporter (COPT1), it is perhaps not surprising that there is considerable inconsistency in the literature and that available soil tests provide inconsistent results. As a consequence of the role of DOM in controlling copper availability, copper does not show the same patterns of pH dependency as seen with manganese, iron, or nickel, and total soil copper contents show only very poor correlation with plant responses. Globally copper deﬁciencies most often occur in sandy, highly leached soils with low total copper (Arrenosols, Ferralsols, Acrisols), calcaereous low organic matter soils (Calcisols), and soils with high organic matter (Histosols, Podzols) (Alloway, 2008). Soil pH inﬂuences copper availability through its effect on both adsorption/desorption from soil minerals and the concentration and characteristics of dissolved organic matter. Thus, high organic matter soils, which may have high total copper content, can have low copper availability due to the formation of an excess of unavailable Cu(II)-DOM complexes. Low organic matter, high pH mineral soils, may have low copper availability due to both low soil mineralization and low levels of Cu(II)DOM, which reduces soluble Cu(II) levels. In large areas of the cereal-growing regions of Europe, the combination of sandy, calcareous, leached soils with high organic matter results in both low total soluble copper and strong complexation of available copper as Cu(II)-DOM. Degryse et al. (2006) noted that at constant Cu2+ ion activity of 1 × 10−12.2 M and constant solution copper (5 μM), copper uptake by spinach in nutrient solution increased as the dissociation rate of the

394

NUTRIENT USE EFFICIENCY IN CROPS

complexes increased. This suggests that the type and concentration of ligand in the DOM is the primary determinant of copper availability in soils. Research conducted with the diffusive-gradient thin-ﬁlm technique (DGT) (Degryse et al., 2009) suggests that copper supply to roots is diffusion limited (not uptake limited) at concentrations normally occurring in soils (Degryse et al., 2006). Additional research on copper and DOM is needed. Copper deﬁciency is a localized but major problem in cereals, especially wheat (T. aestivum L.) and barley (H. vulgare L.), over large acreages in Europe and in south and southwestern Australia (Alloway, 2008). Through soil sampling and greenhouse evaluation, Sillanpää (1990) estimated that up to 14% of agricultural soils globally may be copper deﬁcient, although there has been no subsequent evaluation of this ﬁnding. The full extent of copper deﬁciency globally is difﬁcult to assess since there is no generalized association that can be drawn between soil type, pH, cultivar, and the likelihood of copper response, and existing soil tests are inadequate. Plant analysis and vegetative symptom expression often fail to identify mild deﬁciencies that may result in signiﬁcant grain loss (>20%) with no vegetative symptoms. Typical concentrations of copper in plants is 2–50 μg g−1 DW with deﬁciencies occurring from 1 to 5 μg g−1 DW depending on plant species, plant organ, developmental, stage and nitrogen nutrition (Marschner, 1995). Function and deﬁciency symptoms Copper exists in two oxidation states, Cu+ and Cu2+, and readily participates in oxidation reduction reactions. In photosynthesis, copper is essential for plastocyanin-mediated electron transport, and in mitochondrion, copper occurs in cytochrome c oxidase in respiratory electron transport chains (Hänsch

and Mendel, 2009). More than half of the copper found in plants is found in chloroplasts and participates in photosynthesis. Copper has a high afﬁnity for O2 and is a cofactor for a number of oxidases involved in cell wall metabolism and oxidative stress protection (Marschner, 1995; Burkhead et al., 2009; Yruela Guerrero, 2009). Copper may be essential for molybdenum (Mo) cofactor synthesis and hence may play an indirect role in nitrogen assimilation and abscisic acid (ABA) synthesis (Burkhead et al., 2009). Copper is also involved in the function of the ER-localized ethylene receptor, ETR1 (Rodriguez et al., 1999), and it was recently hypothesized that smallamplitude cytosolic copper oscillations may be involved in synchronization of cellular cycles (Penarrubia et al., 2010). Overexpression of the copper transporters COPT1 or COPT3 substantially reduces the expression of the nuclear circadian clock genes CCA1 and LHY (Andres-Colas et al., 2010). While the function of copper in plastocyanin, ethylene reception, and cytochrome c oxidase are well understood, the function of copper in the other known and putative copper proteins is uncertain. Typical symptoms of copper deﬁciency are stunted growth, apical necrosis, wilting, bleaching of young leaves, and deformation of growing organs including leaves, bark, stems, anthers, and pollen (Marschner, 1995). Reproductive tissues are much more sensitive than vegetative tissues and seed and fruit yield can be decreased signiﬁcantly at copper supply levels that do not cause vegetative symptoms (Marschner, 1995). In addition to visual expressions of copper deﬁciency, low-copper plants show clear reductions in cell wall ligniﬁcation as well as decreases in levels of soluble carbohydrates. Plant requirement for copper is also increased by nitrogen application, and copper deﬁciency inhibits nitrogen ﬁxation.

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

While some of the symptoms of copper deﬁciency can be explained by known functions of copper in plants, others cannot. The deformation of organs, wilting, and impacts on pollen formation under copper deﬁciency occur as a consequence of the disruption of polyphenol oxidase, laccase, amine oxidase, and perhaps ascorbate oxidase, all of which function in cell wall synthesis. Apical necrosis, excessive tillering, and wilting also occur as a consequence of cell wall deformation, and bleaching of leaf tissue may be a consequence of decreased Cu/Zn SOD activity and degradation of chloroplasts (Henriques, 1989; Bernal et al., 2006). It is well demonstrated that dependence on nitrogen ﬁxation and the application of nitrogen fertilizers increases copper demand, although an explanation for this effect has not previously been offered. The discovery that copper is essential for the synthesis of the molybdenum cofactors (Burkhead et al., 2009) may suggest that copper deﬁciency impairs nitrogen metabolism through a disruption of Mo-dependent nitrogenase and nitrate reductase activity. An interaction between copper status and the efﬁciency of molybdenum and nitrogen use in agriculture is likely. Copper is essential for plastocyanin function and more copper is present in plastocyanin that any other molecule. While copper deﬁciency is known to reduce tissue plastocyanin levels and PSI activity, these effects are generally not observed in the early stages of deﬁciency. The apparent insensitivity of plastocyanin to mild copper stress may be explained by recent ﬁndings that copper is preferentially allocated to plastocyanin during early deﬁciency onset. The mechanisms of allocation are discussed below.

395

complex with polypeptides makes copper ions highly toxic. The Irving–Williams series speciﬁes that Cu2+ and Zn2+ typically form more stable complexes than Fe2+ or Mn2+ (Lippard and Berg, 1994). To maintain the essential functions of copper while protecting cellular metabolism from the negative effects of copper ions, plants have developed an array of transport, complexation, and localization mechanisms. Copper, for example, has a greater afﬁnity than manganese for the Mn-dependent periplasmic protein MncA in cyanobacteria such as Synechocystis PCC 6803 (Tottey et al., 2008), and copper incorporation into MncA renders the protein nonfunctional. To avoid substitution of manganese by copper in MncA, protein folding and metal incorporation occur external to the periplasm, where manganese is present at 104 greater molar ratios. Once in the folded conformation, the MncA is exported to the periplasm and copper can no longer replace the incorporated metal. Copper localization and homeostasis is thus tightly regulated and essential for metabolism. With the possible exception of iron, our understanding of the processes governing the transport and homeostasis of copper in plants is greater than for any other microelement. The mechanisms of uptake, transport, and homeostasis have been reviewed in great detail in a number of excellent recent publications and are summarized here (Colangelo and Guerinot, 2006; Pilon et al., 2006; Puig et al., 2007; Burkhead et al., 2009; Giehl et al., 2009; Palmer and Guerinot, 2009; Pilon et al., 2009; Puig and Penarrubia, 2009; Yruela Guerrero, 2009; Penarrubia et al., 2010). A summary of known and putative whole-plant and cellular copper transporters is provided in Figures 17.3 and 17.4.

Uptake, transport, and homeostasis The ability of copper to serve as a redox agent in metabolism, to react with molecular oxygen and generate oxygen radicals, and to

Uptake by roots Estimates of the kinetic parameters (KM, Vmax) of copper uptake derived from soil,

396 Cu+ CO OPT1 Cortex

Mn2+

Endodermis

NRAMP1

HMA5 CAX2 FRD3

Cu+ Mn2+ Citrate Pericycle

Shoot

Mesophyll

CAX2

YSL

IRT1 Mn2+

Mn2+

Mn2+,Cu2+ ,Ni2+ ?

Xylem

YSL?

Ni(II) – malate/citrate/h istidine

NA -Cu2+ NA –Mn2+

COPT1?

Guard Cell

Cu+

Companion cell

YSL 1, 3?

COPT1, COPT3, HMA5

FRO 6,7 ?

Reproductive Organs

Mn2+, Cu2+ ? 2 ? Ni2+

Cu+

Phloem

NA-Cu2+ NA -Mn2+

Ni(II) – malate/citrate/h l t / it t /h istidine

CCH-Cu

reduction is unknown though co-reduction by the ferric reductase FRO2 is possible. Expression analysis suggests that copper loading into leaves and reproductive structures may also utilize COPT transporters. HMA5 appears to be required for copper loading into the xylem, while the YSL transporter may be involved in NA–Cu uptake into leaf and reproductive tissues. The copper chaperone CCH appears to be required for copper movement to seeds during senescence. Manganese: Mn2+ is the sole ionic form of manganese known to be transported in plants and transport occurs through the ZIP, IRT, and NRAMP family transporters at both root surface and into leaf cells. Vascular loading and unloading is mediated by CAX2. Transport into reproductive tissue may involve the YSL transporters of the Mn(I)I-nicotianic acid (Mn–NA) complex. Nickel: Relatively little is known about nickel transport. Uptake likely occurs through the divalent cation transporters (ZIP/IRT/NRAMP). Nickel has a high afﬁnity for histidine, malate, and citrate and is mobile in both the xylem and the phloem. The involvement of the YSL transporters has been proposed. Modiﬁed with permission from Palmer and Guerinot, 2009.

Fig. 17.3. Intercellular copper, manganese, and nickel transport in dicots. Copper: Copper uptake from soils occurs as the reduced Cu+ through COPT1; the mechanism of copper

NRAMP1 Epidermis Root

IRT1

ZIP?

?

F FRO2

Mn2+

?

Ni2+

Cu2+

Cu+

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

397

Cu2+ Cu+ Cu+

Mn2+ ?

NRAMP4

Ni

NRAMP3

Mn2+

CCS CuSOD2

PC

Cu+

CCS

ETR

RAN1 AT X

CuSOD2

IREG2

Ni

CAX2

Mn2+

COPT

Cu+

Mn2+

Chloroplast

Ni-ure?

Mn2+

CCH

Golgi/ER

Mitochondrion COX17 COX11

PVC

Mn2+

Cytc Ox

MTP1 1

MnSOD2

Vacuole

Mn2+ CuSOD3

Peroxisome

Mn2+

Chaperone

Enzyme

Transporter

HMA1

Fig. 17.4. Intracellular metal transport: Copper: Transport into the chloroplast is best characterized for copper, which is transported into the chloroplast by HMA1 and PAA1. PAA2 transports copper across the thylakoid membrane, a mechanism for transport into the chloroplast, and mitochondria are unknown. Cu-chaperones regulating metal insertion into CuSOD (CCS), the ethylene receptor (ATX, CCH), and mitochondrial Cyt C Oxidase (COX11, COX17) have been identiﬁed. Very little is known about transport of copper in and out of the mitochondria. COPT family transporters function at the root plasma membrane, in vascular tissue, cell surface, and tonoplast. Manganese: Mechanisms for transport of Mn2+ have been identiﬁed for mitochondrial transport (MTM1), vacuole (NRAMP3/4) and CAX2, and per-vacuolar and endosomal vesicles (ECAC1, MTP11). Mechanisms for manganese transport into the chloroplast have not been deﬁned but may involve direct tonoplast/chloroplast interaction. Nickel: Nickel transport occurs through divalent metal ion transporters (NRAMP and IREG2). Ni-ure is a nickel urease accessory protein (chaperone) and has been cloned in bacteria with homologs present in plants. Modiﬁed with permission from Palmer and Guerinot (2009).

solution culture, and nutrient-buffered solution culture experiments are highly varied. Reported KM values for copper uptake range from 0.2 to 90 nM, while Vmax varies from 40 to 40,000 ng Cu m−2 s−1 (discussed in Bravin et al., 2010). The inconsistencies in reported values can be attributed to the copper concentrations used and the use of

free Cu2+ as the source and measure of the copper treatment. The majority of copper in soil solutions is present as Cu(II) complexes with dissolved organic matter (Cu(II)DOM); the presence and composition of metal ligands inﬂuences Cu2+ uptake by plants (Degryse et al., 2006; Amery et al., 2010); and free Cu2+ is not present in

398

NUTRIENT USE EFFICIENCY IN CROPS

adequate amounts to support growth (see discussion above). It is estimated from nutrient-buffered solution culture experiments (Bell et al., 1991; Parker and Norvell, 1999) that an available copper concentration (all forms) of about 1 × 10−10 M is required to support plant growth (Degryse et al., 2006; Bravin et al., 2010) and normal plant growth can be maintained in nutrientbuffered solutions with free Cu2+ concentrations of <10−12 M (Bell et al., 1991; Parker and Norvell, 1999). Many uptake experiments, however, have used unrealistic Cu2+ supply in the range of 10−5 to 10−6 M (Bravin et al., 2010) and kinetic parameters determined on the basis of free Cu2+ ion activity are therefore not appropriate. Copper uptake across the plasma membrane occurs primarily as reduced Cu+ (Sancenón et al., 2004) through high-afﬁnity COPT transporters, which are members of the conserved Ctr family (Ctr) (Kampfenkel et al., 1995). There are ﬁve COPT genes expressed in Arabidopsis; COPT1 localizes to the plasma membrane of root tips, embryos, and pollen grains, and COPT1 underexpression depresses plant copper uptake (Sancenón et al., 2004), while both COPT1 and COPT3 overexpression increase sensitivity to high copper (Andres-Colas et al., 2010). Both COPT1 and COPT2 expression are sensitive to copper status, while COPT1, COPT2, COPT3, and COPT5 all complement the yeast ctr1 deletion mutant (Wintz et al., 2003; Sancenon et al., 2003). The localization and function of COPT2, COPT3, COPT5, and COPT6 have not been deﬁned in plants. COPT4 does not appear to be a copper transporter (Puig et al., 2007). Copper uptake by COPT ﬁrst requires reduction of Cu(II)-DOM or free Cu2+ to Cu+. The mechanism of copper reduction is not known, although cell surface ferric reductase FRO2 can reduce copper and facilitate copper uptake. FRO2 is not upreg-

ulated by copper deﬁciency and copper accumulation was not reduced in frd1 mutants grown on plates (Robinson et al., 1999). Copper uptake may also occur as Cu2+ through the ZIP family of divalent cation transporters, although direct evidence and quantiﬁcation is lacking. There is also no evidence of phytosiderophore-mediated uptake of copper, and to date the uptake of copper in a chelated or ligand bound form at the root plasma membrane has not been described. A full list of known and putative copper transporters is provided in two excellent publications (Puig and Penarrubia, 2009; Yruela Guerrero, 2009).

Inter- and intracellular copper transport and homeostasis The primary metabolic functions of copper occur in the chloroplast, ER, apoplast, and mitochondria, and delivery to these organs involves specialized transporters and metal chaperones often working in a coordinated fashion. In the chloroplast, copper is required for plastocyanin in the thylakoid lumen and Cu/Zn SOD (CSD2) in the stroma. Two members of the heavy metal P-type ATPase family, PAA1 (HMA6) and PAA2 (HMA8), are required for delivery of copper to the thylakoidal plastocyanin, while only PAA1 is required for stomal CSD2. PAA1 is localized to the chloroplast envelope inner membrane, while PAA2 is localized to thylakoids (for review see Burkhead et al., 2009; Puig and Penarrubia, 2009). With the exception of HMA1, all plant copper P-type ATPase transporters appear to utilize Cu+; residue analysis of HMA suggests it is a Cu2+ transporter (Yruela Guerrero, 2009). Copper is delivered to the ER-localized ethylene receptor, ETR1 (Rodriguez et al., 1999), by RAN1/HMA7 and presumably other ER-localized copper proteins (Puig and Penarrubia, 2009). The mechanism of

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

399

copper transport into the mitochondrion and apoplastic enzyme systems is unknown. Metal chaperones are low-molecularweight metal-receptors proteins involved in the intracellular trafﬁcking of metal ions and insertion of copper into the active sites of Cu-dependent enzymes (O’Halloran and Culotta, 2000). A number of copper chaperones have been identiﬁed in plants and are described in more detail in several recent reviews (Puig et al., 2007; Burkhead et al., 2009; Yruela Guerrero, 2009). COX11, COX17, and COX 19 are likely transporters of copper to mitochondrial cytochrome c oxidase; copper chaperone for superoxide dismutase 1 (CCS) has been identiﬁed in a number of plant species and is involved in copper delivery to plastid CSD2, and copper delivery into cytosolic CSD1 and CSD3; ATX1 and copper chaperone (CCH) are homologs of yeast Atx1 and may be involved in cytosolic and possibly symplasmic copper transport (Burkhead et al., 2009); and CCH is upregulated during senescence and may be involved in reproductive copper remobilization (Himelblau et al., 1998; Himelblau and Amasino, 2001).

allocation of copper to metalloenzymes under limited conditions is now well understood (see review in (Burkhead et al., 2009; Pilon et al., 2009; Penarrubia et al., 2010). There are at least four micro-RNAs (miR398, miR408, miR397, miR857) that are Cu regulated and act to reduce expression of CSD1 and AtCSD2 laccases, and plantacyanin. Reductions in activities of each of these copper enzymes are observed under copper limitation, while plastocyanin levels remain unchanged. The Cu-regulated microRNAs are in turn regulated by the transcription factor SPL7 (squamosa promoter binding protein). It is hypothesized that the downregulation of CSD laccase, and plantacyanin results in preferential allocation of copper to plastocyanin during early deﬁciency onset. This hypothesis implies that the downregulated Cu-proteins are nonessential or sublethal. The most characteristic symptoms of copper deﬁciency (bleaching, leaf and reproductive deformations, and wilting) do indeed reﬂect perturbations expected from disrupted cell wall synthesis (laccase) and accumulated oxidative damage (Cu/Zn SOD).

Copper homeostasis

Within-plant transport

The essentiality and toxicity of copper has resulted in the development of complex mechanisms to regulate copper uptake and distribution in plants and mechanisms to tolerate limitations in copper supply. Expression of COPT1 and COPT2 are upregulated under copper limitation as are OPT3 and three nicotianamine synthase (NAS) genes (Wintz et al., 2003). Copper-Zinc SOD (CSD1 and CSD2) and the copper chaperone CCS are all downregulated under copper limitation, while FeSOD is upregulated. Copper limitation does not affect plastocyanin mRNA levels, and reduction in Cu-dependent electron transport is not an early effect of copper deﬁciency. The mechanism of this ﬂuidity in

Following root uptake, copper must be delivered to the tissues, organs, and metabolic pathways in which it is required, while limiting potentially toxic copper interactions. Patterns of gene expression of the heavy metal transport ATPase (HMA5) and the accumulation of copper in the roots of the hma5 suggest a role for HMA5 in the loading of copper into the xylem and perhaps also ﬂowers (Andres-Colas et al., 2006; Burkhead et al., 2009). HMA5 functions as an efﬂuxer and likely transports copper as Cu(I)in the root pericycle. There are two groups of HMA proteins in the Arabidopsis genome: HMA1 to HMA4, which may function in divalent cation transport, including

400

NUTRIENT USE EFFICIENCY IN CROPS

Zn2+, Cd2+, and Cu+ or 2+; and HMA5 to HMA8, which presumably function in transport Cu+ ions (Puig et al., 2007). Because of its reactivity, it is presumed that copper exists exclusively in complexes in cells. Once in the xylem, copper likely forms complexes with Cu-nicotianamine (NA) or Cucitrate. Unlike Fe–NA or Mn–NA, the Cu–NA complex is very stable in mildly acidic conditions of the xylem. The mechanism of xylem unloading and uptake of copper in the symplast of the leaves has not been determined but could reasonably involve COPT. If COPT is involved in leaf uptake, then this would imply a requirement for a reduction step that could be performed by FRO6 or FRO7, which are highly expressed in leaves (Burkhead et al., 2009). Very little information is available on copper transport in phloem. NA is abundant in the phloem and is involved in iron and zinc transport to ﬂowers and seeds, and phloem loading and unloading is thought to be performed by yellow stripe-like (YSL) transporters. YSL1 and YSL3 are localized to shoot vasculature and reproductive tissues and ysl1/ysl3 double mutants express decreased copper mobilization from leaves to reproductive tissues (Waters et al., 2006; Waters and Grusak, 2007). In Arabidopsis, much of the copper transport to reproductive tissues appears to be derived directly from roots and not from remobilization (Waters and Grusak, 2008), while in wheat up to 60% of reproductive copper was derived from shoot remobilization (Garnett and Graham, 2005). Prospects for optimizing copper use efﬁciency While copper deﬁciency is a signiﬁcant problem in wheat and barley in Australia and Europe and a scattered problem in other crops, there have been no focused efforts to select or breed crops for enhanced copper

efﬁciency. Copper deﬁciencies can be corrected readily in most environments by fertilization, often with long-term efﬁcacy. Earlier demonstrations that the transfer of the rye 5RL chromosome arm to wheat (Graham et al., 1987; Schlegel et al., 1991) can increase copper efﬁciency in wheat have not been examined further and the molecular mechanism underlying the copper efﬁciency of rye has not been determined. Copper uptake in cultivars of wheat grown under controlled conditions (including those with the rye chromosome) suggests that there is a modest increase in tissue copper in “efﬁcient” cultivars (Graham et al., 1987; Owuoche et al., 1995). Whether increased uptake or improved plant transport resulted in the efﬁciency has not been resolved. Tissue concentrations of copper can also differ markedly between plant species growing in the same environment, although no clear relationship between typical tissue copper and sensitivity to deﬁciency has been established (reviewed in White and Broadley, 2009). Given the inadequacy of current soil and plant tissue tests to predict a copper response, and the potential for yield loss to occur even with no vegetative symptoms (hidden hunger), there may be a rationale for developing cultivars that more efﬁciently utilize soil sources of Cu. Possible strategies include selection for increased root growth and root ﬁneness to reduce diffusion-limited copper supply, increased activity of the COPT transporters, enhanced phloem transport, and enhanced copper remobilization during senescence. Given the sophisticated regulation of copper homeostasis in plants, it appears unlikely that modiﬁcations to any single gene would have a predictable and sustainable impact. Manganese Until quite recently, our understanding of the behavior of manganese (Mn) in soils and

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

the molecular and physiological basis for manganese acquisition and utilization were quite poor. While our understanding of the behavior of manganese in soils has not advanced signiﬁcantly in 20 years, there have been considerable advances in our understanding of the acquisition and utilization of manganese in plants. Research conducted in bacteria, yeast, and fungi suggests that the function of the essential Mncontaining enzymes (chloroplastic PSII, mitochondrial MnSOD2, ER-localized allantoate aminohydrolase (AAH), and cell walllocalized oxalate oxidase) will require the coordinated delivery and incorporation of manganese ions into the enzymatic functional center. Recent discoveries suggest this is coordinated in plants through activity of the intracellular manganese transporters (AtNRAMP3, AtNRAMP4, AtMTM1, AtMTP11, AtECA3). These ﬁndings have highlighted the highly integrated manner in which manganese is regulated within the cell. In contrast with this high degree of intercellular regulation, manganese uptake from the soil appears to be poorly regulated (Clarkson, 1988). Thus, manganese uptake rates are frequently greater than required to support growth, tissue manganese concentrations vary greatly with environment, soil type, and iron deﬁciency, greatly inﬂuencing manganese uptake, and manganese toxicity occurs readily in acid and low eH soils, suggesting inability of many plant species to regulate uptake. The relatively unregulated uptake of manganese is likely a consequence of the promiscuity of iron and zinc uptake proteins (cation exchanger [CAX], natural resistance-associated macrophage protein [NRAMP], ZIP), which are responsible for the majority of manganese uptake under nonlimiting conditions. The imposition of a iron or zinc stress results in greatly enhanced manganese uptake, even causing manganese toxicity in some circumstances. Although manganese can be toxic if accumulated at

401

high concentrations, it is less toxic and disruptive than other divalent cations since it has a lower afﬁnity for ligands and does not compete for uptake or biochemical function (Lippard and Berg, 1994). The primacy of iron and zinc as regulators of the multi-ion transporters (ZIP, NRAMP, etc.) is thus biologically rational. Manganese in soils and plants and occurrence of global deﬁciencies There has been remarkably little recent research on the chemistry of manganese in agricultural soils, and much of our current knowledge is directly drawn from the classic studies conducted between 1940 and 1980 (Leeper, 1947; Heintze and Mann, 1949; Geering et al., 1969; Godo and Reisenauer, 1980). Manganese(II) is the predominant oxidation state of soluble manganese in soils, and chemical equilibrium analysis suggests it is likely to be the sole source of manganese for uptake by plants (Sims, 1986; Warden and Reisenauer, 1991). It has, however, been suggested that iron deﬁciencyinduced ferric chelate reductase can reduce Mn(III), although soluble forms of Mn(III) are present at only very low concentrations in soils at agricultural pH levels (Lindsay, 1991). Manganese(IV) is the predominant oxidation state of solid phase manganese in aerated soils and is present in easily reducible manganese oxides, organic fractions, and carbonates. The reported abundance of soluble manganese in soils varies from 25 to 8000 μg L−1 (Barber, 1984; Lindsay, 1991; Kabata-Pendias and Pendias, 2000). This complexity has confounded the development of broadly applicable soil tests of manganese availability to plants (Warden and Reisenauer, 1991). The solubility of manganese in soils is sensitive to pH and eH, and manganese is easily redistributed between chemical forms (Leeper, 1947; Geering et al., 1969; Godo

402

NUTRIENT USE EFFICIENCY IN CROPS

and Reisenauer, 1980; Sims, 1986). Lindsay and coworkers (1991) have described the pH–eH-dependent chemical equilibrium reactions for many Mn-containing minerals; the utility of these experimental determinations for determination of ﬁeld soil solution manganese concentrations and plant response has not been satisfactory (Kabata-Pendias and Pendias, 2000). The lack of correspondence between predicted and actual soil solution manganese availability is a consequence of the mixed and meta-stable nature of manganese oxides and hydroxides, the complexity of soil organic matter reactions, and the rapidity with which eH–pH and soil moisture can change (Leeper, 1947; Geering et al., 1969; Godo and Reisenauer, 1980; Sims, 1986). Soil pH has a major effect on manganese availability and a decrease in pH will mobilize manganese in most soils. The effect of pH on manganese availability is, however, complex and is determined by relative distribution of manganese in exchangeable, easily reducible manganese oxides, organic fractions, iron oxides, and carbonates (Sims, 1986). Heavy lime application to correct issues of soil acidity or poor structure has frequently resulted in manganese deﬁciency. This effect is not fully explained by pH shifts alone and is inﬂuenced by soil organic matter, soil mineralogy, and moisture (Kabata-Pendias and Pendias, 2000). Rhizosphere acidiﬁcation in response to nitrogen or phosphorus fertilizer source has been reported to either increase or to have no effect on manganese availability depending on the predominant soil manganese pool and soil pH buffering capacity (Marschner, 1995; Tong et al., 1997). Microbiological soil activity has signiﬁcant effects on enzymatic manganese oxidation and reduction (Geering et al., 1969), and practices that inﬂuence soil microbial activity (amendments, tillage practices, etc.) may thus alter manganese availability. Manganese uptake by plants is

also strongly affected by soil moisture and temperature (Moraghan and Mascagni, 1991), and cold wet spring weather frequently aggravates manganese deﬁciency, while increased soil temperatures and soil drying can rapidly alleviate deﬁciencies. The effects of temperature on manganese uptake are a consequence of both enhanced soil manganese availability due to microbial reduction of manganese, and enhanced root exudation as well as increased root activity. The effects of soil moisture on manganese availability are more complex and poorly understood with both short-term drying (oxidizing conditions) and long-term waterlogging (reducing conditions), resulting in an increase in manganese uptake (Moraghan and Mascagni, 1991; Shuman, 1991). The complexity of crop responses to manganese under varying environmental and soil conditions are a consequence of the multiple interacting factors that determine manganese availability in soils. The environment affects manganese uptake by altering soil microbial reduction–oxidation reactions, altering organic matter composition, inﬂuencing root exudation, and changing soil redox and pH-dependent chemical equilibrium. Manganese uptake is also strongly inﬂuenced by plant iron status, which also interacts with environment (Chapter 16 and manganese uptake and transport, this chapter). Manganese deﬁciency is the third most important global micronutrient deﬁciency after zinc and boron, and is most prevalent on calcareous soils with pH >6.5, on soils with low total manganese content (sandy soils and weathered tropical soils) and in high organic matter soils (Alloway, 2008). Manganese deﬁciency is the most important micronutrient problem in large areas of cereal production on light-textured, alkaline, high-carbonate soils of southern Australia (Holloway et al., 2008) and in cool growing regions of Denmark (Hebbern et al., 2005),

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

and is also the most important micronutrient deﬁciency in arable crops in the United Kingdom grown on predominantly neutral high organic matter soils (Sinclair and Edwards, 2008). Although species and cultivars vary dramatically in their ability to grow on low-manganese soils, the critical tissue manganese requirement for most plant species is remarkably uniform (10–20 mg Mn kg−1). Species that are most sensitive to manganese deﬁciency include barley (H. vulgare L.), oat (Avena sativa, L.), wheat (T. aestivum, L.), apple (Malus domestica Borkh.), cherry (Prunus avium L.), raspberry (Rubus spp. L.), pea (Pisum sativum L.), bean (Phaseolus vulgaris L.), sugar beet (B. vulgaris L.), soybean (Glycine max Merr.), and potatoes (Solanum tuberosum L.) (Marschner, 1995; Alloway, 2008). Recent evidence suggests that the C4 NAD malic enzyme species, pearl millet (Pennisetum glaucum (L.) R. Br.) and purple amaranth (Amaranthus hypochondriacus (L.) cv. Plainsman), have a 10–30-fold higher tissue requirement for manganese for optimum growth and photosynthesis than corn (Z. mays (L.) cv. FR 697), grain sorghum (Sorghum bicolor (L.) Moench), wheat (T. aestivum (L.) cv. Ernie), and squash (Cucurbita pepo L. cv. Straighneck) (Kering et al., 2009). Function and deﬁciency symptoms In biological systems, manganese occurs in oxidation states II, III, and IV, and functions in redox reactions and as an activator of a diverse number of enzymes (Marschner, 1995; Hänsch and Mendel, 2009). Manganese functions as a redox element in the catalytic site of (1) the chloroplast-localized oxygenevolving complex of PSII; (2) mitochondrial superoxide dismutase MnSOD; (3) apoplastic oxalate oxidase (OxO), which catalyzes the conversion of oxalate (from Ca oxalate and oxalic acid) and dioxygen to CO2 and

403

H2O2 (Requena and Bornemann, 1999); and (4) ER-localized allantoate amidohydrolase (AAH), which metabolizes allantoate in the ureide pathway (Serventi et al., 2010). Manganese is also essential for malic enzyme activity in bundle sheath chloroplasts of NAD-dependent C4 species (Marschner, 1995; Kering et al., 2009). Manganese is an activator of a large number of different enzymes, such as isocitrate dehydrogenase, PEP carboxykinase, and phenylalanine ammonia lyase, decarboxylases, and dehydrogenases in the tricarboxylic acid cycle and in several glycosyltransferases in the Golgi apparatus. Manganese deﬁciency disrupts the shikimic acid pathway, lignin synthesis, ﬂavanoids, fructans, and indole acetic acid (IAA) metabolism (Marschner, 1995; Bai et al., 2006; Hänsch and Mendel, 2009). Most of the studies demonstrating an effect of Mn2+ on enzyme activation have been conducted in vitro, and in most instances Mg2+ is more effective; given that Mg2+ is present at up to 100 times the concentration of manganese in plants, the relevance of many of these observations to in vivo enzyme activity is unknown. Manganese deﬁciency causes interveinal chlorosis of younger leaves in dicots and gray specks on the basal leaves in cereals. The ﬁrst effect of manganese deﬁciency is a reduction in photosynthesis (Husted et al., 2009), followed by a reduction in soluble carbohydrates and changes in lignin and phenol content and composition. Manganesedeﬁcient plants are more susceptible to pathogen infections (Datnoff et al., 2007), and manganese deﬁciency has been reported to inﬂuence plant response to light, heat, and water stress, and to alter cell wall and root development (Graham et al., 1988; Moraghan and Mascagni, 1991; Tong et al., 1997; Marschner et al., 2003; Yang et al., 2008; Hebbern et al., 2009; Kaur and Sadana, 2010). With the exception of manganese function in PSII, the speciﬁc mechanism underlying these

404

NUTRIENT USE EFFICIENCY IN CROPS

frequently observed deﬁciency effects is unclear in part because our understanding of the metabolic function of the Mn-dependent enzymes, Mn-SOD, OxO, and AAH, is incomplete. The Mn-dependent enzymes in plants, Mn-SOD, OxO, and AAH, are all members of the germin group of proteins. Germin-likeproteins (GLP) are implicated in a wide variety of plant processes including germination, development, pollen formation, cell wall formation, disease resistance, and response to abiotic and biotic stress, which reﬂect the symptoms of manganese deﬁciency (Woo et al., 2000; Dunwell et al., 2004; Dunwell et al., 2008; Davidson et al., 2009; Manosalva et al., 2009). Further consideration of the functions of GLPs in general and Mn-dependent GLPs in particular is warranted. Uptake, transport, and homeostasis Manganese uptake has long been considered poorly regulated (Clarkson, 1988). This conclusion was based on kinetic studies that found manganese uptake rates orders of magnitude greater than required for growth, ﬁeld observation that tissue manganese concentrations vary greatly with environment, soil type, and iron deﬁciency, and the observation that there is a 50–100× range between the critical manganese requirement (10– 20 μg g−1 DW) and toxicity (>1000 μg g−1 DW) in most species (Graham et al., 1988). The occurrence of manganese toxicity in acid and low-eH soils suggests inability of many plant species to regulate uptake under conditions of high Mn(II) solubility. Manganese uptake is strongly enhanced by iron deﬁciency, and several well-described iron and zinc transporters (ZIP and NRAMP family) have been shown to transport manganese, which suggests that much manganese uptake is simply coincident with iron and zinc uptake processes. The relative paucity of manganese uptake studies (in contrast with

iron, zinc, and copper) is perhaps a consequence of these assumptions and, until quite recently, our understanding of the molecular basis of manganese uptake and homeostasis in plants has been quite poor. The observation that manganese uptake is often poorly regulated was incorrectly interpreted as evidence that speciﬁc and highly regulated transport systems do not exist. This conclusion is at odds with knowledge from other organisms and is incompatible with the known functions of manganese in plants. Evidence from bacteria, yeast, and animal systems demonstrate that organelle speciﬁc and highly regulated delivery of manganese is required to support essential manganese functions. An equivalent degree of organelle-speciﬁc and highly regulated delivery of manganese can be expected in plants, as it would be required to support the function of manganese in chloroplasts (PSII), the ER (AAH), mitochondria (MnSOD), and the cell wall (OxO). Observations that species and cultivars growing in the same environment can vary greatly in manganese uptake also imply that mechanisms for the regulation of manganese uptake must exist (Graham et al., 1988; Saberi et al., 1999; KhabazSaberi et al., 2002; Hebbern et al., 2005; Pedas et al., 2005; Jiang, 2006; SayyariZahan et al., 2009). While it has been known since the mid1990s that manganese can be transported through a variety of metal transporter families in plants (NRAMP, cation diffusion facilitator [CDF], ZIP, and cation/H+ antiporters; Williams et al., 2000; Hall and Williams, 2003), evidence for the presence of transporters speciﬁcally responsive to plant manganese status (under nontoxic conditions) has only emerged since 2005 (Pedas et al., 2005; Delhaize et al., 2007; Mills et al., 2008; Pedas et al., 2008). The identiﬁcation of a ﬁrst intercellular Mn2+ transporter (ECA3, a Golgi-localized P2A-type

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

ATPase) essential for plant growth under low manganese supply was identiﬁed in 2008 (Mills et al., 2008; Pedas et al., 2008), and the ﬁrst transporters required for soil manganese uptake under low manganese (AtNRAMP1 and HvIRT1) were not identiﬁed until 2010 (Cailliatte et al., 2010; Lanquar et al., 2010). A summary of known and putative whole-plant and cellular Mn2+ transporters is provided in Figures 17.3 and 17.4. Uptake by roots In their elegant comparison of two barley genotypes differing signiﬁcantly in ability to grow under low manganese supply, Pedas et al. (2005) demonstrated that uptake of manganese under low manganese supply was facilitated by a high-afﬁnity uptake system operating in the low nM concentration range. Subsequently, it was shown that manganese uptake in barley was mediated by HvIRT1, a root-epidermal plasma membranelocalized high-afﬁnity manganese transporter. HvIRT1 was upregulated by both iron and manganese deﬁciency and expression levels and uptake were signiﬁcantly higher in the manganese deﬁciency-tolerant genotype (Pedas et al., 2008). A Km for HvIRT was estimated at 2.7 to 5.4 nM. AtIRT1 transports iron and manganese and is the major high-afﬁnity transporter for Fe2+ and Mn2+ acquisition in iron-limited conditions. However, the irt1-1 mutant can be rescued only by supplying iron and not manganese (Vert et al., 2002). Multiple ZIP transporters with a capacity to transport Mn2+ have also been identiﬁed (MtZIP4 and MtZIP7 from Medicago truncatula; LeIRT1 and LeIRT2 from tomato, and PsIRT1 from pea (see Pittman, 2005; Pedas et al., 2008), but only AtIRT1 and HvIRT1 have been shown to transport manganese in plants. NRAMP transporters are evolutionarily conserved proton/metal symporters with

405

broad capacity to transport divalent metal ions including Fe2+, Mn2+, Zn2+, Cd2+, Co2+, Cu2+, Ni2+, and Pb2+ in many species (Hall and Williams, 2003; Pilon et al., 2009). In Arabidopsis, AtNRAMP1 has been shown to be essential for manganese uptake under low manganese supply (Cailliatte et al., 2010). AtNRAMP1 is a plasma membrane localized high-afﬁnity manganese transporter, with an apparent Km of 28 nM. Expression is restricted to the root and is regulated by manganese availability, and overexpression of NRAMP1 enhances plant growth and tissue manganese concentrations in Mn-limiting conditions. AtNRAMP1 activity is also upregulated by iron deﬁciency and stimulates Co2+, Fe2+, and Zn2+. AtNRAMP1 activity is present throughout the root and is not localized to epidermal layers, suggesting that it functions in manganese uptake from root apoplast. NRAMPs are widely distributed throughout plant species and structural and experimental data suggest many can behave as Mn2+ transporters (Hall and Williams, 2003; Krämer et al., 2007), and it is likely that IRT1 transporters will be found to be important for Mn2+ uptake in other species. The identiﬁcation of distinct high-afﬁnity manganese transporters (HvIRT1 and AtNRAMP1) in barley and Arabidopsis may suggest that multiple coordinated systems of high-afﬁnity manganese uptake exist in plants. Cailliatte et al. (2010) hypothesize that IRT1 and NRAMP1 work in a coordinated fashion with IRT1 functioning at the epidermal layer and NRAMP1 functioning in the apoplast, regulating the storage and utilization of manganese in the cell wall. Inter- and intracellular manganese transport and homeostasis Manganese is an essential component of four enzymes each of which is localized in speciﬁc plant organelles: PSII in chloro-

406

NUTRIENT USE EFFICIENCY IN CROPS

plasts, AAH in the ER, MnSOD in the mitochondria, and OxO in the cell wall. Manganese also has an afﬁnity for a wide array of organic ligands, although it forms far less stable complexes than Cu2+ and Zn2+ (Lippard and Berg, 1994). These characteristics and the evidence of tight regulation of manganese activity in bacterial, animal, and fungal systems suggest that intracellular manganese will be tightly regulated in plants. Transport of manganese into mitochondria to support MnSOD2 activation appears to occur through AtMTM1 a member of the mitochondrial substrate carrier family, and evidence suggests that AtMTM1 activity is regulated directly by the superoxide anion O2− (Su et al., 2007). In Saccharomyces cerevisiae, manganese insertion into SOD2 is only possible with a newly synthesized polypeptide and is closely coupled to Sod2p import. In this context, MTM1 functions in a chaperone-like manner. Given the fundamental role of manganese in photosynthesis, it is surprising that mechanism of Mn2+ delivery to chloroplasts has not been identiﬁed. Lanquar et al. (2010) have recently hypothesized that chloroplastic manganese delivery may be mediated by tonoplast-localized AtNRAMP3/AtNRAMP4 Mn2+ export. The double mutant Atnramp3/ Atnramp4 exhibits strongly increased vacuolar Mn2+ and a decrease in PSII activity with no coincident decrease in MnSOD activity. These results were interpreted as evidence that the vacuole serves a critical function as a site for the cycling and transit of cellular Mn2+ and that NRAMP3/ NRAMP4 are redundant manganese transporters involved in intercellular and probable chloroplastic manganese transport (Lanquar et al., 2010). The role of the vacuole, prevacuolar compartments, and endosomal/Golgi in manganese homeostasis has been highlighted in several recent publications (Delhaize et al.,

2007; Peiter et al., 2007; Li et al., 2008; Mills et al., 2008; Lanquar et al., 2010). In the Mn-tolerant species Stylosanthes hamata, vacuolar manganese transport is mediated by SmMTP1 a member of the CDF family under high manganese supply. SmMTP1 is likely a H+/Mn2+ cotransporter localized to the prevacuolar compartments that function in protein transport through Golgi, endosomal, and vacuolar networks, and functions to sequester cytoplasmic Mn2+ to vacuoles or extracellular spaces to maintain functional cytosolic Mn2+ levels (Delhaize et al., 2007; Peiter et al., 2007). In the mutant Atmtp11, Mn2+ accumulates in the cytosol in toxic concentrations. AtMTP11 functions as a proton antiporter (Delhaize et al., 2007; Peiter et al., 2007). There are four P2A-type ATPases in Arabidopsis, AtECA1, AtECA2, AtECA3, and AtECA4 (ER-localized Ca2+-ATPase); of these, AtECA1 and AtECA3 have been shown to function at the ER in the transport of Ca2+ and Mn2+ (Pittman, 2005; Li et al., 2008). Golgi-localized AtECA3 is involved in the loading of Ca2+ and Mn2+ into a subpopulation of post-Golgi compartments and is critical for highly regulated endosomal trafﬁcking, exocytosis, and secretion under conditions of excess Mn2+. AtECA3 has also been shown to be essential for plant survival under conditions of manganese deﬁciency, and the mutant eca3, but not eca2, exhibited chlorosis and dramatic growth inhibition (Mills et al., 2008). Application of Mn2+ at low but adequate levels recovered the phenotype, indicating that compensatory mechanisms of Mn2+ uptake operate in Arabidopsis (Mills et al., 2008). The failure of eca3 to grow under low Mn2+ was interpreted as evidence that other genes involved in Mn2+ transport (AtMTP11, AtECA1, CAX2) cannot compensate for loss of function of AtECA3 (Mills et al., 2008). While this suggestion is clearly true, it does not imply that the other identiﬁed

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

Mn2+ transporters (NRAMP1, NRAMP3, NRAMP4) do not play essential roles in the uptake and delivery of Mn2+ to essential sites of function.

Within-plant transport Manganese is generally considered phloem immobile, and remobilization is generally very low, except during senescence in a few species (Graham et al., 1988). The mechanism of Mn2+ loading into and Mn(II) transport within the vascular system is unknown. The oligopeptide transporter AtOPT3 is upregulated in root vascular tissue by manganese deﬁciency and rescues the smﬂ Mnsensitive yeast mutant, although a direct role in manganese transport has not been shown (Wintz et al., 2003). YSL NA divalent cation tranporters from a number of species can transport Mn(II)-NA and exhibit vascular expression patterns; a direct role of OsYSL2 in phloem Mn(II)–NA loading has been demonstrated in rice (Koike et al., 2004). Arabidopsis also has a multigene family of CAX genes including the tonoplast-localized cation/H+ antiporter CAX2 (Pittman, 2005). CAX2 GUS reporter gene fusions are strongly expressed in vascular tissue throughout the plant, indicating a possible role of CAX2 in regulating loading or unloading of manganese from the vasculature (Pittman, 2005). CAX-like transporters, however, are low-afﬁnity and hence may only be relevant under conditions of excess manganese. While the past 5 years has seen a remarkable increase in our understanding of manganese transport in plants with new details emerging rapidly, gaps in our knowledge still exist. Our understanding of Mn2+ uptake from soils, manganese delivery and incorporation of Mn2+ into the PSII, delivery of Mn2+ to OxO in the apoplast, the possible function of manganese chaperones, and the

407

mechanisms of manganese signaling and transporter regulation remain inadequate. Prospects for optimizing manganese use efﬁciency There is considerable variability among species and genotypes in tolerance to Mndeﬁcient soils (Graham et al., 1988; Bansal et al., 1991; Tong et al., 1997; Saberi et al., 1999; Khabaz-Saberi et al., 2002; Hebbern et al., 2005; Jiang, 2006; Pedas et al., 2008; Kering et al., 2009). The factors that may contribute to differential manganese efﬁciency include manganese content in the germinating seed; exudation of Mn-chelating and/or Mn-reducing compounds from roots (Marschner et al., 2003; Rengel and Marschner, 2005); manganese uptake kinetics (Hebbern et al., 2005); manganese requirement of Mn-dependent key enzymes (Kering et al., 2009); subcellular compartmentation of Mn; and, ﬁnally, the populations of Mn-oxidizing and Mn-reducing microorganisms in the rhizosphere (Graham et al., 1988; Huang et al., 1996; Saberi et al., 1999; Marschner et al., 2003; Sayyari-Zahan et al., 2009) In some instances, the physiological basis for differences in plant uptake is clear; thus, in the barley varieties Vanessa (efﬁcient) and Antonia (inefﬁcient), differences in tolerance to Mn-deplete soils is a consequence of enhanced activity of a high-afﬁnity Mn2+ transporter, HvIRT1 (Pedas et al., 2005). In a series of experiments conducted in wheat in Australia, it was generally observed that manganese efﬁciency was associated with higher absorption of manganese from lowmanganese soils (reviewed in (Graham et al., 1988). In a comparison of C3 and C4 species, the higher demand for Mn2+ for PSII activity was responsible for the 10 × higher Mn2+ requirement in NAD-malic enzyme C4 species (Kering et al., 2009). Increased internal utilization efﬁciency (speciﬁcally

408

NUTRIENT USE EFFICIENCY IN CROPS

Mn-dependent PSII activity) was also the suggested mechanism for the increased efﬁciency of the wheat variety Maris Butler (Jiang, 2006, 2008). In a comparison of raya (Brassica juncea), wheat (T. aestivum), and oat (A. sativa), differences in efﬁciency were expressed in soil but not in solution culture and were ascribed to enhanced solubility of manganese in the rhizosphere of raya (Sayyari-Zahan et al., 2009). The role of rhizophere processes in manganese availability is, however, ambiguous and inconsistent. The ability of a species to effectively alter Mn2+ by causing rhizosphere changes is highly dependent on the prevailing pH and buffering capacity of the soil. Thus, in the high-pH, high-carbonate soils in which manganese deﬁciency is signiﬁcant in parts of Australia and Europe, changes in rhizosphere pH (Tong et al., 1997) or microbial populations (Rengel and Marschner, 2005) are unlikely to inﬂuence soil manganese availability. The determination that differences in Mn2+ uptake in two cultivars of barley (Pedas et al., 2008) were the consequence of differential expression of the high-afﬁnity Mn2+ transporter HvIRT1 are consistent with long-term experimentation in wheat in Australia that similarly ascribed efﬁciency to enhanced uptake under low Mn. The limited available information also suggests that manganese efﬁciency is a simply heritable characteristic (Graham et al., 1995; Saberi et al., 1999; Khabaz-Saberi et al., 2002). Thus, it appears (at least in cereals) that selection or genetic manipulation activity of IRT1 (but perhaps also NRAMP1) may result in enhanced tolerance to lowmanganese soils. Possible additional targets for manganese transport manipulation include the AtECA3 transporter, which may be involved in manganese storage and trafﬁcking within the cell. The mechanism of manganese storage and remobilization in cell wall, vascular loading, and manganese

remobilization to reproductive structures is not adequately understood. Finally, manganese deﬁciency is frequently expressed transiently during cold wet springs, reﬂecting changes in soil chemical and microbial processes as well as changes in activity of manganese transporters. Spring manganese deﬁciencies can be extremely damaging to productivity; hence, an enhanced understanding of manganese transporter activity during this period may be fruitful. Molybdenum Chronic molybdenum (Mo) deﬁciency has been described in relatively few (though often extensive) regions of the world on acid-leached and iron oxide-rich soils (pH 4.0–5.0). There are large regions of the globe (especially in Africa) in which the need for molybdenum has not been adequately assessed. Molybdenum is essential for the function of bacterial nitrogenase, and four plant enzymes including nitrate reductase. Plants supplied with nitrate as well as nitrogen-ﬁxing legumes are more sensitive to deﬁciencies of molybdenum and have reduced nitrogen use efﬁciency. Moderate deﬁciencies of molybdenum may limit yield as a consequence of impaired nitrogen nutrition and can be hard to identify as molybdenum deﬁciency. Molybdenum uptake occurs through at least two members of the sulfate transport family, although additional transporters likely exist. The mechanisms of within-plant mobility are unknown. Molybdenum is required in only low amounts and fertilizer rates of 50 g ha−1 provided long-term correction of molybdenum deﬁciency in southeastern Australia. Given the low cost, high solubility, longevity, and low toxicity of molybdenum fertilizers, the addition of molybdenum as a seed treatment, or selection of plants with higher seed molybdenum content is a sound strategy. Given the potential impact of molybdenum

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

on productivity and efﬁciency of nitrogen use, preventative molybdenum treatments may be prudent, especially in lowmolybdenum soils and in crops with high molybdenum demand. Molybdenum in soils and plants and occurrence of global deﬁciencies Plants take up molybdenum as the anion molybdate (MoO42–), and molybdenum speciation in soils is pH dependent. Molybdenum is generally available in neutral to alkaline soils and deﬁciencies are more prevalent in acid soils. At pH >5, molybdenum exists primarily as MoO42–, while at lower pH the plant unavailable ions HMoO4− and HMoO40 become dominant. Several excellent reviews on the chemistry and bioavailability of molybdenum are available (Vlek and Lindsay, 1977; Gupta and Lipsett, 1981; Barber, 1984; Barrow, 1985; Kaiser et al., 2005; Hamlin, 2007; Wichard et al., 2008). As an anion, MoO42– forms ionic complexes with positively charged minerals including iron, manganese, and aluminum oxides. Under acidic conditions, these reactions involve ligand formation and are most favorable at pH 4–5. With every increase in pH unit above 4.0, MoO42– availability increases 10- to 100-fold, depending on predominant soil mineralogy (Vlek and Lindsay, 1977). The application of agricultural lime to increase soil pH is commonly used to increase molybdenum availability in acid soils. Organic matter can either increase or decrease soil molybdenum content and availability depending on the speciﬁc form of the organic matter, soil mineralogy, and the prevailing soil pH (Hamlin, 2007). Molybdate forms only weak complexes with most organic ligands with the exception of catechols (Bellenger et al., 2008). The catechol groups of organic matter are able to bind molybdate over a wide pH range, retaining molybdenum in the top layer of the

409

soil (Wichard et al., 2008). Catecholcontaining siderophores are essential for molybdenum acquisition in many bacteria (Wichard et al., 2008). The role of catechol molybdenum complexation in plant uptake is unknown. The average molybdenum content of soils is ∼2.0 mg kg−1 and ranges from 0.013 to 17.0 mg kg−1, although values as high as 300 mg kg−1 may be present in organic rich shales and soils contaminated with sewage sludge or industrial waste (Hamlin, 2007). Soluble molybdenum in the soil ranged from 10−8 to 10−6 M (0.96–96 μg L−1) (Vlek and Lindsay, 1977), although it is usually small (<10 μg L−1). When the level of molybdenum in the soil solution is sustained above 0.04 μmol L−1 (4 μg kg−1), mass ﬂow can usually supply the amount of molybdenum required (Barber, 1984). The critical requirement for molybdenum in plants varies from 0.2 to 2.0 mg kg−1 DM, and species vary greatly in their ability to acquire molybdenum from soils (Hamlin, 2007). Plants supplied with nitrate as the predominant nitrogen form and nitrogenﬁxing legumes are more sensitive to molybdenum deﬁciency and have higher molybdenum requirements. Molybdenum requirements are greater during ﬂowering and seed set in peanuts (Arachis hypogae, L) and corn (Zea mays, L.; Hamlin, 2007), and molybdenum deﬁciency was recently found to impact fruit set in Merlot grapevine (Vitis vinifera, L. cv. Merlot) (Williams et al., 2004). Whether reproductive molybdenum sensitivity is a common trait is unknown. Plants are tolerant of relatively high molybdenum levels and molybdenum toxicity is rare. Globally, responses to molybdenum have been reported in localized but large areas, and molybdenum is likely a signiﬁcant, though under recognized constraint, in many acid soils of Asia and Africa (Alloway, 2008; van der Waals and Laker, 2008). Field deﬁ-

410

NUTRIENT USE EFFICIENCY IN CROPS

ciencies of molybdenum were ﬁrst observed in southern Australia affecting large areas of Fe-rich, moderately acid soils (Holloway et al., 2008). The incorporation of molybdenum into superphosphate fertilizers in Australia in the 1950s and 1960s was responsible for dramatic increases in productivity of legumes and legume-based pastures in southeastern Australia (Kaiser et al., 2005). The prevalence of molybdenum deﬁciency in Western Australia has increased signiﬁcantly in recent years as a result of soil acidiﬁcation (Brennan and Bolland, 2004; Chen et al., 2009). Molybdenum deﬁciencies are most likely on acid soils and highly leached soils. The presence of iron and manganese oxides can reduce soil molybdenum availability, and deﬁciencies are more common in sensitive species including brassicas and legumes, although agriculturally signiﬁcant deﬁciencies are also seen in sunﬂower and wheat. Function and deﬁciency symptoms Molybdenum was established as an essential element in 1939 (Arnon and Stout, 1939) and has subsequently been shown to be essential for nitrogenase in symbiotic nitrogen ﬁxation and four plant enzymes: nitrate reductase, xanthine dehydrogenase, aldehyde oxidase, and suﬁte oxidase (Schwarz and Mendel, 2006; Hamlin, 2007). All enzymes that depend on molybdenum involve the incorporation of molybdate into a metal cofactor; nitrogenase utilizes a Mo–Fe cofactor, while the four plant molybdenum enzymes produce a molybdopterin complex that is required for enzyme function (Schwarz et al., 2009). Within the enzyme, molybdenum shuttles between three oxidation states (+4, +5, and +6), thereby catalyzing twoelectron reduction–oxidation (redox) reactions (Schwarz et al., 2009). There are a very large number of Mo-containing enzymes in biology, and a distinct possibility exists that

additional Mo-containing plant enzymes will be discovered. The most agronomically signiﬁcant impact of molybdenum is on nitrogen metabolism through its role either in nitrogenase or in nitrate reductase, which catalyzes the ﬁrst rate-limiting step in nitrate assimilation. Growth of numerous species under molybdenum deﬁciency reduces activity of nitrate reductase and results in increased tissue levels of nitrate (Kaiser et al., 2005). Molybdenum is present in the Mo–Fe subunit of nitrogenase and is essential for enzyme activity. Molybdenum supply also strongly affects nodule formation, and a deﬁciency of molybdenum results in an increase in the size and number of nodules and an overall reduction in nitrogen ﬁxation (Anderson and Spencer, 1950). Molybdenum is required for the activity of xanthine dehydrogenase, which is essential for purine degradation and possibly stress responses (Schwarz and Mendel, 2006; Schwarz et al., 2009). The role of xanthine dehydrogenase in nitrogen metabolism and the impact of molybdenum deﬁciency on xanthine dehydrogenase activity remain uncertain. Molybdenum is also essential for the function of peroxisomal sulﬁte oxidase, which detoxiﬁes excessive sulﬁte, and aldehyde oxidase, which catalyzes the last step of ABA biosynthesis. Knowledge of the role of these enzymes in plant metabolism and the impact of molybdenum deﬁciency on in vivo enzyme activity is inadequate. In many species, molybdenum deﬁciency is primarily indicated as a nitrogen deﬁciency due to disruption of enzymes involved in nitrogen metabolism (nitrogenase, nitrate reductase, and xanthine dehydrogenase). Changing the amount and source of nitrogen fertilization can reduce symptom expression by reducing plant reliance on nitrogenase or nitrate reductase. Molybdenum deﬁciency can therefore impact the efﬁciency of soil or

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

fertilizer nitrogen use, and molybdenum requirement is higher for plants provided with nitrate-nitrogen or ﬁxing N2 (Hamlin, 2007; Yu et al., 2010). In Brassica crops, symptoms include “whip tail” disorder in which young leaves develop a highly characteristic wavy narrow structure, while in grape (V. vinifera. L. cv. Merlot), bean (P. vulgaris, L.), and a few other dicot species, molybdenum deﬁciency results in leaf cupping that may occur with or without chlorosis or necrosis. The metabolic cause of these symptoms remains obscure. Uptake, transport, and homeostasis Prior to 2007, the molecular mechanisms controlling molybdenum transport in plants were unknown. Earlier physiological data suggested that molybdate transport probably occurred through nonspeciﬁc anion or sulfate transporters. This conclusion was predicated on physical and chemical similarities between molybdate and sulfate in size and charge of the two anions and from numerous laboratory and ﬁeld studies demonstrating that sulfate availability inﬂuenced molybdate uptake (Stout et al., 1951; Leggett and Epstein, 1956; Pasricha and Randhawa, 1972; Bush et al., 1981; Gupta, 1997; Macleod et al., 1997; Alhendawi et al., 2005; Gunes et al., 2009). Both sulfate and phosphate starvation have been shown to increase molybdenum accumulation (Heuwinkel et al., 1992; Alhendawi et al., 2005; Shinmachi et al., 2010). A summary of known and putative whole-plant and cellular molybdenum transporters is provided in Figures 17.1 and 17.2. Due to physical and chemical similarities between molybdate and sulfate, it is likely that molybdenum is transported through transporters with homology to known sulfate transporters. Knowledge of sulfate transporter biology is therefore relevant. The ﬁrst sulfate transporter was cloned from S.

411

hamata (SHST1) (Smith et al., 1995); subsequently, a large number of additional sulfate transporters have been characterized (see Chapter 14). Phylogenetic analysis of available genomes indicates existence of a sulfate transporter gene family with at least ﬁve within-family clusters (Hawkesford, 2003). Members of this gene family exhibit discrete kinetics as well as subcellular, phenological, and environmental expression patterns (Buchner et al., 2010). The role of the sulfate transporter gene family in molybdenum transport has been conﬁrmed (Tejada-Jiménez et al., 2007; Tomatsu et al., 2007; Baxter et al., 2008; Fitzpatrick et al., 2008; Shinmachi et al., 2010), and the effect of sulfur status and sulfate transporter gene activity on molybdenum uptake and patterns of molybdenum distribution in wheat has been described (Shinmachi et al., 2010). While this represents a signiﬁcant advancement, there are some inconsistencies between the results and interpretations from different research groups, and clearly our understanding is incomplete. Tomatsu et al. (2007) identiﬁed a molybdate transporter (MOT1 previously called Sultr5;2), a member of the Group 5 sulfate transporter gene family (Hawkesford, 2003; Shinmachi et al., 2010). MOT1 was described as a high-afﬁnity transporter with Km and Vmax values for uptake of 21 ± 4 nM and 0.5 ± 0.1 μg g−1 DW min−1, respectively. MOT1 did not show any signiﬁcant sulfate transport. GFP fusion localization studies suggested that MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles (Tomatsu et al., 2007). MOT1 expression was reduced under Mo. It was concluded that MOT1 enhances molybdate uptake from soil into root cells for utilization and also for translocation to shoots. Simultaneous with these ﬁndings (Tomatsu et al., 2007), Tejada-Jiménez et al.

412

NUTRIENT USE EFFICIENCY IN CROPS

(2007) reported the identiﬁcation and characterization of the AtMOT1 ortholog in Chlamydomonas reinhardtii. CrMOT1 was shown to be a high-afﬁnity molybdate transporter (Km = 7 nm), but unlike AtMOT1, activity was not regulated by molybdenum concentrations but was strongly regulated by nitrate. The activation of CrMOT1 by nitrate was interpreted as evidence for a role of CrMOT1 in providing molybdenum for nitrate assimilation. Underexpression of CrMOT1, however, did not affect either molybdenum uptake or nitrate reductase activity (Tejada-Jiménez et al., 2007). Utilizing a broad screen of natural variation in tissue molybdenum in Arabidopsis thaliana populations, Baxter et al. (2008) also identiﬁed the AtMOT1 high afﬁnity molybdenum transporter. In agreement with earlier research (Tejada-Jiménez et al., 2007; Tomatsu et al., 2007), MOT1 was shown to be a high-afﬁnity molybdenum transporter (Km = 6 nm). A deletion in the MOT1 promoter reduced shoot molybdenum, GUS localization demonstrated strong root expression, and reciprocal grafting experiments veriﬁed that MOT1 is involved in root uptake and transport of molybdenum to shoots. In contrast to Tomatsu et al. (2007), MOT1 was shown to be localized to mitochondrial membranes and was not expressed in plasma membranes. These differences may be a result of differences in methodology. The localization of MOT1 to mitochondrial membranes may be linked to the requirement for molybdenum in molybdopterin synthesis, part of which is localized to the mitochondria. The strong inﬂuence of tissue nitrate on MOT1 activity seen in CrMOT1 (Tejada-Jiménez et al., 2007) further supports a role for MOT1 in providing molybdenum for nitrate reductase activity. These results are consistent with the hypothesis that mitochondrial molybdenum controls whole-plant molybdenum uptake

(Mendel, 2007). The mechanisms by which this may occur remain unclear. Baxter et al. (2008) hypothesize that MOT1 is involved in transport of MoO42– from the acidic mitochondrial intermembrane space to either the cytoplasm or the matrix. A role for known sulfate transporters in the uptake of MoO42– was recently demonstrated by expression of the SO42– transporter SHST1 in the Saccaromyces cerevisiae sulfate transport mutant Ysd1 (Fitzpatrick et al., 2008). The Stylosanthes hamata sulfate transporter, SHST1, is expressed in roots and the expression is enhanced under sulfur starvation (Smith et al., 1995). SHST1 is a high-afﬁnity H+/SO42– cotransporter with a Km for sulfate of 10 μM. Expression of SHST1 in Ysd1 enhanced the uptake of MoO42– at physiological molybdenum concentrations (80 nM), and uptake of molybdenum was nonsaturating over the range of 1–1000 nM. Presence of SO42– in the uptake solution did not inhibit MoO42– uptake, although MoO42– competitively inhibited SO42–. The afﬁnity of SHST1 for molybdenum has not been determined. The signiﬁcance of sulfate transporters in molybdenum uptake is also indicated by the ﬁnding that sulfur deﬁciency upregulates a number of sulfate transporters (Buchner et al., 2010) and increases molybdenum uptake in wheat (Buchner et al., 2010; Shinmachi et al., 2010) and tomato (Alhendawi et al., 2005; Buchner et al., 2010). The expression of the known sulfate transporters (Sultr1;1, Sultr1;3, Sultr2;1, Sultr4;1, Sultr5;1, and Sultr5;2 [MOT1]) and tissue concentrations of Se, S, and molybdenum were examined at various stages of growth in ﬁeld-grown wheat with adequate or moderate sulfur deﬁciency (–S) (Shinmachi et al., 2010). Under –S conditions, expression of sulfate transporters Sultr1;1 and Sultr4;1 increased and coincided with a 3.7-fold increase in grain molybdenum accumulation compared with

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

+S plants. Sultr5;2 (MOT1), however, did not increase under sulfur deﬁciency and could not have been responsible for the observed enhancement of molybdenum uptake (Shinmachi et al., 2010). Thus, while Sultr5;2 is clearly involved in leaf molybdenum accumulation, it is unlikely to be solely responsible for uptake and distribution of molybdenum in wheat. Taken together, recent ﬁndings demonstrate that the molybdate-speciﬁc transporter MOT1 and the sulfate/molybdate transporter SHST1 are both involved in molybdenum transport in plants. It also appears likely that other sulfate transporter (including Sultr1;1 [SHST1 homolog] and Sultr4;1) also function in molybdenum transport. Further details on the kinetic properties, tissue localization, and regulation of these transporters are required. One of the common paradigms of molybdenum uptake is that molybdate and sulfate compete for uptake at the membrane transporter level (Stout et al., 1951; Marschner, 1995; Macleod et al., 1997; Kaiser et al., 2005). Recent evidence demonstrating that MOT1 is not a sulfate transporter and that sulfate does not compete with molybdenum for uptake through SHST1 calls this longheld truth into question. Indeed, upon reexamination, much of the older literature on this topic appears to have been overinterpreted and was either conducted under unrealistic growth conditions, at unrealistically high-molybdenum applications, does not meet acceptable statistical interpretations, or has been cited by others in a manner never speciﬁed by the authors (Stout et al., 1951; Pasricha and Randhawa, 1972; Bush et al., 1981; Gupta, 1997; Macleod et al., 1997; Leggett and Epstein, 1956; Alhendawi et al., 2005; Gunes et al., 2009). There are also many examples in the literature that contradict the conclusion that sulfate competes with molybdate for uptake (Alhendawi et al., 2005; Tomatsu et al., 2007; Baxter et al.,

413

2008; Fitzpatrick et al., 2008; Gunes et al., 2009). In light of recent discoveries, it seems probable that the reported negative effect of sulfate on molybdate uptake is a consequence of upregulation of sulfate/molybdate transporters under low sulfate supply (Shinmachi et al., 2010). Resupply of sulfate reduces molybdenum uptake by downregulation of sulfate/molybdate transporters and not necessarily by direct uptake competition.

Within-plant transport Molybdenum is generally regarded as highly phloem mobile in most species, although the evidence for this is incomplete (Marschner, 1995). Foliar-applied molybdate is highly mobile (Williams et al., 2004) and is rapidly distributed throughout the plant in many species (Gupta and Lipsett, 1981). Molybdenum appears to be preferentially allocated to nodules, resulting in depletion of other plant organs under low molybdenum conditions (Brodrick and Giller, 1991). There is evidence that molybdenum is not effectively remobilized from senescing leaves to reproductive tissues in wheat (Shinmachi et al., 2010) and the occurrence of reproductive deﬁciencies during ﬂowering and seed set in peanuts (A. hypogae, L), corn (Z. mays, L.; Hamlin, 2007), and Merlot grapevine (V. vinifera, L. cv. Merlot; Williams et al., 2004) may also reﬂect inadequate remobilization. The mechanisms of within-plant transport of molybdenum are unknown.

Prospects for optimizing molybdenum use efﬁciency There are very few examples of cultivar differences in tolerance to molybdenum deﬁciency and we are unaware of any breeding or selection strategy targeting molybdenum

414

NUTRIENT USE EFFICIENCY IN CROPS

deﬁciency tolerance in crop plants. Differences in cultivar efﬁciency for molybdenum have been observed in relatively few species including corn (Z. mays L.), wheat (Triticum aestivum L), poinsettia (Euphorbia pulcherrima Willd ex Kl.), and bean (P. vulgaris) (Brown and Clark, 1974; Brodrick and Giller, 1991; Cox, 1992; Yu et al., 2002; Yu et al., 2010). Where cultivar differences in molybdenum efﬁciency have been observed they appear to be mostly associated with the ability to remobilize molybdenum from vegetative to reproductive tissues. In winter wheat, the highly Mo-efﬁcient cultivar 97003 allocates a higher percentage of plant molybdenum to upper leaves and spikes indicative of a higher phloem mobility (Yu et al., 2002; Yu et al., 2010). A similar enhanced allocation to reproductive tissues was observed in P. vulgaris. L., with the efﬁcient cultivar Kabanima allocating a higher percentage of plant molybdenum to seed production than the inefﬁcient Baseka (Brodrick and Giller, 1991). The extent to which molybdenum limits crop productivity is largely unknown. The limited soils data for sub-Saharan Africa (Sillanpää, 1982; van der Waals and Laker, 2008) suggest molybdenum deﬁciencies may be a signiﬁcant “hidden” hunger particularly in legumebased systems with minimal agrochemical inputs. The extent to which “hidden” molybdenum deﬁciencies can occur is illustrated by the Chinese soils mapping project completed in the late 1990s, which newly identiﬁed an area in excess of 44 million ha of cropping land in China as molybdenum responsive (Zou et al., 2008; Yu et al., 2010). The most effective solution to molybdenum deﬁciency is supplementation of fertilizers or seeds, and given the small amounts of molybdenum required, this is often a relatively low-cost intervention. For many large seeded species, the seed can provide adequate molybdenum for the entire crop cycle (Brodrick and Giller, 1991). Thus, selection

for cultivars with micronutrient-rich seeds and/or the use of micronutrient seed treatments is a promising option, particularly for subsistence agriculture, where the identiﬁcation and correction of molybdenum deﬁciencies remains a formidable challenge. Nickel Nickel was identiﬁed as an essential element in 1987 and in-ﬁeld deﬁciencies were discovered in pecan and other tree species in the southeastern United States in 2004. Foliar applications of nickel enhance crop performance of species grown with urea as a prominent nitrogen source, in some nitrogen-ﬁxing legume species and in species for which ureides are an important nitrogen form. An in-depth analysis of the occurrence of nickel-responsive soils and cropping systems has not been performed. Considerable information on the mechanisms of nickel uptake and remobilization in plants exists, much of it developed in other organisms and in species that tolerate and hyperaccumulate high levels of nickel in the environment. Considerable diversity in nickel demand and acquisition and utilization exists among species, and there are clear opportunities for improvement of micronutrient use efﬁciency. There has been no research to identify or manipulate plant nickel use efﬁciency. Nickel in soils and plants and occurrence of global deﬁciencies Nickel in soils averages 50 mg Ni kg−1 soil and varies from 5 to 500 mg Ni kg−1 soil.Agricultural soils typically contain 3–1000 mg Ni kg−1 soil, while soils derived from basic igneous rocks may contain from 2000 to 6000 mg Ni kg−1 soil (Brown, 2008). At pH >6.7, most nickel exists as insoluble hydroxides, whereas at pH <6.5, most nickel compounds are relatively soluble (Brown et al., 1989). The soil

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

availability of nickel for plant growth has not been well characterized. However, its chemical properties suggest it should behave similarly to iron or manganese, and have lowest availability in alkaline, calcium carbonate-rich soils, and leached, lighttextured soils of low cation exchange capacity. The only known example of nickel-responsive agricultural soils are course textured, poorly drained sand, and sandy loams occurring in the Atlantic coast region of the southeast United States (Wood et al., 2004). The possibility that more extensive nickel-deﬁcient soils exist, however, cannot be discounted, particularly in crops that have higher nickel requirements, which include many nitrogen-ﬁxing species and those that transport ureides as a prominent nitrogen form. Growth of plants in specialized conditions (greenhouses and tissue culture), particularly when urea is used as a nitrogen source, may be especially susceptible to nickel deﬁciency (Nicoulaud and Bloom, 1998; Ruter, 2005b; Gheibi et al., 2009). The nickel concentration in plants grown on uncontaminated soil ranges from 0.05 to 5.0 mg Ni kg−1 DW (Brooks, 1980; Welch, 1981). The adequate range for nickel appears to fall between 0.01 mg Ni kg−1 DW and >10 mg Ni kg−1 DW, which is an extremely wide range as compared with other elements (Gerendas et al., 1999). The critical nickel concentration required for seed germination in barley, shoot growth in oat, barley and wheat, and shoot growth of urea-fed tomato, rice, and zucchini has been estimated independently by two groups to be approximately 100 μg Ni kg−1 (Brown et al., 1987; Gerendas and Sattelmacher, 1997a,b). Function and deﬁciency symptoms Interest in the role of nickel in plant biology was stimulated by the discovery that nickel is a component of the plant enzyme urease

415

in 1975 (Dixon et al., 1975) and subsequent demonstration that tissue-cultured soybean, rice, and tobacco cells could not grow in the absence of nickel when provided with urea as the sole nitrogen source (Polacco, 1977). Eskew et al. (1983) concluded that nickel was an essential element for leguminous plants, although they did not demonstrate a failure of nickel-deﬁcient plants to complete their life cycle. Gerendas et al. (Gerendas et al., 1998; Gerendas and Sattelmacher, 1997a,b; Gerendas et al., 1999) demonstrated a profound effect of nickel deﬁciency on growth of urea-fed tobacco, zucchini, rice, and canola. Brown et al. (1987) demonstrated that barley (H. vulgare L.) seeds from nickel-deprived plants were incapable of germination even when grown on a nonurea nitrogen source and showed a signiﬁcant reduction in shoot growth of barley, wheat, and oats under nickel-deﬁcient conditions when plants where supplied with mineral nitrogen sources. The existence of ﬁeld-level nickel deﬁciency in crops was discovered in pecan (Carya illinoiensis (Wangh.) K. Koch) trees growing in sandy poor draining and low cation exchange soils of southeastern United States (Wood et al., 2004). Responses to foliar and soil applications have been observed in a number of containerized crops, particularly, though not exclusively, when provided with a urea nitrogen source (Ruter, 2005a; Bai et al., 2006; Gheibi et al., 2009) and in species that utilize the ureides as a prominent nitrogen form. Examples of ureide transporting crop genera are Annona, Carya, Diospyros, Juglans, and Vitis (Brown, 2008). Additionally, tropical legumes (e.g., soybean, phaseolus beans, mungbean, and cowpeas) are also relatively sensitive to nickel deﬁciency (Brown, 2008). Nickel is known to be involved in the function of at least nine proteins including methyl-coenzyme M reductase, superoxide dismutase, nickel-dependent

416

NUTRIENT USE EFFICIENCY IN CROPS

glyoxylase, aci-reductone dioxygenase, NiFehydrogenase, carbon monoxide dehydrogenase, acetyl-CoA decarbonylase synthase, and methyleneurease, of which two (urease EC 3.5.1.5; urea amidohydrolase) and the nickel-metallochaperone have identiﬁed roles in plants (Bai et al., 2006; Hänsch and Mendel, 2009). While a function of nickel in urease remains, the only deﬁnitive role for nickel in plants, many observations of plant growth under nickel deﬁciency suggest that additional functions are likely (Brown et al., 1987; Brown et al., 1990; Bai et al., 2006). The review by Li and Zamble (2009) provides an exceptional summary of function and homeostasis of nickel in biology. In legumes and other dicots, nickel deﬁciency results in decreased activity of urease and subsequently results in urea toxicity, exhibited as leaﬂet tip necrosis (Eskew et al., 1983). In graminaceous species, deﬁciency symptoms include chlorosis similar to that induced by iron deﬁciency (Brown et al., 1987), including interveinal chlorosis and patchy necrosis in the youngest leaves. Nickel deﬁciency also results in a marked enhancement in plant senescence and a reduction in tissue iron concentrations. In both monocots and dicots, the accumulation of urea in leaf tips is diagnostic of nickel deﬁciency (Eskew et al., 1983). In early or incipient stages of nickel toxicity, there are no clear symptoms, although shoot and root growth may be reduced. Acute nickel toxicity results in symptoms that have variously been likened to iron deﬁciency (interveinal chlorosis in monocots, mottling in dicots) or zinc deﬁciency (chlorosis and reduced leaf expansion). The clearest agronomic responses to nickel have been observed when nitrogen is supplied as urea or by nitrogen ﬁxation. The most illustrative example of the relationship between nickel and urea metabolism is provided from studies with foliar urea application and tissue culture growth of plants.

Plants without a supply of nickel have low urease activity in the leaves, and foliar application of urea leads to a large accumulation of urea and severe necrosis of the leaf tips (Eskew et al., 1983). Nicoulaud and Bloom (1998) observed that tomato seedlings growing with foliar urea as the only nitrogen source exhibited signiﬁcantly enhanced growth when nickel was added to the nutrient solution. The authors speculated that the effect of nickel was more consistent with its role in urea translocation than as a direct effect on urease activity. This result is in agreement with the ﬁndings of Brown et al. (1987), who suggest that nickel has a role in the transport of nitrogen to the seed, thereby inﬂuencing plant senescence and seed viability. Uptake, transport, and homeostasis In plant systems, most studies have been conducted at high soil nickel concentrations, well above the levels at which deﬁciency might occur. Cataldo et al. (1978), using 63 Ni, indicated that a high afﬁnity Ni2+ carrier functioned at 0.075 and 0.25 μM with a Km of 0.5 μM, which approaches the nickel concentration in uncontaminated soils. Both Cu2+ and Zn2+ competitively inhibit Ni2+ uptake, an observation that is reﬂected in ﬁeld occurrence of nickel deﬁciency in zincand copper -contaminated soils (Wood et al., 2004; Bai et al., 2006). Information on nickel uptake and cellular homeostasis is largely derived from research in bacteria, hyperaccumulating species, and genetic manipulation of metal transport gene families (Li and Zamble, 2009; TejadaJiménez et al., 2009). Three families of transporters, ZIP (ZRT/IRT-like protein; zinc-regulated transporters/iron regulated transporters), NRAMP, and YSL, participate in nickel transport and homeostasis (TejadaJiménez et al., 2009). Transformation of yeast with Thlapsi japonicum ZNT1 or

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

ZNT2 reduced nickel uptake, likely as a result of a competitive increase in zinc inﬂux (Mizuno et al., 2007). Overexpression of the IRT-like membrane protein, AtIREG2, increased tolerance of Arabidopsis to nickel exposure possibly by enhancing vacuolar transport (Colangelo and Guerinot, 2006; Mizuno et al., 2007). The central role of the vacuole in nickel homeostasis is supported by the observation that NRAMP4 overexpression increases nickel sensitivity in yeast by enhancing nickel efﬂux from the vacuole (Mizuno et al., 2005; Colangelo and Guerinot, 2006). A summary of known and putative whole-plant and cellular nickel transporters is provided in Figures 17.3 and 17.4. The bacterial metallochaperone genes (UREd, UREf, and UREg) play a critical role in the activation of the nickel-dependent urease (Witte et al., 2001; Witte et al., 2005). Three putative urease accessory genes (AtUREd, AtUREf, and AtUREg) are required for competent incorporation of nickel into urease (Witte et al., 2005; Li and Zamble, 2009). Nickel, unlike other divalent cations, is readily retranslocated within the plant, likely as a complex with organic acids and amino acids (Tifﬁn, 1971; Tifﬁn and Thompson, 1975; Riesen and Feller, 2005; Krämer et al., 2007). Indeed, up to 70% of nickel in the shoots was transported to the seed of soybean (Tifﬁn, 1971). Nickel forms complexes with malate, citrate, histidine, and nicotianiamine (Ouerdane et al., 2006), which inﬂuences both within-plant nickel mobility and tolerance to nickel excess (Tifﬁn and Thompson, 1975; Mari et al., 2006; Ouerdane et al., 2006). Overexpression of NAS conferred nickel tolerance in Arabidopsis (Pianelli et al., 2005). Prospects for optimizing nickel use efﬁciency There have been no reports of selection, breeding, or molecular manipulation to

417

enhance plant resistance to nickel deﬁciency or increase efﬁciency of nickel use. Given the diversity of functions of nickel in bacterial systems and the lack of systematic examination of the effects of nickel on cropping productivity, there remains a possibility that nickel deﬁciency is a more signiﬁcant problem than currently recognized. The chemistry of nickel and the known functions suggest that deﬁciencies are most likely to occur in leached soils or high-pH soils in which nickel availability is low. Species that utilize ureides and those dependent on urea fertilization or nitrogen ﬁxation will be more susceptible. Knowledge from bacterial and hyperaccumulating species suggests that opportunities exist to manipulate plant nickel uptake, within cell distribution and remobilization. The known plant nickel uptake mechanisms, however, are not nickelspeciﬁc and hence manipulations will inﬂuence the uptake of other essential and nonessential elements, making targeted manipulations impractical. Nickel forms a unique complex with histidine in plants, and manipulation of histidine levels in plants has been shown to alter within-plant distribution and tolerance to high nickel in the environment. The role of histidine metabolism in efﬁciency of nickel use in nickel-limiting environments is unknown. References Alhendawi, R.A., Kirkby, E.A., Pilbeam, D.J. (2005) Evidence that sulfur deﬁciency enhances molybdenum transport in xylem sap of tomato plants. Journal of Plant Nutrition 28, 1347–1353. Alloway, B.J. ed. (2008) Micronutrient Deﬁciencies in Global Crop Production. Springer, Dordrecht. Amery F, Degryse F, Degeling W, Smolders E, & Merckx R (2007) The copper-mobilizing-potential of dissolved organic matter in soils varies 10-fold depending on soil incubation and extraction procedures. Environmental Science & Technology 41, 2277–2281. Amery F, Degryse F, Van Moorleghem C, Duyck M, & Smolders E (2010) The dissociation kinetics of Cu-

418

NUTRIENT USE EFFICIENCY IN CROPS

dissolved organic matter complexes from soil and soil amendments. Analytica Chimica Acta 670, 24–32. Anderson, A.J. & Spencer, D. (1950) Molybdenum in nitrogen metabolism of legumes and non-legumes. Australian Journal of Scientiﬁc Research Series B-Biological Sciences 3, 414. Andres-Colas, N., Sancenon, V., Rodriguez-Navarro, S., et al. (2006) The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxiﬁcation of roots. Plant Journal 45, 225–236. Andres-Colas, N., Perea-Garcia, A., Puig, S., & Penaarubia, L. (2010) Deregulated copper transport affects arabidopsis development especially in the absence of environmental cycles. Plant Physiology 153, 170–184. Arnon, D.I. & Stout, P.R. (1939) Molybdenum and essential element for higher plants. Plant Physiology 14, 599–602. Bai, C., Reilly, C.C., & Wood, B.W. (2006) Nickel deﬁciency disrupts metabolism of ureides, amino acids, and organic acids of young pecan foliage. Plant Physiology 140, 433–443. Bansal, R., Nayyar, V., & Takkar, P. (1991) Field screening of wheat cultivars for manganese efﬁciency. Field Crops Research 29, 107–112. Barber, S.A. (1984) Soil Nutrient Bioavailability. John Wiley, New York. Barrow, N.J. (1985) Reaction of anions and cations with variable-charge soils. Advances in Agronomy 38, 183–230. Bassil, E., Hu, H.N., & Brown, P.H. (2004) Use of phenylboronic acids to investigate boron function in plants. possible role of boron in transvacuolar cytoplasmic strands and cell-to-wall adhesion. Plant Physiology 136, 3383–3395. Baxter, I., Muthukumar, B., Park, H.C., et al. (2008) Variation in molybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter (MOT1). PLoS Genetics 4, 12. Bell, P.F., Chaney, R.L., & Angle, J.S. (1991) Determination of the copper-2+ activity required by maize using chelator-buffered nutrient solutions. Soil Science Society of America Journal 55, 1366–1374. Bellaloui, N., Brown, P.H., & Dandekar, A.M. (1999) Manipulation of in vivo sorbitol production alters boron uptake and transport in tobacco. Plant Physiology 119, 735–741. Bellaloui, N., Yadavc, R.C., Chern, M.S., et al. (2003) Transgenically enhanced sorbitol synthesis facilitates phloem-boron mobility in rice. Physiologia Plantarum 117, 79–84. Bellenger, J.P., Wichard, T., Kustka, A.B., & Kraepiel, A.M.L. (2008) Uptake of molybdenum and vana-

dium by a nitrogen-ﬁxing soil bacterium using siderophores. Nature Geoscience 1, 243–246. Bernal, M., Sanchez-Testillano, P., Risueno, M.D., & Yruela, I. (2006) Excess copper induces structural changes in cultured photosynthetic soybean cells. Functional Plant Biology 33, 1001–1012. Blevins, D.G. & Lukaszewski, K.M. (1998) Boron in plant structure and function. Annual Review of Plant Physiology and Plant Molecular Biology 49, 481–500. Bolanos, L., Esteban, E., Delorenzo, C., et al. (1994) Essentiality of boron for symbiotic dinitrogen ﬁxation in pea (Pisum sativum) rhizobium nodules. Plant Physiology 104, 85–90. Bolanos, L., Brewin, N.J., & Bonilla, I. (1996) Effects of boron on Rhizobium-legume cell-surface interactions and nodule development. Plant Physiology 110, 1249–1256. Bolanos, L., Cebrian, A., Redondo-Nieto, M., Rivilla, R., & Bonilla, I. (2001) Lectin-like glycoprotein PsNLEC-1 is not correctly glycosylated and targeted in boron-deﬁcient pea nodules. Molecular Plant-Microbe Interactions 14, 663–670. Bolanos, L., Lukaszewski, K., Bonilla, I., & Blevins, D. (2004) Why boron? Plant Physiology and Biochemistry 42, 907–912. Bonilla, I., Mergold-Villasenor, C., Campos, M.E., et al. (1997) The aberrant cell walls of borondeﬁcient bean root nodules have no covalently bound hydroxyproline/proline-rich proteins. Plant Physiology 115, 1329–1340. Bravin, M.N., Le Merrer, B., Denaix, L., Schneider, A., & Hinsinger, P. (2010) Copper uptake kinetics in hydroponically-grown durum wheat (Triticum turgidum durum L.) as compared with soil’s ability to supply copper. Plant and Soil 331, 91–104. Brennan, R.F. & Bolland, M.D.A. (2004) Comparing copper requirements of ﬁeld pea and wheat grown on alkaline soils. Australian Journal of Experimental Agriculture 44, 913–920. Brodrick, S.J. & Giller, K.E. (1991) Genotypic difference in molybdenum accumulation affects N2ﬁxation in tropical Phaseolus vulgaris L. Journal of Experimental Botany 42, 1339. Brooks, R.R. (1980) Accumulation of nickel by terrestrial plants. In: Nickel in the Environment (ed. J.O. Nriagu), pp. 407–430. John Wiley, New York. Brown, P.H. (2008) Micronutrient use in agriculture in the united states of america: current practices, trends and constraints. In: Micronutrient Deﬁciencies in Global Crop Production (ed. B.J. Alloway), Springer, Dordrecht. Brown, J.C. & Clark, R.B. (1974) Differential response of 2 maize inbreds to molybdenum stress. Soil Science Society of America Journal 38, 331–333.

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

Brown, P.H. & Hu, H.N. (1994) Boron uptake by sunﬂower, squash and cultured tobacco cells. Physiologia Plantarum 91, 435–441. Brown, P.H. & Hu, H.N. (1997) Does boron play only a structural role in the growing tissues of higher plants? Plant and Soil 196, 211–215. Brown, P.H. & Hu, H. (1998) Phloem boron mobility in diverse plant species. Botanica Acta 111, 331–335. Brown, P.H. & Shelp, B.J. (1997) Boron mobility in plants. Plant and Soil 193, 85–101. Brown, P.H., Welch, R.M., & Cary, E.E. (1987) Nickel: a micronutrient essential for higher plants. Plant Physiology 85, 801. Brown, P.H., Dunemann, L., Schulz, R., & Marschner, H. (1989) Inﬂuence of redox potential and plantspecies on the uptake of nickel and cadmium from soils. Zeitschrift fur Pﬂanzenernahrung und Bodenkunde 152, 85–91. Brown, P.H., Welch, R.M., & Madison, J.T. (1990) Effect of nickel deﬁciency on soluble anion, aminoacid, and nitrogen levels in barley. Plant and Soil 125, 19–27. Brown, P., Bellaloui, N., Hu, H., & Dandekar, A. (1999) Transgenically enhanced sorbitol synthesis facilitates phloem boron transport and increases tolerance of tobacco to boron deﬁciency. Plant Physiology 119, 17. Brown, P.H., Bellaloui, N., Wimmer, M.A., et al. (2002) Boron in plant biology. Plant Biology 4, 205–223. Broyer, T., Carlton, A., Johnson, C., & Stout, P. (1954) Chlorine—a micronutrient element for higher plants. Plant Physiology 29, 526. Brumos, J., Colmenero-Flores, J.M., Conesa, A., et al. (2009) Membrane transporters and carbon metabolism implicated in chloride homeostasis differentiate salt stress responses in tolerant and sensitive Citrus rootstocks. Functional & Integrative. Genomics 9, 293–309. Buchner, P., Parmar, S., Kriegel, A., Carpentier, M., & Hawkesford, M.J. (2010) The sulfate transporter family in wheat: tissue-speciﬁc gene expression in relation to nutrition. Molecular Plant 3, 374– 389. Burkhead, J.L., Reynolds, K.A.G., Abdel-Ghany, S.E., Cohu, C.M., & Pilon, M. (2009) Copper homeostasis. The New Phytologist 182, 799–816. Bush, L.P., Legget, J.E., King, M.J., & Vincent, J.E. (1981) Sulfur and molybdenum nutrition effects in tall fescue. Canadian Journal of Botany-Revue Canadienne De Botanique 59, 536–541. Cailliatte, R., Schikora, A., Briat, J.F., Mari, S., & Curie, C. (2010) High-afﬁnity manganese uptake by the metal transporter nramp1 is essential for Arabidopsis growth in low manganese conditions. Plant Cell 22, 904–917.

419

Cartwright, B., Zarcinas, B.A., & Spouncer, L.R. (1986) Boron toxicity in south australian barley crops. Australian Journal of Agricultural Research 37, 351–359. Cataldo, D., Garland, T., & Wildung, R. (1978) Nickel in plants: I. Uptake kinetics using intact soybean seedlings. Plant Physiology 62, 563. Chen, X., Schauder, S., Potier, N., et al. (2002) Structural identiﬁcation of a bacterial quorumsensing signal containing boron. Nature 415, 545–549. Chen, W., Bell, R.W., Brennan, R.F., et al. (2009) Key crop nutrient management issues in the Western Australia grains industry: a review. Australian Journal of Soil Research 47, 1–18. Christensen, N. & Hayes, P. (2009) Genetics of chloride deﬁciency expression in barley. Communications in Soil Science and Plant Analysis 40, 407–418. Clarkson, D.T. (1988) The uptake and translocation of manganese by plant roots. In: Manganese in Soils and Plants (eds. R.D. Graham, R.J. Hannam & N.C. Uren), pp. 101–111. Kluwer Academic Publishers, Dordrecht, the Netherlands. Colangelo, E. & Guerinot, M. (2006) Put the metal to the petal: metal uptake and transport throughout plants. Current Opinion in Plant Biology 9, 322–330. Colmenero-Flores, J.M., Martinez, G., Gamba, G., et al. (2007) Identiﬁcation and functional characterization of cation-chloride cotransporters in plants. Plant Journal 50, 278–292. Cox, D.A. (1992) Poinsettia cultivars differ in their response to molybdenum deﬁciency. Hortscience 27, 892–893. Dannel, F., Pfeffer, H., & Romheld, V. (2000) Characterization of root boron pools, boron uptake and boron translocation in sunﬂower using stable isotopes 10B and 11B. Australian Journal of Plant Physiology 27, 397–405. Datnoff, L.E., Elmer, W.H., & Huber, D.M. eds. (2007) Mineral Nutrition and Plant Disease. American Phytopathological Society, St Paul, MN. Davidson, R.M., Reeves, P.A., Manosalva, P.M., & Leach, J.E. (2009) Germins: a diverse protein family important for crop improvement. Plant Science 177, 499–510. De Angeli, A., Thomine, S., Frachisse, J.M., Ephritikhine, G., Gambale, F., & Barbier-Brygoo, H. (2007) Anion channels and transporters in plant cell membranes. FEBS Letters 581, 2367–2374. Degryse, F., Smolders, E., & Parker, D.R. (2006) Metal complexes increase uptake of Zn and Cu by plants: implications for uptake and deﬁciency studies in chelator-buffered solutions. Plant and Soil 289, 171–185.

420

NUTRIENT USE EFFICIENCY IN CROPS

Degryse, F., Smolders, E., & Parker, D.R. (2009) Partitioning of metals (Cd, Co, Cu, Ni, Pb, Zn) in soils: concepts, methodologies, prediction and applications—a review. European Journal of Soil Science 60, 590–612. Delhaize, E., Gruber, B.D., Pittman, J.K., et al. (2007) A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance. Plant Journal 51, 198–210. Dixon, N.E., Gazzola, C., Blakeley, R.L., & Zerner, B. (1975)Jack-beanurease(ec3.5.1.5)—metalloenzyme— simple biological role for nickel. Journal of the American Chemical Society 97, 4131–4133. Dordas, C. & Brown, P.H. (2000) Permeability of boric acid across lipid bilayers and factors affecting it. Journal of Membrane Biology 175, 95–105. Dordas, C. & Brown, P.H. (2001a) Evidence for channel mediated transport of boric acid in squash (Cucurbita pepo). Plant and Soil 235, 95–103. Dordas, C. & Brown, P.H. (2001b) Permeability and the mechanism of transport of boric acid across the plasma membrane of Xenopus laevis oocytes. Biological Trace Element Research 81, 127– 139. Dordas, C., Chrispeels, M.J., & Brown, P.H. (2000) Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from squash roots. Plant Physiology 124, 1349–1361. Dunwell, J.M., Purvis, A., & Khuri, S. (2004) Cupins: the most functionally diverse protein superfamily? Phytochemistry 65, 7–17. Dunwell, J.M., Gibbings, J.G., Mahmood, T., & Naqvi, S.M.S. (2008) Germin and germin-like proteins: evolution, structure, and function. Critical Reviews in Plant Sciences 27, 342–375. Engvild, K.C. (1986) Chlorine containing natural compounds in higher-plants. Phytochemistry 25, 781–791. Eskew, D.L., Welch, R.M., & Cary, E.E. (1983) Nickel—an essential micronutrient for legumes and possibly all higher-plants. Science 222, 621–623. Findeklee, P. & Goldbach, H.E. (1996) Rapid effects of boron deﬁciency on cell wall elasticity modulus in Cucurbita pepo roots. Botanica Acta 109, 463–465. Fitzpatrick, K., Tyerman, S., & Kaiser, B. (2008) Molybdate transport through the plant sulfate transporter SHST1. FEBS letters 582, 1508–1513. Fixen, P. (1993) Crop responses to chloride. Advances in Agronomy 50, 107–150. Frommer, W.B. & von Wiren, N. (2002) Plant biology— Ping-pong with boron. Nature 420, 282–283. Garnett, T.P. & Graham, R.D. (2005) Distribution and remobilization of iron and copper in wheat. Annals of Botany 95, 817–826.

Geering, H.R., Hodgson, J.F., & Sdano, C. (1969) Micronutrient cation complexes in soil solution .4. chemical state of manganese in soil solution. Soil Science Society of America Proceedings 33, 81. Gerendas, J. & Sattelmacher, B. (1997a) Signiﬁcance of N source (urea vs. NH4NO3) and Ni supply for growth, urease activity and nitrogen metabolism of zucchini (Cucurbita pepo convar. giromontiina). Plant and Soil 196, 217–222. Gerendas, J. & Sattelmacher, B. (1997b) Signiﬁcance of Ni supply for growth, urease activity and the concentrations of urea, amino acids and mineral nutrients of urea-grown plants. Plant and Soil 190, 153–162. Gerendas, J., Zhu, Z., & Sattelmacher, B. (1998) Inﬂuence of N and Ni supply on nitrogen metabolism and urease activity in rice (Oryza sativa L.). Journal of Experimental Botany 49, 1545–1554. Gerendas, J., Polacco, J.C., Freyermuth, S.K., & Sattelmacher, B. (1999) Signiﬁcance of nickel for plant growth and metabolism. Journal of Plant Nutrition and Soil Science-Zeitschrift fur Pﬂanzenernahrung und Bodenkunde 162, 241–256. Gheibi, M., Malakouti, M., Kholdebarin, B., Ghanati, F., Teimouri, S., & Sayadi, R. (2009) Signiﬁcance of nickel supply for growth and chlorophyll content of wheat supplied with urea or ammonium nitrate. Journal of Plant Nutrition 32, 1440–1450. Giehl, R.F.H., Meda, A.R., & von Wirén, N. (2009) Moving up, down, and everywhere: signaling of micronutrients in plants. Current Opinion in Plant Biology 12, 320–327. Godo, G.H. & Reisenauer, H.M. (1980) Plant effects on soil manganese availability. Soil Science Society of America Journal 44, 993–995. Goldbach, H.E. (1997) A critical review on current hypotheses concerning the role of boron in higher plants: suggestions for further research and methodological requirements. Journal of Trace and Microprobe Techniques 15(1), 51–91. Goldbach, H.E. & Wimmer, M.A. (2007) Boron in plants and animals: is there a role beyond cell-wall structure? Journal of Plant Nutrition and Soil Science-Zeitschrift fur Pﬂanzenernahrung und Bodenkunde 170, 39–48. Goldbach, H.E., Yu, Q., Wingender, R., et al. (2001) Rapid response reactions of roots to boron deprivation. Journal of Plant Nutrition and Soil Science 164, 173–181. Goldberg, S. (1997) Reactions of boron with soils. Plant and Soil 193, 35–48. Gourley, C.J.P., Allan, D.L., & Russelle, M.P. (1994) Plant nutrient efﬁciency: a comparison of deﬁnitions and suggested improvement. Plant Soil 158, 29–37.

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

Graham, R.D. (1984) Breeding characterisitics in cereals. In: Advances in Plant Nutrition Vol 1 (eds. P.B. Tinker & A.E. Lauchli), pp. 57–90. Praeger, New York. Graham, R.D., Ascher, J.S., Ellis, P.A.E., & Shepherd, K.W. (1987) Transfer to wheat of the copper efﬁciency factor carried on rye chromosome arm 5RL. Plant Soil 99, 107–114. Graham, R.D., Hannam, R.J., & Uren, N. (1988) Manganese in Soils and Plants. Kluwer Academic Press, Dordrecht, The Netherlands. Graham, M.J., Nickell, C.D., & Hoeft, R.G. (1995) Inheritance of tolerance to manganese deﬁciency in soybean. Crop Science 35, 1007–1010. Gunes, A., Inal, A., Pilbeam, D.J., & Kadioglu, Y.K. (2009) Effect of sulfur on the yield and essential and nonessential element composition of alfalfa determined by polarized energy dispersive X-ray ﬂuorescence. Communications in Soil Science and Plant Analysis 40, 2264–2284. Gupta, U.C. (1997) Molybdenum in Agriculture. Cambridge University Press, Cambridge. Gupta, U.C. & Lipsett, J. (1981) Molybdenum in agriculture. Advances in Agronomy 34, 73– 115. Guskov, A., Gabdulkhakov, A., Broser, M., Glockner, C., Hellmich, J., Kern, J., Frank, J., Muh, F., Saenger, W., Zouni, A. (2010) Recent progress in the crystallographic studies of photosystem ii. Chemphyschem 11, 1160–1171. Hall, J. & Williams, L. (2003) Transition metal transporters in plants. Journal of Experimental Botany 54, 2601. Hamlin, R.L. (2007) Molybdenum. In: Handbook of Plant Nutrition (eds. A.V. Barker & D. Pilbeam), pp. 375–395. CRC, Boca Raton, FL. Hänsch, R. & Mendel, R. (2009) Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl). Current Opinion in Plant Biology 12, 259–266. Harris, W.R., Amin, S.A., Kupper, F.C., Green, D.H., & Carrano, C.J. (2007) Borate binding to siderophores: structure and stability. Journal of the American Chemical Society 129, 12263–12271. Hawkesford, M.J. (2003) Transporter gene families in plants: the sulphate transporter gene family— redundancy or specialization? Physiologia Plantarum 117, 155–163. Hebbern, C.A., Pedas, P., Schjoerring, J.K., Knudsen, L., & Husted, S. (2005) Genotypic differences in manganese efﬁciency: ﬁeld experiments with winter barley (Hordeum vulgare L.). Plant and Soil 272, 233–244. Hebbern, C.A., Laursen, K.H., Ladegaard, A.H., et al. (2009) Latent manganese deﬁciency increases tran-

421

spiration in barley (Hordeum vulgare). Physiologia Plantarum 135, 307–316. Heckman, J.R. (2007) Chlorine. In: Hankdbook of Plant Nutrition (eds. A.V. Barker & D. Pilbeam), pp. 279–293. CRC Press, Boca Raton, FL. Heintze, S.G. & Mann, P.J.G. (1949) Studies on soil manganese. Journal of Agricultural Science 39, 80–95. Henriques, F.S. (1989) Effects of copper deﬁciency on the photosynthetic apparatus of sugar-beet (Beta vulgaris l). Journal of Plant Physiology 135, 453–458. Heuwinkel, H., Kirkby, E.A., Lebot, J., & Marschner, H. (1992) Phosphorus deﬁciency enhances molybdenum uptake by tomato plants. Journal of Plant Nutrition 15, 549–568. Himelblau, E. & Amasino, R.M. (2001) Nutrients mobilized from leaves of Arabidopsis thaliana during leaf senescence. Journal of Plant Physiology 158, 1317–1323. Himelblau, E., Mira, H., Lin, S.J., Culotta, V.C., Penarrubia, L., & Amasino, R.M. (1998) Identiﬁcation of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis. Plant Physiology 117, 1227–1234. Hodgson, J.F., Lindsay, W.L., & Trierwei, J.F. (1966) Micronutrient cation complexing in soil solution.2. complexing of zinc and copper in displaced solution from calcareous soils. Soil Science Society of America Proceedings 30, 723. Holloway, R.E., Graham, R.D., & Stacey, S.P. (2008) Micronutrient deﬁciencies in Australian ﬁeld crops. In: Micronutrient Deﬁciecnies in Global Crop Production (ed. B.J. Alloway), pp. 63–93. Springer, Dordrecht. Hu, H. & Brown, P.H. (1994) Localization of boron in cell walls of squash and tobacco and its association with pectin. Plant Physiology 105, 681–689. Hu, H., Brown, P.H., & Labavitch, J.M. (1996) Species variability in boron requirement is correlated with cell wall pectin. Journal of Experimental Botany 47, 227–232. Hu, H., Penn, S.G., Lebrilla, C.B., & Brown, P.H. (1997) Isolation and characterization of soluble boron complexes in higher plants. Plant Physiology 113, 649–655. Huang, C.Y., Webb, M.J., & Graham, R.D. (1996) Pot size affects expression of Mn efﬁciency in barley. Plant and Soil 178, 205–208. Husted, S., Laursen, K.H., Hebbern, C.A., et al. (2009) Manganese deﬁciency leads to genotype-speciﬁc changes in ﬂuorescence induction kinetics and state transitions. Plant Physiology 150, 825–833. Isayenkov, S., Isner, J.C., & Maathuis, F.J.M. (2010) Vacuolar ion channels: roles in plant nutrition and signalling. Febs Letters 584, 1982–1988.

422

NUTRIENT USE EFFICIENCY IN CROPS

Ishii, T. & Matsunaga, T. (1996) Isolation and characterization of a boron-rhamnogalacturonan-II complex from cell walls of sugar beet pulp. Carbohydrate Research 284, 1–9. Ishii, T., Matsunaga, T., Pellerin, P., O’Neill, M.A., Darvill, A., & Albersheim, P. (1999) The plant cell wall polysaccharide rhamnogalacturonan II selfassembles into a covalently cross-linked dimer. Journal of Biological Chemistry 274, 13098–13104. Iwai, H., Masaoka, N., Ishii, T., & Satoh, S. (2002) A pectin glucuronyltransferase gene is essential for intercellular attachment in the plant meristem. Proceedings of the National Academy of Sciences of the United States of America 99, 16319–16324. Jamjod, S., Niruntrayagul, S., & Rerkasem, B. (2004) Genetic control of boron efﬁciency in wheat (Triticum aestivum L.). Euphytica 135, 21–27. Jiang, W.Z. (2006) Mn use efﬁciency in different wheat cultivars. Environmental and Experimental Botany 57, 41–50. Jiang, W.Z. (2008) Comparison of responses to Mn deﬁciency between the UK wheat genotypes Maris Butler, Paragon and the Australian wheat genotype C8MM. Journal of Integrative Plant Biology 50, 457–465. Kabata-Pendias, A. & Pendias, H. (2000) Trace Elements in Soils and Plants, 3rd ed. CRC Press, Boca Raton, FL. Kaiser, B., Gridley, K., & Ngaire, B. (2005) The role of molybdenum in agricultural plant production. Annals of Botany 96, 745. Kampfenkel, K., Kushnir, S., Babiychuk, E., Inze, D., & Vanmontagu, M. (1995) Molecular characterization of a putative arabidopsis-thaliana copper transporter and its yeast homolog. Journal of Biological Chemistry 270, 28479–28486. Kato, Y., Miwa, K., Takano, J., Wada, M., & Fujiwara, T. (2009) Highly boron deﬁciency-tolerant plants generated by enhanced expression of NIP5;1, a Boric Acid Channel. Plant and Cell Physiology 50, 58–66. Kaur, A.J. & Sadana, U.S. (2010) Nitrogen source and manganese application effects on manganese dynamics in the rhizosphere of wheat cultivars grown on manganese-deﬁcient soils. Journal of Plant Nutrition 33, 831–845. Kawakami, K., Umena, Y., Kamiya, N., Shen, J.R. (2009) Location of chloride and its possible functions in oxygen-evolving photosystem ii revealed by x-ray crystallography. Proceedings of the National Academy of Sciences of the United States of America 106, 8567–8572. Kering, M.K., Lukaszewska, K., & Blevins, D.G. (2009) Manganese requirement for optimum photo-

synthesis and growth in NAD-malic enzyme C-4 species. Plant and Soil 316, 217–226. Khabaz-Saberi, K., Graham, R.D., Pallotta, M.A., Rathjen, A.J., & Williams, K.J. (2002) Genetic markers for manganese efﬁciency in durum wheat. Plant Breeding 121, 224–227. Kobayashi, M., Matoh, T., & Azuma, J.-I. (1996) Two chains of rhamnogalacturonan II are cross-linked by borate-diol ester bonds in higher plant cell walls. Plant Physiology 110, 1017–1020. Kobayashi, M., Mutoh, T., & Matoh, T. (2004) Boron nutrition of cultured tobacco BY-2 cells. IV. Genes induced under low boron supply. Journal of Experimental Botany 55, 1441–1443. Koike, S., Inoue, H., Mizuno, D., et al. (2004) OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron and expressed in the phloem. Plant Journal 39, 415–424. Koshiba, T., Kobayashi, M., & Matoh, T. (2009) Boron nutrition of tobacco BY-2 Cells. V. oxidative damage is the major cause of cell death induced by boron deprivation. Plant and Cell Physiology 50, 26–36. Krämer, U., Talke, I., & Hanikenne, M. (2007) Transition metal transport. FEBS Letters 581, 2263–2272. Kusunoki, M. (2007) Mono-manganese mechanism of the photosytem II water splitting reaction by a unique Mn4Ca cluster. Biochimica et Biophysica Acta-Bioenergetics 1767, 484–492. Kusunoki, M. (2008) Mono-manganese mechanism of the photosystem II water splitting reaction by a unique Mn4Ca cluster (vol 1767, pg no 484, 2007). Biochimica Et Biophysica Acta-Bioenergetics 1777, 477–477. Lanquar, V., Ramos, M.S., Lelievre, F., et al. (2010) Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required for optimal photosynthesis and growth under manganese deﬁciency. Plant Physiology 152, 1986–1999. Leeper, G.W. (1947) The forms and reactions of manganese in the soil. Soil Science 63, 79–94. Leggett, J.E. & Epstein, E. (1956) Kinetics of sulfate absorption by barley roots. Plant Physiology 31, 222–226. Li, Y.J. & Zamble, D.B. (2009) Nickel homeostasis and nickel regulation: an overview. Chemical Reviews 109, 4617–4643. Li, X.Y., Chanroj, S., Wu, Z.Y., Romanowsky, S.M., Harper, J.F., & Sze, H. (2008) A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiology 147, 1675–1689. Lindsay, W.L. (1991) Inorganic equilibria affecting micronutrients in soils. In: Micronutrients in Agricutlure (eds. J.J. Mortvedt, F.R. Cox, L.M.

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

Shuman & R.M. Welch), pp. 89–112. Soil Science Society of America, Madison, WI. Lippard, S.J. & Berg, J.M. (1994) Principles of Bioorganic Chemistry. University Science Books, Mill Valley, CA. Loneragan, J.F. (1981) Distribution and movement of copper in soils. In: Copper in Soils and Plants (eds. J.F. Loneragan, A.D. Robson & R.D. Graham), pp. 165–188. Academic Press, Cambridge. Loomis, W.D. & Durst, R.W. (1992) Chemistry and biology of boron. Biofactors 3, 229–239. Lukaszewski, K.M. & Blevins, D.G. (1996) Root growth inhibition in boron-deﬁcient or aluminumstressed squash may be a result of impaired ascorbate metabolism. Plant Physiology 112, 1135–1140. Lv, Q., Tang, R., Liu, H., et al. (2009) Cloning and molecular analyses of the Arabidopsis thaliana chloride channel gene family. Plant Science 176, 650–661. Ma, J.F., Tamai, K., Yamaji, N., et al. (2006) A silicon transporter in rice. Nature 440, 688–691. Ma, J.F., Yamaji, N., Mitani, N., et al. (2008) Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proceedings of the National Academy of Sciences of the United States of America 105, 9931–9935. Macleod, J.A., Gupta, U.C., & Stanﬁeld, B. (1997) Molybdenum and sulfur relationships in plants. In: Molybdenum in Agriculture (ed. U.C. Gupta), pp. 229–245. Cambridge University Press, Cambridge. Manosalva, P.M., Davidson, R.M., Liu, B., et al. (2009) A germin-like protein gene family functions as a complex quantitative trait locus conferring broadspectrum disease resistance in rice. Plant Physiology 149, 286–296. Mari, S., Gendre, D., Pianelli, K., et al. (2006) Root-toshoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens. Journal of Experimental Botany 57, 4111–4122. Marmagne, A., Vinauger-Douard, M., Monachello, D., et al. (2007) Two members of the Arabidopsis CLC (chloride channel) family, AtCLCe and AtCLCf, are associated with thylakoid and Golgi membranes, respectively. Journal of Experimental Botany 58, 3385–3393. Marschner, H. (1995) Mineral Nutrition of Higher Plants, 3rd ed. Academic Press, San Diego, CA. Marschner, P., Fu, Q.L., & Rengel, Z. (2003) Manganese availability and microbial populations in the rhizosphere of wheat genotypes differing in tolerance to Mn deﬁciency. Journal of Plant Nutrition and Soil Science-Zeitschrift fur Pﬂanzenernahrung und Bodenkunde 166, 712–718.

423

Matoh, T. & Ochiai, K. (2005) Distribution and partitioning of newly taken-up boron in sunﬂower. Plant and Soil 278, 351–360. Matoh, T., Ishigaki, K., Ohno, K., & Azuma, J. (1993) Isolation and characterization of a boronpolysaccharide complex from radish roots. Plant and Cell Physiology 34, 639–642. Mendel, R.R. (2007) Biology of the molybdenum cofactor. Journal of Experimental Botany 58, 2289–2296. Mills, R.F., Doherty, M.L., Lopez-Marques, R.L., et al. (2008) ECA3, a Golgi-localized P-2A-type ATPase, plays a crucial role in manganese nutrition in Arabidopsis. Plant Physiology 146, 116–128. Miwa, K., Takano, J., & Fujiwara, T. (2006) Improvement of seed yields under boron-limiting conditions through overexpression of BOR1, a boron transporter for xylem loading, in Arabidopsis thaliana. Plant Journal 46, 1084–1091. Miwa, K., Takano, J., Omori, H., Seki, M., Shinozaki, K., & Fujiwara, T. (2007) Plants tolerant of high boron levels. Science 318, 1417. Miwa, K., Tanaka, M., Kamiya, T., & Fujiwara, T. (2010) Molecular mechanisms of boron transport in plants: involvement of Arabidopsis NIP5;1 and NIP6;1. In: Mips and Their Role in the Exchange of Metalloids, Vol 679, pp. 83–96. Springer-Verlag Berlin, Berlin. Mizuno, T., Usui, K., Horie, K., Nosaka, S., Mizuno, N., & Obata, H. (2005) Cloning of three ZIP/ NRAMP transporter genes from a Ni hyperaccumulator plant Thlaspi japonicum and their Ni2+transport abilities. Plant Physiology and Biochemistry 43, 793–801. Mizuno, T., Usui, K., Nishida, S., Unno, T., & Obata, H. (2007) Investigation of the basis for Ni tolerance conferred by the expression of TjZnt1 and TjZnt2 in yeast strains. Plant Physiology and Biochemistry 45, 371–378. Moraghan, J.T. & Mascagni, H.J. (1991) Environmental and soil factors affecting micronutrient deﬁciencies and toxicities. In: Micronutrients in Agriculture (eds. J.J. Mortvedt, F.R. Cox, L.M. Shuman & R.M. Welch), pp. 371–428. Soil Science Society of America, Madison, WI. Moran, N. (2007) Osmoregulation of leaf motor cells. FEBS Letters 581, 2337–2347. Nable, R.O., Banuelos, G.S., & Paull, J.G. (1997) Boron toxicity. Plant and Soil 193, 181–198. Nachiangmai, D., Dell, B., Bell, R., Huang, L., & Rerkasem, B. (2004) Enhanced boron transport into the ear of wheat as a mechanism for boron efﬁciency. Plant Soil 264, 141–147. Nicoulaud, B.A.L. & Bloom, A.J. (1998) Nickel supplements improve growth when foliar urea is the sole nitrogen source for tomato. Journal of the

424

NUTRIENT USE EFFICIENCY IN CROPS

American Society for Horticultural Science 123, 556–559. Noguchi, K., Yasumori, M., Imai, T., et al. (1997) Bor 1-1, an Arabidopsis thaliana mutant that requires a high level of boron. Plant Physiology 115, 901–906. Nolan, A.L., Zhang, H., & McLaughlin, M.J. (2005) Prediction of zinc, cadmium, lead, and copper availability to wheat in contaminated soils using chemical speciation, diffusive gradients in thin ﬁlms, extraction, and isotopic dilution techniques. Journal of Environmental Quality 34, 496–507. Nolan, B.T., Puckett, L.J., Ma, L.W., Green, C.T., Bayless, E.R., & Malone, R.W. (2010) Predicting unsaturated zone nitrogen mass balances in agricultural settings of the United States. Journal of Environmental Quality 39, 1051–1065. Nuttall, C.Y. (2000) Boron tolerance and uptake in higher plants. PhD. University of Cambridge, U.K. Nyomora, A.M.S., Brown, P.H., & Freeman, M. (1997) Fall foliar-applied boron increases tissue boron concentration and nut set of almond. Journal of the American Society for Horticultural Science 122, 405–410. O’Halloran, T.V. & Culotta, V.C. (2000) Metallochaperones, an intracellular shuttle service for metal ions. Journal of Biological Chemistry 275, 25057–25060. O’Neill, M.A., Warrenfeltz, D., Kates, K., et al. (1996) Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester—in vitro conditions for the formation and hydrolysis of the dimer. Journal of Biological Chemistry 271, 22923–22930. O’Neill, M., Eberhard, S., Albersheim, P., & Darvill, A. (2001) Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science 294, 846–849. Ouerdane, L., Mari, S., Czernic, P., Lebrun, M., & Lobinski, R. (2006) Speciation of non-covalent nickel species in plant tissue extracts by electrospray Q-TOFMS/MS after their isolation by 2D size exclusion-hydrophilic interaction LC (SEC-HILIC) monitored by ICP-MS. Journal of Analytical Atomic Spectrometry 21, 676–683. Owuoche, J.O., Briggs, K.G., Taylor, G.J., & Penney, D.C. (1995) Response of 8 canadian spring wheat (triticum-aestivum cultivars to copper—copper content in the leaves and grain. Canadian Journal of Plant Science 75, 405–411. Padmanaban, S., Lin, X.Y., Perera, I., Kawamura, Y., Sze, H. (2004) Differential expression of vacuolar h+-atpase subunit c genes in tissues active in

membrane trafﬁcking and their roles in plant growth as revealed by rnai. Plant Physiology 134, 1514–1526. Palmer, C. & Guerinot, M. (2009) Facing the challenges of Cu, Fe and Zn homeostasis in plants. Nature Chemical Biology 5, 333–340. Pang, Y.Q., Li, L.J., Ren, F., et al. (2010) Overexpression of the tonoplast aquaporin AtTIP5;1 conferred tolerance to boron toxicity in Arabidopsis. Journal of Genetics and Genomics 37, 389–U352. Park, M., Li, Q., Shcheynikov, N., Zeng, W.Z., & Muallem, S. (2004) NaBC1 is a ubiquitous electrogenic Na+-coupled borate transporter essential for cellular boron homeostasis and cell growth and proliferation. Molecular Cell 16, 331–341. Parker, D.R. & Norvell, W.A. (1999) Advances in solution culture methods for plant mineral nutrition research. Advances in Agronomy 65, 151–213. Pasricha, N.S. & Randhawa, N.S. (1972) Interaction effect of sulfur and molybdenum on uptake and utilization of these elements by raya (Brassica juncea l). Plant and Soil 37, 215. Pedas, P., Hebbern, C.A., Schjoerring, J.K., Holm, P.E., & Husted, S. (2005) Differential capacity for highafﬁnity manganese uptake contributes to differences between barley genotypes in tolerance to low manganese availability. Plant Physiology 139, 1411–1420. Pedas, P., Ytting, C.K., Fuglsang, A.T., Jahn, T.P., Schjoerring, J.K., & Husted, S. (2008) Manganese efﬁciency in barley: identiﬁcation and characterization of the metal ion transporter HvIRT1. Plant Physiology 148, 455–466. Peiter, E., Montanini, B., Gobert, A., et al. (2007) A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance. Proceedings of the National Academy of Sciences of the United States of America 104, 8532–8537. Pellerin, P., Doco, T., Vidal, S., Williams, P., Brillouet, J.-M., & O’Neill, M.A. (1996) Structural characterization of red wine rhamnogalacturonan II. Carbohydrate Research 290, 183–197. Penarrubia, L., Andres-Colas, N., Moreno, J., & Puig, S. (2010) Regulation of copper transport in Arabidopsis thaliana: a biochemical oscillator? Journal of Biological Inorganic Chemistry 15, 29–36. Pianelli, K., Mari, S., Marques, L., Lebrun, M., & Czernic, P. (2005) Nicotianamine over-accumulation confers resistance to nickel in Arabidopsis thaliana. Transgenic Research 14, 739–748. Pierce, W.S. & Higinbotham, N. (1970) Compartments and ﬂuxes of K K+, NA+ and CL- in Avena coleoptile. Plant Physiology 46, 666. Pilon, M., Abdel-Ghany, S.E., Cohu, C.M., Gogolin, K.A., & Ye, H. (2006) Copper cofactor delivery in

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

plant cells. Current Opinion in Plant Biology 9, 256–263. Pilon, M., Cohu, C., Ravet, K., Abdel-Ghany, S., & Gaymard, F. (2009) Essential transition metal homeostasis in plants. Current Opinion in Plant Biology 12, 347–357. Pittman, J.K. (2005) Managing the manganese: molecular mechanisms of manganese transport and homeostasis. The New Phytologist 167, 733–742. Polacco, J.C. (1977) Nitrogen-metabolism in soybean tissue-culture.2. urea utilization and urease synthesis require Ni2+. Plant Physiology 59, 827–830. Puig, S. & Penarrubia, L. (2009) Placing metal micronutrients in context: transport and distribution in plants. Current Opinion in Plant Biology 12, 299–306. Puig, S., Andres-Colas, N., Garcia-Molina, A., & Penarrubia, L. (2007) Copper and iron homeostasis in Arabidopsis: responses to metal deﬁciencies, interactions and biotechnological applications. Plant Cell and Environment 30, 271–290. Raven, J.A. (1980) Short-distance and long-distance transport of boric-acid in plants. The New Phytologist 84, 231–249. Redondo-Nieto, M., Rivilla, R., El-Hamdaoui, A., Bonilla, I., & Bolanos, L. (2001) Boron deﬁciency affects early infection events in the pea-Rhizobium symbiotic interaction. Australian Journal of Plant Physiology 28, 819–823. Reguera, M., Espi, A., Bolanos, L., Bonilla, I., & Redondo-Nieto, M. (2009) Endoreduplication before cell differentiation fails in boron-deﬁcient legume nodules. Is boron involved in signalling during cell cycle regulation? The New Phytologist 183, 9–12. Reguera, M., Bonilla, I., & Bolanos, L. (2010) Boron deﬁciency results in induction of pathogenesisrelated proteins from the PR-10 family during the legume-rhizobia interaction. Journal of Plant Physiology 167, 625–632. Reid, R. & Fitzpatrick, K. (2009) Inﬂuence of leaf tolerance mechanisms and rain on boron toxicity in barley and wheat. Plant Physiology 151, 413–420. Rengel, Z. & Marschner, P. (2005) Nutrient availability and management in the rhizosphere: exploiting genotypic differences. New Phytologist 168, 305–312. Reid, R.J., Hayes, J.E., Post, A., Stangoulis, J.C.R., & Graham, R.D. (2004) A critical analysis of the causes of boron toxicity in plants. Plant Cell and Environment 27, 1405–1414. Requena, L.M. & Bornemann, S. (1999) Plant oxalate oxidase is a novel manganese-containing hydrogen peroxide producing enzyme. Journal of Inorganic Biochemistry 74, 275–275.

425

Rerkasem, B. & Jamjod, S. (1997) Genotypic variation in plant response to low boron and implications for plant breeding. Plant and Soil 193, 169–180. Rerkasem, B. & Jamjod, S. (2004) Boron deﬁciency in wheat: a review. Field Crops Research 89, 173–186. Riesen, O. & Feller, U. (2005) Redistribution of nickel, cobalt, manganese, zinc, and cadmium via the phloem in young and maturing wheat. Journal of Plant Nutrition 28, 421–430. Robinson, N.J., Procter, C.M., Connolly, E.L., & Guerinot, M.L. (1999) A ferric-chelate reductase for iron uptake from soils. Nature 397, 694–697. Rodriguez, F.I., Esch, J.J., Hall, A.E., Binder, B.M., Schaller, G.E., & Bleecker, A.B. (1999) A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science 283, 996–998. Roelfsema, M.R.G. & Hedrich, R. (2005) In the light of stomatal opening: New insights into “the watergate.” New Phytologist 167, 665–691. Ruter, J.M. (2005a) Effect of nickel applications disorder on for the control of mouse ear river birch. Journal of Environmental Horticulture 23, 17–20. Ruter, J.M. (2005b) Response of Betula nigra “BNMTF” to foliar and drench applications of nickel. Hortscience 40, 996. Saberi, H.K., Graham, R.D., & Rathjen, A.J. (1999) Inheritance of manganese efﬁciency in durum wheat. Journal of Plant Nutrition 22, 11–21. Sancenon, V., Puig, S., Mira, H., Thiele, D.J., & Penarrubia, L. (2003) Identiﬁcation of a copper transporter family in Arabidopsis thaliana. Plant Molecular Biology 51, 577–587. Sancenón, V., Puig, S., Mateu-Andrés, I., Dorcey, E., Thiele, D., & Peñarrubia, L. (2004) The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development. Journal of Biological Chemistry 279, 15348. Sauve, S., McBride, M.B., Norvell, W.A., & Hendershot, W.H. (1997) Copper solubility and speciation of in situ contaminated soils: effects of copper level, pH and organic matter. Water Air and Soil Pollution 100, 133–149. Sayyari-Zahan, M.H., Sadana, U.S., Steingrobe, B., & Claassen, N. (2009) Manganese efﬁciency and maganese-uptake kinetics of raya (Brassica juncea), wheat (Triticum aestivum), and oat (Avena sativa) grown in nutrient solution and soil. Journal of Plant Nutrition and Soil Science-Zeitschrift fur Pﬂanzenernahrung und Bodenkunde 172, 425–434. Schlegel, R., Werner, T., & Hülgenhof, E. (1991) Conﬁrmation of a 4BL/5RL wheat-rye chromosome translocation line in the wheat cultivar “Viking” showing high copper efﬁciency. Plant Breeding 107, 226–234.

426

NUTRIENT USE EFFICIENCY IN CROPS

Schwarz, G. & Mendel, R.R. (2006) Molybdenum cofactor biosynthesis and molybdenum enzymes. Annual Review of Plant Biology 57, 623–647. Schwarz, G., Mendel, R.R., & Ribbe, M.W. (2009) Molybdenum cofactors, enzymes and pathways. Nature 460, 839–847. Serventi, F., Ramazzina, I., Lamberto, I., Puggioni, V., Gatti, R., & Percudani, R. (2010) Chemical basis of nitrogen recovery through the ureide pathway: formation and hydrolysis of s-ureidoglycine in plants and bacteria. Acs Chemical Biology 5, 203–214. Shinmachi, F., Buchner, P., Stroud, J.L., et al. (2010) Inﬂuence of sulfur deﬁciency on the expression of speciﬁc sulfate transporters and the distribution of sulfur, selenium, and molybdenum in wheat. Plant Physiology 153, 327–336. Shorrocks, V. (1997) The occurrence and correction of boron deﬁciency. In: Boron in Soils and Plants (eds. B. Dell et al.), Kluwer Academic Publishers, Dordrecht, pp. 121–149. Shuman, L.M. (1991) Chemical forms of micronutrients in soils. In: Micronutrients in Agriculture (eds. J.J. Mortvedt, F.R. Cox, L.M. Shuman & R.M. Welch), pp. 113–144. Soil Science Society of America, Madison, WI. Sillanpää, M. (1982) Micronutrients and the nutrient status of soils: a global study. Soils Bulletin 48, 444. Sillanpää, M. (1990) Micronutrient Assessmenent at a Country Level: An International Study, Vol 63, FAO, Rome. Sims, J.T. (1986) Soil-pH effects on the distribution and plant availability of manganese, copper, and zinc. Soil Science Society of America Journal 50, 367–373. Sinclair, A.H. & Edwards, A.C. (2008) Micronutrient deﬁciency problems in Agricultural Crops in Europe. In: Micronutrient Deﬁciencies in Global Crop Production (ed. B.J. Alloway), pp. 225–245. Springer, Dordrecht. Singh, M.V. (2008) Micronutrient deﬁciencies in crops and soils in India. In: Micronutrient Deﬁciencies in Global Crop Production (ed. B.J. Alloway), pp. 93–127. Springer, Dordrecht. Smith, F.W., Ealing, P.M., Hawkesford, M.J., & Clarkson, D.T. (1995) Plant members of a family of sulfate transporters reveal functional subtypes. Proceedings of the National Academy of Sciences of the United States of America 92, 9373–9377. Stangoulis, J., Brown, P., Bellaloui, N., Reid, R., & Graham, R. (2001a) The efﬁciency of boron utilisation in canola. Australian Journal of Plant Physiology 28, 1109–1114. Stangoulis, J.C.R., Reid, R.J., Brown, P.H., & Graham, R.D. (2001b) Kinetic analysis of boron transport in Chara. Planta 213, 142–146.

Stangoulis, J., Tate, M., Graham, R., et al. (2010) The mechanism of boron mobility in wheat and canola phloem. Plant Physiology 153, 876–881. Stout, P.R., Meagher, W.R., Pearson, G.A., & Johnson, C.M. (1951) Molybdenum nutrition of crop plants. 1. The inﬂuence of phosphate and sulfate on the absorption of molybdenum from soils and solution cultures. Plant and Soil 3, 51–87. Su, Z., Chai, M.F., Lu, P.L., An, R., Chen, J., & Wang, X.C. (2007) AtMTM1, a novel mitochondrial protein, may be involved in activation of the manganese-containing superoxide dismutase in Arabidopsis. Planta 226, 1031–1039. Sze, H. (1985) H+-translocating atpases—advances using membrane-vesicles. Annual Review of Plant Physiology and Plant Molecular Biology 36, 175–208. Takano, J., Noguchi, K., Yasumori, M., et al. (2002) Arabidopsis boron transporter for xylem loading. Nature 420, 337–340. Takano, J., Wada, M., Ludewig, U., Schaaf, G., von Wiren, N., & Fujiwara, T. (2006) The Arabidopsis major intrinsic protein NIP5;1 is essential for efﬁcient boron uptake and plant development under boron limitation. Plant Cell 18, 1498–1509. Tanaka, M. & Fujiwara, T. (2008) Physiological roles and transport mechanisms of boron: perspectives from plants. Pﬂügers Archiv European Journal of Physiology 456, 671–677. Tanaka, M., Wallace, I.S., Takano, J., Roberts, D.M., & Fujiwara, T. (2008) NIP6;1 is a boric acid channel for preferential transport of boron to growing shoot tissues in Arabidopsis. Plant Cell 20, 2860–2875. Teakle, N.L. & Tyerman, S.D. (2010) Mechanisms of Cl- transport contributing to salt tolerance. Plant Cell and Environment 33, 566–589. Tejada-Jiménez, M., Llamas, Á., Sanz-Luque, E., Galván, A., & Fernández, E. (2007) A high-afﬁnity molybdate transporter in eukaryotes. Proceedings of the National Academy of Sciences 104, 20126. Tejada-Jiménez, M., Galván, A., Fernández, E., & Llamas, Á. (2009) Homeostasis of the micronutrients Ni, Mo and Cl with speciﬁc biochemical functions. Current Opinion in Plant Biology 12, 358–363. Tifﬁn, L.O. (1971) Translocation of nickel in xylem exudate of plants. Plant Physiology 48, 273. Tifﬁn, L.O. & Thompson, J.F. (1975) Metal chelators in peanut and metal chelate stabilities. Plant Physiology 56, 19–19. Tomatsu, H., Takano, J., Takahashi, H., WatanabeTakahashi, A., Shibagaki, N., & Fujiwara, T. (2007) An Arabidopsis thaliana high-afﬁnity molybdate transporter required for efﬁcient uptake of molyb-

BORON, CHLORINE, COPPER, MANGANESE, MOLYBDENUM, AND NICKEL

date from soil. Proceedings of the National Academy of Sciences 104, 18807. Tong, Y.P., Rengel, Z., & Graham, R.D. (1997) Interactions between nitrogen and manganese nutrition of barley genotypes differing in manganese efﬁciency. Annals of Botany 79, 53–58. Tottey, S., Waldron, K.J., Firbank, S.J., et al. (2008) Protein-folding location can regulate manganesebinding versus copper- or zinc-binding. Nature 455, 1138–1142. van der Waals, J.H. & Laker, M.C. (2008) Micronutrient deﬁciecnies in crops in africa with an emphasis on southern Africa. In: Micronutrient Deﬁciencies in Global Crop Production (ed. B.J. Alloway), pp. 201–225. Springer, Dordrecht. Vert, G., Grotz, N., Dedaldechamp, F., et al. (2002) IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. Plant Cell 14, 1223–1233. Vlek, P.L.G. & Lindsay, W.L. (1977) Thermodynamic stability and solubility of molybdenum minerals in soils. Soil Science Society of America Journal 41, 42–46. von der Fecht-Bartenbach, J., Bogner, M., Krebs, M., Stierhof, Y.D., Schumacher, K., & Ludewig, U. (2007) Function of the anion transporter AtCLC-d in the trans-Golgi network. Plant Journal 50, 466–474. Warden, B.T. & Reisenauer, H.M. (1991) Fractionation of soil manganese forms important to plant availability. Soil Science Society of America Journal 55, 345–349. Warington, K. (1923) The effect of boric acid and borax on the broad bean and certain other plants. Annals of Botany 37, 629–U623. Waters, B.M., Chu, H.H., DiDonato, R.J., et al. (2006) Mutations in Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds. Plant Physiology 141, 1446–1458. Waters, B. & Grusak, M. (2007) Whole-plant mineral partitioning throughout the life cycle in Arabidopsis thaliana ecotypes Columbia, Landsberg erecta, Cape Verde Islands, and the mutant line ysl1ysl3. The New Phytologist 177, 389–405. Waters, B.M. & Grusak, M.A. (2008) Quantitative trait locus mapping for seed mineral concentrations in twoArabidopsis thalianarecombinant inbred populations. The New Phytologist 179, 1033– 1047. Welch, R.M. (1981) The biological signiﬁcance of nickel. Journal of Plant Nutrition 3, 345–356. White, P. & Broadley, M. (2001) Chloride in soils and its uptake and movement within the plant: a review. Annals of Botany 88, 967.

427

White, P.J. & Broadley, M.R. (2009) Biofortiﬁcation of crops with seven mineral elements often lacking in human diets—iron, zinc, copper, calcium, magnesium, selenium and iodine. The New Phytologist 182, 49–84. Wichard, T., Mishra, B., Kraepiel, A.M.L., & Myneni, S. (2008) Molybdenum speciation and bioavailability in soils. Geochimica Et Cosmochimica Acta 72, A1019–A1019. Williams, L., Pittman, J., & Hall, J. (2000) Emerging mechanisms for heavy metal transport in plants. Biochimica et Biophysica Acta (BBA)Biomembranes 1465, 104–126. Williams, C.M.J., Maier, N.A., & Bartlett, L. (2004) Effect of molybdenum foliar sprays on yield, berry size, seed formation, and petiolar nutrient composition of “Merlot” grapevines. Journal of Plant Nutrition 27, 1891–1916. Wintz, H., Fox, T., Wu, Y., et al. (2003) Expression proﬁles of Arabidopsis thaliana in mineral deﬁciencies reveal novel transporters involved in metal homeostasis. Journal of Biological Chemistry 278, 47644. Witte, C.P., Isidore, E., Tiller, S.A., Davies, H.V., & Taylor, M.A. (2001) Functional characterisation of urease accessory protein G (ureG) from potato. Plant Molecular Biology 45, 169–179. Witte, C.P., Rosso, M.G., & Romeis, T. (2005) Identiﬁcation of three urease accessory proteins that are required for urease activation in Arabidopsis. Plant Physiology 139, 1155–1162. Woo, E.J., Dunwell, J.M., Goodenough, P.W., Marvier, A.C., & Pickersgill, R.W. (2000) Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities. Nature Structural Biology 7, 1036–1040. Wood, B., Reilly, C., & Nyczepir, A. (2004) Mouse-ear of pecan: a nickel deﬁciency. HortScience 39, 1238–1242. Woods, W.G. (1996) Review of possible boron speciation relating to its essentiality. Journal of Trace Elements in Experimental Medicine 9, 153–163. Xu, G.H., Magen, H., Tarchitzky, J., & Kafkaﬁ, U. (2000) Advances in chloride nutrition of plants. Advances in Agronomy 68, 97–150. Xu, F., Wang, Y., & Meng, J. (2001) Mapping boron efﬁciency gene in Brassica napus using RFLP and AFLP markers. Plant Breeding 120, 319–324. Xue, J.M., Lin, M.S., Bell, R.W., Graham, R.D., Yang, X., & Yang, Y. (1998) Differential response of oilseed rape (Brassica napus L.) cultivars to low boron supply. Plant and Soil 204, 155–163. Yang, T.J.W., Perry, P.J., Ciani, S., Pandian, S., & Schmidt, W. (2008) Manganese deﬁciency alters the patterning and development of root hairs in

428

NUTRIENT USE EFFICIENCY IN CROPS

Arabidopsis. Journal of Experimental Botany 59, 3453–3464. Yruela Guerrero, I. (2009) Copper in plants: acquisition, transport and interactions. Functional Plant Biology 36, 409–430. Yu, M., Hu, C., & Wang, Y. (2002) Molybdenum efﬁciency in winter wheat cultivars as related to molybdenum uptake and distribution. Plant Soil 245, 287–293. Yu, Q., Baluska, F., Jasper, F., Menzel, D., & Goldbach, H.E. (2003) Short-term boron deprivation enhances levels of cytoskeletal proteins in maize, but not zucchini, root apices. Physiologia Plantarum 117, 270–278. Yu, M., Hu, C.X., Sun, X.C., & Wang, Y.H. (2010) Inﬂuences of Mo on nitrate reductase, glutamine

synthetase and nitrogen accumulation and utilization in Mo-efﬁcient and Mo-inefﬁcient winter wheat cultivars. Agricultural Sciences in China 9, 355–361. Zeng, C., Xu, F., Wang, Y., Hu, C., & Meng, J. (2007) Physiological basis of QTLs for boron efﬁciency in Arabidopsis thaliana. Plant Soil 296, 187–196. Zifarelli, G. & Pusch, M. (2010) CLC transport proteins in plants. FEBS Letters 584, 2122–2127. Zou, C., Gao, X., Shi, R., Fan, X., & Zhang, F. (2008) Micronutrient deﬁciencies in China. In: Micronutrient Deﬁciencies in Global Crop Production (ed. B.J. Alloway), pp. 127–149. Springer, Dordrecht.

Part IV

Specialized Case Studies

Chapter 18

Drought and Implications for Nutrition Eric Ober and Martin A.J. Parry

Abstract The uptake of mineral nutrients by plants depends on the dissociation of ions in the soil solution and movement to the root surface. Therefore, sufﬁcient soil water is critical for adequate plant nutrition. Although in many regions crops are grown under water-limited conditions and drought frequently causes yield losses, nutrient uptake usually keeps pace with the dry matter, which stabilizes the mineral nutrient concentrations in plant tissues. However, there are conditions when this homeostasis is disturbed. These situations provide insight into the regulation of nutrient balance and the role of nutrients in maintaining plant function, particularly under stress. There are new and exciting developments in the area of root-to-shoot communication involving nitrate, regulation of hydraulic conductivity, and stomatal function via abscisic acid (ABA) and K+, and genetic control of root system architecture. Further examination of how nutrient concentrations, growth, and water use are governed and regulated at the molecular level may provide avenues for further crop improvement.

Drought and global food production Plant productivity is primarily limited by the availability of resources (light, CO2, nutrients, and water) and by biotic and abiotic stresses. In agricultural systems, nutrients are supplied to the plants, but in natural systems, availability depends on their abundance in soils, water, and the atmosphere. The uptake of mineral nutrients by plants depends on the dissociation of ions in the soil solution and movement to the root surface. Therefore, sufﬁcient soil water is critical for adequate plant nutrition. The availability of water is a major determinant of plant productivity; this is relevant to food security because a signiﬁcant proportion of crop production worldwide takes place under water-limited conditions (Baldocchi and Valentini, 2004; Eriyagama et al., 2009). Drought causes more yield losses than any other single biotic or abiotic factor (Boyer, 1982). Therefore, plants that are subjected to water deﬁcit can be also nutrient deﬁcient, and nutrient deﬁcient plants often can be more susceptible to drought. However, as the degree of drought stress is not constant

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 431

432

NUTRIENT USE EFFICIENCY IN CROPS

and both the occurrence and severity can vary considerably over the life of a plant, the interactions with nutrition are complex and are affected both by environment and agronomic practice (Parry et al., 2005). For example, when growing cereals where water is limited, the addition of too much early nitrogen can lead to the establishment of a large canopy that cannot be supported by the available water and thus will lead to total crop failure, whereas with the same amount of available water, a later nitrogen application can secure quality and yield. Addressing the challenge of water availability to food production today and for a future world, which in many parts will be warmer and drier (Gornall et al., 2010), is a key research priority for disciplines ranging from agronomy to biotechnology. In this chapter the interactions between drought and plant nutrition are examined, emphasizing the situation for arable crops. Rather than a comprehensive review of the literature, which has been done elsewhere (Viets, 1972; Alam, 1999; Hu and Schmidhalter, 2005; Fleury et al., 2010), a general overview and highlight of certain areas that may provide avenues for crop improvement is provided. Interactions between water and ion ﬂuxes in soils and roots Nutrient uptake depends on soil processes that occur in the rhizosphere and plant processes that occur within the root and shoot. In addition to soil moisture, the availability of mineral nutrients for uptake by roots depends on a wide range of soil conditions including pH, cation exchange capacity, redox potential, soil depth, organic matter content, microﬂora, type and proportion of clay content, and fertilizer application (Marschner, 1995). The rhizosphere is a heterogeneous environment in time and space:

Soil texture and physical structure varies, particularly with depth; the spatial distribution of nutrients and nutrient concentrations can vary enormously; and the occurrence of soil organisms, from earthworms to bacteria, is also variable. Not surprisingly, the excavation of root systems in this subterranean environment often reveals a complex and heterogeneous pattern, the result of root growth responses to local soil conditions and the physiology of the plant itself. Nutrient uptake is related to root biomass (Ehdaie et al., 2010). Roots must arrive where nutrients are located, and nutrients must be able to move into the root. Root growth is affected by supply of assimilates from the shoot and soil physical conditions such as moisture supply, pH, temperature, aeration, and penetration resistance (Waisel et al., 2002; Gregory, 2006). Soils also harden as they dry, so these constraints to root growth interact (Whalley et al., 2006; Whitmore and Whalley, 2009). However, useful variation in the ability of roots to penetrate hard soils has been identiﬁed (Clark et al., 2008). Root growth can also be inhibited in saline soils or soils high in aluminum or other toxic elements, and certain allelopathic organic compounds also restrict growth (Waisel et al., 2002). Mineral nutrients move to the root surface by a combination of mass ﬂow and diffusion (Kramer and Boyer, 1995). Mass ﬂow in fully drained soil occurs when soil water is taken up by roots as plants transpire. This process transports nutrients dissolved in soil solution regardless of concentration gradients present at the root–soil interface. However, when transpiration rates are low, concentration gradient-driven diffusion dominates movement of nutrients in the root–soil interface. Diffusion is slow compared with mass ﬂow, so the ﬂux of water from soil through the plant into the atmosphere in the transpiration stream has a large inﬂuence on nutrient delivery to the root

DROUGHT AND IMPLICATIONS FOR NUTRITION

surface. Water and ion uptake by roots depends on root anatomy, age, and type, and is affected by suberin deposition, and the density and activity of ion transporters (Baxter et al., 2009). Work using seedling roots or excised root segments show that ﬂuxes of ion and water uptake vary with distance from the root apex, but because of technical difﬁculties there is comparatively little data on ion and water uptake in relation to root structure in mature, intact root systems (Varney and Canny, 1993; McCully, 1995; Peterson et al., 1999; Steudle, 2004). Processes governing the uptake and transport of nutrients by roots are covered elsewhere in this volume (see Chapters 2, 10, and 12). Nutrient-limited plants can exhibit decreased rates of photosynthesis, respiration, and growth. With less assimilate translocated from shoots to roots, the low energy status of roots limits the ability of the roots to supply nutrients to the shoot. Cell expansion and ion uptake that occurs against electrical and/or concentration gradients are processes that require energy. Thus, plants with nutrient deﬁciencies of any element, not surprisingly, are often more susceptible to drought because of shallow or weak root systems. In addition, nutrient-starved plants (a rare occurrence in most agricultural systems) show decreased rates of water uptake due to decreased solute potentials in root cells (Clarkson et al., 2000). However, it is interesting that comparisons of tissues of plants subjected to water deﬁcit or irrigated conditions often show small differences in nutrient concentrations. This is because nutrient delivery to the shoot is coupled to dry matter production: decreased demand due to reduced biomass production slows nutrient uptake (Kramer and Boyer, 1995). Under optimum conditions, bulk ﬂow is capable of supplying the demand for nitrogen, calcium, and magnesium, whereas diffusion supplies phosphorus. The sufﬁciency of bulk ﬂow for

433

potassium delivery may depend on soil availability. Therefore, due to this coupling of nutrient delivery with demand resulting from prevailing rates of dry matter accumulation, water deﬁcit has not been observed to have drastic effects on mineral nutrient concentrations in plant tissues. An example of one exception to this general observation is the occurrence of pale green leaves of cereals during water deﬁcits in the spring in the United Kingdom (Fig. 18.1). The low nitrogen status of the crop is most likely due to pellets of NH4NO3 that remain on the dry soil surface and do not become solubilized. However, plant growth continues because roots can access moisture deeper in the soil proﬁle, and plants already have taken up much of the available nitrogen. Thus, tissue expansion outpaces nitrogen uptake and dilutes tissue nitrogen levels, resulting in the pale appearance. Another example is magnesium deﬁciency symptoms that appear in leaves of sugar beet subjected to long-term water deﬁcit (Fig. 18.2). Lower magnesium concentrations were also observed in water-limited wheat plants (Hu et al., 2006). It is probable that water uptake from deep in the soil proﬁle, where nutrient concentrations are very low, sustains some leaf growth while magnesium uptake in fertilized upper soil layers is minimal because of low water content and fewer living roots. However, foliar applications of nutrients, at least in part, may overcome these deﬁciencies. At high transpiration rates, sustained by open stomata and sufﬁcient soil water availability, water ﬂux into roots may exceed rates of ion uptake. This can result in accumulation of ions outside the root surface and dilution of ions in the xylem stream. This is because ion transport at the root cell membrane is governed by energy-dependent processes that are independent of water movement across the membrane. When transpiration rates are low because stomata close in response to water deﬁcit, ion concentrations

434

NUTRIENT USE EFFICIENCY IN CROPS

A

B

Fig. 18.1. (A) Poor nitrogen uptake in rain-fed, water-limited plots of winter wheat (foreground) compared with

darker green irrigated plots (background) during early growth in spring. (B) Plot strips of winter wheat showing differential nitrogen uptake under irrigated (darker green outer strips with drip tape) compared with rain-fed, waterlimited plants in the center of the tunnel structures.

can be depleted at the root–soil interface because ion transport continues unabated even though water ﬂux is diminished. This steepens the concentration gradient, driving increased diffusion from the outer soil solution to the root surface. Another result is that ion concentrations in the xylem can increase considerably because of decreased dilution by water in the transpiration stream.

Aquaporins play an important role in water transport into and out of roots, but their contribution to ion uptake and plant nutrition remains uncertain. However, there is evidence that water channels can mediate uptake of boron and silicon in the form of silicic acid (Clarkson et al., 2000; Maurel et al., 2008). Root hydraulic conductivity, perhaps inﬂuenced by aquaporins, appeared

DROUGHT AND IMPLICATIONS FOR NUTRITION

A

B

(A) Magnesium deﬁciency symptoms in drought-stressed sugar beet. (B) Note the more advanced necrosis and scorching in older leaves. Irrigated plants in nearby plots did not show symptoms.

Fig. 18.2.

to be greater when nitrogen was supplied in the form of NO3− compared with NH4+ (Guo et al., 2002). Root growth under dry conditions Roots continue growing at water potentials that are completely inhibitory to shoot growth (Sharp et al., 2004). The regulation of this phenomenon involves complex interactions between abscisic acid (ABA), ethylene, and cell-wall loosening enzymes sensitive to the redox potential of the apoplast. The net result is that biomass is partitioned in favor of roots over shoots, increasing the shoot to root ratio, limiting water loss to the atmosphere, and maintaining the exploration of soil. This adaptive

435

response helps maintain plant water and nutrient status in challenging environments. Soil drying also causes soils to harden: Increased ability of roots to penetrate hard and compact soils would provide greater access to soil that contains unexploited moisture and nutrients (Whitmore and Whalley, 2009; White and Kirkegaard, 2010). The spatial arrangements of roots, or root system architecture, results from the differential growth of roots in response to local soil conditions. Nutrients and water are often distributed unevenly in the soil proﬁle, and roots exhibit “plastic” behavior that allows plants to exploit these resource-rich patches surrounded by soil relatively depleted in nutrients or water (Ho et al., 2005; Hodge, 2006; Walk et al., 2006). The ability of roots to forage local patches high in nitrogen or phosphorus is well documented, and there is some evidence that roots may exhibit hydrotropism (Ober and Sharp, 2007). There is comparatively less information on how root architecture interacts with multiple conditions, such as patches of moisture and nutrients that are not necessarily colocated. A comparison of two maize cultivars showed different strategies of root placement and investment under combined nitrogen-limiting and waterdeﬁcit conditions (Vamerali et al., 2003). Despite differences in plasticity of root growth, both cultivars showed similar yields. One study of oilseed rape, employing a compartmentalized pot system that permitted application of nutrients and water to isolated sections of the root system, found some unexpected results (Wang et al., 2007). Root growth was greater in sectors given more water or nutrients. However, application of fertilizer in patches resulted in greater soil water extraction from the nonfertilized sectors than from the fertilized sectors, even though root density was greatest in the fertilized sectors. One explanation is that nutrient

436

NUTRIENT USE EFFICIENCY IN CROPS

uptake was high in fertilized sectors but water uptake was relatively low, forcing the balance of water demand to be sourced from the nonfertilized sectors. This again illustrates the predominance of active uptake of ions at the membrane level irrespective of mass ﬂow rates of soil water. These results suggest that foraging for water by roots is stronger than nutrient foraging and that this level of water deﬁcit did not impair the ability to obtain nutrients. In certain cases, partial root zone drying (PRD) or deﬁcit irrigation could be a practical management technique to optimize water and nutrient use efﬁciencies and quality characteristics of harvested parts (Sadras, 2009). Nitrate as a root signal controlling water use

ABA ↑ Cytokinins ↓

ABA ↓ Cytokinins ↑

ABA ↑ Cytokinins ↓?

Xylem/apoplastic sap pH

Leaf growth rate and stomatal conductance

One of the key elements of PRD is that roots in drying soils produce chemical signals that enter the transpiration stream and affect

shoot growth and stomatal behavior (Davies et al., 2005). ABA is synthesized in roots at low water potentials and translocated to shoots where it causes stomata to close, decreasing transpiration and photosynthetic rates. Alkalization of xylem pH under waterdeﬁcit conditions also alters the compartmentation of ABA in leaf tissues, allowing more ABA to be partitioned to the apoplast around stomatal guard cells. Cytokinins also play a role in root-to-shoot signaling (Davies et al., 2005). A fourth root-sourced signal that interacts with ABA, cytokinins, and pH is nitrate (Wilkinson et al., 2007; Fig. 18.3). Uptake of nitrate from the apoplast requires H+ cotransport, which effectively increases xylem pH. This mechanism can begin to explain observations that nitrogen deﬁciency can cause stomatal closure (McDonald and Davies, 1996). The effect of drying soil on xylem nitrate concentrations depends not only on the rate of uptake of nitrate from the soil but also on the degree to which nitrate

DEFICIENT

OPTIMAL

SUPRA-OPTIMAL

Xylem sap nitrate concentration Schematic representation of the interactions between xylem nitrate and other root-sourced signals in the regulation of shoot transpiration and growth. Reprinted with permission from Wilkinson et al. (2007).

Fig. 18.3.

DROUGHT AND IMPLICATIONS FOR NUTRITION

in the xylem is diluted by ﬂux of water into the root and the activity of the assimilatory pathway, speciﬁcally nitrate reductase. Nitrate reductase reduces nitrate to nitrite, which is then converted to NH3 for assimilation into glutamine. Transcriptional and posttranscriptional control of nitrate reductase expression and activity in response to a range of internal and external cues is complex. It is relevant that decreased activity is associated with low tissue water potentials, most likely in response to decreased adenosine triphosphate/ adenosine monophosphate (ATP/AMP) ratios resulting from decreased rates of photosynthesis under stress conditions (Fresneau et al., 2007). In summary, recent evidence demonstrates molecular control points governing the interplay between growth, transpiration, plant nutritional, and water status. Further research may elucidate how these targets could be manipulated to improve crop performance under nutrient- and water-limited conditions.

437

may be indirect effects related to better phosphorus status of plants, and greater water extraction from soil could be due to greater leaf area of phosphorus-improved plants. There is increasing evidence that VAM association with roots can alter expression and activity of water channels (aquaporins) in root cell membranes that modulate the hydraulic conductivity of roots (Maurel et al., 2008). A study of southern beech tree seedlings showed that two species of ectomycorrhyzae contributed marginal improvements to dry matter accumulation under well-watered conditions, but provided a signiﬁcant advantage under water deﬁcit compared with noninoculated plants (Alvarez et al., 2009). The beneﬁcial effect appeared to be through enhanced activity of assimilative enzymes in the infected roots. This is a complex area that needs further research, but there appears to be potential to improve both nutrition and drought tolerance simultaneously through root–fungi associations.

Mycorrhiza There is a substantial volume of literature that records the beneﬁcial effects of root associations with endotrophic (vesicular arbuscular mycorrhizae [VAM], predominantly Glomus spp.) and ectotrophic fungi (Smith and Read, 2008) (Chapter 3). Hyphae extend into the soil and increase solubilization, particularly of phosphorus, and the surface area for absorption (Chapter 12). However, the evidence that mycorrhizal fungi make a positive contribution to the water economy of plants is mixed (Augé, 2001). For example, drought tolerance in pot-grown maize was improved in VAM plants over non-VAM plants (Boomsma and Vyn, 2008), but there were negative interactions between VAM and Rhizobia in Phaseolus under drought conditions (Franzini et al., 2010). The delayed effects of water deﬁcit in VAM-colonized plants

Individual nutrients and plant water relations All the essential nutrients play important roles in maintaining productivity during periods of water deﬁcit. Potassium is a key solute used in the stomatal complex for guard cell opening and closing (MacRobbie, 2006), and for osmotic adjustment in the growing zone of roots at low water potentials (Sharp et al., 1990). Many processes from photonastic leaf movements to phloem loading require K+ as a counterion and for osmotic balance (Marschner, 1995). The potassium concentration in roots affects tissue sensitivity to ABA, which is necessary for root growth maintenance (Sharp et al., 2004), the hyperpolarization response of root cells to decreasing water potentials (Ober and Sharp, 2003), and regulation of root hydraulic conductivity (Quintero et al.,

438

NUTRIENT USE EFFICIENCY IN CROPS

1998). These processes are important during drought conditions but are probably compromised only under extreme conditions of potassium deﬁciency. Nevertheless, high potassium status has been shown to maintain photosynthetic rates during water deﬁcits (Pier and Berkowitz, 1987), probably related to maintenance of high stromal pH in the chloroplast (Gupta et al., 1989). When electron transfer through photosystem II is inhibited during stress conditions, the ability of leaf tissues to handle excess energy and the production of reactive oxygen species is necessary to avoid tissue damage. Under these conditions, the potassium status of leaf tissue was shown to be an important part of the protection mechanism (Cakmak, 2005). Magnesium also affects ion balance and K+ transport across the stromal membrane (Gupta et al., 1989; Berkowitz and Wu, 1993). High magnesium concentrations in chloroplasts can inhibit photophosphorylation via ATPase (Younis et al., 1983; Lawlor and Tezara, 2009). Phosphorous nutrition inﬂuenced photosynthetic rates in moth bean (Garg et al., 2004) and improved growth in soybean under water-limited conditions (Jin et al., 2006). A potential difﬁculty with drought and phosphorus interactions is that root architecture for better drought tolerance (e.g., deep roots) is at odds with the best spatial arrangement for phosphorus acquisition (high density of shallow roots). Plants must balance these trade-offs to optimize biomass allocation to realize the best performance (Walk et al., 2006), which is measured in terms of yield in agricultural settings, or overall ﬁtness in the wild.

there is considerable natural variation in how plants and even cultivars respond to the availability of nutrients and use water, which can be exploited in crop improvement (Barraclough et al., 2010). Both nutrient and water use efﬁciency are complex, quantitative traits that involve multiple genes. However, considerable progress has already been made in deconvoluting these complex traits and also in identifying variation in components traits (e.g., root traits—Clark et al., 2008; Courtois et al., 2009; leaf traits—Khowaja et al., 2009; Xu et al., 2009). Important component traits may have direct effects on the uptake and use of water or nutrients, or affect these processes indirectly, for example, by altering phenology: In some environments, a shorter life cycle may enable a crop to avoid water limitation entirely. Thus, such traits may contribute to yield increases if strategically targeted and effectively selected for (Richards et al., 2010). For example, carbon isotope discrimination by leaves is negatively correlated with transpiration efﬁciency, and low carbon isotope discrimination is being used to identify high water use efﬁciency and yield in rain-fed environments. This has led to the release of commercial wheat cultivars such as “Drysdale.” The individual component traits may be not be determined by multiple but by single genes, which by themselves can be used in marker-assisted breeding strategies (Kuchel et al., 2007) or used to identify novel alleles by TILLING (Parry et al., 2009) or exploited in transgenic approaches (Shrawat et al. 2008; Bi et al. 2009; Saad et al. 2010.

Crop improvement

Conclusion

There is no perfect crop ideotype for all environments; thus, for crop improvement it is necessary to identify speciﬁc ideotypes for speciﬁc crops and environments. Fortunately,

The acquisition of mineral nutrients and water from the soil are intimately interconnected. There have been numerous studies examining the interactions between the

DROUGHT AND IMPLICATIONS FOR NUTRITION

nutritional status of plants and their relative susceptibility to drought conditions, but many of these have compared plants grown under extreme deﬁciency, which may not reﬂect realistic situations in most ﬁeld environments. In general, within the normal range of soil nutrient availability, nutrient uptake keeps pace with the dry matter accumulation of plants, governed by the supply of water, which stabilizes the mineral nutrient concentrations in plant tissues. However, there are conditions when this homeostasis is disturbed. These situations provide insight into the regulation of nutrient balance and the role of nutrients in maintaining plant function, particularly under stress. There are new and exciting developments in the area of root-to-shoot communication involving nitrate, regulation of hydraulic conductivity and stomatal function via ABA and K+, and genetic control of root system architecture. Further examination of how nutrient concentrations, growth, and water use are governed and regulated at the molecular level may provide avenues for further crop improvement. Acknowledgments Rothamsted Research is an institute of the Biotechnology and Biological Sciences Research Council of the United Kingdom. The authors’ research is also supported by DEFRA (An integrated approach to increasing water use efﬁciency and drought tolerance of wheat production in the UK), the European Commission (OPTIWHEAT— Improving the Yield Stability of Durum Wheat under Mediterranean Conditions (EC Contract Number: INCO-CT-2006015460), and the British Beet Research Organisation. References Alam, S.M. (1999) Nutrient uptake by plants under stress conditions. In: Handbook of Plant and Crop

439

Stress (ed. M. Pessarakli), pp. 285–314. Marcel Dekker, New York. Alvarez, M., Huygens, D., Olivares, E., et al. (2009) Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities. Physiologia Plantarum 136, 426–436. Augé, R.M. (2001) Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza 11, 3–42. Baldocchi, D. & Valentini, R. (2004) Geographic and temporal variation of carbon exchange by ecosystems and their sensitivity to environmental perturbations. In: The Global Carbon Cycle: Integrating Humans, Climate, and the Natural World (eds. C.B. Field & M.R. Raupach), pp. 295–316. Island Press, Washington DC. Barraclough, P.B., Howarth, J.R., Jones, J., et al. (2010) Nitrogen efﬁciency of wheat: genotypic and environmental variation and prospects for improvement. European Journal of Agronomy 33, 1–11. Baxter, I., Hosmani, P., Rus, A., et al. (2009) Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis. PLoS Genetics 5, e1000492. Berkowitz, G.A. & Wu, W. (1993) Magnesium, potassium ﬂux and photosynthesis. Magnesium Research 6, 257–265. Bi, Y.M., Kant, S., Clarke, J., et al. (2009) Increased nitrogen-use efﬁciency in transgenic rice plants over-expressing a nitrogen-responsive early nodulin gene identiﬁed from rice expression proﬁling. Plant Cell Environment 32, 1749–1760. Boomsma, C.R. & Vyn, T.J. (2008) Maize drought tolerance: potential improvements through arbuscular mycorrhizal symbiosis? Field Crops Research 108, 14–31. Boyer, J.S. (1982) Plant productivity and environment. Science 218, 443–448. Cakmak, I. (2005) The role of potassium in alleviating detrimental effects of abiotic stresses in plants. Journal of Plant Nutrition and Soil Science 168, 521–530. Clark, L.J., Price, A.H., Steele, K.A., et al. (2008) Evidence from near-isogenic lines that root penetration increases with root diameter and bending stiffness in rice. Functional Plant Biology 35, 1163–1171. Clarkson, D.T., Carvajal, M., Henzler, T., et al. (2000) Root hydraulic conductance: diurnal aquaporin expression and the effects of nutrient stress. Journal of Experimental Botany 51, 61–70. Courtois, B., Ahmadi, N., Khowaja, F., et al. (2009) Rice root genetic architecture: meta-analysis from

440

NUTRIENT USE EFFICIENCY IN CROPS

a QTL database improves resolution to a few candidate genes. Rice 2, 115–128. Davies, W., Kudoyarova, G., & Hartung, W. (2005) Long-distance ABA signaling and its relation to other signaling pathways in the detection of soil drying and the mediation of the plant’s response to drought. Journal of Plant Growth Regulation 24, 285–295. Ehdaie, B., Merhaut, D.J., Ahmadian, S., et al. (2010) Root system size inﬂuences water-nutrient uptake and nitrate leaching potential in wheat. Journal of Agronomy and Crop Science 196, 455–466. Eriyagama, N., Smakhtin, V., & Gamage, N. (2009) Mapping drought patterns and impacts: a global perspective. (IWMI Research Report 133). International Water Management Institute, Colombo, Sri Lanka. Fleury, D., Jefferies, S., Kuchel, H., et al. (2010) Genetic and genomic tools to improve drought tolerance in wheat. Journal of Experimental Botany 61, 3221–3222. Franzini, I.V., Azcon, R., Latanze, M.F., & Aroca, R. (2010) Interactions between Glomus species and Rhizobium strains affect the nutritional physiology of drought-stressed legume hosts. Journal of Plant Physiology 167, 614–619. Fresneau, C., Ghashghaie, J., & Cornic, G. (2007) Drought effect on nitrate reductase and sucrosephosphate synthase activities in wheat (Triticum durum L.): role of leaf internal CO2. Journal of Experimental Botany 58, 2983–2992. Garg, B.K., Burman, U., & Kathju, S. (2004) The inﬂuence of phosphorus nutrition on the physiological response of moth bean genotypes to drought. Journal of Plant Nutrition and Soil Science 167, 503–508. Gornall, J., Betts, R., & Wiltshire, A. (2010). Impacts of climate changes on crop and livestock production. Philosophical Transactions of the Royal Society B Biological Sciences 365, 2973–2989. Gregory, P.J. (2006) Plant Roots: Growth, Activity and Interactions with the Soil. Wiley-Blackwell, London. Guo, S., Brück, H., & Sattelmacher, B. (2002) Effects of supplied nitrogen form on growth and water uptake of French bean (Phaseolus vulgaris L.) plants. Plant and Soil 239, 267–275. Gupta, A.S., Berkowitz, G.A., & Pier, P.A. (1989) Maintenance of photosynthesis at low leaf water potential in wheat: role of potassium status and irrigation history. Plant Physiology 89, 1358– 1365. Ho, M.D., Rosas, J.C., Brown, K.M., et al. (2005) Root architectural tradeoffs for water and phosphorus acquisition. Functional Plant Biology 32, 737–748.

Hodge, A. (2006) Plastic plants and patchy soils. Journal of Experimental Botany 57, 401–411. Hu, Y. & Schmidhalter, U. (2005) Drought and salinity: a comparison of their effects on mineral nutrition of plants. Journal of Plant Nutrition and Soil Science 168, 541–549. Hu, Y., Burucs, Z., & Schmidhalter, U. (2006) Shortterm effect of drought and salinity on growth and mineral elements in wheat seedlings. Journal of Plant Nutrition 29, 2227–2243. Jin, J., Wang, G., Liu, X., et al. (2006) Interaction between phosphorus nutrition and drought on grain yield, and assimilation of phosphorus and nitrogen in two soybean cultivars differing in protein concentration in grains. Journal of Plant Nutrition 29, 1433–1449. Khowaja, F.S., Norton, N.J., Courtois, B., et al. (2009) Improved resolution in the position of droughtrelated QTLs in a single mapping population of rice by meta-analysis. BMC Genomics 10, 276. Kramer, P.J. & Boyer, J.S. (1995) Water Relations of Plants and Soils. Academic Press, London. Kuchel, H., Fox, R., Reinheimer, J., Mosionek, L., et al. (2007) The successful application of a markerassisted wheat breeding strategy. Molecular Breeding 20, 295–308. Lawlor, D.W. & Tezara, W. (2009) Causes of decreased photosynthetic rate and metabolic capacity in waterdeﬁcient leaf cells: a critical evaluation of mechanisms and integration of processes. Annals of Botany 103, 561–579. MacRobbie, E.A.C. (2006) Osmotic effects on vacuolar ion release in guard cells. Proceedings of the National Academy of Sciences of the United States of America 103, 1135–1140. Marschner, H. (1995) Mineral Nutrition of Higher Plants. Academic Press, London. Maurel, C., Verdoucq, L., Luu, D.-T., et al. (2008) Plant aquaporins: membrane channels with multiple integrated functions. Annual Review of Plant Biology 59, 595–624. McCully, M.E. (1995) How do real roots work? Some new views of root structure. Plant Physiology 109, 1–6. McDonald, A.J.S. & Davies, W.J. (1996) Keeping in touch: responses of the whole plant to deﬁcits in water and nitrogen supply. Advances in Botanical Research 22, 229–300. Ober, E.S. & Sharp, R.E. (2003) Electrophysiological responses of maize roots to low water potentials: relationship to growth and ABA accumulation. Journal of Experimental Botany 54, 813–824. Ober, E.S. & Sharp, R.E. (2007) Regulation of root growth responses to water deﬁcit. In: Advances in Molecular-Breeding toward Drought and Salt

DROUGHT AND IMPLICATIONS FOR NUTRITION

Tolerant Crops (eds. M.A. Jenks, P.M. Hasegawa & S.M. Jain), pp. 33–53. Springer, London. Parry, M.A.J., Flexas, J., & Medrano, H. (2005) Prospects for crop production under drought: research priorities and future directions. Annals of Applied Biology 147, 211–226. Parry, M.A.J., Madgwick, P.J., Bayon, C., et al. (2009) Mutation discovery for crop improvement. Journal of Experimental Bot 60, 2817–2825. Peterson, C., Enstone, D., & Taylor, J. (1999) Pine root structure and its potential signiﬁcance for root function. Plant and Soil 217, 205–213. Pier, P.A. & Berkowitz, G.A. (1987) Modulation of water stress effects on photosynthesis by altered leaf K+. Plant Physiology. 85, 655–661. Quintero, J.M., Fournier, J.M., Ramos, J., et al. (1998) K+ status and ABA affect both exudation rate and hydraulic conductivity in sunﬂower roots. Physiologia Plantarum 102, 279–284. Richards, R.A., Rebetzke, G.J., Watt, M., et al. (2010) Breeding for improved water productivity in temperate cereals: phenotyping, quantitative trait loci, markers and the selection environment. Functional Plant Biology 37, 85–97. Saad, R.B., Zouari, N., Ramdhan, W.B., et al. (2010) Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-ﬁnger “AlSAP” gene isolated from the halophyte grass Aeluropus littoralis. Plant Molecular Biology 72, 171–190. Sadras, V. (2009) Does partial root-zone drying improve irrigation water productivity in the ﬁeld? A metaanalysis. Irrigation Science 27, 183–190. Sharp, R.E., Hsiao, T.C., & Silk, W.K. (1990) Growth of the maize primary root at low water potentials. II. Role of growth and deposition of hexose and potassium in osmotic adjustment. Plant Physiology 93, 1337–1346. Sharp, R.E., Poroyko, V., Hejlek, L.G., et al. (2004) Root growth maintenance during water deﬁcits: physiology to functional genomics. Journal of Experimental Botany 55, 2343–2351. Shrawat, A.K., Carroll, R.T., DePauw, M., et al. (2008) Genetic engineering of improved nitrogen use efﬁciency in rice by the tissue-speciﬁc expression of alanine aminotransferase. Plant Biotechnology Journal 6, 722–732. Smith, S.E. & Read, D.J. (2008) Mycorrhizal Symbioses, 3rd ed. Elsevier, London.

441

Steudle, E. (2004) Water uptake by plant roots: an integration of views. Acta Physiologiae Plantarum 26, 77–77. Vamerali, T., Saccomani, M., Bona, S., et al. (2003) A comparison of root characteristics in relation to nutrient and water stress in two maize hybrids. Plant and Soil 255, 157–167. Varney, G.T. & Canny, M.J. (1993) Rates of water uptake into the mature root system of maize plants. The New Phytologist 123, 775–786. Viets, F.G. Jr (1972) Water deﬁcits and nutrient availability. In: Water Deﬁcits and Plant Growth Vol. III: Plant Responses and Control of Water Balance (ed. T.T. Kozlowski), p. 217. Academic Press, New York. Waisel, Y., Eshel, Y., & Kafkaﬁ, U. (2002) Plant Roots: The Hidden Half, 3rd ed. Marcel Dekker, New York. Walk, T.C., Jaramillo, R., & Lynch, J.P. (2006) Architectural tradeoffs between adventitious and basal roots for phosphorus acquisition. Plant and Soil 279, 347–366. Wang, L., de Kroon, H., & Smits, A. (2007) Combined effects of partial root drying and patchy fertilizer placement on nutrient acquisition and growth of oilseed rape. Plant and Soil 295, 207–216. Whalley, W., Clark, L., Gowing, D., et al. (2006) Does soil strength play a role in wheat yield losses caused by soil drying? Plant and Soil 280, 279–290. White, R.G. & Kirkegaard, J.A. (2010) The distribution and abundance of wheat roots in a dense, structured subsoil—implications for water uptake. Plant Cell and Environment 33, 133–148. Whitmore, A.P. & Whalley, W.R. (2009) Physical effects of soil drying on roots and crop growth. Journal of Experimental Botany 60, 2845–2857. Wilkinson, S., Bacon, M., & Davies, W.J. (2007) Nitrate signalling to stomata and growing leaves: interactions with soil drying, ABA, and xylem sap pH in maize. Journal of Experimental Botany 58, 1705–1716. Xu, Y., This, D., Pausch, R.C., et al. (2009) Leaf-level water use efﬁciency determined by carbon isotope discrimination in rice seedlings: genetic variation associated with population structure and QTL mapping. Theoretical and Applied Genetics 118, 1065–1081. Younis, H.M., Weber, G., & Boyer, J.S. (1983) Activity and conformational changes in chloroplast coupling factor induced by ion binding. Formation of a magnesium enzyme phosphate complex. Biochemistry 22, 2505–2512.

Chapter 19

Salt Resistance of Crop Plants: Physiological Characterization of a Multigenic Trait Sven Schubert

Abstract Soil salinity is an increasing hazard for worldwide crop production. Since highquality water for leaching of salts in semiarid and arid areas is scarce, the possibilities for soil amelioration are limited. Thus, efforts are made to improve the salt resistance of crop plants genetically. This requires a profound understanding of the physiological processes that limit plant growth and yield formation. The development of a biphasic model of growth response under salt stress has allowed the separation of osmotic resistance in a ﬁrst phase from ion toxicity in a second phase. Generally, osmotic resistance is not limited by osmotic adjustment and turgor maintenance but by low cell-wall extensibility. Various sodium exclusion strategies as well as sodium inclusion in vacuoles contribute to salt resistance in the second phase. In the generative phase of cereals, kernel set seems to be the key bottleneck for yield formation under salt stress. Introduction Soil salinity is an increasing hazard in many arid and semi-arid regions of the world. When evaporation exceeds precipitation,

dissolved salts move to the upper soil layers, where they accumulate and impair the growth of crop plants. Worldwide nearly 109 ha of land are affected (Szabolcs, 1994), and this situation is being aggravated by improper cultivation practices: Speciﬁcally irrigation may contribute to the import of salts. It is estimated that currently 20% of irrigated lands are affected by salinity problems (Qadir et al., 2006). Although excess irrigation may help to ameliorate soils via the leaching of salts, under poor drainage conditions inappropriate irrigation may also cause additional problems, known as secondary salinity (Szabolcs, 1994). Saline soils are deﬁned as those soils that have an electrical conductivity of at least 4 dS m−1 in the saturation extract. Soils with an exchangeable sodium percentage of more than 15 are classiﬁed as sodic soils (Qadir et al., 2007). Saline–sodic soils suffer both problems and require proper amelioration measures to ensure optimal crop production (Qadir et al., 2001). Plant growth and development are directly affected by salt accumulation and also result from negative effects of salts on soil structure. Depending on the parent soil material, speciﬁc nutrient problems may result in addition: such examples

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 443

444

NUTRIENT USE EFFICIENCY IN CROPS

are toxicities of boron in Turkey, selenium in Southern California, or arsenic in Bangladesh. Ions with a large hydration shell such as sodium, and to some extent also magnesium, may compete with calcium for binding sites on soil colloids when present at high concentrations. Displacement of calcium leads to peptization and impairment of soil structure and as a result rooting and root aerobic metabolism may be inhibited. This in turn inhibits the acquisition of water and nutrients (Qadir and Schubert, 2002), sodium exclusion particularly from shoots (Drew and Läuchli, 1985), and N2 ﬁxation of legumes as a result of insufﬁcient nodulation (Arrese-Igor et al., 1993). These problems are encountered particularly when stagnant moisture develops simultaneously, and require sophisticated soil-amelioration practices (Qadir et al., 2007) or combined salt and waterlogging resistance (Teakle et al., 2010). Amelioration of saline or saline–sodic soils depends on the application of huge amounts of good-quality water, which is scarce in the affected areas. Therefore, the need to improve the salt resistance of crop plants genetically was suggested long ago (Epstein et al., 1980). Despite tremendous scientiﬁc efforts to enhance salt resistance, only moderate progress has been achieved so far (Rozema and Flowers, 2008). This is due to the fact that salt resistance is a multigenic trait that is still poorly understood. In this chapter, important physiological characters are highlighted that contribute to salt resistance. It will be shown that a profound physiological understanding of salt stress is required to improve salt resistance of crop plants, either by means of classical breeding methods and/or by molecular techniques. Although in the international literature the term “salt tolerance” is predominantly used, following Levitt’s deﬁnition (Levitt, 1980), the more general term “salt resistance” is given preference here because avoidance

rather than true tolerance strategies allow glycophytic crop plants to overcome salt stress in most cases. The two-phase model of salt stress An important reason for negligible progress in the development of salt-resistant crops until the beginning of this century was a poor understanding of why salt stress inhibits plant growth. Although detrimental effects of ion toxicity on plant growth were generally assumed, it was difﬁcult to ﬁnd evidence of speciﬁc ion toxicity. For example, no correlation was found between sodium exclusion and salt resistance (Lessani and Marschner, 1978; Schubert and Läuchli, 1986) and the speciﬁc toxicity of sodium was questioned (Cramer et al., 1990). In addition, osmotic resistance and a lack of turgor apparently did not limit plant growth under salt stress (Termaat et al., 1985; Munns, 1988). A big breakthrough was achieved with the postulation of the biphasic model of growth response under salt stress (Munns, 1993). According to this model, salt stress in a ﬁrst phase limits plant growth due to osmotic effects, whereas in a second phase, ions accumulate and lead to ion toxicity (Fig. 19.1). Although being simple, this model proved to be very helpful in identifying important physiological shortcomings during salt stress and formed a basis for progress in the development of salt-resistant wheat and maize genotypes (Xue et al., 2004; Munns et al., 2006; Schubert et al., 2009). Recently, genetically improved breeding lines of durum wheat were tested that demonstrated 25% outperformance in ﬁeld trials under saline conditions (R. Munns, pers. comm.). The model originally developed for wheat was conﬁrmed for maize by Fortmeier and Schubert (1995), who showed that

SALT RESISTANCE OF CROP PLANTS

NaCl Growthrate t

Resistant genotype

Sensitive genotype

Phase I

Phase II

Time Fig. 19.1. Model of the biphasic growth response of

plants under salt stress. Phase I is characterized by osmotic stress, and phase II by ion toxicity (modiﬁed scheme, based on Munns, 1993).

sodium and not chloride is the toxic ion in the second phase of salt stress. This seems to be a general phenomenon in grasses and probably also in other plants. One problem of the model is the differentiation of the two phases by means of the development of toxicity symptoms. In a detailed study, Sümer et al. (2004) showed that ion toxicity comes into play in the ﬁrst phase of salt stress, although the quantitative effects are negligible. On the other hand, potassium deﬁciency as a result of sodium competition with potassium during uptake, translocation, and physiological action was ruled out in that study. In the original model, it was proposed that genotypic differences in salt resistance occur in the second phase of salt stress (Munns, 1993). Numerous experimental data support this statement (e.g., Lauter and Munns, 1986, Schachtman and Munns, 1992; Fortmeier and Schubert, 1995). On the other hand, it was suggested that no genotypic differentiation occurs in the ﬁrst phase of salt stress (Fig. 19.1). Recently, evidence has been presented that, although the differ-

445

ences are small, genotypic variation exists also in the ﬁrst phase of salt stress and can be exploited in breeding programs (Schubert et al., 2009). The biphasic model excludes short-term effects that occur during the ﬁrst several minutes or hours after the imposition of salt stress, that is, before the ﬁrst phase of salt stress; these effects may thus be ascribed to a “phase 0.” This phase 0 comprises transient changes in turgor and growth (Thiel et al., 1988) as well as in membrane potential (Läuchli and Schubert, 1989), and stress during this phase apparently has a profound impact on the chloroplast proteome of saltsensitive plant species such as maize (Zörb et al., 2009). Although these effects may be quantitatively negligible for physiological parameters (in fact, a 25 mM NaCl treatment does not represent a measurable strain for maize growth), they may nevertheless have important implications for the following phases. The biphasic model must be recognized as a conceptual model that has not just a purely temporal dimension, but also takes into account stress intensity and genotypic and developmental differences. It is used to differentiate osmotic and ion effects on plant growth during salt stress. Whereas the model has been used to characterize salt effects during the vegetative phase, only few investigations have been carried out during reproductive growth and development. Osmotic resistance during salt stress: ﬁrst phase Salinity decreases the solute potential of the soil solution and thus the water potential gradient into roots that establishes the driving force for water uptake. Under conditions of low matric potential and/or high water vapor deﬁcit of the atmosphere (including windy conditions), this may contribute to insufﬁcient water supply to the

446

NUTRIENT USE EFFICIENCY IN CROPS

Fig. 19.2. Maize plants (Zea mays L. cv. Pioneer 3906) after 2-month growth in soil culture under control (left) and 11 dS m−1 salinity (right). Plants are still in the ﬁrst phase of salt stress (Photo: Schubert).

plants as is observed under drought stress. Wilting symptoms for leaves of dicots and rolling of leaf blades of grasses may thus be observed under speciﬁc circumstances under salt stress. However, this does not represent the typical reaction of crop plants to saline stress. Rather, stunted growth and occasionally a dark-green color of leaves are observed in the ﬁrst phase of salt stress (Fig. 19.2). In line with these observations, turgor is maintained and osmotic adjustment seems not to limit shoot growth (Termaat et al., 1985; Van Volkenburgh and Boyer, 1985; De Costa et al., 2007). It is generally accepted that roots are able to sense osmotic stress and release signals that control plant growth (Davies and Zhang, 1991). One of these signals may be abscisic acid that is released by root tips and transported via xylem into leaves, where it may

contribute to growth inhibition (Thomas et al., 1992; Montero et al., 1997). Depending on how many root tips sense low water availability, the abscisic acid concentration in the xylem sap may serve as an integrating signal that adjusts growth according to water availability in the rooting zone (Davies and Zhang, 1991). Apart from abscisic acid, xylem pH (Jia and Davies, 2007), hydraulic signals (Chazen and Neumann, 1994), nitrate, and combinations (Felle and Hanstein, 2002; Wilkinson and Davies, 2002) have been suggested as signals to control shoot growth under water stress. Whatever the signal, it is evident that this capability helps to adjust shoot growth under limited water availability of the soil. It has been shown previously that transpiration rates of maize plants, grown in nutrient solution under salt stress, are not necessarily reduced relative to control plants (Schubert, 2009). As shown in Figure 19.3, transpiration rates of two-month-old maize plants grown in soil culture (Fig. 19.2) were not reduced, but even increased under salt stress. This suggests not only that shoot growth is tightly controlled in the ﬁrst phase of salt stress, but also that shoots do not apparently suffer from water deﬁcit, since more than 90% of the water taken up are transpired. Apparently, the decrease in hydraulic conductivity in roots under salt stress (Evlagon et al., 1992) did not limit growth and photosynthetic rates in the maize plants. The fact that transpiration rates may be unchanged under salt stress suggests that photosynthetic rates are not limited by stomatal conductivity. Thus, cell division and cell extension are not limited by carbohydrate supply (Aspinall, 1986). The accumulation of sugars in leaf tissue suggests that the production of sugars in photosynthesis is not limited during the ﬁrst phase of salt stress (De Costa et al., 2007; Hatzig et al., 2010b) and that osmotic adjustment is not a problem even for salt-sensitive plant

SALT RESISTANCE OF CROP PLANTS

447

Transpiration rate (mL cm–2 week–1) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Across

Lector

Pioneer

SR 05

SR 08

SR 15

Transpiration rates of various maize genotypes under control (white columns) and 11 dS m−1 salinity (black columns). Genotypes were Across 8023, Lector, Pioneer 3906, and newly developed salt-resistant (SR) hybrids (Schubert et al., 2009). Plants were grown in soil culture in large containers at 60% of maximum waterholding capacity. Transpiration rates were determined as water lost during the vegetative phase (S. Schubert, unpublished results). Fig. 19.3.

species such as maize (Cramer, 1994). However, osmotic adjustment is related to the synthesis of glycinebetaine (Saneoka et al., 1995) and proline (Kuznetsov and Shevyakova, 1997), which may also serve as cytoplasmic osmoprotectants. Insufﬁcient calcium nutrition and physiological calcium deﬁciency may present a problem for growth in the ﬁrst phase of salt stress (Mizrahi and Pasternak, 1985; He and Cramer, 1992; Hu and Schmidhalter, 2005). It appears that species and cultivars that are more salt-sensitive tend to suffer more from calcium deﬁciency than salt-resistant genotypes (Lynch and Läuchli, 1985; Ehret et al., 1990). It is interesting that substitution of potassium by sodium in the nutrient solution resulted in calcium deﬁciency, and not in potassium deﬁciency of sugar beet (Wakeel et al., 2009). Since generally turgor does not limit shoot growth under salinity stress (see above), according to the Lockhart equation

(Equation 19.1, Lockhart, 1965), growth must be impaired either by cell-wall extensibility or by the growth-effective turgor. As the latter also depends on the cell-wall extensibility, it can be concluded that a more rigid cell wall is mainly responsible for shoot-growth reduction in the ﬁrst phase of salt stress: dV/dt = m × (Ψ p − Y ),

(1)

where dV/dt = growth rate; m = cell wall extensibility; Ψp = turgor; Y = threshold turgor; and Ψp − Y = growth-effective turgor. Although calcium is essential for cellwall structure in its function to bridge carboxyl groups of pectinates, it is unlikely that calcium deﬁciency decreases cell-wall extensibility. Rather an increase of cell-wall extensibility is anticipated when calcium is replaced by monovalent ions such as protons. According to the acid-growth theory, apoplast acidiﬁcation triggers cell extension

448

NUTRIENT USE EFFICIENCY IN CROPS

by increasing the cell-wall extensibility (Hager, 2003). Recently, it has been shown that salt stress resulted in inhibition of in vitro H+ pumping by leaf plasma membrane H+ ATPase in the maize cultivar Pioneer 3906. Shoot-growth reduction in the ﬁrst phase of salt stress was explained in terms of expression of inefﬁcient H+ ATPase isoforms that are not able to sufﬁciently acidify the leaf cell-wall (Zörb et al., 2005). The in vitro results were corroborated in vivo using ratio-imaging techniques (Pitann et al., 2009). At the same time, it was shown that a more salt-resistant maize genotype, SR 03, maintained proton pumping and cell-wall acidiﬁcation in the ﬁrst phase of salt stress (Pitann et al., 2009). This is in line with results of Neves-Piestun and Bernstein (2001), who found no decrease in cell-wall acidiﬁcation of the maize cultivar G.S. 46 under salt stress, and results of Wakeel et al. (2010), who found the same in vitro result for H+ pumping by H+ ATPase from leaves of salt-resistant sugar beet. In contrast, maize hybrid SR 12, which has a similar salt-resistance to SR 03, was not capable of maintaining in vitro proton pumping (Hatzig et al., 2010a). These results indicate that cell-wall acidiﬁcation is an important, but not a sufﬁcient, process to maintain extension growth of leaf cells in the ﬁrst phase of salt stress. Other cell wallstiffening processes such as crosslinking of hemicelluloses by phenolics (N. Uddin, pers. comm..) may in the future explain in more detail genotypic differences of cell extension growth in the ﬁrst phase of salt stress. Sodium exclusion strategies: second phase Testing the biphasic model of growth response to salt stress strictly, it was shown that sodium exclusion contributes to salt resistance in the second phase of salt stress (Fortmeier and Schubert, 1995). It was also

shown that although chloride may synergistically enhance sodium uptake and translocation, it does not directly cause ion toxicity in maize. This ﬁnding may be representative for grasses in general (Gorham et al., 1990). In contrast, chloride toxicity is the primary reason for growth impairment of sensitive crops such as potato, strawberry, citrus, and legumes such as soybean (Martínez Barrosos and Alvarez, 1997, White and Broadley, 2001; Luo et al., 2005). However, a generalization is not possible, as it was shown that the primary toxicity in faba bean is caused by sodium, although chloride toxicity also occurred (Slabu et al., 2009). Various problems of sodium toxicity have been described in the literature. The activation of many enzymes requires high cytoplasmic concentrations of potassium, and displacement of potassium by sodium may thus disturb metabolism (Anil et al., 2007). Hecht-Buchholz et al. (1971) showed that mitochondrial functioning in maize roots was affected by salt stress but respiration was maintained by increasing the number of mitochondria. Plant leaves are particularly sensitive to sodium (Munns, 2002) for two reasons. First, potassium plays an important role in stomatal regulation. Displacement of potassium by sodium can maintain the turgor of guard cells, but due to the poorer membrane mobility of the latter ion, stomatal closure is hampered. This results in uncontrolled water losses and desiccation of leaf tissue, which becomes visible as necrotic spots on the leaf tissues of faba bean (Slabu et al., 2009), and chlorotic and necrotic lesions of maize leaves (Fig. 19.4). Second, chloroplasts are particularly sensitive to sodium, which they readily accumulate when available in the cytosol (Zörb et al., 2009). This results not only in overwhelming changes of the chloroplast proteome at low sodium concentrations (Zörb et al., 2009) but also in chloroplast deformation at higher concentrations (Marschner and Mix, 1973).

SALT RESISTANCE OF CROP PLANTS

449

Plasma membrane Cytosol

Rhizosphere

N + Na

1

H+

Na+

2

Sodium exclusion mechanisms at the plasma membrane. (1) Passive sodium inﬂux of sodium is restricted by selective channels. Sodium inﬂux is driven by the electrochemical gradient. (2) Active sodium efﬂux of sodium via Na+/H+ antiporters is driven by the proton motive force. Fig. 19.5.

Fig. 19.4. Sodium toxicity in maize (Photo: Schubert).

From this it follows that sodium exclusion from the cytoplasm, particularly of leaf cells, is essential. For a complicated organism such as a higher plant, sodium exclusion is realized by various strategies (Tester and Davenport, 2003; Munns and Tester, 2008). The ﬁrst and most important strategy is sodium exclusion at the root surface, which basically restricts sodium entry into the plant and avoids sodium accumulation in the plant (Amtmann et al., 2005). Whereas active sodium efﬂux from cells via SOS1-type Na+/ H+ antiporters was demonstrated for Arabidopsis thaliana (Qiu et al., 2003; Guo et al., 2009) and rice (Matínez-Atienza et al., 2007), no such evidence was found for maize, although active efﬂux was demon-

strated (Schubert and Läuchli, 1988). Genotypic differences in sodium uptake were explained in terms of low passive inﬂux via selective cation channels (Schubert and Läuchli, 1990; Xuan et al., 2010, Fig. 19.5). A second exclusion strategy restricts sodium translocation from roots to shoots. Sodium inclusion in root cell vacuoles via NHX-type Na+/H+ antiporters (Xue et al., 2004) eliminates sodium from transport into the xylem vessels (Fig. 19.6). These tonoplast-located Na+/H+ antiporters have a dual function. They not only contribute to sodium exclusion but also convey sodium tissue tolerance (Zhang and Blumwald, 2001; Queirós et al., 2009), particularly in leaf cells (Davenport et al., 2005; Munns and Tester, 2008). Thus, these antiporters enable sodium to act as a metabolically innocuous solute (Saqib et al., 2005). The overexpression of vacuolar Na+/H+ antiporters in different plant species resulted in higher salt resistance (Apse et al., 1999; Zhang and Blumwald, 2001; Xue et al., 2004).

450

NUTRIENT USE EFFICIENCY IN CROPS

Endodermis

Cortex

Exodermis

K+ K+ Xylem

Rhizosphere

K+ Na+ Na+

Na+

Casparian strip Schematic representation of potassium and sodium uptake and translocation through root tissue to the xylem vessels. Depending on genotypic expression of NHX-type Na+/H+ antiporters, sodium may be actively sequestered into vacuoles and thereby excluded from root-to-shoot translocation. The Casparian strip forces ions to the symplastic way and allows for selectivity. Sodium may also be reabsorbed from xylem by xylem parenchyma cells.

Fig. 19.6.

Salt resistance during the reproductive stage In contrast to the vegetative phase, only a few physiological studies have been performed during the generative phase, which is particularly important for the yield formation of cereals. Ion toxicity in the second phase may directly inhibit photosynthesis and thus yield formation (Yeo et al., 1985). Genetically improved sodium exclusion may therefore enhance yield performance of wheat and maize (Xue et al., 2004; Schubert et al., 2009; R. Munns, pers. comm.). Although genetic variation in the ﬁrst phase of salt stress is limited, some maize inbred lines have been identiﬁed that show slightly superior osmotic resistance. The crossing of these inbred lines yielded maize hybrids that show higher stress resistance in terms of yield formation (Schubert et al., 2009). Since under the experimental conditions used, no stress symptoms of the second phase of salt stress were observed in any of the genotypes tested, it can be concluded that genotypic differences in yield formation were caused by differences in osmotic resistance.

Although drought stress and salt stress in the ﬁrst phase are not identical (see above), they both have in common the root sensing of low water availability and the release of stress signals to shoots. Therefore, some ﬁndings for drought stress may also be relevant for the ﬁrst phase of salt stress, although a validation still is required. Comparable to ﬁndings for faba bean under drought stress (Amede et al., 1999), in the ﬁrst phase of salt stress, kernel set of maize is more sensitive than kernel weight formation (Schubert et al., 2009). Three different hypotheses have been suggested to explain the decrease in kernel number under drought stress: ﬁrst, abscisic acid that controls leaf growth and stomatal aperture may also be responsible for early ﬂower or kernel abortion (Bano et al., 1993; Setter et al., 2001); second, although sugar production in photosynthesis seems not to be limiting under drought stress during vegetative growth and grain ﬁlling stage, the supply of sucrose during ﬂowering may represent a critical factor to establish a high number of kernels (Boyle et al., 1991; Zinselmeier et al., 1999); third, there is strong evidence that a lack of acid invertase in sink organs restricts the

SALT RESISTANCE OF CROP PLANTS

451

Fructose Sucrose

Acid Invertase

Hexose Carrier

Glucose

Phloem Apoplast Sink Cell Phloem unloading of sucrose and active uptake of glucose and fructose by hexose carriers. The sink activity is controlled by acid invertase activity.

Fig. 19.7.

determination of kernels in ovaries (Zinselmeier et al., 1995; Roitsch and González, 2004). It is generally accepted that phloem unloading in cereals occurs via an apoplastic route (Fig. 19.7). According to this model, sucrose leaked from phloem is not retrieved by sucrose/proton cotransport. Instead, acid invertase apoplastically hydrolyzes sucrose (Roitsch and González, 2004). The resultant hexoses cannot be transported into phloem but are imported by sink cells via protondriven speciﬁc hexose carriers. Under drought stress, acid invertase activity is reduced so that hexose import into sink cells is inhibited. There are indications that reduced sink activity due to insufﬁcient acid invertase may also be caused by salt stress (Fukushima et al., 2001; Balibrea et al., 2003). Further experiments are necessary to understand the genotypic differences in salt resistance of cereals during the reproductive stage. Concluding remarks The biphasic model of growth response of crop plants to salt stress has proved to be a powerful theoretical basis to improve salt resistance in wheat and maize germplasm.

Sodium exclusion from shoots was a major step for this improvement. It is expected that this strategy will also be successful in other grasses. In addition, tissue tolerance of sodium may be further improved by increasing vacuolar Na+/H+ antiporter activity. On the other hand, osmotic resistance still has to be raised. A better understanding of the processes of cell elongation is required. It is clear now, however, that osmotic adjustment and production of assimilates are not the main bottleneck. This is apparently also true for the generative phase. In this stage, kernel set mainly limits yield formation, and a better understanding of this process may further add to the development of saltresistant crop plants. Acknowledgment The author is thankful for stimulating discussions with Sarah Hatzig and helpful comments by Dr. Britta Pitann on an early version of the manuscript. References Amede, T., Kittlitz, E.V., & Schubert, S. (1999) Differential drought responses of faba bean (Vicia

452

NUTRIENT USE EFFICIENCY IN CROPS

faba L.) inbred lines. Journal of Agronomy and Crop Science 183, 35–45. Amtmann, A., Bohnert, H.J., & Bressan, R.A. (2005) Abiotic stress and plant genome evaluation. Search for new models. Plant Physiology 138, 127–130. Anil, V.S., Krishnamurthy, H., & Methew, M.K. (2007) Limiting cytosolic Na+ confers salt tolerance to rice cells in culture: a two photon microscopy study of SBFI-loaded cells. Physiologia Plantarum 129, 607–621. Apse, M.P., Aharon, G.S., Snedden, W.A., et al. (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285, 1256–1258. Arrese-Igor, C., Royuela, M., de Lorenzo, C., et al. (1993) Effect of low rhizosphere oxygen on growth, nitrogen ﬁxation and nodule morphology in lucerne. Physiologia Plantarum 89, 55–63. Aspinall, D. (1986) Metabolic effects of water and salinity stress in relation to expansion of the leaf surface. Australian Journal of Plant Physiology 13, 59–73. Balibrea, M.E., Cuartero, J., Bolarín, M.C., et al. (2003) Sucrolytic activities during fruit development of Lycopersicon genotypes differing in tolerance to salinity. Physiologia Plantarum 118, 38–46. Bano, A., Dörfﬂing, K., Bettin, D., et al. (1993) Abscisic acid and cytokinins as possible root-to-shoot signals in xylem sap of rice plants in drying soil. Australian Journal of Plant Physiology 20, 109–115. Boyle, M.G., Boyer, J.S., & Morgan, P.W. (1991) Stem infusion of liquid culture medium prevents reproductive failure of maize at low water potentials. Crop Science 31, 1246–1252. Chasen, O. & Neumann, P.M. (1994) Hydraulic signals from the roots and rapid cell wall hardening in growing maize (Zea mays L.) leaves are primary responses to polyethylene glycol-induced water deﬁcits. Plant Physiology 104, 1385–1392. Cramer, G.R. (1994) Response of maize (Zea mays L.) to salinity. In: Handbook of Plant and Crop Stress (ed. M. Pessarakli), pp. 449–459. Marcel Dekker, New York. Cramer, G.R., Epstein, E., & Läuchli, A. (1990) Effects of sodium, potassium and calcium on salt-stressed barley. I. Growth analysis. Physiologia Plantarum 80, 83–88. Davenport, R., James, R.A., Zakrisson-Plogander, A., et al. (2005) Control of sodium transport in durum wheat. Plant Physiology 137, 807–818. Davies, W.J. & Zhang, J. (1991) Root signals and the regulation of growth and development of plants in drying soil. Annual Review of Plant Physiology and Plant Molecular Biology 42, 55–76.

De Costa, W., Zörb, C., Hartung, W., et al. (2007) Salt resistance is determined by osmotic adjustment and abscisic acid in newly developed maize hybrids in the ﬁrst phase of salt stress. Physiologia Plantarum 131, 311–321. Drew, M.C. & Läuchli, A. (1985) Oxygen-dependent exclusion of sodium ions from shoots by roots of Zea mays (cv. Pioneer 3906) in relation to salinity damage. Plant Physiology 79, 171–176. Ehret, D.L., Redmannn, R.E., Harvey, B.L., et al. (1990) Salinity-induced calcium deﬁciencies in wheat and barley. Plant and Soil 128, 143–151. Epstein, E., Norlyn, J.D., Rush, D.W., et al. (1980) Saline culture of crops: a genetic approach. Science 210, 399–404. Evlagon, D., Ravina, I., & Neumann, P.M. (1992) Effects of salinity stress and calcium on hydraulic conductivity and growth in maize seedling roots. Journal of Plant Nutrition 15, 795–803. Felle, H. & Hanstein, S. (2002) The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors. Journal of Experimental Botany 53, 73–82. Fortmeier, R. & Schubert, S. (1995) Salt tolerance of maize (Zea mays L.): the role of sodium exclusion. Plant, Cell & Environment 18, 1041–1047. Fukushima, E., Arata, Y., Endo, T., et al. (2001) Improved salt tolerance of transgenic tobacco expressing apoplastic yeast-derived invertase. Plant Cell Physiology 42, 245–249. Gorham, J., Wyn Jones, R.G., & Bristol, A. (1990) Partial characterization of the trait for enhanced K+Na+ discrimination in the D genome of wheat. Planta 180, 590–597. Guo, K.-M., Babourina, O., & Rengel, Z. (2009) Na+/ H+ antiporter activity of the SOS1 gene: lifetime imaging analysis and electrophysiological studies on Arabidopsis seedlings. Physiologia Plantarum 137, 155–165. Hager, A. (2003) Role of the plasma membrane H+ATPase in auxin-induced elongation growth: historical and new aspects. Journal of Plant Research 116, 483–505. Hatzig, S., Hanstein, S., & Schubert, S. (2010a) Apoplast acidiﬁcation is not a necessary determinant for the resistance of maize in the ﬁrst phase of salt stress. Journal of Plant Nutrition and Soil Science 173, 559–562. Hatzig, S., Kumar, A., Neubert, A., et al. (2010b) PEPcarboxylase activity: a comparison of its role in a C4 and a C3 species under salt stress. Journal of Agronomy and Crop Science 196, 185–192. He, T. & Cramer, G.R. (1992) Growth and mineral nutrition of six rapid-cycling Brassica species in response to seawater salinity. Plant and Soil 139, 285–294.

SALT RESISTANCE OF CROP PLANTS

Hecht-Buchholz, C., Pﬂüger, R., & Marschner, H. (1971) Einﬂuss von Natriumchlorid auf die Mitochondrienzahl und Atmung von Maiswurzelspitzen. Zeitschrift für Pﬂanzenphysiologie 65, 410– 417. Hu, Y. & Schmidhalter, U. (2005) Drought and salinity: a comparison of their effects on mineral nutrition of plants. Journal of Plant Nutrition and Soil Science 168, 541–549. Jia, W. & Davies, W.J. (2007) Modiﬁcation of leaf apoplastic pH in relation to stomatal sensitivity to root-sourced abscisic acid signals. Plant Physiology 143, 68–77. Kuznetsov, V.V. & Shevyakova, N.I. (1997) Stress responses of tobacco cells to high temperature and salinity. Proline accumulation and phosphorylation of polypeptides. Physiologia Plantarum 100, 320–326. Läuchli, A. & Schubert, S. (1989) The role of calcium in the regulation of membrane and cellular growth processes under salt stress. In: Environmental Stress in Plants. Biochemical and Physiological Mechanisms (ed. J.H. Cherry), pp. 131–138. Springer, Berlin. Lauter, D.J. & Munns, D.N. (1986) Salt resistance of chickpea genotypes in solutions salinized with NaCl or Na2SO4. Plant and Soil 95, 271– 279. Lessani, H. & Marschner, H. (1978) Relation between salt tolerance and long-distance transport of sodium and chloride in various crop species. Australian Journal of Plant Physiology 5, 27–37. Levitt, J. (1980) Responses of Plants to Environmental Stresses. Vol. I. Chilling, Freezing, and High Temperature Stresses (ed. T.T. Kozlowski) Academic Press, Orlando, FL. Lockhart, J.A. (1965) Cell extension. In: Plant Biochemsitry (eds. J. Bonner & J.E. Varner), pp. 826–849. Academic Press, New York. Luo, Q., Yu, B., & Liu, Y. (2005) Differential sensitivity to chloride and sodium ions in seedlings of Glycine max and G. soja under NaCl stress. Journal of Plant Physiology 162, 1003–1012. Lynch, J. & Läuchli, A. (1985) Salt stress disturbs the calcium nutrition of barley (Hordeum vulgare L.). New Phytologist 99, 345–354. Marschner, H. & Mix, G. (1973) Einﬂuss von Natriumchlorid und Mycostatin auf den Mineralstoffgehalt im Blattgewebe und die Feinstruktur der Chloroplasten. Zeitschrift für Pﬂanzenernährung und Bodenkunde 136, 203–219. Martínez Barrosos, M.C. & Alvarez, C.E. (1997) Toxicity symptoms and tolerance of strawberry to salinity in the irrigation water. Scientia Horticulturae 71, 177–188.

453

Matínez-Atienza, J., Jiang, X., Garciadeblas, B., et al. (2007) Conservation of the salt overly sensitive pathway in rice. Plant Physiology 143, 1001–1012. Mizrahi, Y. & Pasternak, D. (1985) Effect of salinity on quality of various agricultural crops. Plant and Soil 89, 301–307. Montero, E., Cabot, C., Barceló, J., et al. (1997) Endogenous abscisic acid levels are linked to decreased growth of bush bean plants treated with NaCl. Physiologia Plantarum 101, 17–22. Munns, R. (1988) Why measure osmotic adjustment? Australian Journal of Plant Physiology 15, 717–726. Munns, R. (1993) Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant, Cell & Environment 16, 15–24. Munns, R. (2002) Comparative physiology of salt and water stress. Plant, Cell & Environment 25, 239–250. Munns, R. & Tester, M. (2008) Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651–681. Munns, R., James, R.A., & Läuchli, A. (2006) Approaches to increasing the salt tolerance of wheat and other cereals. Journal of Experimental Botany 57, 1025–1043. Neves-Piestun, B.G. & Bernstein, N. (2001) Salinityinduced inhibition of leaf elongation in maize is not mediated by changes in cell wall acidiﬁcation capacity. Plant Physiology 125, 1419–1428. Pitann, B., Schubert, S., & Mühling, K.H. (2009) Decline in leaf growth under salt stress is due to an inhibition of H+-pumping activity and increase in apoplastic pH of maize leaves. Journal of Plant Nutrition and Soil Science 172, 535–543. Qadir, M. & Schubert, S. (2002) Degradation processes and nutrient constraints in sodic soils. Land Degradation & Development 13, 275–294. Qadir, M., Schubert, S., Ghafoor, A., et al. (2001) Amelioration strategies for sodic soils: a review. Land Degradation & Development 12, 357– 386. Qadir, M., Noble, A.D., Schubert, S., et al. (2006) Sodicity induced land degradation and its sustainable management: problems and prospects. Land Degradation & Development 17, 661–676. Qadir, M., Oster, J.D., Schubert, S., et al. (2007) Phytoremediation of sodic and saline-sodic soils. Advances in Agronomy 96, 197–247. Qiu, Q.-S., Barkla, B.J., Vera-Estrella, J.-K., et al. (2003) Na+/H+ exchange activity in the plasma membrane of Arabidopsis. Plant Physiology 132, 1041–1052. Queirós, F., Fontes, N., Silva, P., et al. (2009) Activity of tonoplast proton pumps and Na+/H+ exchange in

454

NUTRIENT USE EFFICIENCY IN CROPS

potato cell cultures is modulated by salt. Journal of Experimental Botany 60, 1363–1374. Roitsch, T. & González, M.-C. (2004) Function and regulation of plant invertases: sweet sensations. Trends in Plant Science 9, 606–613. Rozema, J. & Flowers, T.J. (2008) Crops for a salinized world. Science 322, 1478–1480. Saneoka, S., Nagasaka, C., Hahn, D.T., et al. (1995) Salt tolerance of glycinebetaine-deﬁcient and -containing maize lines. Plant Physiology 107, 631–638. Saqib, M., Zörb, C., Rengel, Z., et al. (2005) The expression of the endogenous vacuolar Na+/H+ antiporters in roots and shoots correlates positively with the salt resistance of wheat (Triticum aestivum L.). Plant Science 169, 959–965. Schachtman, D.P. & Munns, R. (1992) Sodium accumulation in leaves of Tritcum species that differ in salt tolerance. Australian Journal of Plant Physiology 19, 331–340. Schubert, S. (2009) Advances in alleviating growth limitations of maize under salt stress. Proceedings of the XVI International Plant Nutrition Colloquium, Sacramento, USA. Available at: http://escholarship. org/uc/item/891457b8?query=Schubert#page - 1 (accessed May 3, 2011). Schubert, S. & Läuchli, A. (1986) Na+ exclusion, H+ release, and growth of two different maize cultivars under NaCl salinity. Journal of Plant Physiology 126, 145–154. Schubert, S. & Läuchli, A. (1988) Metabolic dependence of Na+ efﬂux from roots of intact maize seedlings. Journal of Plant Physiology 133, 193– 198. Schubert, S. & Läuchli, A. (1990) Sodium exclusion mechanisms at the root surface of two maize cultivars. Plant and Soil 123, 205–209. Schubert, S., Neubert, A., Schierholt, A., et al. (2009) Development of salt-resistant maize hybrids: the combination of physiological strategies using conventional breeding methods. Plant Science 177, 196–202. Setter, T.L., Flannigan, B.A., & Melkonian, J. (2001) Crop Science 41, 1530–1540. Slabu, C., Zörb, C., Steffens, D., et al. (2009) Is salt stress of faba been (Vicia faba L.) caused by Na+ or Cl- toxicity? Journal of Plant Nutrition and Soil Science 172, 644–650. Sümer, A., Zörb, C., Yan, F., et al. (2004) Evidence of sodium toxicity for the vegetative growth of maize (Zea mays L.) during the ﬁrst phase of salt stress. Journal of Applied Botany 78, 135–139. Szabolcs, I. (1994) Soils and salinization. In: Handbook of Plant and Crop Stress (ed. M. Pessarakli), pp. 3–17. Marcell Dekker, New York.

Teakle, N.L., Amtmann, A., Real, D., et al. (2010) Lotus tenuis tolerates combined salinity and waterlogging: maintaining O2 transport to roots and expression of an NHX1-like gene contribute to regulation of Na+ transport. Physiologia Plantarum 139, 358–374. Termaat, A., Passioura, J.B., & Munns, R. (1985) Shoot turgor does not limit shoot growth of NaClaffected wheat and barley. Plant Physiology 77, 869–872. Tester, M. & Davenport, R. (2003) Na+ tolerance and Na+ transport in higher plants. Annals of Botany 91, 503–527. Thiel, G., Lynch, J., & Läuchli, A. (1988) Short-term effects of salinity stress on the turgor and elongation of growing barley leaves. Journal of Plant Physiology 132, 38–44. Thomas, J.C., McElwain, E.F., & Bohnert, H.J. (1992) Convergent induction of osmotic stress-responses. Abscisic acid, cytokinin, and the effects of NaCl. Plant Physiology 100, 416–423. Van Volkenburgh, E. & Boyer, J.S. (1985) Inhibitory effects of water deﬁcit on maize leaf elongation. Plant Physiology 77, 190–194. Wakeel, A., Abd-El-Motadally, F., Steffens, D., et al. (2009) Sodium-induced calcium deﬁciency in sugar beet during substitution of potassium by sodium. Journal of Plant Nutrition and Soil Science 172, 254–260. Wakeel, A., Hanstein, S., Pitann, B., et al. (2010) Hydrolytic and pumping activity of H+-ATPase from leaves of sugar beet (Beta vulgaris L.) as affected by salt stress. Journal of Plant Physiology 167, 725–731. White, P.J. & Broadley, M.R. (2001) Chloride in soils and its uptake and movement within the plant: a review. Annals of Botany 88, 967–988. Wilkinson, S. & Davies, W.J. (2002) ABA-based chemical signalling: the co-ordination of responses to stress in plants. Plant, Cell & Environment 25, 195–210. Xuan, Y., Horie, T., Xue, S., et al. (2010) Differential sodium and potassium transport selectivities of the rice OsHKT2;1 and OsHKT2;2 transporters in plant cells. Plant Physiology 152, 341–355. Xue, Z.-Y., Zhi, D.-Y., Xue, G.-P., et al. (2004) Enhanced salt tolerance of transgenic wheat (Triticum aestivum L.) expressing a vacuolar Na+H+ antiporter gene with improved grain yield in saline soils in the ﬁeld and a reduced level of Na+. Plant Science 167, 849–859. Yeo, A.R., Caporn, S.J.M., & Flowers, T.J. (1985) The effect of salinity upon photosynthesis in rice (Orzyza sativa L.): gas exchange by individual leaves in relation to their salt content. Journal of Experimental Botany 36, 1240–1248.

SALT RESISTANCE OF CROP PLANTS

Zhang, H.X. & Blumwald, E. (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nature Biotechnology 19, 765–768. Zinselmeier, C., Westgate, M.E., & Schussler, J.R. (1995) Low water potential disrupts carbohydrate metabolism in maize (Zea mays L.) ovaries. Plant Physiology 107, 385–391. Zinselmeier, C., Yeong, B.-R., & Boyer, J.S. (1999) Starch and the control of kernel number in maize at low water potentials. Plant Physiology 121, 25–35.

455

Zörb, C., Stracke, B., Tramnitz, B., et al. (2005) Does H+ pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the ﬁrst phase of salt stress? Journal of Plant Nutrition and Soil Science 168, 550–557. Zörb, C., Herbst, R., Forreiter, C., et al. (2009) Shortterm effects of salt exposure on the maize chloroplast pattern: a proteomic study. Proteomics 9, 4209–4220.

Chapter 20

Legumes and Nitrogen Fixation: Physiological, Molecular, Evolutionary Perspectives, and Applications Muthusubramanian Venkateshwaran and Jean-Michel Ané

Abstract In order to meet the ever-growing food, feed, and biofuel demand, there has been an increasing dependence on intensive agriculture. These nonsustainable practices may lead to the deterioration of soil quality and require the production of nitrogen fertilizers at the expense of nonrenewable fossil fuels. Apart from increasing the cost of cultivation, the excessive use of fertilizers is also responsible for the damage to many ecosystems. Legumes have the ability to establish a mutualistic association with soil bacteria known as rhizobia, which form root nodules, inside of which atmospheric dinitrogen is reduced into assimilable forms and supplied to host plants. A high level of species speciﬁcity exists in rhizobia–legume interactions, which are initiated by a signal exchange between the two partners. The infection process and the development of root nodules require a set of highly coordinated events at the root epidermal and cortical cells. Over the past decades, elegant genetic and biochemical studies have been conducted on legume nodulation to decipher the intrica-

cies of this unique plant–microbe association. This chapter highlights the biochemical, physiological, molecular, and evolutionary features of legume nodulation, and explores the applications of nature’s unique gift toward the sustainability of our agriculture.

Introduction Soil fertility is a major limiting factor for crop production in developed and developing countries. Intensive agricultural practices to meet ever-growing food, feed, and biofuel demands are depleting soil quality at an alarming rate. The excessive use of fertilizers not only increases the cost of cultivation but also depletes nonrenewable energy sources and causes irreparable damage to the environment. Enriching soil nutrient levels by biological means seems to offer a much more sustainable solution. Among land plants, legumes are unique as they can enter into symbiotic association with nitrogenﬁxing bacteria known as rhizobia. This interaction results in the formation of new organs called nodules on legume roots, in

The Molecular and Physiological Basis of Nutrient Use Efﬁciency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 457

458

NUTRIENT USE EFFICIENCY IN CROPS

which the rhizobia thrive and ﬁx atmospheric nitrogen in a process known as biological nitrogen ﬁxation (BNF). Hence, legumes have the ability to grow even in nitrogen-deprived soils. In agricultural ecosystems worldwide, legumes provide an amount of assimilable nitrogen roughly equivalent to that produced by the chemical fertilizer industries. Crop rotations and intercropping with legumes are practiced in many parts of the world to reduce the cost of nitrogen fertilizer applications. Being cultivated in arid and semi-arid tropics, legumes serve as a major source of protein in dietary supplements of developing countries. Worldwide, legumes are grown over 71 million hectares, with a total production of 61.5 million tonnes (FAOSTAT, 2010), with major contributions from Asian and African countries. In the United States, legumes are grown over 1 million hectares, with a total production of 2.2 million tonnes (FAOSTAT, 2010). Despite global industrialization, the area under legume cultivation is constantly growing (FAOSTAT, 2010) due to the importance of legumes as a food, fodder, and manure crop. The symbiosis between legumes and rhizobia is a sophisticated example of coordinated development between bacteria and eukaryotes culminating in the organogenesis of root nodules (Oldroyd and Downie, 2008). Being the third largest plant family, legumes display a huge diversity comprising trees, shrubs, herbs, and climbers. Likewise, rhizobia are a paraphyletic group comprising members of two classes, α- and βproteobacteria. Rhizobiales, which belong to the most studied α-proteobacteria group, have been grouped at the generic level into Azorhizobium, Bradyrhizobium, Devosia, Mesorhizobium, Methylobacterium, Ochrobactrum, Phyllobacterium, Shinella, Rhizobium, and Sinorhizobium (Ensifer), each of which have multiple species, biovars, and a plethora of strains. Members of the

β-proteobacteria group, such as Burkholderia, Cupriavidus (Ralstonia), and Herbaspirillum, are also potential symbionts of legumes (Amadou et al., 2008). Amidst this diversity, there exists a high level of species speciﬁcity among legume species and their symbiotic partners. Although root nodule symbiosis (RNS) is a unique association between legumes and rhizobia, one exception to this “rule” is the mutualistic interaction between the nonlegume Parasponia (Ulmaceae) and nitrogen-ﬁxing rhizobia (Trinick, 1979). Apart from legumes (Fabales), members of the plant orders Fagales, Cucurbitales, and Rosales are able to establish symbiotic interaction with the nitrogen-ﬁxing actinobacteria Frankia. This symbiotic interaction is referred to as actinorhizal symbiosis (ARS). This chapter focuses on biochemical, physiological, molecular, and evolutionary aspects of legume–rhizobial interaction, with special interest on its immediate and long-term ﬁeld applicability. Biological nitrogen ﬁxation and nitrogenase biology BNF is the reduction of inert atmospheric nitrogen (N2) into ammonia (NH3) through biological means. The general chemical reaction for the biological ﬁxation of nitrogen is N 2 + 8e − + 16 MgATP + 8H + → 2 NH 3 + H 2 + 16 MgADP + 16 Pi , where Pi is inorganic phosphate. This reaction is catalyzed by nitrogenase, a molybdenum-dependent enzyme, which comprises two component proteins called the Fe protein and the MoFe protein (Burgess and Lowe, 1996). The role of Fe protein is to deliver electrons from its [4Fe-4S] cluster to the MoFe protein one at a time. This elec-

LEGUMES AND NITROGEN FIXATION

tron transfer is coupled to the hydrolysis of a minimum of two magnesium–adenosine triphosphate (Mg–ATP) molecules (Lanzilotta et al., 1998). The MoFe protein (an α2ß2 heterotetramer) consists of α- and β-subunits and two metal clusters called the P cluster and the MoFe cofactor (Chan et al., 1993). The P cluster, which in turn is made up of 8Fe-7S, is positioned at the interface of, and with, α and β-subunits on each side (Lanzilotta et al., 1998). The role of the P cluster is likely to be to accept electrons from the Fe protein and to deliver it to MoFe cofactor, which is deeply buried within each of the α-subunits. The molecular composition of MoFe cofactor is quite intriguing, as it is composed of 7Fe-9S-Mo-X-homocitrate and serves as the site of nitrogen reduction (Einsle et al., 2002). A water-ﬁlled substrate channel that extends from the solventexposed surface to a speciﬁc face of the MoFe cofactor provides a pathway for substrate to bind to the active site (Barney et al., 2009). Several genes are involved in the synthesis and assembly of functional nitrogenase in rhizobia, albeit with high level of variations among different species. Photosynthetic Bradyrhizobium and Azorhizobium caulinodans carry about 15 nif genes required for nitrogenase synthesis, while Rhizobium and Sinorhizobium possess eight and nine genes, respectively. Some of the core nif genes, which are conserved in majority of the rhizobia are nifH (nitrogenase reductase), nifDK (α-and β-subunits of nitrogenase), nifEN (scaffold for MoFe cofactor), nifB (P cluster), and the regulatory gene, nifA (Masson-Boivin et al., 2009). The high energy requirement (16 moles of ATP for each mole of N2 reduced) of nitrogenase in rhizobia demands high levels of photosynthates as fuel from the plants. The extreme sensitivity of the rhizobia nitrogenase enzyme to oxygen may seem surprising, rhizobia being mostly aerobic bacteria.

459

Thus, legume plants synthesize leghemoglobin (Lb) to scavenge excess oxygen and provide a microaerobic environment (less than 10 nM of free O2) required for nitrogen ﬁxation. Lb are hemoproteins that have a high afﬁnity for oxygen due to their extremely low oxygen–dissociation constant (Garrocho-Villegas et al., 2007). Legumes contain several symbiotic Lb genes, which are essential for the formation of functional nodules but do not seem necessary for general plant growth and development (Ott et al., 2005; Ott et al., 2009). Wild-type nodules exhibit a steep oxygen gradient from the surface toward the center of the nodule. Legume plants silenced for Lb genes exhibit a very high level of oxygen (more than 4.5% of ambient O2) at the central and infected zone, in contrast to much lower levels in wild-type nodules (Ott et al., 2005). Thus, the high level of intricacy and sophistication in the functionality of nitrogenase and Lb in nitrogen ﬁxation has enabled legumes to occupy a unique niche among land plants. Nodule development and physiology Preinfection and infection Nodule development may be divided into three overlapping stages: preinfection, nodule initiation, and nodule differentiation. The preinfection stage commences with the release, by the host plants, of ﬂavonoids and isoﬂavonoids, which act as chemoattractants for the rhizobial symbionts (Peters et al., 1986). Flavonoids are polyphenolic compounds that constitute one of the most diverse classes of compounds in higher plants, with more than 4000 identiﬁed. Unlike ﬂavonoids, the distribution of isoﬂavonoids is limited to few taxonomic groups including the Leguminosae. Flavonoids and isoﬂavonoids allow the rhizobia to

460

NUTRIENT USE EFFICIENCY IN CROPS

recognize their potential host. For instance, Sinorhizobium meliloti speciﬁcally recognizes ﬂavonoids such as luteolin and crysoeriol released by alfalfa (Maxwell et al., 1989; Hartwig et al., 1990a). Similarly, Bradyrhizobium japonicum speciﬁcally responds to isoﬂavonoids, daidzein, and genistein secreted by soybean (Kosslak et al., 1987; Zhang and Smith, 1995). Upon perception, ﬂavonoids and isoﬂavonoids bind to NodD proteins, which are transcriptional regulators in rhizobia. This activates the expression of several nod genes (Hartwig et al., 1990b; Perret et al., 2000; D’Haeze and Holsters, 2002). Enzymes encoded by rhizobial genes such as nod, noe, and nol are required for the synthesis of nodulation factors, referred to as “Nod factors.” Chemically, Nod factors are lipochitooligosaccharide molecules with a backbone of β-1,4linked N-acetyl-D-glucosamine residues. They are N-acylated at the nonreducing terminal residue with acyl chains of different lengths and saturation levels (Dénarié et al., 1996). This chemical core of Nod factors is synthesized by enzymes encoded by genes of the nodABC operon. However, speciesspeciﬁc decorations such as the addition of fucosyl, suphuryl, acetyl, methyl, carbamoyl, and arabinosyl are encoded by enzymes encoded by other nod genes, as well as noe and nol genes (Perret et al., 2000; Oldroyd and Downie, 2008). These decorations provide great variation in the ﬁnal chemical makeup not only between different species of rhizobia but also within the same species (Perret et al., 2000; Oldroyd and Downie, 2004; Oldroyd and Downie, 2008). Nod factors are required for rhizobial infection and nodule development, are active at very low concentrations (10−9–10−12 M), and able to initiate responses in host plants similar to those elicited by the rhizobia themselves. The earliest responses elicited by Nod factors are ion ﬂuxes (Ca2+ inﬂux; H+, K+, and Cl− efﬂux), which alter the plasma mem-

brane potential in the root hair cells (Ehrhardt et al., 1996; Felle et al., 1999). From 10 to 20 min after addition of Nod factors, oscillations of calcium concentration and calcium spiking occur around and inside of the nucleus (Ehrhardt et al., 1996; Wais et al., 2000; Shaw and Long, 2003; Sieberer et al., 2009). Puriﬁed Nod factors induce cytoskeleton rearrangements that lead to root hair deformations, such as root hair waving, swelling, and branching (Heidstra et al., 1994; Spaink, 1995; Dénarié et al., 1996; Stougaard, 2000). Localized application of Nod factors by the rhizobia induces a curling of root hairs that entraps the bacteria and results in the formation of a microcolony inside the “Shepherd’s crook,” from which an infection thread originates (Fig. 20.1A). Infection threads are thin tubules ﬁlled with proliferating rhizobia and matrix growing inside plant cells and always separating the rhizobia from the plant cytoplasm. Infection threads reach the root cortex and release the bacteria into dividing cortical cells. In coordination with infection thread formation, Nod factors stimulate root cortical cells to reenter mitosis, resulting in the formation of a nodule primordium. The freshly dividing cortical cells internalize the invading bacteria through an endocytosis-like process using a host-derived membrane called the peribacteroid membrane, which results in the formation of symbiosomes. This Nod factor-dependent nodulation strategy is used by the phylogenetically distant symbionts, Methylobacterium nodulans (Jourand et al., 2005; Renier et al., 2008) and Cupriavidus taiwanensis (Amadou et al., 2008), demonstrating its widespread occurrence. In Andrea and other woody legumes, internalization of the rhizobia is not observed, and nitrogen ﬁxation takes place in suberized infection threads known as ﬁxation threads (Hirsch, 1992; Sprent and James, 2007). These ﬁxation threads are restricted to an individual host cell.

LEGUMES AND NITROGEN FIXATION

461

Two different modes of entry and nodulation strategies in rhizobia. (A) Nod factor-dependent root hair entry involves root hair contact with rhizobia, root hair curling, entrapping rhizobia, proliferation and infection thread growth via root hair to reach the cortical cell, and release of rhizobia in the newly divided nodule meristem. (B) Crack entry mode of infection involves entry of rhizobia via wounds or cracks formed due to the emergence of lateral roots. This mode of entry can be either Nod factor dependent or Nod factor independent. Nod factor-dependent crack entry involves early signaling and nodule organogenesis. Nod factor-independent crack entry bypasses the early signaling and triggers nodule organogenesis by cytokinin-dependent pathway.

Fig. 20.1.

Along with Nod factors, rhizobial exopolysaccharides (EPSs), lipopolysaccharides (LPSs), capsular polysaccharides (CPSs), and cyclic β-glucans are often required for infection thread formation and successful plant infection, possibly by suppressing plant defense responses (Leigh and Coplin, 1992; D’Haeze and Holsters, 2004). Among the various EPSs, succinoglycan (EPS1), a polymer of octasaccharide with succinyl, acetyl, and pyruvyl modiﬁcations, and galactoglucan (EPSII), are the subjects of intense studies. The role of EPS in rhizobial infection and nodule development was reported by several groups in various Galegoid legumes, such as Medicago sativa, Medicago truncatula, Pisum sativum, Trifolium pretense, and Leucaena leucocephala (Leigh et al., 1985; Pellock et al., 2000; Mazur et al., 2002; Jones et al., 2007; Jones et al., 2008). In M. truncatula, succinoglycan-deﬁcient (exo) mutants of S.

meliloti are symbiotically defective, resulting in the formation of small nodules that are devoid of bacteria (Leigh et al., 1985; Jones et al., 2008). The role of a type III secretion system in rhizobial infection and suppression of legume defense responses was proposed by several groups (Freiberg et al., 1997; Viprey et al., 1998; Kaneko et al., 2000; Gottfert et al., 2001; Marie et al., 2001; Zehner et al., 2008). Unlike Rhizobium NGR234, Mesorhizobium loti, or B. japonicum, the alfalfa partner S. meliloti lacks the type III secretion system (Marie et al., 2001). Therefore, the use of the type III secretion system to infect plant cells is not a common mechanism among rhizobia but instead may play a role in host speciﬁcity (Perret et al., 2000). Similarly, VirB/D4 type IV secretion system found in M. loti strain R7A is necessary for nodulation in Lotus carniculatus and enables this strain to nodulate a nonhost, Leucaena leucocephala (Hubber et al.,

462

NUTRIENT USE EFFICIENCY IN CROPS

2004). Another mechanism utilized by rhizobia to invade the host root hair is by degrading the cellulose present in the cell wall. A cellulase-encoding gene celC2 in Rhizobium leguminosarum was found to be crucial for root hair penetration (Robledo et al., 2008). Nod factor-dependent nodulation is not a universal strategy in legume–rhizobia interactions. Certain rhizobial strains, such as Bradyrhizobium BTAi1 and ORS278, form root and stem nodules on restricted species of Aeschynomene, although they lack the core nodABC genes required for the synthesis of Nod factors (Giraud et al., 2007). Since many species of Bradyrhizobium produce cytokinins (or equivalent purine derivatives), the recent identiﬁcation of cytokinin receptors in legumes has led to the hypothesis that these purine derivatives may play a key role in these Nod factorindependent symbiotic events (Chaintreuil et al., 2001; Gonzalez-Rizzo et al., 2006; Giraud et al., 2007; Murray et al., 2007). The role of cytokinins in nodule development is discussed later in this chapter. Entry through root hairs is not the only mechanism of invasion by rhizobia. In several legume species, rhizobia enter via cracks at lateral root bases, wounds, or natural openings on the epidermis (Fig. 20.1B). This method of intercellular invasion, also known as “crack entry,” is observed in several legumes, including Arachis, Sesbania, Andrea, and other woody legumes (Sprent, 2008). In Papilionoideae such as Arachis hypogaea, crack entry has been known for several years, but the molecular mechanisms involved in this mode of entry are still poorly understood. Sesbania rostrata, which usually grows in waterlogged conditions, displays both crack entry and Nod factor-dependent root hair entry of rhizobia. Hence, S. rostrata is an excellent system to dissect the molecular mechanism mediating “crack entry” invasion (Capoen et

al., 2005; Capoen et al., 2009). Apart from Nod factor-dependent infection thread formation, infection by crack entry also depends on rhizobial EPS. An EPS-defective mutant of rhizobia failed to form functional nodules in peanut, resulting in nodule-like structures that were devoid of bacteroids (Morgante et al., 2005; Morgante et al., 2007). Unlike in the Galegoids, the role of EPS in nodule organogenesis in Phaseoloids is still controversial (Hirsch, 1992; Parniske et al., 1994; Parveen et al., 1997; D’Haeze and Holsters, 2004). Nodule organogenesis Upon perception of Nod factors, along with rhizobial infection and invasion, cortical cell divisions mark the commencement of nodule organogenesis. Root cortical cells below the point of infection de-differentiate and reenter the cell cycle. The site of cell division (outer or inner cortex of the root depending on the host plant) determines the type of nodule that forms. Unlike the cell divisions involved in lateral root formation, the cell divisions that initiate nodule primordium are initially anticlinal (Hirsch, 1992). Some of the cortical cells are arrested in the G2 phase and allow passage of inward progressing infection threads. The rest of the cells that complete their cell cycle resume cell division to form the rest of the nodule where infection and nitrogen ﬁxation will occur. The bacteria released from the inward progressing infection thread are endocytosed into a subset of cells where they differentiate into nitrogen-ﬁxing bacteroids. The vascular bundles present in the nodule parenchyma of mature nodules are connected to the root vasculature. The different cells and tissue types within a mature nodule can be distinguished by their cytological, anatomical, and functional attributes, and, to some extent, with molecular markers (Hirsch, 1992; Stougaard, 2000).

LEGUMES AND NITROGEN FIXATION

A

463

B

Fig. 20.2. The two major nodule types observed in legume plants. (A) Indeterminate nodules on the roots of M. truncatula. (B) Determinate nodules on the roots of L. japonicus. Scale bar: 2 mm.

Genetic analyses show that rhizobial infection and nodule development are two separable processes, although they are tightly synchronized and coordinated for the making of a functional nodule. Legumes display a huge diversity with respect to nodule organogenesis. Based on the origin, anatomy, histology, host, and shape, legume nodules can be grouped into four types: indeterminate, determinate, aeschynomenoid, and lupinoid nodules (Hirsch, 1992; Sprent, 2008; Guinel, 2009). Although the structural features of these nodule types are discrete, there are a few unifying patterns observed among them with respect to their vasculature. They all possess vascular bundles sheathed by a vascular endodermis running through the uninfected parenchymatous cells, which encircles the infected tissue (Guinel, 2009). Indeterminate nodules are characterized by a persistent meristem that arises from the root inner cortex. Anticlinal cell divisions occur opposite to the protoxylem pole, suggesting a role for the vascular stele in conducting hormones regulating this event (Hirsch, 1992). The long-lived meristem makes the nodule elongated or club shaped, as newly dividing cells are added consistently at its distal end. Members of the

legume tribes Trifoliae and Viciae, such as Medicago, Trifolium, Pisum, and Vicia, form such indeterminate nodules (Fig. 20.2A). Mature nodules comprise distinct zones, such as zone I (meristematic), zone II (preﬁxation), interzone II–III, zone III (ﬁxation), and zone IV (senescence zone). The actively dividing meristematic cells are present in zone I, which gives rise to fully prepared cells with the ability to accommodate the invading infection thread and the released bacteria (zone II) and nitrogen-ﬁxing bacteroids (zone III). Interzone II–III is characterized by the presence of a heterogeneous mixture of cells, which are rich in starch and Lb transcripts (Perlick et al., 1996). A senescence zone is observed in fully mature nodules, where both host cells and rhizobial symbionts degenerate. In fully mature indeterminate nodules, an additional saprophytic zone (zone V) is often observed (Timmers et al., 2000). This saprophytic zone is characterized by the presence of nondifferentiated rhizobia, termed rhizoboids, which are more saprophytic than mutualistic (Timmers et al., 2000; Guinel, 2009). Generally, indeterminate nodules transport assimilated nitrogen as amides, whereas determinate nodules transport both amides (Glycine, Phaseolus, Vigna) and ureides

464

NUTRIENT USE EFFICIENCY IN CROPS

(Lotus) (Sprent and James, 2007). The bacteroids in indeterminate nodules are of three types: bacteroids in the invasion zone (zone I) are rod-shaped, still dividing and surrounded by a peribacteroid membrane (type 1), while more elongated and differentiated bacteroids (type 2) are present in the preﬁxation zone (zone II). The interzone II–III comprises type 3 bacteroids, which are fully elongated and display cytoplasmic heterogeneity (Vasse et al., 1990; Hirsch, 1992). These type 3 bacteroids are longer than freeliving rhizobia (5–10 μm), Y-shaped, and polynucleoid, with an average of 24C (C being the haploid DNA content). The symbiosomes are formed by a single bacteroid and are distributed perpendicular to the cell wall. Bacteroids in these types of nodules are terminally differentiated and are digested by plants during senescence and therefore cannot be subcultured from indeterminate nodules (Vasse et al., 1990; Mergaert et al., 2006; Den Herder and Parniske, 2009). This terminal differentiation of bacteria is mediated by nodule-speciﬁc cysteine-rich (NCR) peptides encoded by the host and processed by a recently identiﬁed signal peptidase complex (Van de Velde et al., 2010; Wang et al., 2010b). Determinate or desmodioid nodules lack a persistent meristem and are spherical in shape. Legumes such as Phaseolus, Glycine, Vigna, and Lotus form such determinate nodules that contain a central and a peripheral tissue (Fig. 20.2B). The peripheral tissue comprises the nodule cortex and the nodule parenchyma, which are separated by the nodule endodermis. Nodule parenchyma in determinate nodules encircles the central infected tissue and is embedded with vascular bundles. The ﬁrst cell divisions in the formation of desmodioid nodules are anticlinal, which are conﬁned to the outer cortex, but later extend to the pericycle and inner cortex of the root.

Cell division activity ceases 12–18 days after inoculation with rhizobia. Some cells in the central tissue are invaded by infection threads. Infected cells at the center are large and dense due to the presence of released bacteria and differentiating bacteroids (Hirsch, 1992). These infected cells are interspersed with highly vacuolated uninfected cells. Bacteroids in determinate nodules do not differ in their shape and size from free-living bacteria, being 1–2 μm in size, rod-shaped, and uninucleate or binucleate. Bacteroids in determinate nodules accumulate excess carbon obtained from the legume plants as poly-β-hydroxybutyrate (PHB), a polyester reserve of carbon. This highly reduced product is an important source of oxidizable substrates that help maintain the respiratory demand of the bacteroids and support nitrogen ﬁxation when the supply of photosynthate from the host is reduced (Kim and Copeland, 1996). Interestingly, bacteroids of indeterminate nodules do not accumulate PHB (Povolo et al., 1994; Cevallos et al., 1996). The symbiosomes in determinate nodules comprise multiple bacteroids and are distributed randomly within the infection zone. Bacteroids in determinate nodules are not terminally differentiated and can therefore be isolated and subcultured from a mature nodule (Muller et al., 2001; Mergaert et al., 2006; Den Herder and Parniske, 2009). The other two types of nodules, aeschynomenoid and lupinoid, differ in the mode of infection, morphology, and anatomy. Aeschynomenoid nodules are observed in peanuts, while lupinoid nodules are present in Lupinus sp. In aeschynomenoid nodules, infection takes place by crack entry through wounds that arise from lateral root emergence or through tufted hairs that originate from the epidermis of the lateral root primordium. These nodules do not arise from the tissue of primary roots but from the axils

LEGUMES AND NITROGEN FIXATION

of lateral roots. In lupinoid nodules, rhizobia penetrate the root in a similar way through the interstices found between the base of a root hair and its adjacent epidermal cell (Gonzalez-Sama et al., 2006). Both nodules have infected cells at the center with no adjoining uninfected cells (Sprent, 2008). Although both nodule types have broad attachments to the root, the aeschynomenoid nodule has a narrow base compared with the lupinoid nodule, which is the largest of all types. These nodules do not have lenticels on their surface. Bacterial proliferation in these two nodule types occur by successive division of infected cells at the center. With respect to the meristem, aeschynomenoid nodules have a nonpersistent meristem and are therefore small and oblate in shape. Lupinoid nodules have several lateral meristems of indeterminate growth (Guinel, 2009). Molecular mechanism of rhizobia infection and nodule organogenesis Early symbiotic signaling Genetic and genomic analyses in model legumes such as M. truncatula and Lotus japonicus have identiﬁed many genes involved in bacterial infection and nodule organogenesis. The species speciﬁcity associated with legume–rhizobia interaction relies on the high level of speciﬁcity between the Nod factor structure and their receptors. Receptor-like kinases (RLKs) located on the plasma membrane, such as Nod factor receptor 1 (NFR1) and NFR5 in L. japonicus are required for all responses to Nod factors and can recognize speciﬁc substitutions on the Nod factors (Madsen et al., 2003; Radutoiu et al., 2003; Radutoiu et al., 2007). In M. truncatula, Nod factor perception (NFP) is a homolog of Lotus NFR5 and is a compo-

465

nent of a signaling receptor required for all responses to Nod factors (Amor et al., 2003; Arrighi et al., 2006). In contrast, Medicago LYK3/HCL (homolog of NFR1) is required for infection thread formation, but not for cortical cell divisions, calcium spiking, or expression of some nodulin genes such as NIN, and is hypothesized to be part of an entry receptor (Wais et al., 2000; Catoira et al., 2001; Limpens et al., 2003; Smit et al., 2007). A similar system with a low stringency signaling receptor and a high stringency entry receptor probably exists in pea, but there is no evidence so far for different receptor complexes in L. japonicus (Ardourel et al., 1994; Limpens et al., 2003). Lectin ecto-apyrases (LNPs) are a group of proteins that bind to Nod factors with high afﬁnity, and regulate early nodulation signaling and nodule organogenesis. The role of LNPs in nodulation was initially demonstrated in Dolichos biﬂorus and subsequently in soybean (GS52), Medicago (apy1 and apy4), and Lotus (Etzler et al., 1999; Day et al., 2000; Cohn et al., 2001; McAlvin and Stacey, 2005; Govindarajulu et al., 2009). Their speciﬁc role is to hydrolyze nucleoside triphosphates and nucleoside diphosphates to nucloside monophosphate and orthophosphates, suggesting a role for extracellular ATP in legume nodulation. Another RLK with leucine rich repeats (LRR), known as NORK or DMI2 in Medicago and SYMRK in L. japonicus, also resides on the plasma membrane and plays a critical role in symbiotic signal transduction (Endre et al., 2002; Stracke et al., 2002; Limpens et al., 2005). NORK is also involved in actinorhizal symbiosis, suggesting conserved genetic machinery between these two types of nodule symbioses (Gherbi et al., 2008). To date, three interactors of NORK/ SYMRK have been identiﬁed: 3-hydroxyl 3-methylglutaryl coenzyme A reductase

466

NUTRIENT USE EFFICIENCY IN CROPS

(HMGR1), which interacts with NORK; a DNA-binding protein, SIP1, which interacts with SYMRK; and symbiotic remorin, SYMREM1, which interacts with NFP, LYK3, and NORK (Kevei et al., 2007; Zhu et al., 2008; Lefebvre et al., 2010). HMGR1 is required for nodule development, infection thread formation, and calcium spiking, indicating its role in the Nod factor signaling pathway at the same level as NORK (Kevei et al., 2007; J.M. Ané, unpublished data). SIP1 binds to the promoter region of the early nodulin gene NIN in L. japonicus, suggesting the existence of a calcium spikingindependent pathway for the activation of nodulin expression (Zhu et al., 2008). SymREM1 localizes on the host plasma membrane surrounding the bacteria and controls infection and release of rhizobia into the host cytoplasm (Lefebvre et al., 2010). The nucleus plays a central role in Nod factor signaling. Several nuclear proteins have been identiﬁed as required for calcium spiking initiation and transduction. Among them, two ion channels, DMI1/POLLUX and CASTOR, localized on the nuclear envelope mediate Nod factor-induced calcium spiking in root hair cells (Ané et al., 2004; Imaizumi-Anraku et al., 2005; Peiter et al., 2007; Riely et al., 2007; Charpentier et al., 2008). Mutants in two nucleoporins, NUP133 and NUP85, are also affected in calcium spiking, rhizobial infection, and nodule development (Kanamori et al., 2006; Saito et al., 2007). Although the precise role of these nucleoporins has not yet been resolved, they probably help trafﬁcking symbiotic proteins or symbiotic signals across the nuclear envelope. The nuclear envelope is probably the calcium source for intra- and perinuclear calcium spiking. However, the perception and transduction of this signal seem to take place only inside of the nucleus. A calciumand calmodulin-dependent protein kinase, DMI3/CCaMK, and its interactor, IPD3/

CYCLOPS, which are required for Nod factor signaling and very likely calcium spiking sensing, are both localized inside of the nucleus (Lévy et al., 2004; Mitra et al., 2004; Tirichine et al., 2006a,b; Messinese et al., 2007; Yano et al., 2008). Constitutively active alleles of DMI3/CCaMK have shown that this protein is not only required but also sufﬁcient for nodulin gene expression and nodule development (Tirichine et al., 2006a,b; Gleason et al., 2006). In addition, two GRAS (gibberellin-insensitive [GAI], repressor of gal-3 [RGA] and SCARECROW [SCR]) family transcriptional regulators, nodulation signaling pathway 1 (NSP1) and NSP2, are indispensable for Nod factorinduced activation of early nodulin genes (Kaló et al., 2005; Smit et al., 2005; Heckmann et al., 2006; Murakami et al., 2006). NSP2 interacts with DMI3, while NSP1 binds to the promoter region of ENOD11 and NIN and regulates their expression in a Nod factor-dependent manner (Oldroyd and Downie, 2008; Hirsch et al., 2009). Infection thread formation and nodule organogenesis Mutants affected in later events, such as infection thread formation and nodule development, have also been identiﬁed in model legumes. M. truncatula ERN1 (BIT1) and ERN2, which are AP2/ERF-like transcription factors, play a role downstream of DMI3 in Nod factor-induced transcriptional activation of ENOD11 (Andriankaja et al., 2007; Middleton et al., 2007). ERN3 acts as a negative regulator (repressor) of ERN1/ ERN2-mediated transcriptional activation. BIT1/ERN1 mutants undergo normal root hair curling upon contact with rhizobia, but form aberrant infection threads, which cannot penetrate through the epidermal layers into the dividing cortical cells (Andriankaja et al., 2007; Middleton et al.,

LEGUMES AND NITROGEN FIXATION

2007). A transmembrane-containing transcription factor, NIN, was identiﬁed initially in L. japonicus and later in M. truncatula as required for both infection thread formation and cortical cell division. However, nin mutants display normal responses for early Nod factor signaling, indicating a role after NSP1 and NSP2 (Schauser et al., 1999, 2005; Murakami et al., 2006; Marsh et al., 2007). Medicago ﬂotillin-like genes, FLOT2 and FLOT4, which are localized to membrane microdomains, play a critical role in infection thread initiation and nodule formation (Haney and Long, 2010); speciﬁcally, FLOT4 seems to be involved in polar growth of the infection thread, as silencing of FLOT4 affects infection thread elongation (Haney and Long, 2010). M. truncatula LIN (lumpy infections) encodes an E3 ubiquitin ligase with U-box and WD40 repeat domains, and the corresponding mutants are characterized by a reduction in the number of infections, aborted infection threads, and nodule primordia (Kuppusamy et al., 2004; Kiss et al., 2009). In M. truncatula dnf1 mutants, bacterial release into the host cell takes place; however, subsequent differentiation of the bacteria into bacteroids is blocked (Wang et al., 2010b). The DNF1 complex is an important host determinant, involved in the processing of NCR peptides, which mediate symbiosome development and terminal differentiation of bacteria in indeterminate nodules (Van de Velde et al., 2010; Wang et al., 2010b). Several other M. truncatula mutants, such as nip (numerous infections and polyphenolics), latd (lateral root organ-defective), api, rit1, and rpg (rhizobium-directed polar growth); L. japonicus itd1 (infection thread deﬁcient 1), sym7, itd3, itd4, and lot1 (low nodulation and trichome distortion) and crincke; and P. sativum mutants sym7, sym34, sym37, and sym38, all show arrested infection (Veereshlingam et al., 2004; Bright et al.,

467

2005; Ooki et al., 2005; Lombardo et al., 2006; Starker et al., 2006; Jones et al., 2007; Arrighi et al., 2008; Oldroyd and Downie, 2008; Teillet et al., 2008). Nodule organogenesis commences with programmed mitotic cell divisions in cortical cells to accommodate the invading rhizobia. Cortical cell divisions leading to nodule primordium always occur across from protoxylem poles (Timmers et al., 1999). Hence, regulators of the cell cycle obviously play a crucial role in nodule development (Cebolla et al., 1999). Characterization of plant mutants showed that rhizobial infection and cortical cell division can be uncoupled. The transcriptional regulation and developmental processes that occur in the cortex are different from those that occur in the epidermis. Several nodulins, ENOD40, ENOD2, ENOD12, and ENOD20, are speciﬁcally expressed in the cortex during nodule organogenesis (Crespi et al., 1994; Journet et al., 1994, 2001; Charon et al., 1999). ENOD40 plays a direct role in cell dedifferentiation and division of cortical cells during nodule formation (Charon et al., 1999). The HAP2-1 transcription factor plays a pivotal role during nodule development by controlling meristem persistence and rhizobial release (Combier et al., 2006). The spatial and temporal expression of the HAP2-1 transcription factor is controlled by a microRNA, miR169, in M. truncatula (Combier et al., 2006). A schematic illustration of genetic components involved in the symbiotic signaling pathway and the entry pathway, leading to coordination of infection and cortical cell division as shown in Figure 20.3. Hormonal regulation of nodulation Plant hormones are not only master regulators of general plant growth and development, but

468

LEGUMES AND NITROGEN FIXATION

also control nodule development in legumes. Different groups of hormones affect the nodulation process either by positively or by negatively regulating these events (Oldroyd and Downie, 2008; Mukherjee and Ané, 2011). Auxins and cytokinins Elegant studies demonstrate that the auxin transport and auxin/cytokinin ratio is crucial for successful nodule formation (Mathesius et al., 1998; Mathesius, 2008). Exogenous application of Nod factors results in a transient inhibition of auxin transport to the roots of Vicia sativa and Trifolium repens (Mathesius et al., 1998; Boot et al., 1999). Auxin reporter constructs were used to study auxin transport and accumulation during root nodule formation (Mathesius et al., 1998; Pacios-Bras et al., 2003). Similarly, application of auxin transport inhibitors (1-naphthylphthalamic acid [NPA]) induces pseudonodules and expression of early nodulin genes, such as ENOD2, ENOD12, and ENOD40, which are associated with the establishment of nodule primordia (Hirsch

469

et al., 1989; Hirsch and Fang, 1994; Fang and Hirsch, 1998). Interestingly, auxin homeostasis during nodulation appears to differ between determinate and indeterminate nodule types (Subramanian et al., 2007). In the roots of indeterminate noduleforming plants, such as Medicago, auxin transport is arrested at the site of infection by rhizobia, which is mediated by ﬂavonoids. Flavonoid-deﬁcient roots in these plants failed to inhibit auxin transport at the site of rhizobial inoculation and were defective for nodule organogenesis, while control roots showed a clear inhibition in auxin transport (Wasson et al., 2006). In plants forming determinate nodules, such as Glycine or Lotus, auxin transport is not arrested at the site of rhizobial entry (PaciosBras et al., 2003; Subramanian et al., 2006). RNAi silencing of the isoﬂavone biosynthesis pathway resulted in increased auxin transport and defective nodule formation. However, exogenous application of isoﬂavones inhibited auxin transport but failed to restore nodules (Subramanian et al., 2006). This suggests that isoﬂavones modulate auxin transport in plants forming determinate-type

Schematic illustration of genetic components involved in the symbiotic signaling pathway and entry pathway leading to coordination of infection and cortical cell division. The genetic components are grouped based on their functional hierarchy and their site of residence at the subcellular level. Nod factors are perceived by Nod factor receptors NFR1 and NFR5 (or NFP), which reside on the plasma membrane. This leads to root hair deformations and early calcium inﬂux. The signals are transduced to NORK/SYMRK. Interacting proteins, such as remorin (SymREM1), 3-hydroxyl 3-methylglutaryl coenzyme A reductase (HMGR1), and SYMRK interacting protein 1 (SIP1) may be involved in this early signal transduction from LysM receptor kinases to LRR receptor kinases. It is also hypothesized that these interacting proteins may connect the genetic components in the plasma membrane to components in the nucleus. The nucleus acts as a central player in the symbiotic signaling. M. truncatula DMI1 (Does not Make Infections 1) and nucleoporins NUP85 and NUP133 reside on the nuclear envelope and are required for Nod factor-induced calcium spiking. These signals are decoded by DMI3, a calcium- and calmodulin-dependent protein kinase (CCamK) and its interactor IPD3 (Interacting Protein of DMI3). A set of transcriptional regulators in the nucleus govern the expression of early nodulin genes and cortical cell division in coordination with rhizobial infection and proliferation. It is hypothesized that the entry pathway comprises M. truncatula LysM receptor kinase (LYK3), IPD3, and nodule inception (NIN), as mutants of these genes are affected for rhizobial infection, but not in the cortical cell division and meristem formation. Coordinated infection and cortical cell division is needed for successful nodule formation.

Fig. 20.3.

470

NUTRIENT USE EFFICIENCY IN CROPS

nodules, but the role of isoﬂavones in auxin transport is not necessary for nodule formation. M. truncatula cell division cycle 16 (CDC16), a core component of the anaphasepromoting complex, plays a key role in auxin signaling. Partial silencing of CDC16 results in reduced sensitivity to auxin, leading to reduced lateral root formation and a fourfold increase in the number of nodules (Kuppusamy et al., 2009). Cytokinins play a crucial role in various phases of legume nodulation including infection thread formation, induction of early nodulin genes, and cortical cell divisions. The role of cytokinins in the crack entry mode of infection in Bradyrhizobium defective for Nod factor synthesis was discussed earlier. Nod factor-defective rhizobia gain the ability to form nodules when engineered to produce transzeatin (Cooper and Long, 1994). Cytokinins secreted by rhizobia like Bradyrhizobium are perceived by cytokinin receptors in legumes, leading to the onset of nodule organogenesis (Giraud et al., 2007). Cytokinins are expressed in the dividing cortical cells and periphery of the emerging and young nodules in L. japonicus. However, their presence is not detected in the mature nodule (Lohar et al., 2004). Mutation in the cytokinin receptor gene, Lotus histidine kinase 1 (LHK1), results in a failure to form nodule primordia, although these mutants are unaffected for rhizobial infection. Similar effects were observed when an LHK1 ortholog in M. truncatula, CRE1, was silenced by an RNAi-based gene knockdown strategy (Gonzalez-Rizzo et al., 2006). Mutation in LHK1 is strong enough to suppress the supernodulation phenotype observed in har1 mutants. Reciprocally, a gain-of-function mutation in LHK1 resulted in the spontaneous formation of pseudonodules in L. japonicus, even in the absence of rhizobia (Murray et al., 2007; Tirichine et al., 2007).

Gibberellic acid and brassinosteroids In addition to auxins and cytokinins, gibberellic acid (GA) and brassinosteroids (BR) positively regulate nodulation. Application of GA induced pseudonodule structure in a nitrogen-sensitive manner (0–5 mM NO3 permissible and more than 10 mM NO3 inhibitory) in L. japonicus (Kawaguchi et al., 1996). Pea mutants that are affected in the GA and BR regulatory pathways show a reduction in nodule organogenesis (Ferguson et al., 2005). Exogenous application of 10−6 M GA to deﬁcient mutants completely restored nodulation comparable to wild-type plants, but not at higher concentrations (10−3 M). Grafting experiments suggested that both the shoot and the root control GA signaling, while only the shoot controls the BR level pertaining to nodulation (Ferguson et al., 2005). However, exogenous application of GA (10−6 to 10−9 M) blocked Nod factor-induced root hair deformations, nodulin gene expression, and nodule organogenesis in L. japonicus. This effect was counteracted by the application of a GA biosynthesis inhibitor, uniconazole-P. The number of spontaneous pseudonodules induced by gain-of-function mutations in DMI3/CCaMK and LHK1 was reduced on exogenous application of GA. Overexpression of the gain-offunction mutant, L. japonicus SLEEPY1 (sly1-d), a positive regulator of GA signaling, results in reduced nodule number, demonstrating a negative role of SLY1 in nodulation (Maekawa et al., 2009). Hence, GA acts as a positive regulator in Medicago and pea, while negative regulator in L. japonicus.

Ethylene In most legumes, ethylene acts as a negative regulator for the formation of nodules. Ethylene appears to regulate multiple steps,

LEGUMES AND NITROGEN FIXATION

such as Nod factor-induced calcium spiking, early nodulin gene expression, growth of the infection thread, nodule number, and nodule positioning. One possible mechanism of ethylene inhibition is by decreasing the sensitivity of epidermal cells to Nod factors, leading to the loss of ENOD11 expression and Nod factor-induced calcium spiking (Oldroyd et al., 2001b; Oldroyd and Downie, 2008). Therefore, it has been hypothesized that ethylene-sensitive components necessary for the perpetuation of symbiotic signaling are positioned either upstream or at the site of origin of nuclear calcium spiking. Application of 1 - aminocyclopropane - 1 - carboxylic - acid (ACC), an ethylene precursor, on Medicago and Lotus affects calcium spiking, ENOD11 expression, and polar growth of infection thread through the cortex, ultimately resulting in a reduced number of nodules (Oldroyd et al., 2001a). This suggests that ethylene regulates the function of a genetic component involved in the Nod factor signaling cascade upstream of calcium spiking (Oldroyd and Downie, 2008). In contrast, application of aminoethoxyvinylglycine (AVG), an ethylene synthesis inhibitor, promotes these symbiotic responses (Oldroyd et al., 2001b). The role of ethylene as a negative regulator of nodulation was demonstrated using the ethylene-insensitive skl mutant in M. truncatula (Penmetsa and Cook, 1997). Skl mutants are also affected in auxin transport; suggesting that cytokinin and auxin signaling are ethylene-dependent (Prayitno et al., 2006; Penmetsa et al., 2008). SKL is a homolog of ethylene-insensitive 2 (EIN2), a key member of the ethylene signal transduction pathway in Arabidopsis thaliana (Penmetsa et al., 2008). Medicago skl mutants showed an increased number of infection events with successful infection thread formation and a 10-fold increase in nodules (Penmetsa and Cook, 1997; Oldroyd et al., 2001b). A similar negative regulatory

471

role of ethylene was observed in P. sativum and L. japonicus but not in soybean (Glycine max) (Schmidt et al., 1999; Guinel and Geil, 2002; Nukui et al., 2004). Interestingly, S. rostrata, which allows root hair entry under dry conditions and crack entry under waterlogged conditions, possesses a switching mechanism controlled by ethylene concentration. While root hair entry requires low ethylene concentration, the crack entry mode of infection is possible at high ethylene levels (Goormachtig et al., 2004). Jasmonic acid and methyl jasmonate Like ethylene, jasmonic acid (JA), or methyl jasmonate, is a negative regulator of nodulation that affects both early signaling and nodule organogenesis. JA induces Nod factor expression and secretion in rhizobia, such as R. leguminosarum and B. japonicum (Rosas et al., 1998; Mabood et al., 2006). However, JA decreases the responsiveness of root hair cells to Nod factors, and reduces the frequency of calcium spiking. JA also changes the expression pattern of early nodulin genes such as RIP1 and ENOD11 (Sun et al., 2006). JA inhibits nodulation in a dose-dependent manner when applied exogenously and complete inhibition occurs at a 10 μM concentration in M. truncatula. However, such a high concentration does not affect rhizobial growth or Nod factor production (Sun et al., 2006). Abscisic acid In legume nodulation, abscisic acid (ABA) acts at multiple levels by independently regulating both Nod factor signaling in the epidermis, cytokinin-induced cell division in the cortex and nitrogen ﬁxation in mature nodules (Cho and Harper, 1993; Suzuki et al., 2004; Liang et al., 2007; Ding et al.,

472

NUTRIENT USE EFFICIENCY IN CROPS

2008; Tominaga et al., 2009). Unlike JA, ABA inhibits Nod factor-induced calcium spiking and early nodulin genes, such as RIP1 and ENOD1, in an ethyleneindependent manner (Ding et al., 2008). ABA negatively regulates not only nodule formation but also nitrogen ﬁxation. The L. japonicus mutant, enhanced nitrogen ﬁxation 1 (enf1), displays low sensitivity to ABA, increased root nodule number, and also enhanced nitrogen ﬁxation by decreasing the nitric oxide production in the nodules. This mutant also had a lower concentration of endogenous ABA than that of wild-type seedlings, indicating the negative role of ABA in nodule organogenesis and nitrogen

ﬁxation (Tominaga et al., 2009). Similar effects were observed in wild-type plants which were suppressed for ABA signaling, either by the overexpression of the dominant negative allele abscisic acid insensitive 1 (abi1-1) from A. thaliana, or by treatment with abamine, an inhibitor of ABA biosynthesis (Ding et al., 2008; Tominaga et al., 2009). In Medicago, mutants of sensitivity to ABA (sta-1) show reduced sensitivity to ABA and are hypernodulators (Ding et al., 2008). A schematic illustration of hormonal regulation of Nod factor signaling, nodule organogenesis, and nitrogen ﬁxation is presented in Figure 20.4.

Schematic illustration of hormonal regulation of Nod factor signaling, nodule organogenesis, and nitrogen ﬁxation. Auxins, cytokinins, gibberellic acid (GA), and brassinosteroids (BR) act as positive regulators in nodule organogenesis. Ethylene, jasmonic acid (JA), abscissic acid (ABA), and salicylic acid (SA) act as negative regulators and affect early signaling, nodulin gene expression, meristem formation, and nitrogen ﬁxation. In Lotus, GA is shown to negatively regulate nodulin gene expression and nodule meristem formation.

Fig. 20.4.

LEGUMES AND NITROGEN FIXATION

Evolutionary perspectives The diversity and high level of host speciﬁcity in legume–rhizobia interactions suggest that legumes exert a high selection pressure on rhizobia. The evolution of rhizobia might have involved different strategies. Horizontal gene transfer of nodulation and nitrogen ﬁxation-clustered genes within a “nod-nif kit” is considered a prime factor in the emergence of rhizobia. One of the rhizobial traits that may have favored such an event is the facultative saprophytic nature of rhizobia, which allows the exchange of genetic material with other soil bacteria (Sullivan et al., 1995; Barcellos et al., 2007). The acquisition of nod-nif genes allows a soil bacterium to infect, nodulate, and ﬁx nitrogen in legumes. The nod-nif kit may have been transferred to a diverse group of soil bacteria via plasmids, phages, or even naked DNA (Sullivan et al., 1995; Rogel et al., 2001; Barcellos et al., 2007). Long-term evolution optimized the symbiotic performances, host range, and competitiveness of rhizobia (Martinez-Romero, 2009). Some of the proposed molecular events leading to the evolution of rhizobia include (1) integration of newly acquired nod-nif genes to ﬁt in the preexisting regulatory circuits of recipient bacteria (Dixon and Kahn, 2004); (2) recruitment of genes necessary for infection, such as, exo, bacA, eps, lps, and celC2, which show restricted distribution in soil bacteria communities (Amadou et al., 2008; Robledo et al., 2008); (3) mechanisms that suppress plant defense responses; (4) duplication and integration of glmS and cysD in nod-box to improve nodulation efﬁciency; (5) incorporation of allelic variation in nodABC genes for Nod factor length and acylation reﬁnement; (6) recruitment of genes for new Nod factor decorations (nodZ for fucosylation) and a type III secretion system for host range modulation; and (7) replacement of incoming nif genes by their endogeneous counter-

473

parts by the recipient bacteria (Masson-Boivin et al., 2009). The emergence of rhizobia from nonrhizobial soil bacteria is well supported by several observations. The phylogenetic tree based on 16S rDNA sequences from selected α-, β-, and γ-proteobacteria contains a mixed population of both rhizobia and nonrhizobia (Masson-Boivin et al., 2009). Essential nod and nif genes are located on symbiotic plasmids or genomic islands that are readily transferable, and, once transferred, these symbiotic genes confer the ability to nodulate legumes to the recipient bacteria both in ﬁeld and laboratory conditions (Sullivan et al., 1995; Brom et al., 2004). Finally, the core symbiotic genes nodABC are monophylgenetic in origin (Laguerre et al., 2001; Chen et al., 2003). Several species of Burkholderia nodulate legumes with varying levels of host speciﬁcity (Sprent, 2007). Some are capable of ﬁxing nitrogen ex planta (Elliott et al., 2007). Although all the early reports were from Mimosoid legumes, there is now evidence that Papilionoid legumes such as Cyclopia (Elliott et al., 2007) and Dalbergia (Rasolomampianina et al., 2005) can also be nodulated with Burkholderia sp. Legumes evolved to occupy a unique niche in the plant kingdom due to their ability to form RNS with rhizobia. Fossil and genetic evidence suggests that legumes evolved about 60 million years ago, possibly by hijacking preexisting mycorrhizal and phagocytosis machineries and exploiting them to infect legume roots. The key mechanisms that legumes may have seized and ﬁne-tuned from their predecessors include (1) the ability to recognize their symbiotic partners; (2) the ability to turn off defense responses against invading symbionts; (3) pollen tube growth, which shares commonality with that of the rhizobia infection thread growth through root hairs in plants; (4) hormonal regulation of cell differentiation; and

474

NUTRIENT USE EFFICIENCY IN CROPS

(5) acquiring genes for Lb. The current understanding of the infection mechanisms suggests that Nod factor-dependent root hair entry is evolutionarily younger than crack entry. Rhizobia and other bacteria can penetrate the roots of a variety of plants through cracks; for example, rhizobia are able to enter the cells of potato tissue (Spencer et al., 1994). Similar modes of entry into host tissue was demonstrated in the nonnodulating Caesalpinioid legume Gleditsia triacanthos (Bryan et al., 1996), where the invading bacteria were conﬁned by a hostderived membrane compartment comparable to infection thread material, but failed to penetrate the cells. This infection process is considered the ﬁrst stage in the evolution of nodulation. Mimosoid legumes, Neptunia plena and Mimosa scabrella, which lack root hair infection, showed intercellular infection threads. In these cases, the mode of entry is through wounds present at the emergence sites of lateral or adventitious roots (James et al., 1992; Sprent, 2007). As with legume nodulation, arbuscular mycorrhization (AM) is a root endosymbiotic association of land plants with glomeromycetous fungi. AM evolved about 400 million years ago and paved the way for the emergence of land plants (Kistner and Parniske, 2002; Wang et al., 2010a). Genetic dissection of AM events in the model legumes, M. truncatula and L. japonicas, indicates that at least seven genes are shared between legume nodulation and AM (Banba et al., 2008; Markmann et al., 2008; Markmann and Parniske, 2009). This set of genes, collectively termed the common symbiotic pathway (CSP) genes in L. japonicus, includes NORK/SYMRK, CASTOR, POLLUX, NUP133, NUP85, DMI3/CCaMK, and IPD3/CYCLOPS. Mutants of these genes in the model legumes are defective for both symbioses. Homologs of NORK/ SYMRK, CASTOR, POLLUX, DMI3/CCaMK, and IPD3/CYCLOPS are conserved in the

majority of land plants and show vertical inheritance (Zhu et al., 2006; Wang et al., 2010a). Among the CSP genes, the NORK/ SYMRK receptor-like kinase has striking structural and functional divergence with respect to its potential to confer symbiotic abilities. Three distinct version of NORK have been identiﬁed in angiosperms (Markmann et al., 2008). A full version of NORK in the Rosids lineage possesses a conserved protein kinase domain, a transmembrane domain, a leucine-rich repeat (LRR) domain, a conserved extracellular domain, and an N-terminal region of unknown function. Variations have been observed with respect to the length of the LRR domain and the presence or absence of an N-terminal region across the plant kingdom. Members of the Rosids lineage establish RNS, ARS, and AM associations, while those higher plants with shorter versions of NORK only undergo AM association. Nonleguminous orthologs of other CSP genes, such as the two-ion channels CASTOR and POLLUX, DMI3/CCaMK, and the RNS-speciﬁc GRAS domain transcription factor NSP1, were able to partially restore nodulation in rescue assays (Gleason et al., 2006; Godfroy et al., 2006; Heckmann et al., 2006; Banba et al., 2008; Chen et al., 2008, 2009). Similarly, DMI3 homologs from lower plants such as liverworts and hornworts could rescue the AM phenotype in a dmi3 mutant of M. truncatula, indicating a functional conservation of these vertically inherited genes (Wang et al., 2010a). In parallel, legumes probably modiﬁed the lateral root genetic program to invent nodule organogenesis. The role of cytokinins and the cytokinin receptor LHK1/ CRE1 in nodule organogenesis is discussed earlier in this chapter. Cytokinin receptors (LHK1–3) are conserved in higher plants and are involved in developmental processes, such as the emergence of the lateral root, root length, and root vasculature,

LEGUMES AND NITROGEN FIXATION

which structurally resemble actinorhizal nodules. It is hypothesized that legumes acquired the AM symbiosis machinery from their non-nodulating ancestors to mediate symbiont recognition and intracellular infection, and lateral root machinery to control nodule organogenesis, and ﬁnetuned it to form functional nodules (Markmann et al., 2008). Applied agricultural implications Rhizobia bioinoculants and Nod factors applications Application of rhizobia as bioinoculants to enhance nodulation in legume plants is a common practice across the world (Herridge et al., 2002). About 2000 tons of rhizobia inoculants worth $50 million are produced annually, and it is estimated to meet the requirement of 20 million hectares of legumes (Singleton et al., 1997). By far, the United States stands as the largest producer of rhizobial inoculant with an annual production of about 1000 tons (Singleton et al., 1997). Seed treatment is the most popular mode of rhizobia inoculation, but other methods such as soil application to seedbed or seedling application as root dip are also practiced (Dart, 1988; Jha et al., 1994). Although rhizobia are ubiquitous in legumegrowing regions, many soils used for legume cultivation lack adequate numbers of highly effective rhizobial strains. Absence of a symbiotically related legume in the recent history of the land, poor nodulation when the same crop was grown on the land previously, legume crop following a nonlegume in a rotation, and land undergoing reclamation are few factors that necessitate rhizobial inoculation (Allen and Allen, 1961). The general recommendation is to apply rhizobial inoculum to the ﬁelds with no history of legume cultivation in the past 3–5 years, a

475

soil pH of less than 6.0, sandy texture, low organic matter, or ﬂooded for more than a week (Abendroth and Elmore, 2006). Fields planted with soybean for the ﬁrst time require inoculation of rhizobia to ensure nodulation and to enhance the rhizobial population in the soil (Nelson et al., 1978; Hiltbold et al., 1980). The ability of soybean to form nodules decreased with the length of time since the last cultivation with soybean (Larson and Siemann, 1998). In several instances, the application of rhizobial inoculants signiﬁcantly increased the crop yield where legumes had never been produced (Schulz and Thelen, 2008; Ruiz Diaz et al., 2009). However, no signiﬁcant increase in soybean yield was observed due to the application of rhizobia to the ﬁelds where soybean was grown in the previous season (Nelson et al., 1978; De Bruin et al., 2010). In addition to the direct applications of rhizobia as bioinoculants to improve legume nutrition, simple application of Nod factors (or lipophilic chitin oligosaccharides [LCOs]) has signiﬁcant impacts on crop cultivation. In poor soil with a limited rhizobial population, treatments with Nod factors increase the number of nodules per plant (Macchiavelli and Brelles-Mariño, 2004). In the United States, LCO promoter technology®, which utilizes the beneﬁcial effects of rhizobial Nod factor molecules, has been widely popularized by EMD Crop BioSciences (Milwaukee, WI) for promoting growth in economic crops, such as soybean (Optimize 400), peanuts (Optimize LIFT), forage crops (Optimize Gold, Nitragin), and other pulses (Optimize Pulse). Nod factors are capable of triggering responses in crop plants in amounts as low as picomolar concentrations (Spaink, 1995, 1996). Nod factor-induced lateral root formation, which results in root proliferation in the model legume M. truncatula, is dependent on the DMI1/DMI2-mediated signaling pathway (Oláh et al., 2005).

476

NUTRIENT USE EFFICIENCY IN CROPS

Breeding legumes for enhanced nitrogen ﬁxation Enhancing the efﬁciency of BNF in legumes through conventional breeding programs has been pursued over the past decades with some success (Phillips et al., 1971; Mytton, 1984; Nutman, 1984; Herridge and Rose, 2000). There are a few success stories in the breeding strategies for improving nitrogen ﬁxation in legumes. As early as the 1980s, ﬁve common bean germplasm lines with superior nitrogen-ﬁxing abilities were registered. The backcross inbred method of population development was utilized to develop superior varieties in common beans with enhanced nitrogen ﬁxation (Bliss and Hardarson, 1993). In this approach, the inbred backcross families were developed by hybridization of an adapted cultivar with a superior nitrogen-ﬁxing parent, with few initial backcrosses followed by successive single seed descent. Similarly, divergent selection for speciﬁc nodule enzymes, such as aspartate aminotransferase and asparagine synthetase, was attempted in alfalfa (Degenhart et al., 1992). Recurrent selection was used for improving seed yield and nitrogen ﬁxation in the common bean (Barron et al., 1999). Although the ongoing genetic dissection of model legumes, economically important oilseeds, and grain legumes signiﬁcantly enriches our knowledge of the molecular mechanism behind nitrogen ﬁxation, their ﬁeld applications seem to be in the distant future. A tremendous revival of legume breeding programs for the selection of agronomic traits favoring enhanced nitrogen ﬁxation is an urgent need. The research areas that focus on identifying the agronomic traits and developing successful breeding strategies include (1) investigation of carbon and nitrogen allocation in plant tissues; (2) legume– rhizobium interactions and host speciﬁcity for superior rhizobial strains; (3) develop-

ment of a simple, rapid, nondestructive, ﬁeld-based, and cost-effective assays for nitrogen ﬁxation; (4) enhancing symbiotic nitrate tolerance, early nodulation, and delayed nodule senescence; (5) root colonization and saprophytic competence; and (6) stress tolerance and its impact on interactions between the partners (Herridge and Rose, 2000; Graham et al., 2004). In addition to selecting traits for enhanced nitrogen ﬁxation, legume breeding programs must also incorporate other agronomic traits, such as yield attributes, crop stand, adaptability, and disease and insect resistance. Regional preferences for cultivars, cropping systems, day length, and seed characteristics must also be considered (Bliss and Hardarson, 1993). Several studies have investigated variations among species and cultivars regarding traits associated with nitrogen ﬁxation. Legumes, such as soybean, common bean, clover, and alfalfa, show cultivar differences with respect to nitrogen ﬁxation traits, such as nodule number and mass, speed of nodulation, lateral root formation, nodulation postﬂowering, nitrogen accumulation, acetylene reduction activity, allantoic acid production, and nodule enzymes (Hardy et al., 1973; Rennie and Kemp, 1983; Nutman, 1984; Jessen et al., 1988; Degenhart et al., 1992; Pazdernik et al., 1996; Graham et al., 2004). Of the several approaches, breeding in nitrogen-poor soils for improving symbiotic ability is considered a promising and practical one (Graham et al., 2004). Transfer of root nodule symbiosis to nonlegumes: a feasibility study For several decades, it has been the dream of scientists to develop transgenic cereals and other nonlegumes that can form root nodules with rhizobia. One of the long-term goals of deciphering the molecular mechanism of RNS is to reconstitute the symbiotic

LEGUMES AND NITROGEN FIXATION

signaling machinery in nonleguminous plants. Albeit extremely challenging, such a transfer of RNS machinery to nonleguminous crops could tremendously help minimize the exogenous application of energy-expensive nitrogen fertilizers. Apart from reducing the cost of cultivation for food, feed, and bioenergy, this strategy would also provide protection for the environment against pollution from chemical fertilizers. This task will be extremely challenging, but the availability of advanced molecular tools in rhizobia and host plants makes this goal more realistic now than ever before. Several approaches could help in reaching this goal. The commonalities between legume RNS and ARS should be considered to identify key components and missing links in non-nodulating plants (Markmann and Parniske, 2009). Likewise, the molecular dissection of dissimilarities between nodulating and non-nodulating legumes seems a promising approach to identify master regulators of RNS, which are missing in nonleguminous plants (Sprent and James, 2007). Molecular dissection of RNS machinery in the nonleguminous plant Parasponia could also provide clues as to the master players in the symbiotic signaling, which non-nodulating plants lack. Attempts to transfer RNS to nonleguminous plants, both mono- and dicotyledonous, have been made since the 1980s (Ridge et al., 1993). Artiﬁcial induction of nodulelike structures was obtained in partially macerated rice roots exposed to rhizobia, as well as in Brassica napus (Al-Mallah et al., 1989, 1990). Another attempt was made to transfer RNS to rice by transformation (Zhang et al., 2001). Genes encoding a pea lectin and Parasponia hemoglobin were cloned into a plant expression vector and introduced by particle bombardment into rice calli from immature embryos (Zhang et al., 2001). Although these early attempts to transfer the RNS pathway to cereals were unsuccess-

477

ful, they provide a platform to explore further possibilities for successful transfer of root nodule symbiotic machinery to nonlegumes. The high speciﬁcity of legume–rhizobia interactions makes it difﬁcult to nodulate one group of legumes with the symbiotic partner of other group. Most rhizobia have a very narrow host range, making studies across species difﬁcult. However, the rhizobial strain NGR234, with a wide host range of 353 legume species representing 122 genera, seems an interesting candidate for the transfer of RNS to nonlegume crops (Pueppke and Broughton, 1999). To reconstitute RNS machinery in nonleguminous plants, the genetic makeup of the host needs to be ﬁne-tuned, to allow major nodulation events such as infection thread formation, intracellular uptake, and the formation of root nodules (Markmann and Parniske, 2009). Prevalence of CSP genes in the majority of land plants suggests that they could be adjusted to accommodate RNS. The homolog of NORK/SYMRK is necessary for actinorhizal interaction in Casuarina glauca and in the cucurbit Datista glomerata (Gherbi et al., 2008; Markmann et al., 2008). Events leading to the intracellular accommodation of the symbiont in all three of these mutualistic interactions share remarkable similarities. Prior to the invasion by the respective endosymbiont, the host cell prepares to accommodate the invading partner by cytoskeletal and organellar rearrangements and nuclear movements (Genre et al., 2005, 2008). The formation of the prepenetration apparatus (PPA) in AM symbiosis, and preinfection threads (PIT) in RNS and ARS present major similarities. Since AM symbiosis is prevalent in the majority of land plants, the components necessary for intracellular accommodation of symbionts may exist in nonleguminous crops already. Therefore, in our opinion, engineering a receptor-mediated perception of nitrogen-ﬁxing symbionts in

478

NUTRIENT USE EFFICIENCY IN CROPS

nonleguminous plants is not just a dream and will be possible in the future. The recognition of symbiotic partners by the host is a key factor for the establishment of mutualistic interactions. Such symbiont recognition is mediated by receptor kinases in plants. LysM receptor kinases NFR1 and NFR5 in Lotus and NFP, and LYK3 in Medicago are proven Nod factor receptors. The introduction of Lotus Nod factor receptors into Medicago enables the latter to enter into a symbiotic association with the Lotus symbiont, M. loti. Such an extreme speciﬁcity of receptors toward Nod factors needs to be relaxed by genetic manipulations, which would enable the host plant to recognize a wide array of rhizobial species. Homologs of LysM receptor kinases are present in nonleguminous crop plants, such as A. thaliana and rice, where they trigger the defense reaction upon perception of chitin oligomers, a common elicitor of fungal pathogens. Utilizing such preexisting recognition machineries without compromising their defenserelated role presents additional challenges. Alternatively, Nod factor-independent infection strategies, like those observed in certain species of photosynthetic Bradyrhizobia, have also been considered as an avenue for nonleguminous plants. Although it is not clear that LysM receptor kinases might play a role in recognition of yet unknown actinorhizal factors, the dual infection in Parasponia (Ulmaceae) by both Frankia and Nod factor-dependent rhizobia suggests this possibility (Trinick, 1979). The occurrence of endophytic colonization of plants roots by rhizobia has been demonstrated several times over the past decade (GutierrezZamora and Martinez-Romero, 2001; Cocking, 2003; Chi et al., 2005; Singh et al., 2009). This suggests that rhizobia can utilize the preexisting infection machinery in nonlegume plants to colonize them as endophytes. Rhizobial infection and nodule organogenesis are two separable events in RNS.

Genes involved in the spatiotemporal synchronization of these two distinct events are mandatory for successful RNS. Further characterization of mutants such as IPD3/CYCLOPS or NIN might help in identifying the coordinators of epidermal (infection) and cortical (infection and nodule organogenesis) events. In addition to having the genes necessary for rhizobial infection, nodule organogenesis and their coordination, legumes possess symbiosis-speciﬁc Lb genes for the protection and function of nitrogenase activity. Homologs of Lb genes are present in the majority of land plants, including the evolutionarily older Bryophytes and Pteridophytes (Garrocho-Villegas et al., 2007). In nonleguminous plants, including rice and maize, homologs of Lb are present as nonsymbiotic hemoglobins (nsHbs). Such nsHbs from Parasponia are expressed in the nodules formed during rhizobial interaction and facilitate the diffusion of oxygen to bacteroids (Appleby et al., 1983; Gibson et al., 1989). The role of nsHbs in symbiosis in actinorhizal plants, such as Causarina glauca, Alnus glutinosa, and Myrica gale, is still unknown (Fleming et al., 1987; Pathirana and Tjepkema, 1995; Suharjo and Tjepkema, 1995). Similarly, Lb genes are induced during AM, but their role in this widespread ancestral symbiosis is unknown. These observations provide encouraging evidence that nsHbs can be utilized in the assembly of the nodulation pathway in nonlegume plants. Together these observations suggest that reconstitution of root nodule symbiotic machinery in nonlegume plants is a feasible yet distant goal. Perspectives With the ability to increase soil nitrogen levels, legumes are a key component in many agroecological environments. Their rich diversity and adaptability make them a

LEGUMES AND NITROGEN FIXATION

primary choice for crop rotations, intercropping, mixed cropping, or relay-cropping systems. The beneﬁts of incorporating legumes in cropping systems rely heavily on their ability to establish efﬁcient nitrogenﬁxing symbioses with rhizobia. However, under biotic and abiotic stresses, legumes struggle to develop efﬁcient symbiotic associations. Increased knowledge on the mechanisms allowing the establishment of these symbioses will provide practical remedies to enhance the utility of legumes in suboptimal environments. Continued and focused efforts on developing improved rhizobial inoculums, and breeding methods for superior legume cultivars with enhanced nitrogen ﬁxation, are urgent needs. The ongoing genetic and genomic approaches aiming at deciphering the mechanisms of symbiotic signaling are paving the way for transferring the symbiotic abilities of legumes to nonlegume crops, which will be a major venture for sustainable agriculture in the future. Acknowledgments The authors sincerely thank Kari Forshey and Maxime Magne for critically reviewing this manuscript and Ronald Crandall for his help with the preparation of ﬁgures. The ﬁnancial support for this work was provided by Hatch grant and National Science Foundation grant to JMA. References Abendroth, L.J. & Elmore, R.W. (2006) Soybean Inoculation: Applying the facts to Your Fields, G1622 ed. University of Nebraska-Lincoln Ext., Lincoln. Allen, E.K. & Allen, O.N. (1961) The scope of Nodulation in the Leguminosae. In: Recent Advances in Botany. Vol I, Proceedings of the Ninth International Botanical Congress, pp. 585–588, University of Toronto Press, Toronto. Al-Mallah, M.K., Davey, M.R., & Cocking, E.C. (1989) Formation of nodular structures on rice seedlings by rhizobia. Journal of Experimental Botany 40, 473–478.

479

Al-Mallah, M.K., Davey, M.R., & Cocking, E.C. (1990) Nodulation of oilseed rape (Brassica napus) by rhizobia. Journal of Experimental Botany 41, 1567–1572. Amadou, C., Pascal, G., Mangenot, S., et al. (2008) Genome sequence of the beta-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia. Genome Research 18(9), 1472–1483. Amor, B.B., Shaw, S.L., Oldroyd, G.E.D., et al. (2003) The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium ﬂux and root hair deformation. The Plant Journal 34(4), 495–506. Andriankaja, A., Boisson-Dernier, A., Frances, L., et al. (2007) AP2-ERF transcription factors mediate Nod factor dependent Mt ENOD11 activation in root hairs via a novel cis-regulatory motif. Plant Cell 19(9), 2866–2885. Ané, J.M., Kiss, G.B., Riely, B.K., et al. (2004) Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes. Science 303(5662), 1364–1367. Appleby, C.A., Tjepkema, J.D., & Trinick, M.J. (1983) Haemoglobin in a Non-leguminous plant, Parasponia: possible genetic origin and function in nitrogen ﬁxation. Science (New York, N.Y.) 220(4600), 951–953. Ardourel, M., Demont, N., Debelle, F., et al. (1994) Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses. The Plant Cell 6(10), 1357–1374. Arrighi, J.F., Barre, A., Ben Amor, B., et al. (2006) The Medicago truncatula lysin [corrected] motifreceptor-like kinase gene family includes NFP and new nodule-expressed genes. Plant Physiology 142(1), 265–279. Arrighi, J.F., Godfroy, O., de Billy, F., Saurat, O., Jauneau, A., & Gough, C. (2008) The RPG gene of Medicago truncatula controls Rhizobium-directed polar growth during infection. Proceedings of The National Academy of Sciences of The United States of America 105(28), 9817–9822. Banba, M., Gutjahr, C., Miyao, A., et al. (2008) Divergence of evolutionary ways among common sym genes: CASTOR and CCaMK show functional conservation between two symbiosis systems and constitute the root of a common signalling pathway. Plant and Cell Physiology 49(11), 1659–1671. Barcellos, F.G., Menna, P., Batista, J.S., & Hungria, M. (2007) Evidence of horizontal transfer of symbiotic genes from a Bradyrhizobium japonicum inoculant strain to indigenous diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a

480

NUTRIENT USE EFFICIENCY IN CROPS

Brazilian Savannah soil. Applied and Environmental Microbiology 73(8), 2635–2643. Barney, B.M., Yurth, M.G., Dos Santos, P.C., Dean, D.R., & Seefeldt, L.C. (2009) A substrate channel in the nitrogenase MoFe protein. Journal of Biological Inorganic Chemistry: JBIC: A Publication of the Society of Biological Inorganic Chemistry 14(7), 1015–1022. Barron, E.J., Pasini, R.J., Davis, D.W., Stuthman, D.D., & Graham, P.H. (1999) Response to selection for seed yield and nitrogen (N2) ﬁxation in common bean. Field Crops Research 62(119), 128. Bliss, F.A. & Hardarson, G. (1993) Enhancement of biological nitrogen ﬁxation of common bean in Latin America. Plant Soil 152, 1–160. Boot, K.J.M., van Brussel, A.A.N., Tak, T., Spaink, H.P., & Kijne, J.W. (1999) Lipochitinoligosaccharides from Rhizobium leguminosarum bv. viciae reduce auxin transport capacity in Vicia sativa subsp. nigra roots. Molecular Plant-Microbe Interactions 12, 839–844. Bright, L.J., Liang, Y., Mitchell, D., & Harris, J. (2005) The LATD gene of Medicago truncatula is required for both nodule and root development. Molecular Plant-Microbe Interactions 18, 521–532. Brom, S., Girard, L., Tun-Garrido, C., et al. (2004) Transfer of the symbiotic plasmid of Rhizobium etli CFN42 requires cointegration with p42a, which may be mediated by site-speciﬁc recombination. Journal of Bacteriology 186(22), 7538–7548. Bryan, J.A., Berlyn, G.P., & Gordon, J.C. (1996) Towards a new concept of the evolution of symbiotic nitrogen ﬁxation in the Leguminosae. Plant and Soil 186, 151–159. Burgess, B.K. & Lowe, D.J. (1996) Mechanism of molybdenum nitrogenase. Chemical Reviews 96(7), 2983–3012. Capoen, W., Goormachtig, S., De Rycke, R., Schroeyers, K., & Holsters, M. (2005) SrSymRK, a plant receptor essential for symbiosome formation. Proceedings of the National Academy of Sciences of the United States of America 102(29), 10369–10374. Capoen, W., Oldroyd, G.E., Goormachtig, S., & Holsters, M. (2009) Sesbania rostrata: a case study of natural variation in legume nodulation. The New Phytologist 186, 340–345. Catoira, R., Timmers, A.C.J., Maillet, F., et al. (2001) The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling. Development 128(9), 1507–1518. Cebolla, A., Vinardell, J.M., Kiss, E., et al. (1999) The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants. The EMBO Journal 18, 4476–4484.

Cevallos, M.A., Encarnacion, S., Leija, A., Mora, Y., & Mora, J. (1996) Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate. Journal of Bacteriology 178(6), 1646–1654. Chaintreuil, C., Boivin, C., Dreyfus, B., & Giraud, E. (2001) Characterization of the common nodulation genes of the photosynthetic Bradyrhizobium sp. ORS285 reveals the presence of a new insertion sequence upstream of nodA. FEMS Microbiology Letters 194(1), 83–86. Chan, M.K., Kim, J., & Rees, D.C. (1993) The nitrogenase FeMo-cofactor and P-cluster pair: 2.2 A resolution structures. Science (New York, N.Y.) 260(5109), 792–794. Charon, C., Sousa, C., Crespi, M., & Kondorosi, A. (1999) Alteration of enod40 expression modiﬁes Medicago truncatula root nodule development induced by Sinorhizobium meliloti. The Plant Cell 11, 1953–1965. Charpentier, M., Bredemeier, R., Wanner, G., Takeda, N., Schleiff, E., & Parniske, M. (2008) Lotus japonicus CASTOR and POLLUX are ion channels essential for perinuclear calcium spiking in legume root endosymbiosis. The Plant Cell 20(12), 3467–3479. Chen, W.M., Moulin, L., Bontemps, C., Vandamme, P., Béna, G., & Boivin-Masson, C. (2003) Legume symbiotic nitrogen ﬁxation by beta-proteobacteria is widespread in nature. Journal of Bacteriology 185(24), 7266–7272. Chen, C., Ané, J.M., & Zhu, H. (2008) OsIPD3, an orthologue of the Medicago truncatula DMI3 interacting protein IPD3, is required for mycorrhizal symbiosis in rice. The New Phytologist 180, 311–315. Chen, C., Fan, C., Gao, M., & Zhu, H. (2009) Antiquity and function of CASTOR and POLLUX, the twin ion channel-encoding genes key to the evolution of root symbioses in plants. Plant Physiology 149, 306–317. Chi, F., Shen, S.H., Cheng, H.P., Jing, Y.X., Yanni, Y.G., & Dazzo, F.B. (2005) Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of beneﬁts to rice growth physiology. Applied and Environmental Microbiology 71(11), 7271–7278. Cho, M.J. & Harper, J.E. (1993) Effect of abscisic acid application on root isoﬂavonoid concentration and nodulation of wild-type and nodulation mutant soybean plants. Plant Soil 153, 145–149. Cocking, E.C. (2003) Endophytic colonization of plant roots by nitrogen-ﬁxing bacteria. Plant and Soil 252(169), 175. Cohn, J.R., Uhm, T., Ramu, S., et al. (2001) Differential regulation of a family of apyrase genes from

LEGUMES AND NITROGEN FIXATION

Medicago truncatula. Plant Physiology 125(4), 2104–2119. Combier, J.P., Frugier, F., de Billy, F., et al. (2006) MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula. Genes and Development 20(22), 3084–3088. Cooper, J.B. & Long, S.R. (1994) Morphogenetic rescue of Rhizobium meliloti nodulation mutants by trans-zeatin secretion. Plant Cell 6, 215–225. Crespi, M., Jurkevitch, E., Poiret, M., et al. (1994) Enod40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth. The EMBO Journal 13, 5099–5112. Dart, P. (1988) Nitrogen ﬁxation in tropical forestry and the use of Rhizobium. In: Tropical Forest Ecology and Management in the Asia-Paciﬁc Region (eds. P. Kapoor-Vijay, S. Appanah & S.M. Saulei), Proceedings of Regional Workshop held at Lae, Papua New Guinea, Commonwealth Science Council. U.K., pp. 142–154. Day, R.B., McAlvin, C.B., Loh, J.T., et al. (2000) Differential expression of two soybean apyrases, one of which is an early nodulin. Molecular PlantMicrobe Interactions 13, 1053–1070. De Bruin, J.L., Pedersen, P., Conley, S.P., et al. (2010) Probability of yield response to inoculants in ﬁelds with a history of soybean. Crop Science 50, 265–272. Degenhart, N.R., Barnes, D.K., & Vance, C. (1992) Divergent selection for nodule aspartate aminotransferase and asparagine synthetase activities in alfalfa. Crop Science 32, 313–317. Den Herder, G. & Parniske, M. (2009) The unbearable naivety of legumes in symbiosis. Current Opinion in Plant Biology 12(4), 491–499. Dénarié, J., Debellé, F., & Promé, J.C. (1996) Rhizobium lipo-chitooligosaccharide nodulation factors: signalling molecules mediating recognition and morphogenesis. Annual Review of Biochemistry 65, 503–535. D’Haeze, W. & Holsters, M. (2002) Nod factor structures, responses, and perception during initiation of nodule development. Glycobiology 12(6), 79. D’Haeze, W. & Holsters, M. (2004) Surface polysaccharides enable bacteria to evade plant immunity. Trends in in Microbiology 12(12), 555–561. Ding, Y., Kaló, P., Yendrek, C., et al. (2008) Abscisic acid coordinates nod factor and cytokinin signalling during the regulation of nodulation in Medicago truncatula. The Plant Cell 20(10), 2681–2695. Dixon, R. & Kahn, D. (2004) Genetic regulation of biological nitrogen ﬁxation. Nature Reviews Microbiology 2(8), 621–631.

481

Ehrhardt, D.W., Wais, R., & Long, S.R. (1996) Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell 85(5), 673–681. Einsle, O., Tezcan, F.A., Andrade, S.L., et al. (2002) Nitrogenase MoFe-protein at 1.16 A resolution: a central ligand in the FeMo-cofactor. Science (New York, N.Y.) 297(5587), 1696–1700. Elliott, G.N., Chen, W.M., Chou, J.H., et al. (2007) Burkholderia phymatum is a highly effective nitrogen-ﬁxing symbiont of Mimosa spp. and ﬁxes nitrogen ex planta. The New Phytologist 173(1), 168–180. Endre, G., Kereszt, A., Kevei, Z., Mihacea, S., Kaló, P., & Kiss, G.B. (2002) A receptor kinase gene regulating symbiotic nodule development. Nature 417(6892), 962–966. Etzler, M.E., Kalsi, G., Ewing, N.N., Roberts, N.J., Day, R.B., & Murphy, J.B. (1999) A Nod Factor binding lectin with apyrase activity from legume roots. Proceedings of the National Academy of Sciences of the United States of America 96(10), 5856–5861. Fang, Y. & Hirsch, A.M. (1998) Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in alfalfa. Plant Physiology 116, 53–68. FAOSTAT (2010) Food and Agricultural Organization Statistical Year Book 2010. FAO Statistical Division, Rome, Italy. Felle, H.H., Kondorosi, E., Kondorosi, A., & Schultze, M. (1999) Elevation of the cytosolic free [Ca2+] is indispensable for the transduction of the Nod factor signal in alfalfa. Plant Physiology 121(1), 273–280. Ferguson, B.J., Ross, J.J., & Reid, J.B. (2005) Nodulation phenotypes of gibberellin and brassinosteroid mutants of pea. Plant Physiology 138(4), 2396–2405. Fleming, A.I., Wittenberg, J.B., Wittenberg, B.A., Dudman, W.F., & Appleby, C.A. (1987) The puriﬁcation, characterization and ligand-binding kinetics of haemoglobin from root nodules of the nonleguminous Casuarina glauca-Frankia symbiosis. Biochim Biophys Acta 911, 209–220. Freiberg, C., Fellay, R., Bairoch, A., Broughton, W.J., Rosenthal, A., & Perret, X. (1997) Molecular basis of symbiosis between Rhizobium and legumes. Nature 387(6631), 394–401. Garrocho-Villegas, V., Gopalasubramaniam, S.K., & Arredondo-Peter, R. (2007) Plant haemoglobins: what we know six decades after their discovery. Gene 398(1–2), 78–85. Genre, A., Chabaud, M., Facio, A., Barker, D.G., & Bonfante, P. (2008) Prepenetration assembly precedes and predicts the colonization patterns of

482

NUTRIENT USE EFFICIENCY IN CROPS

arbuscular mycorrhizal fungi within root cortex of both Medicago truncatula and Daucus carota. The Plant Cell 20, 1407–1420. Genre, A., Chabaud, M., Timmers, T., Bonfante, P., & Baker, G.D. (2005) Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection. The Plant Cell 17, 3489–3499. Gherbi, H., Markmann, K., Svistoonoff, S., et al. (2008) SymRK deﬁnes a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankia bacteria. Proceedings of the National Academy of Sciences of the United States of America 105(12), 4928–4932. Gibson, Q.H., Wittenberg, J.B., Wittenberg, B.A., Bogusz, D., & Appleby, C.A. (1989) The kinetics of ligand binding to plant haemoglobins. Structural implications. The Journal of Biological Chemistry 264(1), 100–107. Giraud, E., Moulin, L., Vallenet, D., et al. (2007) Legume symbioses: absence of Nod genes in photosynthetic bradyrhizobia. Science 316, 1307–1312. Gleason, C., Chaudhuri, S., Yang, T., Muñoz, A., Poovaiah, B.W., & Oldroyd, G.E. (2006) Nodulation independent of rhizobia induced by a calciumactivated kinase lacking autoinhibition. Nature 441(7097), 1149–1152. Godfroy, O., Debelle, F., Timmers, T., & Rosenberg, C. (2006) A rice calcium- and calmodulin-dependent protein kinase restores nodulation to a legume mutant. Molecular Plant-Microbe Interactions 19(5), 495–501. Gonzalez-Rizzo, S., Crespi, M., & Frugier, F. (2006) The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti. The Plant Cell 18, 2680–2693. Gonzalez-Sama, A., Pena, T.C., Kevei, Z., et al. (2006) Nuclear DNA endoreduplication and expression of the mitotic inhibitor Ccs52 associated to determinate and lupinoid nodule organogenesis. Molecular Plant-Microbe Interactions 19(2), 173–180. Goormachtig, S., Capoen, W., & Holsters, M. (2004) Rhizobium infection: lessons from the versatile nodulation behaviour of water-tolerant legumes. Trends in in Plant Science 9(11), 518–522. Gottfert, M., Rothlisberger, S., Kundig, C., Beck, C., Marty, R., & Hennecke, H. (2001) Potential symbiosis-speciﬁc genes uncovered by sequencing a 410-kilobase DNA region of the Bradyrhizobium japonicum chromosome. Journal of Bacteriology 183(4), 1405–1412. Govindarajulu, M., Kim, S.Y., Libault, M., et al. (2009) GS52 ecto-apyrase plays a critical role during soybean nodulation. Plant Physiology 149, 994–1004.

Graham, P.H., Hungria, M., & Tlusty, B. (2004) Breeding for better nitrogen ﬁxation in grain legumes: where do the rhizobia ﬁt in? Crop Management doi: 10.1094/CM-2004-0301-02-RV. Guinel, F.C. (2009) Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types. Botany 87, 1117–1138. Guinel, F.C. & Geil, R.D. (2002) A model for the development of the rhizobial and arbuscular mycorrhizal symbioses in legumes and its use ito understand the roles of ethylene in the establishment in these two symbioses. Canadian Journal of Botany 80, 695–720. Gutierrez-Zamora, M.L. & Martinez-Romero, E. (2001) Natural endophytic association between Rhizobium etli and maize (Zea mays L.). Journal of Biotechnology 91(2–3), 117–126. Haney, C.H. & Long, S.R. (2010) Plant ﬂotillins are required for infection by nitrogen-ﬁxing bacteria. Proceedings of the National Academy of Sciences of the United States of America 107(1), 478– 483. Hardy, R.W.F., Burns, R.C., & Holsten, R.D. (1973) Applications of the acetylene-ethylene assay for measurement of nitrogen ﬁxation. Soil Biology & Biochemistry 5, 47–81. Hartwig, U.A., Maxwell, C.A., Joseph, C.M., & Phillips, D.A. (1990a) Chrysoeriol and luteolin released from alfalfa seeds induce nod genes in Rhizobium meliloti. Plant Physiology 92(1), 116–122. Hartwig, U.A., Maxwell, C.A., Joseph, C.M., & Phillips, D.A. (1990b) Effect of alfalfa nod geneinducing ﬂavonoids on nodABC transcription in Rhizobium meliloti strains containing different nodD genes. Journal of Bacteriology 172(5), 2769–2773. Heckmann, A.B., Lombardo, F., Miwa, H., et al. (2006) Lotus japonicus requires two GRAS domain regulators, one of which is functionally conserved in a non-legume. Plant Physiology 142, 1739– 1750. Heidstra, R., Geurts, R., Franssen, H., Spaink, H.P., Van Kammen, A., & Bisseling, T. (1994) Root hair deformation activity of nodulation factors and their fate on Vicia sativa. Plant Physiology 105(3), 787–797. Herridge, D. & Rose, I. (2000) Breeding for enhanced nitrogen ﬁxation in crop legumes. Field Crops Research 65(229), 248. Herridge, D., Gemell, G., & Hartley, E. (2002) Legume inoculants and quality control. In: Inoculants and Nitrogen Fixation of Legumes in Vietnam (ed. D. Herridge), pp. 105–115. ACIAR Proceedings. Hiltbold, A.E., Thurlow, D.L., & Skipper, H.D. (1980) Evaluation of commercial soybean inoculants by

LEGUMES AND NITROGEN FIXATION

various techniques. Agronomy Journal 72, 675–681. Hirsch, A.M. (1992) Developmental biology of legume nodulation. The New Phytologist 122, 211–237. Hirsch, A.M. & Fang, Y. (1994) Plant hormones and nodulation: what’s the connection? Plant Molecular Biology 26, 5–9. Hirsch, A.M., Bhuvaneswari, T.V., Torrey, J.G., & Bisseling, T. (1989) Early nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport inhibitors. Proceedings of the National Academy of Sciences of the United States of America 86, 1246–1248. Hirsch, S., Kim, J., Muñoz, A., Heckmann, A.B., Downie, J.A., & Oldroyd, G.E. (2009) GRAS proteins form a DNA binding complex to induce gene expression during nodulation signalling in Medicago truncatula. The Plant Cell 21, 545–557. Hubber, A., Vergunst, A.C., Sullivan, J.T., Hooykaas, P.J., & Ronson, C.W. (2004) Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system. Molecular Microbiology 54(2), 561–574. Imaizumi-Anraku, H., Takeda, N., Charpentier, M., et al. (2005) Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. Nature 433(7025), 527–531. James, E.K., Sprent, J.I., Sutherland, J.M., McInroy, S.G., & Minchin, F.R. (1992) The structure of nitrogen-ﬁxing root nodules in the aquatic mimosoid legume Neptunia plena. Annals of Botany 14, 173–180. Jessen, D.L., Barnes, D.K., & Vance, C.P. (1988) Bidirectional selection in alfalfa for activity of nodule nitrogen and carbon assimilating enzymes. Crop Science 28, 18–22. Jha, P.K., Nair, S., & Babu, C.R. (1994) Development of an inexpensive legume-Rhizobium inoculation technology which may be used in aerial seeding. Journal of Basic Microbiology 34(4), 231–243. Jones, K.M., Kobayashi, H., Davies, B.W., Taga, M.E., & Walker, G.C. (2007) How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nature Reviews in Microbiology 5(8), 619–633. Jones, K.M., Sharopova, N., Lohar, D.P., Zhang, J.Q., VandenBosch, K.A., & Walker, G.C. (2008) Differential response of the plant Medicago truncatula to its symbiont Sinorhizobium meliloti or an exopolysaccharide-deﬁcient mutant. Proceedings of the National Academy of Sciences of the United States of America 105(2), 704–709. Jourand, P., Renier, A., Rapior, S., et al. (2005) Role of methylotrophy during symbiosis between Methylobacterium nodulans and Crotalaria podo-

483

carpa. Molecular Plant-Microbe Interactions 18(10), 1061–1068. Journet, E.P., Pichon, M., Dedieu, A., de Billy, F., Truchet, G., & Barker, D.G. (1994) Rhizobium meliloti Nod factors elicit cell-speciﬁc transcription of the ENOD12 gene in transgenic alfalfa. Plant Journal 6, 241–249. Journet, E.P., El-Gachtouli, N., Vernoud, V., et al. (2001) Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells. Molecular Plant-Microbe Interactions 14(6), 737–748. Kaló, P., Gleason, C., Edwards, A., et al. (2005) Nodulation signalling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 308(5729), 1786–1789. Kanamori, N., Madsen, L.H., Radutoiu, S., et al. (2006) A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proceedings of the National Academy of Sciences of the United States of America 103(2), 359–364. Kaneko, T., Nakamura, Y., Sato, S., et al. (2000) Complete genome structure of the nitrogen-ﬁxing symbiotic bacterium Mesorhizobium loti. DNA Research 7(6), 331–338. Kawaguchi, M., Imaizumi-Anraku, H., Fukai, S., & Syono, K. (1996) Unusual branching in the seedlings of Lotus japonicus-Gibberellins reveal the nitrogen sensitive cell divisions within the pericycle on roots. Plant Cell Physiology 37, 461–470. Kevei, Z., Lougnon, G., Mergaert, P., et al. (2007) 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula. The Plant Cell 19(12), 3974–3989. Kim, S.A. & Copeland, L. (1996) Enzymes of Poly(beta)-Hydroxybutyrate metabolism in soybean and chickpea bacteroids. Applied and Environmental Microbiology 62(11), 4186–4190. Kiss, E., Olah, B., Kalo, P., et al. (2009) LIN, a novel type of U-box/WD40 protein, controls early infection by rhizobia in legumes. Plant Physiology 151(3), 1239–1249. Kistner, C. & Parniske, M. (2002) Evolution of signal transduction in intracellular symbiosis. Trends in in Plant Science 7(11), 511–518. Kosslak, R.M., Bookland, R., Barkei, J., Paaren, H.E., & Appelbaum, E.R. (1987) Induction of Bradyrhizobium japonicum common nod genes by isoﬂavones isolated from Glycine max. Proceedings of the National Academy of Sciences of the United States of America 84(21), 7428–7432. Kuppusamy, K.T., Endre, G., Prabhu, R., et al. (2004) LIN, a Medicago truncatula gene required for

484

NUTRIENT USE EFFICIENCY IN CROPS

nodule differentiation and persistence of rhizobial infections. Plant Physiology 136, 3682–3691. Kuppusamy, K.T., Ivashuta, S., Bucciarelli, B., Vance, C.P., Gantt, J.S., & Vandenbosch, K.A. (2009) Knockdown of CELL DIVISION CYCLE16 reveals an inverse relationship between lateral root and nodule numbers and a link to auxin in Medicago truncatula. Plant Physiology 151(3), 1155–1166. Laguerre, G., Nour, S.M., Macheret, V., Sanjuan, J., Drouin, P., & Amarger, N. (2001) Classiﬁcation of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiology (Reading, England) 147(Pt 4), 981–993. Lanzilotta, W.N., Christiansen, J., Dean, D.R., & Seefeldt, L.C. (1998) Evidence for coupled electron and proton transfer in the [8Fe-7S] cluster of nitrogenase. Biochemistry 37(32), 11376–11384. Larson, J.L. & Siemann, E. (1998) Legumes may be symbiont-limited during old-ﬁeld succession. The American Midland Naturalist 140, 90–95. Lefebvre, B., Timmers, T., Mbengue, M., et al. (2010) A remorin protein interacts with symbiotic receptors and regulates bacterial infection. Proceedings of the National Academy of Sciences of the United States of America 107(5), 2343–2348. Leigh, J.A. & Coplin, D.L. (1992) Exopolysaccharides in plant-bacterial interactions. Annual Review of Microbiology 46, 307–346. Leigh, J.A., Signer, E.R., & Walker, G.C. (1985) Exopolysaccharide-deﬁcient mutants of Rhizobium meliloti that form ineffective nodules. Proceedings of the National Academy of Sciences of the United States of America 82(18), 6231–6235. Lévy, J., Bres, C., Geurts, R., et al. (2004) A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses. Science 303(5662), 1361–1364. Liang, Y., Mitchell, D.M., & Harris, J.M. (2007) Abscisic acid rescues the root meristem defects of the Medicago truncatula latd mutant. Developmental biology 304, 297–307. Limpens, E., Franken, C., Smit, P., Willemse, J., Bisseling, T., & Geurts, R. (2003) LysM domain receptor kinases regulating rhizobial Nod factorinduced infection. Science 302(5645), 630–633. Limpens, E., Mirabella, R., Fedorova, E., et al. (2005) Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by DMI2. Proceedings of The National Academy of Sciences of The United States of America 102(29), 10375–10380. Lohar, D.P., Schaff, J.E., Laskey, J.G., Kieber, J.J., Bilyeu, K.D., & Bird, D.M. (2004) Cytokinins play opposite roles in lateral root formation, and nema-

tode and Rhizobial symbioses. Plant Journal 38, 203–214. Lombardo, F., Heckmann, A.B., Miwa, H., et al. (2006) Identiﬁcation of symbiotically defective mutants of Lotus japonicus affected in infection thread growth. Molecular Plant-Microbe Interactions 19(12), 1444–1450. Mabood, F., Souleimanov, A., Khan, W., & Smith, D.L. (2006) Jasmonates induce Nod factor production by Bradyrhizobium japonicum. Plant Physiology and Biochemistry 44, 759–765. Macchiavelli, R.E. & Brelles-Mariño, G. (2004) Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti. Journal of Experimental Botany 55(408), 2635–2640. Madsen, E.B., Madsen, L.H., Radutoiu, S., et al. (2003) A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature 425(6958), 637–640. Maekawa, T., Maekawa-Yoshikawa, M., Takeda, N., Imaizumi-Anraku, H., Murooka, Y., & Hayashi, M. (2009) Gibberellin controls the nodulation signalling pathway in Lotus japonicus. Plant Journal 58, 183–194. Marie, C., Broughton, W.J., & Deakin, W.J. (2001) Rhizobium type III secretion systems: legume charmers or alarmers? Current opinion in plant biology 4(4), 336–342. Markmann, K. & Parniske, M. (2009) Evolution of root endosymbiosis with bacteria: how novel are nodules? Trends in Plant Science 14(2), 77–86. Markmann, K., Giczey, G., & Parniske, M. (2008) Functional adaptation of a plant receptor-kinase paved the way for the evolution of intracellular root symbioses with bacteria. PLoS Biology 6(3), e68. Marsh, J.F., Rakocevic, A., Mitra, R.M., et al. (2007) Medicago truncatula NIN is essential for rhizobialindependent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase. Plant Physiology 144(1), 324–335. Martinez-Romero, E. (2009) Coevolution in Rhizobiumlegume symbiosis? DNA and cell biology 28(8), 361–370. Masson-Boivin, C., Giraud, E., Perret, X., & Batut, J. (2009) Establishing nitrogen-ﬁxing symbiosis with legumes: how many rhizobium recipes? Trends in in Microbiology 17(10), 458–466. Mathesius, U. (2008) Auxin: at the root of nodule development? Functional Plant Biology 35, 651–668. Mathesius, U., Schlaman, H.R., Spaink, H., Sautter, C., Rolfe, B., & Djordjevic, M.A. (1998) Auxin transport inhibition precedes root nodule formation in

LEGUMES AND NITROGEN FIXATION

white clover roots and is regulated by ﬂavanoids and derivatives of chitin oligosaccharides. Plant Journal 14, 23–34. Maxwell, C.A., Hartwig, U.A., Joseph, C.M., & Phillips, D.A. (1989) A chalcone and two related ﬂavonoids released from alfalfa roots induce nod genes of Rhizobium meliloti. Plant Physiology 91(3), 842–847. Mazur, A., Krol, J.E., Wielbo, J., Urbanik-Sypniewska, T., & Skorupska, A. (2002) Rhizobium leguminosarum bv. trifolii PssP protein is required for exopolysaccharide biosynthesis and polymerization. Molecular Plant-Microbe Interactions 15(4), 388–397. McAlvin, C.B. & Stacey, G. (2005) Transgenic expression of the soybean apyrase in Lotus japonicus enhances nodulation. Plant Physiology 137(4), 1456–1462. Mergaert, P., Uchiumi, T., Alunni, B., et al. (2006) Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis. Proceedings of the National Academy of Sciences of the United States of America 103(13), 5230–5235. Messinese, E., Mun, J.H., Yeun, L.H., et al. (2007) A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula. Molecular PlantMicrobe Interactions 20(8), 912–921. Middleton, P.H., Jakab, J., Penmetsa, R.V., et al. (2007) An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction. Plant Cell 19, 1221–1234. Mitra, R.M., Gleason, C.A., Edwards, A., et al. (2004) A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: gene identiﬁcation by transcript-based cloning. Proceedings of the National Academy of Sciences of The United States of America 101(13), 4701–4705. Morgante, C., Angelini, J., Castro, S., & Fabra, A. (2005) Role of rhizobial exopolysacharides in crack entry/ intercellular infection of peanut. Soil Biology & Biochemistry 37, 1436–1444. Morgante, C., Castro, S., & Fabra, A. (2007) Role of rhizobial EPS in the evasion of peanut defence response during the crack-entry infection process. Soil Biology & Biochemistry 39, 1222–1225. Mukherjee, A. & Ané, J.M. (2011) Plant hormones and initiation of legume nodulation and arbuscular mycorrhization. In: Environmental Aspects of Plant Nitrogen Metabolism (eds. J. Polacco & C. Todd) John Wiley & Sons, Hoboken, NJ, pp. 354–396. Muller, J., Wiemken, A., & Boller, T. (2001) Redifferentiation of bacteria isolated from Lotus

485

japonicus root nodules colonized by Rhizobium sp. NGR234. Journal of Experimental Botany 52(364), 2181–2186. Murakami, Y., Miwa, H., Imaizumi-Anraku, H., et al. (2006) Positional cloning identiﬁes Lotus japonicus NSP2, a putative transcription factor of the GRAS family, required for NIN and ENOD40 gene expression in nodule initiation. DNA Research 13, 255–265. Murray, J.D., Karas, B.J., Sato, S., Tabata, S., Amyot, L., & Szczyglowski, K. (2007) A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis. Science 315(5808), 101–104. Mytton, L.R. (1984) Developing a breeding strategy to exploit quantitative variation in symbiotic nitrogen ﬁxation. Plant Soil 82, 329–335. Nelson, D.W., Swearingin, M.L., & Beckham, L.S. (1978) Response of soybeans to commercial soilapplied inoculants. Agronomy Journal 70, 517–518. Nukui, N., Ezura, H., & Minamisawa, K. (2004) Transgenic Lotus japonicus with an ethylene receptor gene Cm-ERS1/H70A enhances formation of infection threads and nodule primordia. Plant and Cell Physiology 45, 427–435. Nutman, P.S. (1984) Improving nitrogen ﬁxation in legumes by plant breeding: the relevance of host selection experiments in red clover (Trifolium pratense L.) and subterranean clover (T. subterraneum L.). Plant Soil 82, 285–301. Oláh, B., Brière, C., Bécard, G., Dénarié, J., & Gough, C. (2005) Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/ DMI2 signalling pathway. The Plant Journal 44(2), 195–207. Oldroyd, G.E. & Downie, J.A. (2004) Calcium, kinases and nodulation signalling in legumes. Nature Reviews Molecular Cell Biology 5(7), 566–576. Oldroyd, G.E. & Downie, J.A. (2008) Coordinating nodule morphogenesis with rhizobial infection in legumes. Annual Review of Plant Biology 59, 519–546. Oldroyd, G.E., Engstrom, E.M., & Long, S.R. (2001b) Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula. The Plant Cell 13, 1835–1849. Oldroyd, G.E., Mitra, R.M., Wais, R.J., & Long, S.R. (2001a) Evidence for structurally speciﬁc negative feedback in the Nod factor signal transduction pathway. The Plant Journal 28(2), 191–199. Ooki, Y., Banba, M., Yano, K., et al. (2005) Characterization of the Lotus japonicus symbiotic mutant lot1 that shows a reduced nodule number

486

NUTRIENT USE EFFICIENCY IN CROPS

and distorted trichomes. Plant Physiology 137, 1261–1271. Ott, T., van Dongen, J.T., Gunther, C., et al. (2005) Symbiotic leghaemoglobins are crucial for nitrogen ﬁxation in legume root nodules but not for general plant growth and development. Current Biology 15, 531–535. Ott, T., Sullivan, J., James, E.K., et al. (2009) Absence of symbiotic leghaemoglobins alters bacteroid and plant cell differentiation during development of Lotus japonicus root nodules. Molecular PlantMicrobe Interactions 22(7), 800–808. Pacios-Bras, C., Schlaman, H.R., Boot, K., et al. (2003) Auxin distribution in Lotus japonicus during root nodule development. Plant Molecular Biology 52(6), 1169–1180. Parniske, M., Schmidt, P., Kosch, K., & Müller, P. (1994) Plant defence responses of host plants with determinate nodules induced by exopolysaccharidedefective exoB mutants of Bradyrhizobium japonicum. Molecular Plant–Microbe Interaction 7, 631–638. Parveen, N., Webb, D., & Borthakur, D. (1997) The symbiotic phenotypes of exopolysaccharidedefective mutants of Rhizobium sp. strain TAL1145 do not differ on determinate and indeterminate nodulating tree legumes. Microbiology 143, 1959–1967. Pathirana, S.M. & Tjepkema, J.D. (1995) Puriﬁcation of haemoglobin from the actinorhizal root nodules of Myrica gale L. Plant Physiology 107(3), 827–831. Pazdernik, D.L., Graham, P.H., Vance, C.P., & Orf, J.H. (1996) Host genetic variation in the early nodulation and dinitrogen ﬁxation of soybeans. Crop Science 36, 1102–1107. Peiter, E., Sun, J., Heckmann, A.B., et al. (2007) The Medicago truncatula DMI1 protein modulates cytosolic calcium signalling. Plant Physiology 145(1), 192–203. Pellock, B.J., Cheng, H.P., & Walker, G.C. (2000) Alfalfa root nodule invasion efﬁciency is dependent on Sinorhizobium meliloti polysaccharides. Journal of Bacteriology 182(15), 4310–4318. Penmetsa, R.V. & Cook, D. (1997) A legume ethyleneinsensitive mutant hyperinfected by its rhizobial symbiont. Science 275, 527–530. Penmetsa, R.V., Uribe, P., Anderson, J., et al. (2008) The Medicago truncatula orthologue of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations. Plant Journal 55, 580–595. Perlick, A.M., Albus, U., Stavridis, T., Fruhling, M., Kuster, H., & Puhler, A. (1996) The Vicia faba lipoxygenase gene VfLOX1 is expressed in the root

nodule parenchyma. Molecular Plant-Microbe Interactions 9(9), 860–863. Perret, X., Staehelin, C., & Broughton, W.J. (2000) Molecular basis of symbiotic promiscuity. Microbiology and Molecular Biology Reviews 64(1), 180–201. Peters, N.K., Frost, J.W., & Long, S.R. (1986) A plant ﬂavone, luteolin, induces expression of Rhizobium meliloti nodulation genes. Science 233(4767), 977–980. Phillips, D.A., Torrey, J.G., & Burris, R.H. (1971) Extending symbiotic nitrogen ﬁxation to increase man’s food supply. Science (New York, N.Y.) 174(4005), 169–170. Povolo, S., Tombolini, R., Morea, A., Anderson, A.J., Casella, S., & Nuti, M.P. (1994) Isolation and characterization of mutants of Rhizobium meliloti unable to synthesize poly-b-hydroxybutyrate. Canadian Journal of Microbiology 40, 823–829. Prayitno, J., Rolfe, B.G., & Mathesius, U. (2006) The ethylene-insensitive sickle mutant of Medicago truncatula shows altered auxin transport regulation during nodulation. Plant Physiology 142(1), 168–180. Pueppke, S.G. & Broughton, W.J. (1999) Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges. Molecular Plant-Microbe Interactions 12(4), 293–318. Radutoiu, S., Madsen, L.H., Madsen, E.B., et al. (2003) Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425(6958), 585–592. Radutoiu, S., Madsen, L.H., Madsen, E.B., et al. (2007) LysM domains mediate lipochitinoligosaccharide recognition and Nfr genes extend the symbiotic host range. The EMBO Journal 26(17), 3923–3935. Rasolomampianina, R., Bailly, X., Fetiarison, R., et al. (2005) Nitrogen-ﬁxing nodules from rose wood legume trees (Dalbergia spp.) endemic to Madagascar host seven different genera belonging to alpha- and beta-Proteobacteria. Molecular Ecology 14(13), 4135–4146. Renier, A., Jourand, P., Rapior, S., et al. (2008) Symbiotic properties of Methylobacterium nodulans ORS2060: a classic process for an atypical symbiont. Soil Biology & Biochemistry 40, 1404–1412. Rennie, R.J. & Kemp, G.A. (1983) Nitrogen ﬁxation in ﬁeld beans quantiﬁed by 15N isotope dilution.2. Effect of cultivars of beans. Agronomy Journal 75, 645–649. Ridge, R.W., Ride, K.M., & Rolfe, B.G. (1993) Nodulelike structures induced on the roots of rice seedlings by addition of the synthetic auxin 2,4-

LEGUMES AND NITROGEN FIXATION

dichlorophenoxyacetic acid. Australian Journal of Plant Physiology 20, 705–717. Riely, B.K., Lougnon, G., Ané, J.M., & Cook, D.R. (2007) The symbiotic ion channel homologue DMI1 is localized in the nuclear membrane of Medicago truncatula roots. The Plant Journal 49(2), 208–216. Robledo, M., Jimenez-Zurdo, J.I., Velazquez, E., et al. (2008) Rhizobium cellulase CelC2 is essential for primary symbiotic infection of legume host roots. Proceedings of the National Academy of Sciences of the United States of America 105(19), 7064–7069. Rogel, M.A., Hernandez-Lucas, I., Kuykendall, L.D., Balkwill, D.L., & Martinez-Romero, E. (2001) Nitrogen-ﬁxing nodules with Ensifer adhaerens harboring Rhizobium tropici symbiotic plasmids. Applied and Environmental Microbiology 67(7), 3264–3268. Rosas, S., Soria, R., Correa, N., & Abdala, G. (1998) Jasmonic acid stimulates the expression of nod genes in Rhizobium. Plant Molecular Biology 38, 1161–1168. Ruiz Diaz, D.A., Pedersen, P., & Sawyer, J.E. (2009) Soybean response to inoculation and nitrogen application following long-term grass pasture. Crop Science 49, 1058–1062. Saito, K., Yoshikawa, M., Yano, K., et al. (2007) NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotus japonicus. The Plant Cell 19(2), 610–624. Schauser, L., Roussi, A., Stiller, J., & Stougaard, J. (1999) A plant regulator controlling development of symbiotic root nodules. Nature 402, 191–195. Schauser, L., Wieloch, W., & Stougaard, J. (2005) Evolution of NIN-like proteins in Arabidopsis, rice and Lotus japonicus. Journal of Molecular Evolution 60, 229–237. Schmidt, J.S., Harper, J.E., Hoffman, T.K., & Bent, A.F. (1999) Regulation of soybean nodulation independent of ethylene signalling. Plant Physiology 119, 951–959. Schulz, T.J. & Thelen, K.D. (2008) Soybean seed inoculant and fungicidal seed treatment effects on soybean. Crop Science 48, 1975–1983. Shaw, S.L. & Long, S.R. (2003) Nod factor inhibition of reactive oxygen efﬂux in a host legume. Plant Physiology 132(4), 2196–2204. Sieberer, B.J., Chabaud, M., Timmers, A.C., Monin, A., Fournier, J., & Barker, D.G. (2009) A nucleartargeted cameleon demonstrates intranuclear Ca2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors. Plant Physiology 151, 1197–1206.

487

Singh, M.K., Kushwaha, C., & Singh, R.K. (2009) Studies on endophytic colonization ability of two upland rice endophytes, Rhizobium sp. and Burkholderia sp., using green ﬂuorescent protein reporter. Current Microbiology 59(3), 240–243. Singleton, P.W., Boonkerd, N., Carr, T.J., & Thompson, J.A. (1997) Technical and market constraints limiting legume inoculant use in Asia. In: Extending Nitrogen Fixation Research to Farmers’ Fields (eds. O.P. Rupela, C. Johansen & D.F. Herridge), pp. 17–38. ICRISAT, Patancheru, AP, India. Smit, P., Raedts, J., Portyanko, V., et al. (2005) NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription. Science 308(5729), 1789–1791. Smit, P., Limpens, E., Geurts, R., et al. (2007) Medicago LYK3, an entry receptor in rhizobial nodulation factor signalling. Plant Physiology 145(1), 183–191. Spaink, H.P. (1995) The molecular basis of infection and nodulation by rhizobia—the ins and outs of sympathogenesis. Annual Review of Phytopathology 33, 345–368. Spaink, H.P. (1996) Regulation of plant morphogenesis by lipo-chitin oligosaccharides. Critical Review of Plant Science 15, 559–582. Spencer, D., James, E.K., Ellis, G.J., Shaw, J.E., & Sprent, J.I. (1994) Interaction between rhizobia and potato tissue. Journal of Experimental Botany 45, 1475–1482. Sprent, J.I. (2007) Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation. The New Phytologist 174(1), 11–25. Sprent, J.I. (2008) 60Ma of legume nodulation. What’s new? What’s changing? Journal of Experimental Botany 59(5), 1081–1084. Sprent, J.I. & James, E.K. (2007) Legume evolution: where do nodules and mycorrhizas ﬁt in? Plant Physiology 144(2), 575–581. Starker, C.G., Parra-Cohnenares, A.L., Smith, L., Mitra, R.M., & Long, S.R. (2006) Nitrogen-ﬁxing mutants of Medicago truncatula fail to support plant and bacterial symbiotic gene expression. Plant Physiology 140, 671–680. Stougaard, J. (2000) Regulators and regulation of legume root nodule development. Plant Physiology 124, 531–540. Stracke, S., Kistner, C., Yoshida, S., et al. (2002) A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature 417(6892), 959–962. Subramanian, S., Stacey, G., & Yu, O. (2006) Endogenous isoﬂavones are essential for the establishment of symbiosis between soybean and

488

NUTRIENT USE EFFICIENCY IN CROPS

Bradyrhizobium japonicum. Plant Journal 48, 261–273. Subramanian, S., Stacey, G., & Yu, O. (2007) Distinct, crucial roles of ﬂavonoids during legume nodulation. Trends in Plant Science 12, 282–285. Suharjo, U.K.J. & Tjepkema, J.D. (1995) Occurrence of haemoglobin in the nitrogen-ﬁxing root nodules of Alnus glutinosa. Physiologia Plantarum 95(247), 252. Sullivan, J.T., Patrick, H.N., Lowther, W.L., Scott, D.B., & Ronson, C.W. (1995) Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment. Proceedings of the National Academy of Sciences of the United States of America 92(19), 8985–8989. Sun, J., Cardoza, V., Mitchell, D.M., Bright, L., Oldroyd, G.E., & Harris, J.M. (2006) Crosstalk between jasmonic acid, ethylene and Nod factor signalling allows integration of diverse inputs for regulation of nodulation. Plant Journal 46, 961–970. Suzuki, A., Akune, M., Kogiso, M., et al. (2004) Control of nodule number by the phytohormone abscisic Acid in the roots of two leguminous species. Plant & Cell Physiology 45(7), 914–922. Teillet, A., Garcia, J., de Billy, F., et al. (2008) api, A novel Medicago truncatula symbiotic mutant impaired in nodule primordium invasion. Molecular Plant-Microbe Interactions 21(5), 535–546. Timmers, A.C.J., Auriac, M.C., & Truchet, G. (1999) Reﬁned analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements. Development 126, 3617–3628. Timmers, A.C., Soupene, E., Auriac, M.C., et al. (2000) Saprophytic intracellular rhizobia in alfalfa nodules. Molecular Plant-Microbe Interactions 13(11), 1204–1213. Tirichine, L., Imaizumi-Anraku, H., Yoshida, S., et al. (2006a) Deregulation of Ca2+/calmodulin-dependent kinase leads to the spontaneous nodule development. Nature 441, 1153–1156. Tirichine, L., James, E.K., Sandal, N., & Stougaard, J. (2006b) Spontaneous root-nodule formation in the model legume Lotus japonicus: a novel class of mutants nodulates in the absence of rhizobia. Molecular Plant-Microbe Interactions 19, 373–382. Tirichine, L., Sandal, N., Madsen, L.H., et al. (2007) A gain-of-function mutation in a cytokinin receptor triggers spontaneous root nodule organogenesis. Science 315(5808), 104–107. Tominaga, A., Nagata, M., Futsuki, K., et al. (2009) Enhanced nodulation and nitrogen ﬁxation in the

abscisic acid low-sensitive mutant enhanced nitrogen ﬁxation1 of Lotus japonicus. Plant Physiology 151(4), 1965–1976. Trinick, M.J. (1979) Structure of nitrogen-ﬁxing nodules formed by Rhizobium on roots of Parasponia andersonii Planch. Canadian Journal of Microbiology 25, 565–578. Van de Velde, W., Zehirov, G., Szatmari, A., et al. (2010) Plant peptides govern terminal differentiation of bacteria in symbiosis. Science (New York, N.Y.) 327(5969), 1122–1126. Vasse, J., de Billy, F., Camut, S., & Truchet, G. (1990) Correlation between ultrastructural differentiation of bacteroids and nitrogen ﬁxation in alfalfa nodules. Journal of Bacteriology 172(8), 4295–4306. Veereshlingam, H., Haynes, J.G., Penmetsa, R.V., Cook, D.R., Sherrier, D.J., & Dickstein, R. (2004) nip, A symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defence-like response. Plant Physiology 136, 3692–3702. Viprey, V., Del Greco, A., Golinowski, W., Broughton, W.J., & Perret, X. (1998) Symbiotic implications of type III protein secretion machinery in Rhizobium. Molecular Microbiology 28(6), 1381–1389. Wais, R.J., Galera, C., Oldroyd, G.E., et al. (2000) Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula. Proceedings of the National Academy of Sciences of the United States of America 97(24), 13407–13412. Wang, B., Yeun, L.H., Xue, J.Y., Liu, Y., Ané, J.M., & Qiu, Y.L. (2010a) Presence of three mycorrhizal genes in the common ancestor of land plants suggests a key role of mycorrhizas in the colonization of land by plants. The New Phytologist 186, 514–525. Wang, D., Grifﬁtts, J., Starker, C., et al. (2010b) A nodule-speciﬁc protein secretory pathway required for nitrogen-ﬁxing symbiosis. Science (New York, N.Y.) 327(5969), 1126–1129. Wasson, A.P., Pellerone, F.I., & Mathesius, U. (2006) Silencing the ﬂavonoid pathway in Medicago truncatula inhibits root nodule formation and prevents auxin transport regulation by rhizobia. Plant Cell 18, 1617–1629. Yano, K., Yoshida, S., Muller, J., et al. (2008) CYCLOPS, a mediator of symbiotic intracellular accommodation. Proceedings of the National Academy of Sciences of the United States of America 105, 20540–20545. Zehner, S., Schober, G., Wenzel, M., Lang, K., & Göttfert, M. (2008) Expression of the Bradyrhizobium japonicum type III secretion system in legume

LEGUMES AND NITROGEN FIXATION

nodules and analysis of the associated tts box promoter. Molecular Plant-Microbe Interactions 21(8), 1087–1093. Zhang, F. & Smith, D.L. (1995) Preincubation of Bradyrhizobium japonicum with genistein accelerates nodule development of soybean at suboptimal root zone temperatures. Plant Physiology 108(3), 961–968. Zhang, J.X., Wang, Y.P., Shen, S.H., et al. (2001) Transformation of pea lectin gene and Parasponia

489

haemoglobin gene into rice and their expressions. Acta Botanica Sinica 43(3), 267–274. Zhu, H., Riely, B.K., Burns, N.J., & Ané, J.M. (2006) Tracing nonlegume orthologues of legume genes required for nodulation and arbuscular mycorrhizal symbioses. Genetics 172(4), 2491–2499. Zhu, H., Chen, T., Zhu, M., et al. (2008) A novel ARID DNA-binding protein interacts with SymRK and is expressed during early nodule development in Lotus japonicus. Plant Physiology 148(1), 337–347.

Index

acquisition efficiency, 10–13 Aegilops tauschii, 132 Aegilops longissima, 326 AlaAT, see alanine aminotransferase alanine, 181–182, 184 alanine aminotransferase (AlaAT), 14, 144, 165–191 alkaline soil(s), 338 Allium spp., 296 alternative oxidase, 243 Amaranthus cruentus, 76 amino acid(s), 90, 106, 113 metabolism, 89, 141–143 transporter(s), 90, 205 ammonia-oxidizing bacteria, 52 ANR1, 30 Arabidopsis thaliana, 21, 21, 26, 30, 87–89, 93, 144, 150, 152–153, 170–182, 196, 201–204, 230–238, 244, 248–249, 252, 272, 279, 314, 317, 319, 385–387, 391, 406, 412, 449, 471–472 Arachis hypogaea, 76, 409, 413 462, 464 Arcadia Bioscience, 184 AS, see asparagine synthetase asparagine, 113–114 asparagine synthetase, 170 aspartate amino transferase (AspAT), 170 AspAT, see aspartate amino transferase Aspergillus nidulans, 176 auxin(s), 27, 245, 469, 470 -ethylene interactions, 236

AXR2, 235 AXR4, 30 Azorhizobium spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis Azorhizobium caulinodans, 459 Azospirillum spp., 52 β-glucan(s), 113 β-oxidation, 88 Banksia grandis, 238 barley, 112, 183, 324. See also Hordeum vulgare iron uptake, 317 bioavailability of nutrients, 11 biofortification, 325, 327, 335, 351 biofuels, 140, 169 biological nitrification inhibitor, 132 biological nitrogen fixation (BNF), 458–459 nitrogenase, 458–459 nodulation factor(s) (Nod factor(s)), 460–462, 465 biomass, 68 boron, 377, 379–388 availability, 380 crop improvement, 387–388 deficiency symptoms, 380–382 efficiency, 387 function, 380–382 RGII, 382 rhizobium-legume symbiosis, 382 in soils, 380

The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops, First Edition. Edited by Malcolm J. Hawkesford, Peter Barraclough. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 491

492

INDEX

boron (continued) transport, 382–384 BOR1, 386 channel-mediated transport, 385–386 translocation, 386–387 uptake, 382–386 vacuolar, 385 Bradyrhizobium spp., 458–459, 462, 470, 478. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis Bradyrhizobium japonicum, 460–461 Brassica spp., 183, 296, 302, 410, 435 Brassica rapa, 22 Brassica napus, 183, 296, 302, 410, 435 Burkholderia spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis C3, 14 C4, 14, 69 calcium, 265–280 disorders, 276 nutrition, 277, 285–277 occurrence and availability in soil, 274 physiological function(s), 274–275 salt (sodic) stress, 447–448 structural, 274–275 signaling, 275 transporter(s), 276 uptake and distribution, 275–280 cellular partitioning, 279–280 radial transport, 278 symplastic movement, 278 translocation and distribution, 278–279 uptake, 275–278 Calvin cycle, 76 canopy, 65–82 architecture, 71 fractional interception, 69 nitrogen, 67, 74 photosynthetic capacity, 67, 69 radiation capture, 68–76 radiation interception, 68 rubisco, 74 carbon dioxide, 134, 311, 322 Carex acutiformis, 75 Carya illinoiensis 415 Chamydomonas reinhardtii 323, 412 chelator(s)/chelation, 56, 313 metal, 318

chloride channels (CLCs), 193, 200 chlorine, 388–392 crop improvement, 391–392 deficiency and symptoms of, 388–390, 393–394 function, 389–390, 394 abscisis acid (ABA) synthesis, 394 homeostasis, 390 translocation, 390 antiporters (CLCs), 390 cation chlorine transporter (CCC), 390 zinc iron premeases (ZIP), 392 uptake, 390 chloroplast, 88 CIMMYT, 95, 124, 132–133 CIPK, 206 CKX, see cytokinin oxidase CKX2, 177 climate change, 134, 166, 324 comparative genomics, 150 convection, 28 copper, 392–400 circadian clock genes, 394 complexed with dissolved organic matter ((CuII)-DOM), 392 crop improvement, 400 deficiency, 392 efficiency, 400 ethylene, 394, 397 homeostasis, 395, 398–399 transport, 395, 397–399 transporter(s), 392 COPT, 392, 394, 396, 398 YSL, 400 uptake, 395–397 Crambe abysinnica, 22 crop breeding, 304 drought, 438; see also drought improvement, 5, 7, 6, 211, 326, 351, 387, 391, 400, 413–414, 417, 438, 451, 475 production, 6, 443, 457 Cupriavidus (Ralstonia) spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis 458 Cupriavidus taiwanensis, 460 cysteine, 14, 301 cytokinin, 92, 177, 237, 245, 436, 469–471, 474 cytokinin oxidase (CKX), 237

INDEX

decision support systems, 212 MANAGE RICE, 212 MANAGE-N, 212 denitrification, 51, 212 Devosia spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis diet, 106, 140, 325–326, 350 diffusion, 432 Dionaea muscipula, 268 dof1, 145, 178 doubled haploid line(s) (DHL), 141 dough extensibility, bread making and baking, 105, 108, 129 drought, 431–441; see also water crop improvement, 438 global food production, 431–432 mycorrhiza, 437 nitrate, 436–437 nutrition, 431–432 relationship between nutrients and plant water, 437–438 magnesium, 438 osmotic balance, 437–438 phosphorus, 438 root growth, 435–436 compaction, 435 partial root zone drying (PRD), 436 transport, 434 water-limited plots, 434 DuPont-Pioneer, 141 economic margin, 126 edaphic environment, 22. See also soil epistasis, 151 Escherichia coli, 176 essential trace elements, 114 ethylene, 92, 237, 245, 394, 397, 470–471 -auxin reactions, 236, 243 exudates, 51–53, 55, 57, 353 fatty acid degeneration, 88 fertilizer management, 168 placement, 36–37 use efficiency, 126, 212 flowering, 86 Flour Fortification Initiative, 325 fossil fuels, 65 Frankia spp., 458

493

Galderia partita, 74 GC-MS, 153 GDH, 176 genetics, 128–138 forward, 143 gene networks, 152 improvement, 15 reverse, 143 variability/variation, 33, 146, 354 Gigaspora rosea, 32 gibberellic acid (GA), 470 gibberellins, 27 gliadins, 108–109, 129 Glomus caledonium, 32 Glomus intraradices, 32 glutamine, 90, 202 synthetase (GS), 13, 142, 144, 147, 149, 156, 166, 179 glutamate synthetase, 142 glutathione S-transferases, 296 glutenins, 108 Glycine max, 76 glycolytic pathways, 242 GOGAT, 142, 144, 169–170, 184–185, 220 Gpc-B1, 73, 93, 131 grain, 103–120 nitrogen concentration (GNC), 128 protein concentration (GPC), 123–124, 128–129, 131 storage proteins, 106, 128; see also seed storage compounds yields, 9, 213 Green Revolution, 169, 325 Grevillea robusta, 238 Hakea serica, 241 harvest index (HI), 7, 14–15, 219 Herbaspirillum spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis Hereward wheat, 109–110 heterosis, 129 hexose, 176 HI, see harvest index high molecular mass polymer(s), 110 Hordeum vulgare, 113, 183, 345, 386, 394, 415; see also barley malting, 212 hormone(s), 152, 177, 436, 472 human population, 140

494

INDEX

Illinois High Protein (IHP) maize, 128 Illinois Low Protein (ILP) maize, 128 indole-3-acetic acid (IAA), 236 inputs to organic cropping system(s), 9 iron, 14, 50, 311–334 biofortification, 325, 327 agronomic practice(s), 326 intercropping, 326 plant breeding, 326–327 chelation, 313 nicotinamine synthase (NAS), 313 compartmentation, 318, 320 distribution, 318 ferrirrigation, 324 homeostasis, 320 ferritin, 322 frataxin, 322 interaction(s) with CO2, 322, 324 interaction(s) with light, 322 nutrition, 311 photosynthesis, 323 production, 311 plant nutritional quality, 311 reduction, 314 transcription factor(s), 316 bHLH family, 316–317 FIT1, 316–317 IDEF1 and IDEF2, 316 IRO2, 318–319 transporter(s), 314, 321 FPN2, 318 IRT, 314–316 NRAMP, 320 YSL, 319 ZIP, 315 uptake, 312–315 barley, 317 regulation, 316 rice, 317 Strategy I, 55 Strategy II, 55 use efficiency, 55 Lactuca sativa, 170 landrace, 85 lateral root primordium (LRP), 26 leaf area index, 77 lamina, 72

legumes, 52, 104, 457, 458, 459, 560, 461–463 actinobacteria (Frankia), 458 crop improvement, 475–476 Leguminosae, 459 nitrogen fixation, 141, 182, 410, 457–489 nonlegumes, 476–478 Nod factor(s), 460–462, 470–472 Nod factor perception (NFP), 465, 468, 478 lectin ecto-apyrase(s) (LNPs), 465 nodule, 459, 464–465 aeschynomenoid, 464 determinate (or desmodioid), 464 development, 459–460 evolutionary perspectives, 473–475 indeterminate, 463 lupinoid, 464–465 organogenesis, 462, 465–467, 470, 478 abscisic acid (ABA), 471–472 brassinosteroids (BR), 470 gibberellic acid (GA), 470 hormonal regulation, 467–469 jasmonic acid (JA), 471 physiology, 459–460 nonlegume bacteria, 458, 476 rhizobial infection, 465, 476–477 entry pathway, 469, 478 ethylene, 471 flotillin-like genes, 467–468 infection thread formation, 466, 468, 471 symbiotic signaling, 465–466, 469 soil fertility, 457 symbiosis, 474 arbuscular mycorrhization (AM), 474–475 Leymus racemosus, 132 light extinction coefficient, 69, 71 lin1, 197 localized nutrient supply, 30 Lotus japonicas, 326 low phosphate root (LPR), 234, 249 LPR, see low phosphate root LRP, see lateral root primordium Lupinus spp., 22, 244 Lupinus angustifolius, 23, 31 Lycopersicon esculentum, 170 lysine, 14, 107 macronutrients, see calcium; magnesium; potassium magnesium, 265, 280–287 limitation, 282–283 nutrition, 285–287

INDEX

occurrence and availability in soil(s), 280 physiological functions, 280 cofactor, 281 photosynthesis, 280 transporter(s), 282–283 uptake, 281, 283–284 partitioning, 284–285 translocation, 284 maize, 69, 40, 145, 147, 409, 413, 446, 448, 450 Illinois High Protein (IHP), 128 Illinois Low Protein (ILP), 128 manganese, 49–50, 400–408 crop improvement, 407 deficiency and symptoms of, 401–403 efficiency and variation of, 403 function, 403 homeostasis, 404 in soils, 401–402 translocation, 405, 407 transport, 404 transporter(s), 396 MTM, 401 NRAMP, 401, 405–406 uptake, 404, 407 by roots, 405 use efficiency, 56, 407 vacuolar transport, 406 MapMan, 152 marker-assisted selection (MAS), 151 mass flow, 28–29, 338, 432 MCO, see multicopper oxidase gene Medicago spp., 465, 467, 471–472, 478 Medicago sativa 461 Medicago truncatula, 231, 326, 405, 461, 466–467 Mesorhizobium spp., 458, 461. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis Mesorhizobium loti, 461, 478 methionine, 14 Methylobacterium spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis Methylobacterium nodulans, 460 micronutrients, 377; see also boron; chlorine; copper; manganese; molybdenum; nickel micronutrient use efficiency (MUE), 377–379 definition, 378 microorganisms, 52, 55

495

microRNA, 93, 251 milling, 114 miRNA399, 229 molecular markers, 154 molybdenum, 394, 408–414 crop improvement, 413–414 deficiency and symptoms of, 409–410 efficiency, 413 function, 410 ABA biosynthesis, 410 nitrogenase, 458 nitrogen metabolism, 410 sulphite oxidase, 410 xanthine dehydrogenase, 410 homeostasis, 411 in soils, 409–410 translocation, 413 transport, 411 transporter(s), 411 MOT1, 411–412 sulfate, 411–412 uptake, 411–413 variation, 412 Monsanto, 141, 184 multicopper oxidase gene (MCO), 234 mycorrizha, 10, 32–33, 53, 55, 205, 230, 437 15

N-labeling, 156 NADH-dependent GDH, 179 NADH-GOGAT, 220 NAM-B1, 73, 93, 131 NAR2 (NRT3), 198 NHI, see nutrient harvest index nickel, 414–417 crop improvement, 417 deficiency, 415–416 function, 415–416 urea metabolism, 416 homeostasis, 416–417 in soils, 414–415 transport, 416–417 transporter(s), 396, 416–417 NRAMP, 416 YSL, 416 ZIP, 416 uptake, 416–417 use efficiency, 417 nitrate, 12–13, 194 control of water use in roots, 436–437 efflux, 201 transporter(s), 193–210

496

INDEX

nitrate reductase (NR), 142, 147, 156, 180 nitrification, 11, 132 nitrogen; see also nitrogen uptake/capture efficiency; nitrogen use efficiency; nitrogen utilization efficiency canopy, 67 effect on functional properties of wheat, 108 on grain protein nutrition, 103–120 on grain quality, 103–120 on prolamin (hordein) composition, 112 on protein composition, 108 fertilization, 105, 109–110 fertilizer, 65, 105, 139, 166 fixation, 141, 182, 410, 457–489 biological nitrogen fixation (BNF), 458–459 legumes, 457–458 nodule development and physiology, 459–460 rhizobial bacteria, 458 form of, 50 harvest index, 219 high inputs, 131 late acquisition, 131 leaching, 124 leaf, 216 metabolism, 52, 141, 171, 410 mineralization, 171, 215 nutrition, 103 optimal distribution, 75 optimum, 133 partitioning, 71, 75 pools, 72 remobilization, 71, 77, 86–87, 179 storage, 179 sulfur interactions, 303 underlying physiological mechanisms, 215 nitrogen uptake/capture efficiency (NUpE), 10, 124–125, 141,166, 180, 184, 187 nitrogen use efficiency (NUE), 7, 10, 16, 47, 51, 65, 123, 128–129, 130, 141, 165, 211, 215 compensation, 211 crop improvement, 8, 212–214 definition of, 123, 139 genetic improvement in wheat, 125, 128 genetic progress, 166 genetic variation, 129, 218 heterosis, 139–164

molecular genetics, 168 partial factor productivity (PFPN), 10, 15, 124–125 nitrogen utilization efficiency (NUtE), 10, 15, 124–125, 130, 141, 180 Nitromonas spp., 132 nitrous oxide, 65, 166 NMR, 154 non-protein amino acids, 104 NR, see nitrate reductase NRT1, 12, 30, 180, 194, 200, 204 NRT2, 196–197, 200 NUE, see nitrogen use efficiency NUpE, see nitrogen uptake/capture efficiency NUtE, see nitrogen utilization efficiency nutrient(s) availability, 10,13, 32, 51 capture, 10–11, 28, 33, 36; see also nitrogen uptake/capture efficiency remobilization, 83–84, 86 response curve(s), 7 uptake from soils, 21, 29, 57, 432–434 utilization efficiency, 13 nutrient harvest index (NHI), 15 nutrient use efficiency, 5–19, 123–138 O-acetyl-L-serine (thiol)lyase, 301 Ochrobactrum spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis “omics,” 139 proteomics, 152 transcription factors MYB, 248 PHR1, 248 transcriptomics, 152–154, 245 Oryza sativa, see rice OsENOD93–1, 145, 177 Panicum miliaceum, 182–183 Parasponia spp., 458, 478 partial factor productivity, 10, 15, 124–125, 168 partitioning dry matter, 72 nitrogen, 71 penetrometer, 26 PEPC, 176, 179–180 Phaseolus spp., 464 Phaseolus vulgaris, 411

INDEX

phosphorus, 11–12, 27, 29, 48, 205, 229–264 acquisition, 27 bioavailability, 11, 33, 39 deficiency, 230, 231, 232 233, 234, 237 fertilizer, 230 mobilization, 55 phosphorus limitation, 243, 245 stress miR399, 253 sugars and microRNA crosstalk, 251–253 ubiquitin-conjugating E2 enzyme (UBC24), 252 uptake, 39, 233 use efficiency, 53 zinc, 340 photosynthesis, 65, 71, 211, 216, 229, 311, 323, 433, 446 photosynthetic capacity, 69 photosynthetic rate, 70 Phyllobacterium spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis phytochelatins, 296 PII, 180 plant(s) growth, 6 breeding, 7, 133, 326 plastid transport, 204, 300 pollution, 65, 166, 230 potassium, 48, 265–271 biochemical roles, 267–268 biophysical roles, 268–269 cytoplasmic charge balancing, 268 electrical signaling, 269 homeostasis, 268 efflux, 271 nutrition, 285–287 physiological functions, 267 occurrence and availability in soil, 265–267 translocation and distribution, 271 AKT2,3, 272 SKOR, 272 sub-cellular partitioning, 272 uptake from soil, 270–271 prolamin box, 115 promoter(s), 186 protease, 88 proteasome pathway, 94 proteolysis, 87

497

quantitative trait(s), 125 loci (QTL), 15, 21, 31, 133–135, 146–147, 149, 150–151, 248 low phosphate root, 234, 249 recombinant inbred line (RIL), 141 respiration, 77 rhizobial bacteria, 458–466, 475. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis Rhizobium spp., 458–459, 461. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis Rhizobium-legume symbiosis, 141, 382, 457–459, 465–466, 477 Rhizobium leguminosarum, 462 Rhizopus arrhizus, 56 Rhizosphere, 37–39, 47–61, 205, 239, 313, 353–354, 402 availability of carbon, 51 availability of water, 49 nutrient transport, 48 pH, 50 redox potential, 49 rice, 76, 89, 113, 140, 144, 170, 184, 195, 211–225, 315, 319, 353, 415 breeding, 219 genotypic variation, 217 harvest index, 219 hybrid rice, 217 ion uptake, 317 world production, 213 RNA antisense RNA, 144 interference RNA (RNAi), 144 root(s), 21–45, 47, 432, 435–436 architecture, 10–11, 33–34, 179, 187, 220, 231–232, 234, 237, 353, 435 cluster roots, 238–240, 244 depth, 22–23 extracellular phosphatases, 233 hairs, 55, 235, 238, 462 mutants, 235 lateral, 236, 238, 314, 470, 474 microbe-soil interactions, 32 nutrient uptake, 57, 278, 395, 397, 405 via mycorrizha, 10 via plasma membranes, 10, 385 response to localized nutrient supply, 30

498

INDEX

root(s) (continued) response to phosphorus deficiency, 230 response to salt (sodic) stress, 446 rubisco, 14, 67, 74, 77, 87, 144, 179, 211, 215–216, 220 salinity, 443–455 accumulation in soil, 443–444 crop improvement, 451 H+ATPase, 448 nutrient problems, 443–444 resistance, 451 during reproductive stage, 450 sodic soils, 443–444 two-phase model of salt stress, 444–445 osmotic resistance, 445–446 sodium exclusion strategies, 448–449 hexose, 450 NHX transporter(s), 449 seed storage compounds, 103–120 selenium, 14, 114, 303–304 agronomic fortification, 114 senescence, 83–102 abscisic acid (ABA), 91, 394, 450, 471 hormones, 91, 93 proteolysis, 87 proteosome pathway, 94 regulation of, 91, 93 Shinella spp., 458. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis shuttle breeding, 133 sinks, 14, 179 carbon, 243 iron, 14 selenium 14, zinc, 14 Sinorhizobium spp. (Ensifer), 458–459. See also legumes; nitrogen, fixation; Rhizobium-legume symbiosis Sinorhizobium meliloti, 460 SLN, see specific leaf nitrogen soil, 22, 47, 432; see also edaphic environment fertility, 457–458 limitations to growth, 25 nutrient availability, 10, 48 pH, 338, 402 structure, 48 Sorghum bicolour, 76 specific leaf nitrogen (SLN), 69–70, 74 stay-green, 72–73, 85, 177 stem, 72

storage proteins, 129 gene expression, 115 sulfur, 12, 103, 108, 110–111, 295–309 assimilatory pathways, 298, 300–301 availability in barley, 113 demand, 296 distribution, 298, 301 effect on prolamin (hordein) composition, 112 fertilizer, 296 in grain, 112 limitation, 302–303 nitrogen interaction, 303 nutrition, 103, 295–296 selenium, 303 sulfur-rich protein(s), 111 transporter(s), 12, 297–298 uptake, 298 regulation, 301 use efficiency, 305 Syngenta, 141 synteny, 150 systems biology, 151 TCA, see tricarboxylic cycle acid Thlaspi arvense, 344 Thlaspi caerulescens, 319 Thlaspi japonicum, 416 tillers, 217 transcription factors NAC, 94 SCARECROW, 238, 466 WRKY, 93 transcriptome, 16, 152 transgenic crops, 143, 172–176, 327 transport, 90, 176, 193 peribacteroid membrane, 203 plastid, 204, 300 transporter(s) amino acid, 90, 205 ammonia, 203 ammonium, 201 uptake by roots, 202 boron, 386 copper, 392 dicarboxylic acid, 194 iron, 314 FPN2, 318 IRT family, 314–316 phytosiderophore complexes, 313 yellow stripe 1 (YSL), 313, 319, 335 ZIP, 315

INDEX

MATE, 12, 30, 180, 194, 198, 200 molybdenum, 299, 411 MOT1, 416–417 nickel, 416 NRAMP, 416 YSL, 416 ZIP, 193–210 nitrate (NRTs), 193–210 NRT1, 12, 30, 180, 194–196, 200, 204 NRT2, 196–197 post-translational regulation, 199 two-component, 198 regulation, 198 vacuolar, 200 peptide, 194, 196 phosphate, 12, 90, 233, 241 sulfate, 12, 297–298 vacuolar antiporters, 279, 390 boron, 385 calcium, 279 potassium, 266, 269, 279 CHX, 272 NHX, 272 sulfate, 299 zinc phytosiderophore complexes, 344 zinc iron premeases (ZIP), 344 tricarboxylic acid (TCA), pathways, 176, 242 trichoblasts, 235 Triticum spp., see also wheat Triticum aestivum, 394 Triticum durum, 132 Triticum turgidum spp. durum, 73 Triticum turgidum var. dicoccoides, 131 vacuoles, 13, 200, 203 VfAAP1, 177 Vicia faba, 177 Vitis vinifera, 409, 411, 413 water, 49 drought, 431–441 hydraulic conductivity, 434 interaction with ion fluxes, 432–433 in soils, 432–434 in roots, 432–433 wheat, 71, 73, 103, 111, 123, 127, 131–132, 140, 345, 394; see also Triticum spp. breeding, 123 functional properties, 111

499

genetic improvement of NUE, 123 yields, 127 xenobiotics, 296 yield, 7 gap, 7 grain, 9, 213 potential, 7 quality in grain, 9, 212 YSL, 319, 327, 335, 345, 400, 416 zinc, 14, 335–375 availability, 339 biofortification, 351 crop improvement, 351, 360 deficiency, 345–346 correction of, 346 drought tolerance, 349–350 efficiency, 351–353, 355 genetics of, 359, 361 screening for, 356–358 upregulated genes, 360 enzymes, 347 fertilizers, 336, 346–349, 356 functions, 336, 342–343 genotypic variation in seeds, 355 heavy metal ATPase (HMA) family, 344 homeostasis, 354 metal response element-binding transcription factor-1 (MTF1), 354 interactions with other soil nutrients, 340–341 limitation, 335, 337, 344 nutrition, 350 phosphorus interactions, 340 phytate, 360 phytosiderophore complexes, 335, 344 reactive oxygen species (ROS), 343 in soils, 337 fractions, 337 organic matter, 339 pH, 338–339 predicting deficiency, 342 superoxide dismutase (SOD), 350 translocation, 343 uptake, 338, 343 yellow stripe 1 (YSL), 335 zinc iron permease (ZIP), 344