Advances in
BOTANICAL RESEARCH Series Editors JEAN-CLAUDE KADER
Laboratoire Physiologie Cellulaire et Mole´culaire des Plantes, CNRS, Universite´ de Paris, Paris, France
MICHEL DELSENY
Laboratoire Ge´nome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France
Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW17BY, UK 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands First edition 2011 Copyright ß 2011, Elsevier Ltd. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
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CONTRIBUTORS TO VOLUME 57
MARIS P. APSE Arcadia Biosciences, Davis, California, USA EDUARDO BLUMWALD Department of Plant Sciences, University of California, Davis, California, USA ALAIN BOUCHEREAU Universite´ de Rennes 1, Campus de Beaulieu, Baˆtiment 14A, Rennes Cedex, France HIKMET BUDAK Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey MARIA M. CHAVES Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Av. da Repu´blica, Oeiras, Portugal; CBAA, Instituto Superior de Agronomia, Universidade Te´cnica de Lisboa, Tapada da Ajuda, Lisboa, Portugal J. MIGUEL COSTA Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Av. da Repu´blica, Oeiras, Portugal; CBAA, Instituto Superior de Agronomia, Universidade Te´cnica de Lisboa, Tapada da Ajuda, Lisboa, Portugal TIJEN DEMIRAL Department of Biology, Faculty of Science and Arts, Harran University, Sanlıurfa, Turkey JILL M. FARRANT Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa ABDELWAHED GHORBEL Biotechnology Center, Borj Cedria Science and Technology Park, Route Touristique Borj Ce´dria-Soliman, Hammam-Lif, Tunisia TAKASHI HIRAYAMA Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama, Japan ARCHANA JOSHI-SAHA Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, UPR 2355, Gif-sur-Yvette 91198 Cedex, France MELDA KANTAR Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey ´ CS Institute of Plant Biology, Biological Research HAJNALKA KOVA Center, Szeged, Hungary TAKASHI KUROMORI Gene Discovery Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa, Japan JEFFREY LEUNG Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, UPR 2355, Gif-sur-Yvette 91198 Cedex, France
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CONTRIBUTORS
STUART J. LUCAS Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey ALEX MACKAY School of Agricultural Science, University of Tasmania, Hobart, Tas, Australia KANA MIYATA Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki, Japan YUTAKA MIYAZAWA Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai, Japan TSUYOSHI MIZOGUCHI Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki, Japan TEPPEI MORIWAKI Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai, Japan MONIQUE MORSE Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa RANA MUNNS CSIRO Plant Industry, Canberra, and School of Plant Biology, The University of Western Australia, Perth, Australia YU NATSUI Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki, Japan RIM NEFISSI Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki, Japan PETER M. NEUMANN Department of Environmental, Water and Agricultural Engineering, Technion Israel Institute of Technology, Haifa, Israel ZVI PELEG Department of Plant Sciences, University of California, Davis, California, USA MOHAMED S. RAFUDEEN Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa NELSON J. MADEIRA SAIBO Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Av. da Repu´blica, Oeiras, Portugal A. HEDIYE SEKMEN Department of Biology, Faculty of Science and Arts, Harran University, Sanlıurfa, Turkey SERGEY SHABALA School of Agricultural Science, University of Tasmania, Hobart, Tas, Australia KAZUO SHINOZAKI Gene Discovery Research Group, RIKEN Plant Science Center, Tsukuba, Ibaraki, Japan; Gene Discovery Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa, Japan ´ SZLO ´ SZABADOS Institute of Plant Biology, Biological Research LA Center, Szeged, Hungary ISMAIL TURKAN Department of Biology, Faculty of Science and Arts, Harran University, Sanlıurfa, Turkey HIDEYUKI TAKAHASHI Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai, Japan
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TAISHI UMEZAWA Gene Discovery Research Group, RIKEN Plant Science Center, Tsukuba, Ibaraki, Japan CHRISTIANE VALON Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, UPR 2355, Gif-sur-Yvette 91198 Cedex, France TOMOKAZU YAMAZAKI Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai, Japan AVIAH ZILBERSTEIN Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel
PREFACE
Drought and soil salinity are major abiotic stresses that adversely affect crop productivity and quality. Their harmful effects are increasing due to global warming, and human activities such as overutilization of water resources, over-irrigation, improper drainage, besides natural causes such as entry of seawater in coastal areas and salt accumulation in the root zone in arid and semi-arid regions due to high evaporation rates. Drought and salinity have osmotic, ionic and nutritional constraint effects on plants. These effects lead to growth retardation, metabolic disturbances and oxidative stress. Plants may tolerate and adapt to these stressors by mechanisms including changed leaf architecture, osmotic adjustment, ion exclusion and compartmentalization, and more efficient reactive oxygen species (ROS) scavenging systems. Recent advances in genetic analysis of model plant Arabidopsis thaliana mutants are providing new insights into drought and salt stress signalling and tolerance mechanisms that will help to breed plants with an increased tolerance to stress. These advancements include sensing and signalling of drought and salinity, osmotic homeostasis, ROS scavenging, Naþ efflux and ion homeostasis, cytosolic calcium signals and abscisic acid (ABA)-mediated regulation of stress proteins. The impacts of global plant analytical tools like metabolomics and proteomics are providing new information on these processes, but to link genomics with the metabolic regulation of these processes and their output in terms of growth and development under stress requires an understanding of the biochemistry and physiology behind these processes. Quantitative measurements of genetic variation in traits known or predicted to be important adaptive mechanisms will enable links back to the genome and provide a platform for genetic manipulation and molecular plant breeding. Hence, in this volume, developments in understanding of plant responses to drought and salinity in a postgenomic are described and evaluated by worldwide-known experts. Rana Munns reviews specific traits for drought and salinity tolerance and experimental methods that could distinguish drought and salinity adaptations. She also points out the importance of the use of new phenomics techniques combined with rapidly advancing molecular tools that provides a powerful impetus to identify key traits and genes for stress tolerance, and new methods to introduce these genes into important food crops. One of the latest known about drought and salinity is the extracellular (apoplastic) region of plants, that is, plant cell walls, xylem and environmental boundary layers at root and leaf surfaces each of which can make direct ‘post-genomic’ contributions to the regulation of whole plant growth and
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development under optimal or stressful conditions. Peter M. Neumann in his chapter emphasizes apoplastic changes that are involved in the regulation of cell, organ and whole plant growth responses to salinity and water stress. He also presents an elective perspective in growth inhibition by water deficits, as caused by salinity, drought and, most recently, novel plant interactions with aqueous colloids in soil solutions. The current understanding of the effect of drought and salinity on photosynthesis, a highly sensitive process to these stresses and a major determinant of plant’s growth and yield, is addressed by Maria M. Chaves et al. They represent a comprehensive and up-to-date review on the CO2 diffusive limitations to photosynthesis under water deficits and the underlying regulatory mechanisms of stomatal behaviour and photosynthetic metabolism. Recent molecular advances are also described, in particular, those related to stomatal development and guard cell signalling by giving a special emphasis to the effects of ABA signalling on stomatal regulation under water deficits. Knowledge on leaf gas exchange limitations caused by drought and high salinity for future breeding strategies is also addressed. In general, osmoprotective compounds can accumulate to very high concentrations in extremophile plants in saline or dry environments, suggesting that these metabolites contribute considerably to the adaptation of these plants to the harsh environment. Besides their main role in osmotic adjustment, they have also showed a protective role functioning in the stabilization of cellular structures, photosynthetic complexes, specific enzymes and other macromolecules; the scavenging of ROS; or acting as metabolic signals in stress conditions. In this volume, the importance of osmoprotective compounds for the adaptation to extreme environmental conditions is reviewed by La´szlo´ Szabados et al. In their chapter, they mention numerous studies obtained with natural variants, mutants or transgenic plants with different capabilities to accumulate these metabolites. Due to their remarkable ability to tolerate and even benefit from excessive salt concentrations that kill most other plant species, halophytic plants have attracted attention of plant biologists who consider that they may provide genes that confer salinity tolerance to crops. Sergey Shabala and Alex Mackay review current knowledge of physiological mechanisms regulating ion uptake and sequestration in halophytes. Their chapter includes specific anatomical and morphological features of halophytes, tissue- and organspecific ion compartmentalization, mechanisms of osmotic adjustment in halophytes, radial ion transport in halophyte roots, mechanisms of Naþ and Kþ loading into the xylem, Naþ sequestration in vacuoles in roots and leaf cells, ion transport in guard cells, control of ion fluxes into salt glands and bladders, and oxidative signalling and damage repair in halophytes. ABA has an important function in various biological processes in plants, especially in the regulation of seed maturation, dormancy, and abiotic stress responses, which have all strong relationships with crop yields. Recently,
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some of the breakthrough studies unravelled identification of ABA receptors and the establishment of a major ABA signalling pathway and the transport activity of ABA among tissues. Taishi Umezawa et al. describe the recent progress in our knowledge of ABA biosynthesis/catabolism, intercellular transport and intracellular signalling which open new avenues to understanding hormonal response, not only in cells but also in the whole organism, and for the improvement of crop yields. Another up-to-date review on ABA is given by Archana Joshi-Saha et al. In their chapter, they relate some of the most recent, insightful and exciting findings in the signalling network that orchestrate ABA-dependent adaptive processes. They also extend their discussion in applying ABA research, not on the more obvious agricultural benefits but as a novel and potent modulator of the immune system in humans. Many effective protection systems exist in plants that allow them to perceive, respond to, and appropriately adapt to a range of stress signals, and a variety of genes and gene products have been identified that involve responses to drought and high salinity stress. Understanding the mechanisms by which the plant perceives drought and the intracellular signalling pathways that are engaged in initiating the drought response in target cells is vital to cope with stressors. In this volume, the most recent advances in revealing ROS-mediated signalling under salinity and drought stresses are focused and the regulatory circuits that allow plants to cope with stress are presented by Tijen Demiral et al. In their chapter, some examples were cited of how osmotic change is sensed and relayed. The role of some signalling components covering of ROS and ABA have also been discussed. In their chapter, Monique Morse et al. review the current understanding of desiccation tolerance in the vegetative tissues of resurrection plants by presenting an overview of the stresses associated with desiccation and the physiological and biochemical protection reported to result in amelioration of these stresses. They also discuss the contribution of the genomics era in furthering our understanding of these protection systems in the attainment of desiccation tolerance along with the advances made in proteomics and give a brief overview of recent contributions in the field of metabolomics that have contributed to the understanding of desiccation tolerance. In contrast to the physiological and molecular mechanisms contributing to drought tolerance, the mechanisms underlying drought avoidance are much less well understood. Yutaka Miyazawa et al. in this volume describe what happens to cells faced with a water deficit and then outline the molecular mechanisms underlying different tropisms, with particular emphasis on the molecular mechanism contributing to root hydrotropism. In many prokaryotic and eukaryotic organisms including plants, the response of several physiological and molecular processes to diurnal changes in environmental conditions such as light quality and quantity, temperature and humidity is mediated by biological clocks which persist with a period
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close to 24 h in the absence of any environmental time cue. Rim Nefissi et al. in their chapter summarize a recent progress on understanding molecular mechanisms underlying the regulation of flowering time and organ elongation. They also discuss possible roles of clock genes such as ELF3, PRRs, LHY and CCA1 in response to environmental stresses. After stressing the use of new technologies provided by high-resolution transcript profiling, the identification of large-scale specific protein networks and their association with the plant responses to environmental perturbations, Zvi Peleg et al. address recent advances in engineering drought and salinity tolerance in crop plants with emphasis on yield and the needs to close the gaps between the laboratory and the field conditions. They have also focused on efforts towards the improvement of drought and salinity stress tolerance in crop plants with emphasis on field trials. A large number of genes and gene products have been implicated in the drought response, but identifying which are most useful for breeding drought-resistant crop varieties remains a significant technical challenge. Therefore, Melda Kantar et al. survey the molecular methods that are currently in use for drought research and ways in which they can be applied to accelerate breeding for drought resistance after summarizing the current state of knowledge of the molecular events that take place when a plant is under drought stress. In their chapter, particular focus is given to postgenomic techniques—transcriptomics, proteomics and metabolomics— assessing the relative strengths and weaknesses of each approach and how to make use of the large datasets they produce. Finally, as described above, our understanding of drought and salinity stress responses has taken a big leap forward in a post-genomic era. Advances in genetic analysis of model plant, A. thaliana mutants, as well as completion of full genome sequencing of rice, poplar and some other plants are providing new insight into drought and salt stress signalling and tolerance. Therefore, I hope this volume, containing new insights along with existing information, will help to not only close the gap but also document latest research findings in a very hot topic such as drought and salinity both of which limits productivity of crop plants worldwide. We hope that this volume will be very beneficial to the academics such as graduate students and researchers, mostly from plant science including botany, agriculture and forestry. It will also be of interest to environmental scientists, microbiologists, biochemists, biophysicists and chemists. Professionals working in seed and food companies and crop growers will also be among the benefited. ISMAIL TURKAN
CONTENTS OF VOLUMES 35–56 Series Editor (Volumes 35–44) J.A. CALLOW School of Biosciences, University of Birmingham, Birmingham, United Kingdom
Contents of Volume 35 Recent Advances in the Cell Biology of Chlorophyll Catabolism H. THOMAS, H. OUGHAM and S. HORTENSTEINER The Microspore: A Haploid Multipurpose Cell A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS The Seed Oleosins: Structure Properties and Biological Role J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY Compartmentation of Proteins in the Protein Storage Vacuole: A Compound Organelle in Plant Cells L. JIANG and J. ROGERS Intraspecific Variation in Seaweeds: The Application of New Tools and Approaches C. MAGGS and R. WATTIER Glucosinolates and Their Degradation Products R. F. MITHEN
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Contents of Volume 36 PLANT VIRUS VECTOR INTERACTIONS Edited by R. Plumb Aphids: Non-Persistent Transmission T. P. PIRONE and K. L. PERRY Persistent Transmission of Luteoviruses by Aphids B. REAVY and M. A. MAYO Fungi M. J. ADAMS Whitefly Transmission of Plant Viruses J. K. BROWN and H. CZOSNEK Beetles R. C. GERGERICH Thrips as Vectors of Tospoviruses D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL, A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN Virus Transmission by Leafhoppers, Planthoppers and Treehoppers (Auchenorrhyncha, Homoptera) E. AMMAR and L. R. NAULT Nematodes S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN Other Vectors R. T. PLUMB
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Contents of Volume 37 ANTHOCYANINS IN LEAVES Edited by K. S. Gould and D. W. Lee Anthocyanins in Leaves and Other Vegetative Organs: An Introduction D. W. LEE and K. S. GOULD Le Rouge et le Noir: Are Anthocyanins Plant Melanins? G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD Anthocyanins in Leaves: History, Phylogeny and Development D. W. LEE The Final Steps in Anthocyanin Formation: A Story of Modification and Sequestration C. S. WINEFIELD Molecular Genetics and Control of Anthocyanin Expression B. WINKEL-SHIRLEY Differential Expression and Functional Significance of Anthocyanins in Relation to Phasic Development in Hedera helix L. W. P. HACKETT Do Anthocyanins Function as Osmoregulators in Leaf Tissues? L. CHALKER-SCOTT The Role of Anthocyanins for Photosynthesis of Alaskan Arctic Evergreens During Snowmelt S. F. OBERBAUER and G. STARR Anthocyanins in Autumn Leaf Senescence D. W. LEE A Unified Explanation for Anthocyanins in Leaves? K. S. GOULD, S. O. NEILL and T. C. VOGELMANN
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Contents of Volume 38 An Epidemiological Framework for Disease Management C. A. GILLIGAN Golgi-independent Trafficking of Macromolecules to the Plant Vacuole D. C. BASSHAM Phosphoenolpyruvate Carboxykinase: Structure, Function and Regulation R. P. WALKER and Z.-H. CHEN Developmental Genetics of the Angiosperm Leaf C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE and R. A. MARTIENSSEN A Model for the Evolution and Genesis of the Pseudotetraploid Arabidopsis thaliana Genome Y. HENRY, A. CHAMPION, I. GY, A. PICAUD, A. LECHARNY and M. KREIS
Contents of Volume 39 Cumulative Subject Index Volumes 1–38
Contents of Volume 40 Starch Synthesis in Cereal Grains K. TOMLINSON and K. DENYER The Hyperaccumulation of Metals by Plants M. R. MACNAIR Plant Chromatin — Learning from Similarities and Differences J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI, P. ZIELENKIEWICZ and A. JERZMANOWSKI
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The Interface Between the Cell Cycle and Programmed Cell Death in Higher Plants: From Division unto Death D. FRANCIS The Importance of Extracellular Carbohydrate Production by Marine Epipelic Diatoms G. J. C. UNDERWOOD and D. M. PATERSON Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins A. K. CHARNLEY
Contents of Volume 41 Multiple Responses of Rhizobia to Flavonoids During Legume Root Infection JAMES E. COOPER Investigating and Manipulating Lignin Biosynthesis in the Postgenomic Era CLAIRE HALPIN Application of Thermal Imaging and Infrared Sensing in Plant Physiology and Ecophysiology HAMLYN G. JONES Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and Transposable Elements CELIA HANSEN and J. S. HESLOP-HARRISON
Role of Plasmodesmata Regulation in Plant Development ARNAUD COMPLAINVILLE and MARTIN CRESPI
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Contents of Volume 42 Chemical Manipulation of Antioxidant Defences in Plants ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON and IAN CUMMINS The Impact of Molecular Data in Fungal Systematics P. D. BRIDGE, B. M. SPOONER and P. J. ROBERTS Cytoskeletal Regulation of the Plane of Cell Division: An Essential Component of Plant Development and Reproduction HILARY J. ROGERS Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol ALYSON K. TOBIN and CAROLINE G. BOWSHER
Contents of Volume 43 Defensive and Sensory Chemical Ecology of Brown Algae CHARLES D. AMSLER and VICTORIA A. FAIRHEAD Regulation of Carbon and Amino Acid Metabolism: Roles of Sucrose Nonfermenting-1-Related Protein Kinase-1 and General Control Nonderepressible-2-Related Protein Kinase NIGEL G. HALFORD Opportunities for the Control of Brassicaceous Weeds of Cropping Systems Using Mycoherbicides AARON MAXWELL and JOHN K. SCOTT Stress Resistance and Disease Resistance in Seaweeds: The Role of Reactive Oxygen Metabolism MATTHEW J. DRING Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus ANNA AMTMANN, JOHN P. HAMMOND, PATRICK ARMENGAUD and PHILIP J. WHITE
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Contents of Volume 44 Angiosperm Floral Evolution: Morphological Developmental Framework PETER K. ENDRESS Recent Developments Regarding the Evolutionary Origin of Flowers MICHAEL W. FROHLICH Duplication, Diversification, and Comparative Genetics of Angiosperm MADS-Box Genes VIVIAN F. IRISH Beyond the ABC-Model: Regulation of Floral Homeotic Genes LAURA M. ZAHN, BAOMIN FENG and HONG MA Missing Links: DNA-Binding and Target Gene Specificity of Floral Homeotic Proteins RAINER MELZER, KERSTIN KAUFMANN ¨ NTER THEIßEN and GU Genetics of Floral Development in Petunia ANNEKE RIJPKEMA, TOM GERATS and MICHIEL VANDENBUSSCHE Flower Development: The Antirrhinum Perspective BRENDAN DAVIES, MARIA CARTOLANO and ZSUZSANNA SCHWARZ-SOMMER Floral Developmental Genetics of Gerbera (Asteraceae) TEEMU H. TEERI, MIKA KOTILAINEN, ANNE UIMARI, SATU RUOKOLAINEN, YAN PENG NG, URSULA MALM, ¨ NEN, SUVI BROHOLM, ROOSA LAITINEN, ¨ LLA EIJA PO PAULA ELOMAA and VICTOR A. ALBERT Gene Duplication and Floral Developmental Genetics of Basal Eudicots ELENA M. KRAMER and ELIZABETH A. ZIMMER
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Genetics of Grass Flower Development CLINTON J. WHIPPLE and ROBERT J. SCHMIDT Developmental Gene Evolution and the Origin of Grass Inflorescence Diversity SIMON T. MALCOMBER, JILL C. PRESTON, RENATA REINHEIMER, JESSIE KOSSUTH and ELIZABETH A. KELLOGG Expression of Floral Regulators in Basal Angiosperms and the Origin and Evolution of ABC-Function PAMELA S. SOLTIS, DOUGLAS E. SOLTIS, SANGTAE KIM, ANDRE CHANDERBALI and MATYAS BUZGO The Molecular Evolutionary Ecology of Plant Development: Flowering Time in Arabidopsis thaliana KATHLEEN ENGELMANN and MICHAEL PURUGGANAN A Genomics Approach to the Study of Ancient Polyploidy and Floral Developmental Genetics JAMES H. LEEBENS-MACK, KERR WALL, JILL DUARTE, ZHENGUI ZHENG, DAVID OPPENHEIMER and CLAUDE DEPAMPHILIS Series Editors (Volume 45– ) JEAN-CLAUDE KADER Laboratoire Physiologie Cellulaire et Mole´culaire des Plantes, CNRS, Universite´ de Paris, Paris, France MICHEL DELSENY Laboratoire Ge´nome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France
Contents of Volume 45 RAPESEED BREEDING History, Origin and Evolution S. K. GUPTA and ADITYA PRATAP
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Breeding Methods B. RAI, S. K. GUPTA and ADITYA PRATAP The Chronicles of Oil and Meal Quality Improvement in Oilseed Rape ABHA AGNIHOTRI, DEEPAK PREM and KADAMBARI GUPTA Development and Practical Use of DNA Markers KATARZYNA MIKOLAJCZYK Self-Incompatibility RYO FUJIMOTO and TAKESHI NISHIO Fingerprinting of Oilseed Rape Cultivars ´ ˇ URN and JANA ZˇALUDOVA VLADISLAV C Haploid and Doubled Haploid Technology L. XU, U. NAJEEB, G. X. TANG, H. H. GU, G. Q. ZHANG, Y. HE and W. J. ZHOU Breeding for Apetalous Rape: Inheritance and Yield Physiology LIXI JIANG Breeding Herbicide-Tolerant Oilseed Rape Cultivars PETER B. E. MCVETTY and CARLA D. ZELMER Breeding for Blackleg Resistance: The Biology and Epidemiology W. G. DILANTHA FERNANDO, YU CHEN and KAVEH GHANBARNIA Development of Alloplasmic Rape MICHAL STARZYCKI, ELIGIA STARZYCKI and JAN PSZCZOLA Honeybees and Rapeseed: A Pollinator–Plant Interaction D. P. ABROL
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Genetic Variation and Metabolism of Glucosinolates NATALIA BELLOSTAS, ANNE DORTHE SØRENSEN, JENS CHRISTIAN SØRENSEN and HILMER SØRENSEN Mutagenesis: Generation and Evaluation of Induced Mutations SANJAY J. JAMBHULKAR Rapeseed Biotechnology VINITHA CARDOZA and C. NEAL STEWART, JR. Oilseed Rape: Co-existence and Gene Flow from Wild Species RIKKE BAGGER JØRGENSEN Evaluation, Maintenance, and Conservation of Germplasm RANBIR SINGH and S. K. SHARMA Oil Technology ¨ US BERTRAND MATTHA
Contents of Volume 46 INCORPORATING ADVANCES IN PLANT PATHOLOGY Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features Influencing Root Growth and Development ´ NICA CREUS, MARI´A CELESTE MOLINA-FAVERO, CECILIA MO LUCIANA LANTERI, NATALIA CORREA-ARAGUNDE, MARI´A CRISTINA LOMBARDO, CARLOS ALBERTO BARASSI and LORENZO LAMATTINA How the Environment Regulates Root Architecture in Dicots ´ RIE LEFEBVRE, PHILIPPE MARIANA JOVANOVIC, VALE LAPORTE, SILVINA GONZALEZ-RIZZO, CHRISTINE LELANDAIS-BRIE`RE, FLORIAN FRUGIER, CAROLINE HARTMANN and MARTIN CRESPI
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Aquaporins in Plants: From Molecular Structure to Integrated Functions OLIVIER POSTAIRE, LIONEL VERDOUCQ and CHRISTOPHE MAUREL Iron Dynamics in Plants JEAN-FRANC ¸ OIS BRIAT Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the Early Steps of Symbiotic Interactions VIVIENNE GIANINAZZI-PEARSON, NATHALIE SE´JALON-DELMAS, ANDREA GENRE, SYLVAIN JEANDROZ and PAOLA BONFANTE Dynamic Defense of Marine Macroalgae Against Pathogens: From Early Activated to Gene-Regulated Responses AUDREY COSSE, CATHERINE LEBLANC and PHILIPPE POTIN
Contents of Volume 47 INCORPORATING ADVANCES IN PLANT PATHOLOGY The Plant Nucleolus ´ EZ-VA ´ SQUEZ AND FRANCISCO JAVIER MEDINA JULIO SA Expansins in Plant Development DONGSU CHOI, JEONG HOE KIM AND YI LEE Molecular Biology of Orchid Flowers: With Emphasis on Phalaenopsis WEN-CHIEH TSAI, YU-YUN HSIAO, ZHAO-JUN PAN, CHIACHI HSU, YA-PING YANG, WEN-HUEI CHEN AND HONG-HWA CHEN
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Molecular Physiology of Development and Quality of Citrus ´ S, JOSE´ M. FRANCISCO R. TADEO, MANUEL CERCO COLMENERO-FLORES, DOMINGO J. IGLESIAS, MIGUEL A. NARANJO, GABINO RI´OS, ESTHER CARRERA, OMAR RUIZ-RIVERO, IGNACIO LLISO, RAPHAE¨ L MORILLON, PATRICK OLLITRAULT AND MANUEL TALON Bamboo Taxonomy and Diversity in the Era of Molecular Markers MALAY DAS, SAMIK BHATTACHARYA, PARAMJIT SINGH, TARCISO S. FILGUEIRAS AND AMITA PAL
Contents of Volume 48 Molecular Mechanisms Underlying Vascular Development JAE-HOON JUNG, SANG-GYU KIM, PIL JOON SEO AND CHUNG-MO PARK Clock Control Over Plant Gene Expression ANTOINE BAUDRY AND STEVE KAY Plant Lectins ELS J. M. VAN DAMME, NAUSICAA LANNOO AND WILLY J. PEUMANS Late Embryogenesis Abundant Proteins MING-DER SHIH, FOLKERT A. HOEKSTRA AND YUE-IE C. HSING
Contents of Volume 49 Phototropism and Gravitropism in Plants MARIA LIA MOLAS AND JOHN Z. KISS
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Cold Signalling and Cold Acclimation in Plants ERIC RUELLAND, MARIE-NOELLE VAULTIER, ALAIN ZACHOWSKI AND VAUGHAN HURRY Genome Evolution in Plant Pathogenic and Symbiotic Fungi GABRIELA AGUILETA, MICHAEL E. HOOD, GUISLAINE REFRE´GIER AND TATIANA GIRAUD
Contents of Volume 50 Aroma Volatiles: Biosynthesis and Mechanisms of Modulation During Fruit Ripening BRUNO G. DEFILIPPI, DANIEL MANRI´QUEZ, ´ LEZ-AGU ¨ ERO KIETSUDA LUENGWILAI AND MAURICIO GONZA Jatropha curcas: A Review NICOLAS CARELS You are What You Eat: Interactions Between Root Parasitic Plants and Their Hosts LOUIS J. IRVING AND DUNCAN D. CAMERON Low Oxygen Signaling and Tolerance in Plants FRANCESCO LICAUSI AND PIERDOMENICO PERATA Roles of Circadian Clock and Histone Methylation in the Control of Floral Repressors RYM FEKIH, RIM NEFISSI, KANA MIYATA, HIROSHI EZURA AND TSUYOSHI MIZOGUCHI
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Contents of Volume 51 PAMP-Triggered Basal Immunity in Plants ¨ RNBERGER AND BIRGIT KEMMERLING THORSTEN NU Plant Pathogens as Suppressors of Host Defense ´ TRAUX, ROBERT WILSON JACKSON, JEAN-PIERRE ME ESTHER SCHNETTLER AND ROB W. GOLDBACH From Nonhost Resistance to Lesion-Mimic Mutants: Useful for Studies of Defense Signaling ANDREA LENK AND HANS THORDAL-CHRISTENSEN Action at a Distance: Long-Distance Signals in Induced Resistance MARC J. CHAMPIGNY AND ROBIN K. CAMERON Systemic Acquired Resistance R. HAMMERSCHMIDT Rhizobacteria-Induced Systemic Resistance ¨ FTE DAVID DE VLEESSCHAUWER AND MONICA HO Plant Growth-Promoting Actions of Rhizobacteria STIJN SPAEPEN, JOS VANDERLEYDEN AND YAACOV OKON Interactions Between Nonpathogenic Fungi and Plants M. I. TRILLAS AND G. SEGARRA Priming of Induced Plant Defense Responses UWE CONRATH Transcriptional Regulation of Plant Defense Responses MARCEL C. VAN VERK, CHRISTIANE GATZ AND HUUB J. M. LINTHORST
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Unexpected Turns and Twists in Structure/Function of PR-Proteins that Connect Energy Metabolism and Immunity MEENA L. NARASIMHAN, RAY A. BRESSAN, MATILDE PAINO D’URZO, MATTHEW A. JENKS AND TESFAYE MENGISTE Role of Iron in Plant–Microbe Interactions P. LEMANCEAU, D. EXPERT, F. GAYMARD, P. A. H. M. BAKKER AND J.-F. BRIAT Adaptive Defense Responses to Pathogens and Insects LINDA L. WALLING Plant Volatiles in Defence MERIJN R. KANT, PETRA M. BLEEKER, MICHIEL VAN WIJK, ROBERT C. SCHUURINK AND MICHEL A. HARING Ecological Consequences of Plant Defence Signalling MARTIN HEIL AND DALE R. WALTERS
Contents of Volume 52 Oxidation of Proteins in Plants—Mechanisms and Consequences LEE J. SWEETLOVE AND IAN M. MØLLER Reactive Oxygen Species: Regulation of Plant Growth and Development HYUN-SOON KIM, YOON-SIK KIM, KYU-WOONG HAHN, HYOUK JOUNG AND JAE-HEUNG JEON Ultraviolet-B Induced Changes in Gene Expression and Antioxidants in Plants S. B. AGRAWAL, SURUCHI SINGH AND MADHOOLIKA AGRAWAL
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Roles of -Glutamyl Transpeptidase and -Glutamyl Cyclotransferase in Glutathione and Glutathione-Conjugate Metabolism in Plants NAOKO OHKAMA-OHTSU, KEIICHI FUKUYAMA AND DAVID J. OLIVER The Redox State, a Referee of the Legume–Rhizobia Symbiotic Game DANIEL MARINO, CHIARA PUCCIARIELLO, ALAIN PUPPO AND PIERRE FRENDO
Contents of Volume 53 Arabidopsis Histone Lysine Methyltransferases FRE´DE´ RIC PONTVIANNE, TODD BLEVINS, AND CRAIG S. PIKAARD Advances in Coffea Genomics ALEXANDRE DE KOCHKO, SE´LASTIQUE AKAFFOU, ALAN ANDRADE, CLAUDINE CAMPA, DOMINIQUE CROUZILLAT, ROMAIN GUYOT, PERLA HAMON, RAY MING, LUKAS A. MUELLER, VALE´RIE PONCET, CHRISTINE TRANCHANTDUBREUIL, AND SERGE HAMON Regulatory Components of Shade Avoidance Syndrome JAIME F. MARTI´NEZ-GARCI´A, ANAHIT GALSTYAN, ´ S CIFUENTES-ESQUIVEL, MERCE`SALLA-MARTRET, NICOLA ´ MARC¸ AL GALLEMI, AND JORDI BOU-TORRENT Responses of Halophytes to Environmental Stresses with Special Emphasis to Salinity KSOURI RIADH, MEGDICHE WIDED, KOYRO HANS-WERNER, AND ABDELLY CHEDLY Plant Nematode Interaction: A Sophisticated Dialogue PIERRE ABAD AND VALERIE M. WILLIAMSON
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Optimization of Nutrition in Soilless Systems: A Review ´ NGELES CALATAYUD ELISA GORBE AND A
Contents of Volume 54 Pollen Germination and Tube Growth HUEI-JING WANG, JONG-CHIN HUANG, AND GUANG-YUH JAUH Molecular Mechanisms of Sex Determination in Monoecious and Dioecious Plants GEORGE CHUCK The Evolution of Floral Symmetry HE´LE`NE CITERNE, FLORIAN JABBOUR, SOPHIE NADOT, AND CATHERINE DAMERVAL Protein Turnover in Grass Leaves LOUIS JOHN IRVING, YUJI SUZUKI, HIROYUKI ISHIDA, AND AMANE MAKINO
Contents of Volume 55 Carpel Development ´ NDIZ, CHLOE´ FOURQUIN, CRISTINA FERRA NATHANAEL PRUNET, CHARLIE P. SCUTT, EVA SUNDBERG, CHRISTOPHE TREHIN, AND AURE´LIE C. M. VIALETTE-GUIRAUD Root System Architecture PAUL A. INGRAM AND JOCELYN E. MALAMY
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Functional Genomics of Cacao FABIENNE MICHELI, MARK GUILTINAN, KARINA PERES GRAMACHO, MIKE J. WILKINSON, ANTONIO VARGAS DE ´ LIO CE´ZAR DE MATTOS CASCARDO, OLIVEIRA FIGUEIRA, JU SIELA MAXIMOVA, AND CLAIRE LANAUD The Ecological Water-Use Strategies of Succulent Plants R. MATTHEW OGBURN AND ERIKA J. EDWARDS
Contents of Volume 56 Nodule Physiology and Proteomics of Stressed Legumes M. I. QURESHI, S. MUNEER, H. BASHIR, J. AHMAD, AND M. IQBAL Molecular Aspects of Fragrance and Aroma in Rice APICHART VANAVICHIT AND TADACHI YOSHIHASHI Miscanthus: A Promising Biomass Crop EMILY A. HEATON, FRANK G. DOHLEMAN, A. FERNANDO MIGUEZ, JOHN A. JUVIK, VERA LOZOVAYA, JACK WIDHOLM, OLGA A. ZABOTINA, GREGORY F. MCISAAC, MARK B. DAVID, THOMAS B. VOIGT, NICHOLAS N. BOERSMA, AND STEPHEN P. LONG
Plant Adaptations to Salt and Water Stress: Differences and Commonalities
RANA MUNNS1
CSIRO Plant Industry, Canberra, and School of Plant Biology, The University of Western Australia, Perth, Australia
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Whole Plant Responses to Water and Salt Stress: Commonalities and Differences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Mechanisms of Drought and Salinity Tolerance. . . . . . . . . . . . . . . . . . . . . . . . . . . A. Commonalities ................................................................. B. Long-distance Signals Controlling Leaf Expansion and Stomatal Conductance........................................................ C. Changes in Leaf Morphology and Root System Architecture.......... D. Turgor Maintenance and Osmotic Adjustment........................... E. Differences—Salt Specific .................................................... IV. Adaptations, Traits and QTLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Drought ......................................................................... B. Salinity .......................................................................... C. Salt-Specific traits.............................................................. D. Traits in Common with Drought ........................................... E. QTL Analysis and Gene Discovery......................................... V. Growth Studies and Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Water Stress .................................................................... B. Salinity .......................................................................... C. Experiments to Distinguish Water Stress from Salt-Specific Effects ..
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Corresponding author: E-mail:
[email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00001-1
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VI. Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT Drought and salinity are the most significant abiotic stresses to limit the production of the world’s staple food crops. Knowledge about physiological traits, and new molecular tools to identify key genes or to provide molecular markers, has the potential to increase yield over the present limits. The employment of molecular knowledge needs appropriate experimental design and accurate phenotyping. Testing the value of physiological traits and key candidate genes is crucial for progress towards crop improvement on dry and saline land. This chapter reviews specific traits for drought and salinity tolerance, and experimental methods that could distinguish drought and salinity adaptations. The use of new phenomics techniques combined with rapidly advancing molecular tools provides a powerful impetus to identify key traits and genes for stress tolerance, and new methods to introduce these genes into important food crops.
I. INTRODUCTION Water and salt stress due to drought and soil salinity are the two most intractable abiotic stresses that limit the production of the world’s staple food crops, wheat and rice. Drought cannot be avoided, and salinity can only be temporarily reduced. Scarcity of water will increase in the near future in many regions of the world due to climate change (IPCC, 2007) and also to urbanization. World population is predicted to double in the next 50 years, so greater yields must be extracted from the current agricultural areas along with more marginal areas (Chaves and Davies, 2010). The need for more water for cities as the population grows means less is available for irrigation, so a greater proportion of the world’s food will come from drought-prone areas. Productivity of crops on the best land in the most favourable environment is approaching a theoretical maximum, so to meet the increasing world food requirement, productivity in less favourable environments is needed (Passioura and Angus, 2010). Salinity is also increasing, due to either salts naturally present in the soil, or irrigation or the clearing of land for dryland agriculture. Over 6% of land throughout the world are salt affected, either by salinity or by the associated condition of sodicity (Munns, 2005). Most of this salinity, and all of the sodicity, is natural. However, a significant proportion of recently cultivated agricultural land has become saline due to land clearing or irrigation. For example, in Australia, about 2 million ha of the 17 million
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ha of land farmed by dryland agriculture are affected by secondary salinity to varying degrees and a further 8 million ha are at risk of salinisation in the next 40 years (Munns and James, 2003). A report by FAO (reported by Munns, 2003) stated that of the 230 million ha of irrigated land worldwide (much of which is in Asia where rice is the main cereal crop), 45 million ha are salt affected (20%). Irrigated land is only 15% of total cultivated land, but as irrigated land has at least twice the productivity of rainfed land, it produces one-third of the world’s food. In Pakistan alone, 1 million ha of the ricegrowing area is affected by salt (Ul Haq et al., 2010). Natural or ‘primary salinity’ is more widespread than first realised (Rengasamy, 2006, 2010a), while ‘secondary salinity’, due to land clearing or irrigation, continues to grow. Hence, increased salt tolerance of crops and horticultural species is needed to sustain increases in food production in many regions in the world. Increased salt tolerance of perennial species used for fodder or fuel production is a key component in reducing the spread of secondary salinity, while increased salt tolerance of crops will directly improve production in soils with primary salinity. Advances in crop productivity come from better management of the land and from new varieties. Genetic engineering is one way of increasing genetic gain, but a single gene may have limited effect. There are tens of thousands of accessions in international seed banks providing a huge genetic diversity, but selecting is a daunting task. Understanding the importance of various traits, and the molecular basis will provide a focused approach. This review summarises the physiological understanding of mechanisms of adaptation and the traits that confer it, and raises the question of how best to screen for these traits. It also points out the strong commonality between adaptation to water and salt stress. Conventional plant breeding with good agronomic practices has doubled the production of crops in regions that are not limited by water. For example, the yield of wheat in UK has doubled from 4 to 8 tonnes per hectare over the past 50 years, but average yield in Australia has increased only from about 1 to 1.5 tonnes per hectare (Richards et al., 2010). This has come about by empirical selection for yield, which has proved extremely effective, but shows signs of reaching a plateau. To complement empirical breeding for yield in water-limited environments, physiological breeding including the use of molecular markers has been proposed (Cattivelli et al., 2008; Richards et al., 2002). Knowledge about traits and their mechanistic basis at both the physiological and molecular level has the potential to take yield improvement further. It is therefore important to recognise and understand the processes that allow plants to adapt to water and salinity stress, and to allow an increase in biomass or seed yield for food production.
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With the knowledge about physiological traits, new molecular tools can be employed to identify key genes or to provide molecular markers. To do this, appropriate and accurate phenotyping is needed. Experimental methods that could distinguish specific traits for drought and salinity adaptations, and quantify the effect of these traits, will allow QTLs (quantitative trait loci) to be identified, and from this, the possibility of identification of candidate genes or least linked molecular markers. Previous reviews have covered various aspects of the similarities and contrasts in the plant response to drought and salt stress, from the whole plant (Munns, 2002) to the metabolic (Bartels and Sunkar, 2005) and molecular level (Pardo, 2010), and the use of physiological traits in breeding (Munns and Richards, 2007). This review focuses on measurable traits for drought and salinity tolerance, and methods that can be used to select new germ plasm for improving the growth and yield of crops in dry and saline soil.
II. WHOLE PLANT RESPONSES TO WATER AND SALT STRESS: COMMONALITIES AND DIFFERENCES Soil salinity like drought reduces the soil water potential and the ability of plants to take up water, and this quickly reduces the rate of cell expansion in growing tissues. The slower formation of photosynthetic leaf area in turns reduces the flow of assimilates to the meristematic and growing tissues of the plant, both leaves and roots, although leaves are often more affected than roots (Munns and Sharp, 1993). At the same time, both water and salt stress reduce stomatal conductance in the older leaves, which limits their photosynthetic rate. It is difficult to gauge whether this causes an additional reduction in growth rate, or the reduction in growth rate is in balance with the reduction in photosynthesis rate, as carbohydrate status of the growing tissue is usually not affected and sometimes is higher indicating unused assimilate. With time, salt may exert an additional effect on growth. If excessive amounts of Naþ or Cl enter the plant it may rise to toxic levels in the older transpiring leaves. This injury, added to an already reduced leaf area, will then further limit the flow of carbon compounds to meristems and growing zones in leaves. The plant may die before seed is produced. The rate of leaf death is crucial for the survival of the plant. If new leaves are continually produced at a rate greater than that at which old leaves die, then there are enough photosynthesising leaves for the plant to produce flowers and seeds, although in reduced numbers. However, if old leaves die faster than new ones develop, then the plant may not survive to produce seed. For an annual plant, there is a race against time to initiate flowers and form
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seeds, while the leaf area is still adequate to supply the necessary photosynthate. For perennial species, there is an opportunity to enter a state akin to dormancy, and thus survive the stress. The effects of the different components of salinity stress on plant growth can be visually distinguished. The water stress affects the growth rate of the younger leaves, the toxic effect causes premature death of older leaves and can be seen by premature yellowing. The two effects can be quantitatively measured over time. In a laboratory experiment, when salt is introduced to a constant level, a two-phase effect on plant growth is evident (Fig. 1). The first phase of the growth response results from the effect of salt outside the plant. The salt in the soil solution reduces leaf growth and to a lesser extent root growth (Munns, 1993). The cellular and metabolic processes involved are in common to drought-affected plants. Neither Naþ nor Cl builds up in the growing tissues at concentrations that inhibit growth: meristematic tissues are fed largely in the phloem from which salt is effectively excluded, and rapidly elongating cells can accommodate the salt that arrives in the xylem within their expanding vacuoles. The second phase of the growth response results from the toxic effect of salt inside the plant. The salt taken up by the plant concentrates in the old
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Fig 1. Growth of two accessions of the diploid wheat progenitor Triticum tauschii in control solution (closed symbols) and in 150 mM NaCl with supplemental Ca2þ (open symbols). Circles denote the tolerant accession, triangles the sensitive one. The arrow marks the time at which symptoms of salt injury could be seen on the sensitive accession; at that time the proportion of dead leaves was 10% for the sensitive and 1% for the tolerant accession. Adapted from Munns et al., 1995.
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leaves: continued transport into transpiring leaves over a long period of time eventually results in very high Naþ and Cl concentrations, and they die. The cause of the injury is probably the salt load exceeding the ability of the cells to compartmentalise salts in the vacuole. This distinction was illustrated by contrasting genotypes in Fig. 1. Another way of showing it, in the absence of contrasting genotypes, is to compare different osmotica with and without NaCl. Non-ionic compounds such as polyethylene glycol (PEG) are unsuitable for longer term experiments, as it is taken up over time (reviewed in Munns et al., 2010). The least toxic osmotica is a balance salt concentration, with the macronutrients in Hoagland’s solution (Rengasamy, 2010b; Termaat and Munns, 1986). Figure 2 shows the difference in wheat growth between NaCl versus Hoagland solution at 7 and 30 dS/m.
III. MECHANISMS OF DROUGHT AND SALINITY TOLERANCE A. COMMONALITIES
Mechanisms controlling leaf and root growth are in common to drought and salinity; they are due to factors associated with water stress. This is supported by the evidence that Naþ and Cl are below toxic concentrations in the growing cells themselves, in leaves (Fricke, 2004; Hu and Schmidhalter, 1998) and roots (Jeschke, 1984, Jeschke et al., 1986).
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The understanding of plant responses to water deficits from the physiological to the molecular level has been summarised by Chaves et al. (2003, 2009). Many of the traits that explain plant adaptation to drought—such as phenology, root size and depth, hydraulic conductivity and the storage of reserves—are associated with plant development and structure and are constitutive rather than stress induced (Chaves et al., 2003). But a large part of plant resistance to drought is the ability to dissipate excess radiation, a concomitant stress under natural conditions. The effects of drought and salt stresses on photosynthesis are either direct (as the diffusion limitations through the stomata and the mesophyll and the alterations in photosynthetic metabolism) or secondary, such as the oxidative stress arising from the superimposition of multiple stresses (Chaves et al., 2009). The carbon balance of a plant during a period of salt/water stress and recovery may depend as much on the velocity and degree of photosynthetic recovery, as it depends on the degree and velocity of photosynthesis decline during water depletion. B. LONG-DISTANCE SIGNALS CONTROLLING LEAF EXPANSION AND STOMATAL CONDUCTANCE
Whether water status, hormonal regulation or supply of photosynthate exerts dominant control over growth of plants in dry or saline soil is an issue that has been hotly debated. Over the time scale of days, there is much evidence to suggest that hormonal signals rather than water relations are controlling growth in saline soils. The evidence for this is that leaf expansion in saline soil at the time scale of days does not respond to an increase in leaf water status (summarised in Munns, 2002). These experiments indicated that there are chemical signals coming from roots in dry or saline soil that reduce leaf growth. These are commonly referred to as ‘root signals’. Abscisic acid (ABA) is the obvious candidate for this signal, as it is found in xylem sap, and increases after drought and salinity stress (reviewed by Munns and Cramer, 1996). However, there is still no conclusive proof that ABA is the only signal from the roots; other hormones are involved (reviewed by Dodd, 2005; Pe´rezAlfocea et al., 2010; Ghanem et al., 2011). Moreover, the origin of the ABA in the xylem sap is not known, for it moves readily in the phloem and recirculates from leaves to roots (reviewed by Munns and Cramer, 1996). When roots sense a soil water deficit, root cells change in growth rate and differentiation, and the root system architecture changes in the degree of branching or rate of branch root elongation. They also transmit a signal to the shoot. Although root elongation rate decreases, the decrease is less than of shoot growth. In Arabidopsis, the proportion of roots as a fraction of whole plant biomass can increase significantly in drying soil, by at least 30%
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(Hummel et al., 2010). In grapevines in the field, roots in drying soil continue to grow into deeper wetter layers, whereas the roots of irrigated plants proliferate in the topsoil (Lovisolo et al., 2010). Analysis of Arabidopsis of the impact of water deficit on the activity on carbon assimilation and of 30 enzymes central to carbon metabolism showed that drought stress decreased rosette expansion more than photosynthesis, while root growth was promoted. All C metabolites showed a diurnal turnover that increased under water deficit, but with no fundamental reprogramming (Hummel et al., 2010), indicating quantitative shifts in C metabolism but no induction or repression of novel genes. Grapevine genotypes differ in their responses to water stress, exhibiting either isohydric or anisohydric behaviour, which is linked to stomatal and non-stomatal effects. Stomatal regulation of grapevine is under ABA and hydraulic control, the latter linked to embolism formation and recovery. Xylem embolism occurs and repairs during diurnal cycles under ABA control. Aquaporins are essential mediators of radial transport of water both across the roots into the xylem, and out from the xylem in leaves. They may explain the differences in iso- and anisohydric behaviours. Stomatal sensitivity has genotypic variation in grapevine (Beis and Patakas, 2010), and can be altered by irrigation practices (Collins et al. 2010). When roots sense a saline soil, the responses are the same, except that embolisms do not occur. ABA is not the only signal, at least in species other than grapevine, as cytokinins, auxins and the ethylene precursor ACC are implicated (Pe´rez-Alfocea et al., 2010). Hormonal signals may coordinate assimilate production and use in the competing sinks of roots, leaves and lateral shoots. The hormonal regulation of source–sink relations during the osmotic phase of salinity, the phase when growth rate and development is reduced and before ions build up to toxic levels in leaves, affects whole plant energy balance, and is critical to delay the accumulation of ions to toxic levels (Pe´rez-Alfocea et al., 2010). Productivity in saline as well as dry soil depends on maintaining a high growth rate of young leaves, while at the same time delaying the senescence of older leaves. C. CHANGES IN LEAF MORPHOLOGY AND ROOT SYSTEM ARCHITECTURE
Hormonal control of cell division and differentiation is clear from the appearance of leaves, which are smaller in area but often thicker, indicating that cell size and shape have changed (James et al., 2002). Leaves from salttreated plants have a higher weight:area ratio, which means that their transpiration efficiency is higher (more carbon fixed per water lost), a feature that is common in plants adapted to dry and saline soil.
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Recently, the concept of phenotypic plasticity has re-emerged, partly because of a clearer understanding of the influence of epigenetic regulations on gene expression. Phenotypic plasticity illustrates the range of phenotypes that an organism can express as a function of its environment (Schlichting, 1986), that is the resultant component of the G E (genotype by environment) interaction. Plasticity is more readily seen in morphological traits such as variation in leaf size or stem length in response to environmental influences such as shade or herbivory, but can also be measured in complex physiological traits such as water use efficiency (Nicotra and Davidson, 2010). The genetics and molecular biology underlying such adaptive phenotypic plasticity are exciting areas for future research. Hormonal control of cell division and elongation is also evident in roots. Several studies have shown that salinity has differential effects on root elongation rates and lateral root initiation that is due to the osmotic effect of the saline soil (Munns and Cramer, 1996; Rahnama et al., 2011). D. TURGOR MAINTENANCE AND OSMOTIC ADJUSTMENT
Turgor maintenance is essential for cell viability as well as for elongating cells and stomata. The commonality of traits in halophytes and glycophytes is emphasised by Flowers et al. (2010), particularly the ability for osmotic adjustment. Osmotic adjustment is of course essential for adapting plants to soils of low water potential, but may bring penalties in terms of carbon allocation for the rapidly growing phase of rapidly growing plants such as wheat and barley (Munns, 1988). Compatible solute synthesis comes with an energy cost and hence involves a potential growth penalty (Munns, 2005; Munns and Tester, 2008). For example, in leaf cells, approximately 7 moles of ATP are needed to accumulate 1 moles of NaCl as an osmoticum, whereas the amount of ATP required to synthesise 1 mole of an organic compatible solute is an order of magnitude higher (Raven, 1985). The synthesis of these compounds occurs at the expense of plant growth, but may allow the plant to survive and recover from the period without water. E. DIFFERENCES—SALT SPECIFIC
The critical issue for tolerance of a saline soil is to keep Naþ and Cl concentrations in the cytoplasm below toxic levels. Naþ must be partitioned within cells so that concentrations in the cytoplasm are kept low, possibly as low as 10–30 mM (Munns and Tester, 2008). However, the concentration at which Naþ becomes toxic is not well defined. In vitro studies showed Naþ
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starts to inhibit most enzymes at concentrations approaching 100 mM, although some enzymes are sensitive to lower concentrations (reviewed in Munns and Tester, 2008). The concentration at which Cl becomes toxic in the cytoplasm is even less well defined, but is probably similar to that for Naþ (Munns and Tester, 2008; Teakle and Tyerman, 2010). Mechanisms for keeping cytoplasmic concentrations of Naþ and Cl below toxic levels are of two main types: those minimising the entry of salt into the root and its transport through the plant, and those minimising the concentration of salt in the cytoplasm by sequestration in vacuoles. Roots must exclude most of the Naþ and Cl dissolved in the soil solution or the salt will gradually build up with time in the shoot to toxic levels. Plants transpire about 50 times more water than they retain in their leaves (Munns, 2005). If a plant lets in only 1/50 of the salt in the soil solution, that is, it excludes 98%, the concentration of salt in the shoot as a whole would never increase over that in the soil and the plant could survive indefinitely in saline soil (Munns, 2005). Most plants in fact do exclude about 98% of the salt in the soil solution, allowing only 2% to be transported in the xylem to the shoots. A comparison between cereal genotypes with contrasting rates of Naþ uptake when grown in 50 mM NaCl showed that bread wheat excludes more than 98% of the Naþ in the soil solution, and the concentrations do not build up in leaves to more than 50 mM (summarised in Munns, 2005). The concentrations of Naþ in the xylem were 1–2 mM. Barley, however, had higher concentrations in the xylem, about 3 mM, which means it excludes less than 98% of the Naþ in the soil solution, and concentrations in leaves reach very high levels, up to 500 mM (e.g. Rawson et al., 1988). Cl exclusion also differs between species, being 1–2 mM for bread wheat and about 5 mM for barley (Munns, 2005). This high degree of exclusion of Naþ and Cl from the leaves is achieved by (i) tightly controlled uptake from the soil, the epidermis of the roots forming a virtual ‘barrier’ to entry of salt into the roots (La¨uchli et al., 2008) and (ii) regulated movement in the xylem, by controlled loading of Cl into the xylem (Tregeagle et al., 2010) or by retrieval of Naþ as it moves in the transpiration stream to the leaves (James et al., 2006). Export from leaves in the phloem could conceivably help to maintain low salt concentrations. However, there appears to be relatively little retranslocation of salt from leaves, in relation to the import in the transpiration stream (Munns, 2005). For example, estimates of xylem and phloem fluxes indicated that in barley, phloem export from a leaf was only about 10% of the import in the xylem (Munns et al., 1986). Exclusion of salt from the phloem ensures that salt is not redirected to growing tissues of the shoot. Although salt that is
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loaded into the phloem in lower leaves may be translocated down to the roots, the phloem from younger leaves may go up to the meristematic and elongating tissues in the shoot. As shown by C14 urea feeding studies, lower leaves feed carbon to the root, upper leaves feed the shoot apex and midposition leaves feed both shoot apex and root (Layzell et al., 1981). Low rates of ion accumulation in leaves could also be influenced by the shoot:root ratio, or the relative growth rates, either of which would affect the rate at which the ion accumulates in shoots (Munns et al., 2006). Naþ exclusion from leaves is associated with salt tolerance in cereal crops including rice (Asch et al., 2000; Ul Haq et al., 2010), durum wheat (Munns and James 2003), bread wheat (Cuin et al., 2009, 2010), barley (Shavrukov et al., 2010), pearl millet (Krishnamurthy et al., 2007) and wild relatives such as Hordeum species (Garthwaite et al., 2005), tall wheatgrass (Colmer et al., 2006) and Triticum tauschii (Schachtman et al., 1991). Most studies focus on Naþ rather than Cl, as genotype comparisons within most species link Naþ accumulation in leaves negatively with salt tolerance. This is particularly so for species that accumulate high concentrations of Cl and not Naþ in leaves, such as soybean, woody perennials such as avocado, and species that are routinely grown on Cl-excluding rootstocks such as grapevines and citrus. For these species, Cl toxicity is more likely to occur than Naþ toxicity. However, this statement does not imply that Cl is more metabolically toxic than Naþ, rather these species are better at excluding Naþ from the leaf blades than Cl. For example, Naþ does not increase in the leaf blade of grapevines until after several years of exposure to saline soil, then the exclusion within the root, stem and petiole breaks down, and Naþ starts to accumulate in the leaf blade, whereas leaf blade Cl concentrations increase progressively (Prior et al., 2007). Cl exclusion is critical for production of certain species on saline soil, and Cl excluding rootstocks are used in the citrus and grapevine industry (Storey and Walker, 1999; Teakle and Tyerman, 2010; Tregeagle et al., 2010). The mechanisms of Naþ transport at the molecular, electrophysiological and whole plant level have been summarised by Tester and Davenport (2003) and Munns and Tester (2008). In contrast to Naþ, mechanisms of Cl transport in plants are poorly understood (Teakle and Tyerman, 2010).
IV. ADAPTATIONS, TRAITS AND QTLS With the knowledge of adaptations and the mechanisms behind them, quantitative traits can be identified or hypothesised. These traits can be physiologically complex but easily measured, like leaf shape or time of flowering,
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which can be measured with a ruler or a visual record. They can be complex but need expensive equipment like water use efficiency which is measured as carbon isotope discrimination by a mass spectrometer. Or they could be physiologically simple but difficult to measure like leaf rolling. If a trait can be easily and inexpensively measured as a quantitative measurement (not like presence or absence of leaf rolling), then a large number of genotypes can be screened. If there is genetic variation, then a QTL analysis can be performed, by crossing the contrasting parents, screening the F2 generation for the trait (the phenotype) and mapping with molecular markers. The QTL should be validated in a different population. If the QTL is robust and repeatable in different genetic backgrounds, fine mapping can be attempted with the aid of a model species, and a candidate gene may be revealed. If not, the original linked marker can be used for breeding purposes. A. DROUGHT
When considering traits that are useful in plant breeding in drought-prone environments, the term ‘drought tolerance’ is not appropriate. It is not useful that a plant tolerates a drought, that is, it does not die; it is only useful that it continues to develop and produce grain. Desiccation tolerance is only applicable to plants that can undergo and survive a prolonged dry season, many months or many years. ‘Drought resistance’ is a nebulous term. There are no units for ‘drought resistance’. Likewise, there are no units for ‘drought tolerance’. It is more effective to ask, given a fixed and limiting water supply, what is the best growth rate or maximum biomass that can be produced. This focuses on the efficiency of water use, that is, water use when the plants have water, not when they do not (Passioura and Angus, 2010). Traits for grain production under drought have been comprehensively reviewed and discussed by Richards et al. (2002, 2010), Reynolds et al. (2007) and Reynolds and Tuberosa (2008). Richards et al. (2010) reviews the key ‘best-bet’ traits for water-limited environments. These are summarised in Table I. Measurable adaptive characters can be considered in four classes: whole plant growth and development, root architecture, leaf architecture and longevity and biochemical properties. Some of these traits have QTLs that are reliable and useful for breeding, and some have candidate genes; however, most have only a phenotype (see Table I). Traits involving whole plant growth and shoot development are thoroughly reviewed by Richards et al. (2010). These are:
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TABLE I Summary of the Most Important Traits and the Selection Method for Improving Yield of Temperate Cereals in Water-Limited Environments
Trait Seedling establishment Shoot vigour Root vigour Root depth Transpiration efficiency (CID) Stem carbohydrate remobilisation (WSC) Tillering Glaucousness Leaf rolling Floret sterility
Environment in which trait selection is most effective
Markers or genomic regions identified
Favourable
Yes
Favourable Favourable Favourable soil moisture at depth Favourable
Yes Yes No
Phenotype and marker Phenotype Phenotype Phenotype
Yes
Phenotype
Favourable
Yes
Phenotype
Favourable
Yes
Favourable Favourable Non-droughted
Yes No No
Phenotype or marker Phenotype Phenotype Phenotype
Most efficient selection method
Reproduced with permission from Richards et al. (2010); doi 10.1071/FP09219.
. . . . .
seedling establishment shoot vigour tiller regulation phenology and early flowering floret sterility
Traits involving root vigour and architecture have attracted less interest, largely because they are so difficult to measure. Root system vigour describes the variation in rate of root growth that results in the capture of greater volumes of soil water and nutrients (Palta and Watt, 2009). Deeper roots can access more water, maintain high stomatal conductance and hence photosynthesis, and are indicated by cooler canopies (Lopes and Reynolds, 2010). Root systems research has been hampered by the difficulty and time-consuming methods of measurement, and so molecular markers would provide a new impetus for their selection and use in breeding. The fast-growing grass Brachypodium distachyon has a small and fully sequenced genome and as a model for wheat and other cereal crops should provide a breakthrough in the use of genomics and other genetic technologies (Watt et al., 2009)
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Traits involving leaf architecture have been reviewed by Richards et al. (2010). The main traits are: . . . .
maintenance of green leaf area (also called stay green) osmotic adjustment leaf rolling glaucousness (leaf wax properties to reflect light)
Biochemically based properties, which are complex but for which there are markers (Richards et al., 2010) or candidate genes, are: . . .
transpiration efficiency stem carbohydrate storage and remoblisation osmotic adjustment and compatible solutes
B. SALINITY
Traits that increase productivity on dry land would also increase productivity in saline land. Salinity as well as drought is a common feature of arid and semi-arid zones, so early vigour, rapid phenology, avoidance of floret sterility and transpiration efficiency would be as important in saline soils as well as in dry soils. In saline as in dry land, the term ‘salt-tolerance’ is not a useful concept for crop growth and yield. A more useful concept is the efficiency of use of the available water. The term may be applicable only for perennial species. In most evergreen shrubs, survival and not growth performance is the primary outcome of ‘salt-tolerance’ mechanisms (Tattini et al., 2009).
C. SALT-SPECIFIC TRAITS
Traits for saline soils were summarised by Colmer et al. (2005) and listed in Table II. The first traits listed are salt specific, the last four in common with drought. For Naþ exclusion, and maintenance of high Kþ/Naþ ratios in leaves, HKT genes are considered important in the regulation of Naþ and Kþ in higher plants, and in mediating salinity tolerance in plants (Horie et al., 2009; Munns and Tester, 2008; Pardo, 2010). HKT genes are important for cellular Naþ and Kþ homeostasis and some family members are responsible for
TABLE II Key Traits for Salt Tolerance in Wheat and Barley. Recommendations on Approaches, Plant Stages and other Considerations for Screening
Trait þ
Measurement þ
Amenable to large scale evaluation?
Plant stage
Comment
Yes
1
Na ‘exclusion’
Leaf blade Na concentration
At defined leaf age (days after emergence) and stage of plant development (leaf number)
2
Kþ/Naþ discrimination
Leaf blade Naþ and Kþ
As above
3
Sheath retention of ions
As above
4
Tissue tolerance
Seedling stage
Laborious to phenotype
No
5
Ion partitioning into different-aged leaves
Pre- and post-stem elongation
See also (1) above
Yes
6
Osmotic adjustment
Naþ and/or Cl concentration in sheath and blade Leaf injury, coupled with Naþ and/or Cl accumulation Leaf blade Naþ concentration of different leaves Turgor or concentration of specific solutes
The most useful trait; requires only small tissue samples (i.e. non-destructive); Cl ‘exclusion’ is much less studied, and for wheat showed little correlation with genotypic differences in salt tolerance Leaf Naþ alone is sufficient Not widely studied
Seedling stage
Assessing tissue water relations is laborious; screening organic solutes requires a highthroughput analytical system
Turgor—no Solutes— yes
Yes
Yes
(continues)
Table II
Trait
Measurement
(continued )
Plant stage
7
Enhanced vigour
Area of first leaves
Seedling stage
8
Water use efficiency
12
Late vegetative stage
9
Early flowering
Flowering date
Flowering
C:13C discrimination (low or 13C)
Reproduced with permission Colmer et al. (2005); doi 10.1071/EA04162.
Amenable to large scale evaluation?
Comment Enhances water uptake early in season, and reduces evaporation from soil Reduces water uptake later in the season; might be useful for transient salinity only May reduce yield potential
Yes
Yes
Yes
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Naþ exclusion from leaves. The transporters encoded by these HKT genes can remove Naþ from the xylem and result in lower Naþ in leaves and a higher K/Naþ ratio which is important to maintain ion homeostasis of the cytoplasm (Munns and Tester, 2008). The Arabidopsis gene AtHKT1;1 is a Naþ transporter that limits Naþ transport from root to shoots by removing Naþ from the xylem (Davenport et al., 2007). Overexpression of this gene with a stele-specific promoter reduced the rate of transport of Naþ to the leaves and improved the salt tolerance of Arabidopsis (Moller et al., 2009). Although dicots have only one family member, monocots have several, and there are nine in rice, wheat and barley (Huang et al., 2008). HKT members of sub-family 1 have a much higher selectivity for Naþ over Kþ than do members of sub-family 2 (Pardo, 2010; Hauser and Horie, 2010). Major genes with QTLs have been found for five Naþ excluding genes— Nax1 and Nax2 in durum wheat, Nax3 and Nax4 in barley and Kna1 in bread wheat as described below. In durum wheat, Nax1 and Nax2 enhanced removal of Naþ from the xylem, leading to low Naþ concentrations in leaves (James et al., 2006). Nax1 removes Naþ from the xylem in roots and the lower parts of leaves, the leaf sheaths, while Nax2 removes Naþ from the xylem only in the roots (James et al., 2006). Nax1 has a unique phenotype of a high sheath: blade ratio of Naþ concentration. Nax2 has the same phenotype as Kna1, the QTL for Naþ exclusion and enhanced K/Naþ selectivity in bread wheat, Triticum aestivum (Dvorˇa´k et al., 2004). Nax2 was shown to be homoeologous to Kna1 in T. aestivum, namely TaHKT8 (TaHKT1;5) (Byrt et al., 2007). Modern dwarf rice is less tolerant of salinity than wheat, but the original landraces Pokkali and NonaBokra are relatively tolerant. This is due largely to the presence of an orthologue of TmHKT1;5-A, namely OsHKT1;5 (Ren et al., 2005). However, no QTL for Naþ exclusion has been found on chromosome 4, the syntenic region to wheat chromosome 2A containing the Nax1 gene. Yet the gene sequence for HKT1;4 is present on the rice genome (Huang et al., 2006). It is possible that this gene is silenced in rice, or for whatever reason its expression is prevented, leading to the interesting idea that transformation of rice with TmHKT1;4 might increase its salt tolerance. This might be particularly beneficial when the soil is flooded and insufficient oxygen reaches the roots, despite the aerenchyma, to maintain ion transport activity in roots. The family of HKT genes are not the only genes controlling Naþ transport to the shoot. Studies with Na22 showed that durum wheat lacking the Nax
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genes was able to withdraw about 50% of the Na22 flowing to the shoot (James et al., 2006). The presence of either Nax1 or Nax2 increased this withdrawal from the xylem to about 90%. QTLs for Naþ exclusion in leaves of barley revealed two other major genes, named Nax3 (Shavrukov et al., 2010) and Nax4 (Rivandi et al., 2011). The latter locus contains a SOS3 homologue. Efflux of Naþ across the plasma membrane to the apoplast and ultimately to the soil solution is by Naþ/Hþ antiporters (Blumwald et al., 2000; Pardo, 2010; Pardo et al., 2006). SOS1 exchanger is regulated through the SOS2–SOS3 kinase complex in Arabidopsis, and a similar pathway is important in salt tolerance of this (Shi et al., 2003) and other species (reviewed by Pardo, 2010). NHX1, the vacuolar Naþ/Hþ antiporter controls Naþ sequestration in cells (Blumwald et al., 2000), leading to the concept of ‘tissue tolerance’—the ability of some species to tolerate high concentrations of Naþ in their leaves, presumably because it is largely sequestered in the vacuole (Flowers and Yeo, 1986). Undoubtedly, there are genotypic differences, but it is difficult to measure. We found that there was a relation between the Naþ concentration at which the leaf died, or lost 50% of its chlorophyll content, but that this quantitative relationship differed for leaves of different ages (R. Munns and R. James, unpublished data). This means that just linking the increase in Naþ concentration with a decrease in chlorophyll is inconclusive. In the list in Table II, Cl exclusion as a trait is not mentioned, because the chapter was focused only on wheat and barley, for which species there is no evidence of Cl toxicity, and no correlation between genotypic differences in Cl uptake and salinity tolerance. Genes important in the regulation of Cl transport in saline soil are reviewed by Teakle and Tyerman (2010). No QTLs for these genes have been identified. A decrease in leaf chlorophyll content is an indicator of tolerance, and an easily measurable phenotype, but there is a problem of assigning cause. A decrease in chlorophyll could be due to lower tolerance of tissue Naþ or Cl concentrations, or premature senescence caused by signals from the roots. Maintenance of a high Kþ concentration in leaves and roots, and a ratio of þ K /Naþ greater than 1, is also crucial for salt tolerance (Hauser and Horie, 2010; Shabala and Cuin, 2008. The ability of a plant to retain Kþ in the roots exposed to high NaCl concentrations is a critical feature of tolerance in many species (reviewed by Shabala and Cuin, 2008), and genetic variation in NaClinduced Kþ efflux from roots correlates with genetic variation in grain yield of barley (reviewed by Shabala and Cuin, 2008) but not of wheat (Cuin et al., 2009). In wheat, measurements of shoot sap Kþ concentration in non-salinised plants, along with the changes of chlorophyll concentration in a given leaf after 6 weeks of NaCl treatment correlated with % grain yield in saline soil and were proposed as an efficient screening tool (Cuin et al., 2010).
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D. TRAITS IN COMMON WITH DROUGHT
The last four traits listed in Table II are in common with drought. Osmotic adjustment occurs readily because of the high uptake of Naþ and Cl , and in species like wheat and barley is regulated so that it does not become toxic. Turgor is perfectly maintained (Boyer et al., 2008). Compatible solutes are needed to balance the Naþ and Cl in the vacuole as their concentration there may be only 30 mM, as mentioned earlier in this chapter (Section III.E). If Naþ and Cl are sequestered in the vacuole of a cell, organic solutes that are compatible with metabolic activity even at high concentrations (hence ‘compatible solutes’) must accumulate in the cytosol and organelles to balance the osmotic pressure of the ions in the vacuole (Flowers et al., 1977). The compounds that accumulate most commonly are sucrose, proline, and glycine betaine, although other molecules can accumulate to high concentrations in certain species. In many halophytes, proline or glycine betaine occurs at sufficiently high concentrations in leaves (over 40 mM on a tissue water basis) to contribute to the osmotic pressure (over 0.1 MPa) in the cell as a whole (Flowers et al., 1977). In glycophytes, the concentrations of compatible solutes that accumulate are not so high, on the order of 10 mM, but if partitioned exclusively to the cytoplasm, they could generate a significant osmotic pressure and function as an osmolyte. At low concentrations, these solutes presumably have another role, perhaps in stabilising the tertiary structure of proteins, and function as osmoprotectants (Rhodes et al., 2002). Most of the other traits in Table II are covered in the above section on ‘Drought’ (Section IV.A). Not listed in that table are root properties and floral sterility. 1. Root properties Durum wheat (Triticum turgidum L. ssp. durum Desf.) is relatively salt sensitive compared to bread wheat (T. aestivum L.), and yields poorly on saline soil (Munns and James, 2003). Field studies indicate that roots of durum wheat do not proliferate as extensively as bread wheat in saline soil. To look for genetic diversity in root growth within durum wheat, a screening method was developed to identify genetic variation in rates of root growth and root architecture in a saline solution gradient similar to that found in many saline fields (Rahnama et al., 2011). Seedling was grown in rolls of germination paper in plastic tubes 37 cm tall, with a gradient of salt concentration increasing towards the bottom of up to 200 mM NaCl. A NaCl concentration of 150 mM at the bottom was found suitable to identify
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genetic variation in seminal axile root length as well as branch root length. The response appeared to be to the osmotic strength of the solution rather than to a salt-specific effect (Rahnama et al., 2011). 2. Sterility Ghanem et al. (2009) showed that the failure of the tomato inflorescence to develop normally under salt stress was best explained in terms of altered source–sink relationships than accumulation of ions to toxic concentrations. Using laser ablation inductively coupled plasma mass spectrometry microanalysis to measure Naþ concentrations is reproductive tissues including the tapetum, they concluded that the decrease in soluble carbohydrate was the most likely cause of the sterility. E. QTL ANALYSIS AND GENE DISCOVERY
The significance of knowing the essential traits is to use this information for gene discovery, and one way is via a QTL. For this, an unambiguous and quantitative phenotype, and an experimental design that is realistic and reproducible are needed. Genetic technologies for identifying genes important in environmental stress responses have been summarised by Papdi et al. (2009). These are as follows: traditional mutagenesis (using forward or reverse screens), insertion mutagenesis (forward or reverse screens), QTL mapping and cDNA transformation (forward screen) (summarised in Figure 7 of Papdi et al., 2009). All require a physiologically based genetic analysis at some stage (Papdi et al., 2009). QTL mapping requires a quantitative physiologically based screen, although just biomass production can be successful. All studies require a sound experimental design that can quantify growth responses under water deficit or salinity stress, not easy in the laboratory or when using genetic material that has different intrinsic growth rates or phenology.
V. GROWTH STUDIES AND EXPERIMENTAL DESIGN A. WATER STRESS
The search for the best medium for growing plants to impose a controlled water deficit has been going on for decades, without a clear resolution. There is no perfect medium—all have limitations including pots containing soil (Munns et al., 2010).
PLANT ADAPTATIONS TO SALT AND WATER STRESS
21
Soil drying is hard to control especially when comparing genotypes of different vigour or rates of development. Even a drying soil, as well as being very difficult to maintain at a uniform and constant water potential through the whole soil profile, may exert specific effects; for example, transmission of nutrients through the soil will be reduced at low soil water potentials. Another problem with soil is drainage, and easily become saturated at the bottom (Passioura, 2006). Soils should drain quickly; otherwise, the ‘controls’ are waterlogged. The addition of perlite or vermiculite, or the use of ‘inorganic soils’ such as fritted or calcined clay can overcome the problems of soil with a high clay content, which does not drain quickly or a predominantly sandy soil which holds little water and releases it quickly as the soil dries. Hydroponics avoids problems of drainage. A variety of non-ionic osmotica have been used to mimic a decrease in soil water potential, such as mannitol. However, a percentage of these small molecules enter roots and move in the xylem to the shoots, either through cracks in roots or through membranes that are not completely impermeable to neutral solutes of this size. High-molecular-weight PEG (MW 6000) has been examined in many early studies that attempted to impose a controlled water deficit. Its main problem is its viscosity that decreases O2 movement to roots so that the roots become O2 deficient. The latter can be overcome by bubbling with O2 rather than air; however, the experiments must be limited to a short period of time as the PEG can enter the roots and reduce the hydraulic conductivity (Munns et al., 2010). Concentrated mixed salts, such as the macronutrients used in Hoagland’s solution, are preferable to NaCl as plants are less likely to take up any one ion to toxic concentrations (Munns, 2010a, in PrometheusWiki). When hydroponics are used to simulate a ‘drought’ or soil water deficit, the effect on plant growth may be quite different, and a lot less, than with a drying soil. Hydroponics ensure that there is no nutrient deficit, but in a dry soil the access to nutrients decreases and the plants may suffer N or P deficiency (summarised in Munns et al., 2010). Experiments in drying soil are difficult when comparing genotypes of different sizes such as mutants or transformants whose size or relative growth rate is less than that of the wild type, or when comparing modern versus old cultivars whose size has been reduced by the introduction of dwarfing genes. When genetically modified lines are compared with their wild type, the soil water status must be measured and their performance compared at the same soil water potential; otherwise, the small plants use the soil water more slowly and stay greener longer, and appear to be more ‘drought tolerant’ when photographs are taken. Rewatering should be avoided, as it favours the small plants.
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Three treatment approaches can be taken (Munns, 2010b, in PrometheusWiki). The most common and convenient one is solution culture, which can be supported by solid material such as fine gravel or high-density plastic beads. A recirculating nutrient solution using a modification of the original Hoagland’s solution (Munns and James, 2003) is applied using aeration in pots or subirrigation in tanks. A second method is sand culture, when the sand is irrigated with Hoagland’s nutrient solution. A third method is to use soil as the medium, which is likely to best mimic field conditions, but in small pots the soil needs to flushed periodically with salt-nutrient solution (making is similar to sand culture but with reduced drainage) or rewatered by replacing the water transpired but in this case it creates pockets of low-salinity soil in high-salinity background (Munns, 2010b). Experiments should be conducted over a reasonable time period, and the salt increased in gradual steps to avoid severe osmotic shock. Plant responses in the short term are primarily osmotic, and only in the longer term (days, weeks or months, depending on the species) does the salt rise to toxic concentrations in leaves and the salt-specific effect is seen (Munns, 2002). Experiments that have compared hydroponics and soil at a range of salinities have found quite different qualitative and quantitative differences. Differences between two barley cultivars in growth, moisture content and Naþ accumulation were not apparent in hydroponics but significant differences occurred in soil (Tavakkoli et al., 2010) due to salt moving in the soil as it was watered, probably. Ca2þ deficiency is an artefact generated in hydroponics. Kopittke et al. (2010) provide a comprehensive analysis of the effect of any one cation on the activities of other cations in solution and the effect on plant growth rates. Short-term (2d) growth of cowpea seedling to Cl salts of Naþ, Kþ, Ca2þ and Mg showed that growth was poorly related to activities in the bulk solution but closely related to activities at the outer surface of the plasma membrane. The addition of Mg Naþ or Kþ resulted in Ca2þ deficiency in roots at Ca2þ concentrations less than 1.6 mM.
C. EXPERIMENTS TO DISTINGUISH WATER STRESS FROM SALT-SPECIFIC EFFECTS
To distinguish the effects of water stress, as in a dry soil, from salt-specific effects, can most easily be done in a time course. The osmotic effect of the soil salinity, as does the dehydrating effect of the soil, has an immediate effect on growth rate (Fig. 2). The effect of toxicity on the older leaves takes time to
PLANT ADAPTATIONS TO SALT AND WATER STRESS
23
show up (Munns, 2002; Munns et al., 1995). This concept led to Rajendran et al., 2009 showing that it could be quantified within 5–7 days by nondestructive leaf area measurements, and genotypic differences in Triticum monococcum were distinguished. The ‘salt transcriptome’ was studied over time in a salt-tolerant Poplar species along with physiological changes, in order to identify genes important in the acclimation to stress—rather than just the response to the osmotic shock (Brinker et al., 2010). Over 24 h, three distinct phases of salt stress: dehydration (due to water withdrawal and osmotic shock), salt accumulation (rapid uptake) and osmotic restoration (regaining osmotic or ionic homeostasis). The initial dehydration was more marked in leaves than in roots and resulted in changes in transcript levels of mitochondrial and photosynthetic genes indicating adjustments of energy metabolism. Stress-specific genes responded only when leaves had osmotically adjusted by salt accumulation. Another way of distinguishing osmotic from salt-specific effects is to compare the growth of plants in NaCl with that in other salts, with mixed salts, or in non-ionic media. A variety of non-ionic osmotica have previously been used to mimic a decrease in soil water potential, including mannitol, melibiose and sorbitol. However, all these small molecules eventually enter roots and move in the xylem to the shoots because membranes are permeable to neutral solutes of this size (reviewed in Munns et al., 2010). High-molecular-weight PEG has been examined in many early studies that attempted to impose a controlled water deficit. Its main problem is that its viscosity limits O2 diffusion so that the roots become O2 deficient (Verslues et al., 1998). When PEG solutions were bubbled with O2 (not vigorously with air, which breaks roots), the rate of root growth increased and the partial pressure of O2 at the root tip increased (Verslues et al., 1998). A problem with solution culture is that lateral roots break when the solution is changed and the roots are unsupported, and solution enters through the damaged junction between a lateral root and the seminal axis. This allows a pulse of salt into the plant (own observations) and also provides a mode of entry for PEG or other osmotica (Munns et al., 2010). This problem can be overcome by using solid media such as quartz gravel, which supports the roots when the solution is removed or aerated (e.g. Munns and James, 2003). Mixed salts, for example, high concentrations of the macronutrients in Hoagland’s solution, are a better osmotica than any of the above, as their rate of uptake is tightly regulated by transporters that have evolved to deal with variable concentrations in soils, and they do not support bacterial growth. Concentrated macronutrients were shown by Termaat and Munns (1986), Rengasamy (2010a) and Tavakkoli et al. (2010) to be a good
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surrogate for water deficit caused by osmotic stress. Figure 2 shows that growth of wheat was reduced by 7 dS/m of NaCl, about 0.25 MPa, more than by mixed salts (derived from concentrating the macronutrients of Hoagland’s solution) of the same total concentration. However, at the higher concentration of 30 dS/m or 1.1 MPa, there was less difference, indicating the osmotic effect became the overriding effect on growth at the higher osmotic strength. The figure also illustrates that differences did not show up for 25 days of treatment. In other more salt-sensitive species, which do not have such a tight regulation of Naþ or Cl transport to the leaves, differences will show up earlier. To distinguish Naþ from Cl toxicity is surprisingly difficult. One might consider that comparing growth rates in different salts should be informative. For example, the specific effect of Cl could be tested by comparing NaNO3 or Na2SO4 versus NaCl, and specific effect of Naþ by comparing CaCl2 or KCl versus NaCl. These experiments have been tried for over 50 years and have not produced unambiguous results. The main reason is that the osmotic effect overwhelms the specific salt effect (Kopittke et al., 2010; Rengasamy, 2010a). Furthermore, the effect of any single salt can be toxic, more so than NaCl. Plants have evolved to exclude Naþ and Cl, as they are the most prolific soluble salts in the soil, and when presented with high concentrations of NO3, SO4 or Kþ, they take up large amounts. Ca2þ salts are least toxic (Rengasamy, 2010a) but they do not dissociate as fully in solution as other salts and allowance must be made for their lower osmotic strength for a given concentration.
VI. PHENOTYPING Many of the traits for drought and salt tolerance have a clear phenotype, and many of these have molecular markers, if not perfect markers derived from the candidate genes. However, some do not or the technique is too expensive and slow, for example, transpiration efficiency. In this case, a measurement of plant growth rate is more cost effective. Also, we may have insufficient knowledge of traits and their interactions, and plant breeders still select on yield, so a measurement of growth rate, biomass or yield, empirical though it is, may be the most successful. Selection techniques thus can make use of modern ‘phenomics’ tools. Nondestructive, high-throughput crop evaluation in controlled environments and the field is being developed (Berger et al., 2010; Furbank, 2009). This enables a correlation of gene function with plant performance and environmental response, with high resolution and speed.
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Infrared thermography as a surrogate for measurements of transpiration and photosynthetic response to stress shows great promise in high-throughput plant phenomics and can be used both in controlled environments (Sirault et al., 2009) and in the field (Jones et al., 2009). The power of this approach is that it allows high-throughput screening at the seedling level to be validated at the canopy level in a field environment, using the same genotypes and tools. Spectroscopic techniques that quantify photosynthetic performance and can be used at the leaf and canopy level are described briefly by Furbank (2009) and Munns et al. (2010). Spectral reflectance and absorption measurements can potentially provide plant phenomics with a tool to non-invasively delve into plant function and chemical composition scalable from the tissue to the canopy level. For example, high-throughput chlorophyll fluorescence imaging using a 9 8 grid array can rapidly identify mutants in photorespiration (Badger et al., 2009). Determining root function in soil and screening for optimising root structure and growth has long been a challenging field (reviewed by Gregory et al., 2009). Access to water at depth for cereal species growing on stored soil moisture is of great importance for drought tolerance and screening of model species to elucidate genes responsible for root characters is underway. Recently developed small, short-lifecycle crop models more appropriate to cereal species are excellent systems for phenomic screening (Watt et al., 2009). Deeper roots can access more water, maintain high stomatal conductance and hence photosynthesis and be indicated by cooler canopies (Lopes and Reynolds 2010), a phenotype less laborious than soil coring (Watt et al., 2005). Infrared thermography (Jones et al., 2009), canopy greenness or carbon isotype discrimination have the potential to provide a non-destructive phenotype (Richards et al., 2010). Looking ahead, new non-destructive methods to measure soil moisture should become available such as ground penetrating radar and electrical resistance thermography, and become sufficiently sensitive to detect genotypic differences. Another application of modern phenomics is in gene discovery. A significant proportion of even the Arabidopsis genome is annotated as ‘gene of unknown function’ or annotated using only loose sequence homology-based clues. Reverse genetic approaches to disrupt gene function often result in the unsatisfactory conclusion ‘no visible phenotype’ (Furbank, 2009). The recent developments of chlorophyll fluorescence techniques combined with infrared thermography provide information on photosynthetic and stomatal functions that can reveal new knowledge on the functions of key genes that ultimately control plant growth.
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VII. CONCLUSIONS The future of gene-based approaches to increasing growth and yield under drought and salinity depends on physiological understanding and realistic experiments. The significance of knowing the essential traits is to use this information for gene discovery, via a QTL. For this, an unambiguous and quantitative phenotype, and an experimental design that is realistic and reproducible are needed. QTLs need to be validated in different populations. Other approaches of traditional or insertion mutagenesis (using forward or reverse screens) and cDNA transformation (forward screen) also require good experimental design, and the information is enhanced if a trait is hypothesised. Traits in controlled environment chambers need to be validated in the field. The field brings many other factors—atmospheric particularly vapour pressure deficit and temperature, and the soil will undoubtedly contain other constraints such as poor structure physical such as compaction or chemical such as acidity or sodicity. There are many different categories of saline soil, arising from different mineral compositions and different types of salinisation process, all affecting plant responses to the increased osmotic pressure or specific ion concentrations (Rengasamy, 2010b). Salinity can be transient (Rengasamy, 2006) as can waterlogging, and the salinity–waterlogging interaction can severely inhibit plant growth (Colmer et al., 2005). Drought and salinity limit the production of the world’s staple food crops. Conventional plant breeding based on yield in target environments has increased production; however, physiologically based approaches utilising molecular tools to identify key genes or provide molecular markers have the potential to take it further. Accurate and selective phenotyping will enable the best use of mechanistic and molecular understanding of plant responses to drought and salinity, and mechanisms of adaptation.
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Recent Advances in Understanding the Regulation of Whole-Plant Growth Inhibition by Salinity, Drought and Colloid Stress
PETER M. NEUMANN1
Department of Environmental, Water and Agricultural Engineering, Technion Israel Institute of Technology, Haifa, Israel
I. II. III. IV.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant Growth and Survival During Moderate and Severe Salinity Stress . . . Plant Growth During Moderate Water Stress Episodes . . . . . . . . . . . . . . . . . . . . . Whole-Plant Water Availability and Growth Can also be Limited by Colloid Stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT This chapter presents an eclectic perspective, based largely on research findings from our laboratory on biochemical and biophysical mechanisms involved in growth inhibition by water deficits, as caused by salinity (Section II), drought (Section III) and recently reported plant interactions with aqueous colloids found in soil solutions or xylem sap (Section IV). Much attention in each section is given to the roles of plant cell walls in regulating whole-plant growth inhibition under stressful conditions. One conclusion is that ongoing research into genomic and epigenetic changes that participate in the regulation of cell wall changes, cellular antioxidant status and plant hydraulics may provide new approaches for limiting the plant growth inhibition or mortality associated with salinity and drought. In Section IV, it is concluded that the ‘colloid stress’ that results from the inhibitory effects on water transport of physical
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Corresponding author: E-mail:
[email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00002-3
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interactions between plant cell walls and environmental or internal colloids is a novel stress-factor that can affect plant water relations and further limit plant ability to resist salinity and drought.
I. INTRODUCTION Abiotic stress can be defined as an adverse situation resulting from plant exposure to sub- or supra-optimal levels of environmental inputs such as water, light, temperature or solutes. Abiotic stress resulting from water deficits or excessive salinity generally leads to reductions in photosynthesis, transpiration and growth. Such stresses are a major, world-wide-factor limiting the ability of mankind to produce adequate supplies of plant food, fibre and fuel for an expanding world population. Ongoing advances in botanical research into plant responses to abiotic stresses, in addition to increasing basic knowledge, may facilitate advances in our practical ability to maintain and increase plant productivity in stressful agricultural or natural environments. Botany, or that branch of biology that concerns the scientific study of plant life, has evolved rapidly since Watson and Crick (1953) published their seminal paper on DNA structure. The genomic era they signalled has been characterised by the introduction of increasingly sophisticated analytical technologies that now facilitate ‘deep’ genomic, transcriptomic, proteomic and metabolomic investigations. Most recently, there has been increasing interest in hereditable epigenetic changes involving DNA methylation, histone-modification and microRNA. These developments in cell molecular biology are leading to a post-genomic era where systems biology seeks to integrate understanding of the processes regulating metabolism into holistic models of development, albeit mainly at the cellular level. Parallel advances in research into transport and communication processes that are associated with development at the level of tissues, organs and whole organisms, are also needed for fuller understanding and more successful modifications of whole-plant responses to environmental change. An important area in which further study is certainly needed is the extracellular (apoplastic) region of plants, that is, plant cell walls, xylem and environmental boundary layers at root and leaf surfaces. Each of these can make direct ‘post-genomic’ contributions to the regulation of whole-plant growth and development under optimal or stressful conditions. An example of apoplastic changes that may regulate plant development is given by what happens as tension, that is, negative hydrostatic pressure, increases in the xylem water columns of plants facing water deficits. When critical tensions are reached, the formation of gaseous embolisms may disrupt xylem hydraulic continuity and thereby restrict essential xylem-transport of soil derived
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water and solutes to the shoot. Such physical reductions in water transport can then adversely affect leaf functioning and hence, plant growth and development (McDowell et al., 2008; Sperry et al., 2002). Much of the emphasis in this chapter is on apoplastic changes that are involved in the regulation of cell, organ and whole-plant growth responses to salinity and water stress. Such stresses can be moderate, that is, non-lethal, or extreme, that is, lethal. A central theme is that the molecular changes induced in plant cells by moderate environmental stresses will usually lead to rapid decrease in rates of growth of tissues and organs, be they roots, stems, leaves, flowers, fruits or seeds. Stress-induced changes in plant growth rates, tissue mechanics and phenology can therefore provide integrated reflections of underlying molecular changes (Hauben et al., 2009; Neumann, 1997; Nicotra and Davidson, 2010; Uyttewaal et al., 2010). The practical consequences of stress-induced decreases in the growth of plants can be viewed in different ways. For example, limiting new leaf area production under water stress not only will limit plant potential for photosynthesis but will also limit overall leaf transpiration. It may therefore extend the availability of limited soil water reserves and hence plant ability to survive long enough to produce (a limited amount of) seeds. Albeit, in an agricultural context and in the viewpoint of this chapter, stress-induced reductions in growth rates of crop plants will generally be associated with unwanted reductions in economic yields. Thus, stress acclimation that involves growth inhibition and associated increases in plant survival may still be viewed as a hindrance by farmers. In order to review the plant growth reductions induced by environmental stresses, it is necessary to first consider what exactly is meant by the term growth. Growth can be defined as an irreversible increase in size. In plants, vegetative and reproductive growth is always based on the expansion of young daughter cells produced by ongoing meristematic divisions. Growth is a prerequisite for plant development, and its regulation may occur via changes in the cell division cycle and in rates or duration of cell expansion (cf. Lu and Neumann, 1998; Skirycx and Inze, 2010). Note, however, that even cell division is dependent on a limited amount of prior cell expansion. The processes involved in the cell expansion that is primarily responsible for size increases at tissue, organ and whole-plant levels can be conveniently described by the Lockhart (1965) model equations. The first equation states that relative rates of cell expansion growth (RGR) are limited by hydraulic factors which control the entry of water required for cell volume increases. One such hydraulic factor is the maintenance of cytoplasmic water potential (Cc) at a level which is more negative than that of the external water source (Co). The resultant water potential gradient provides the driving force for
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water uptake and resultant flow is in turn, modulated by an additional factor, that is, the hydraulic conductivity, L, of the water conducting pathways leading into the cytoplasm. RGR ¼ LðCo Cc Þ
ð1Þ
The second Lockhart equation indicates that relative rates of cell growth are also co-regulated by interactions between cell turgor pressure (P), the mechanical extensibility of the cell walls (m) and the minimum turgor pressure required to start cell expansion, that is, the cell wall yield threshold (Y). RGR ¼ mðP Y Þ
ð2Þ
An often overlooked but important point is that each of the cellular parameters involved (Cc, L, P, m, Y) and hence cell growth itself, can be metabolically regulated. For example, L can be influenced by the opening or closure of aquaporin water channels in the membranes (e.g., Lu and Neumann, 1999; Postaire et al., 2010), Cc by solute accumulation (Munns and Tester, 2008), m and Y by the activities of wall enzymes (Cosgrove, 2005) and P by interactions with any one of the preceding values. Both water deficits and excess salinity in the rhizosphere have been shown to induce reductions in cell and whole-plant growth. In the short term (hours), these may be associated with reductions in cell turgor pressure. Stress-induced losses in turgor pressure can be reversed by increases in solute accumulation, that is, osmotic adjustment. However, even after complete turgor recovery in expanding tissues, growth may continue to be limited by ongoing reductions in rates of cell production and expansion. These may in turn be related to reductions in wall extensibility parameters. This chapter presents an eclectic perspective, based largely on research findings from our laboratory on biochemical and biophysical mechanisms involved in growth inhibition by water deficits, as caused by salinity (Section II), drought (Section III) and most recently, by novel plant interactions with aqueous colloids in soil solutions (Section IV; Fig. 1). Much attention in each section is given to the often-overlooked roles of plant cell walls in regulating whole-plant growth inhibition under stressful conditions. Better understanding of such mechanisms could ideally point to ways of improving the ability of crop plants to maintain growth and hence yield stability. For many additional aspects of plant responses to salt and water stress, the reader is directed to the other chapters of this volume and to many fine review articles that have appeared in recent years, for example, Munns and Tester (2008), Neumann (2008), Walter et al. (2009), Mittler and Blumwald (2010), Skirycx and Inze (2010), Urano et al. (2010) and Yang et al., (2010).
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Sub- or supra-optimal environmental inputs
Sensing mechanisms
Local and long distance signals
Signal transduction
Genomic and post-genomic responses
Alterations in cytoplasmic and apoplastic metabolism
Altered growth and development at level of cell, organ and organism
Acclimation or stress-induced death
Fig. 1. A holistic view of plant interactions with stressful environmental conditions.
II. PLANT GROWTH AND SURVIVAL DURING MODERATE AND SEVERE SALINITY STRESS Excess salinity in the soil solution to which plant roots are exposed can be derived from geochemical sources, sea water infiltration of coastal ground waters, sea water salts in wind and rain, excess fertilizer application to soils or supplementary irrigation with salt-containing irrigation waters (the water evaporates leaving salts to accumulate in the soil). Excessive accumulation of salts in the rhizosphere can lead to growth inhibition, leaf necrosis, accelerated onset of senescence, wilting and death. Different physiological mechanisms can be involved. An osmotic mechanism involves the build-up of salts in the rhizosphere or in the small volume of fluids in the apoplastic cell
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wall compartments. This leads to more negative water potentials which can decrease or even reverse the inwardly directed water potential gradients responsible for water uptake, turgor maintenance and cell expansion (see Eq. (1)). Decreases in water uptake can be gradually reversed, in the case of cells by cytoplasmic accumulation of additional solutes through the process of osmotic adjustment (Evlagon et al., 1990; Munns and Tester, 2008; Neumann et al., 1988) or, in the case of root water uptake, by the development of more negative xylem water potentials. However, growth of maize seedling roots and leaves (but not of bean or rice leaves cf. Lu and Neumann, 1999; Neumann et al., 1988), may continue to be reduced, by parallel salinityinduced reductions in the physical extensibility of the expanding cell walls, even after full osmotic adjustment (cf. Neumann, 1993; Neumann et al., 1994). Interestingly, reduced cell wall extensibility (sometimes termed ‘wall stiffening’) induced by salinity in growing maize leaves can also be induced by exposure of maize seedling roots to osmotic stress alone; thus, toxic effects of sodium or other ions are not necessarily involved. Instead, the regulatory involvement of root to leaf hydraulic signals in initiating decreases in wall extensibility of growing tissues (and stomatal closure) has been postulated (e. g., Chazen and Neumann, 1994; Chazen et al., 1995; Christmann et al., 2007). Excessive salinity in the rhizosphere can also cause hydraulic limitations to leaf growth by inducing regulated decreases in root hydraulic conductivity (Azaizeh and Steudle, 1991; Chazen et al., 1995; Evlagon et al., 1990; Lu and Neumann, 1999). Thus, ongoing research into genomic and epigenetic changes that regulate either cell wall mechanics or plant hydraulics may provide useful approaches for potentially limiting the plant growth inhibition that occurs during water and salinity stress. Another mechanism involved in adverse plant-responses to salinity is associated with excessive, concentration-dependent uptake of salts into affected cells. This can lead to toxic symptoms associated with ionic and hormonal imbalances (Munns and Tester, 2008). The toxicity associated with excess salt accumulation in the cytoplasm has also been mechanistically related to increased generation of reactive oxygen species (ROS). Increased levels of ROS, aside of potential signaling roles, can have damaging effects on essential cellular components such as membranes, proteins and nucleic acids (see reviews by Halliwell and Gutteridge, 1989; Miller et al., 2010). Endogenous or salt-induced increases in levels of antioxidant enzymes and their associated substrates may more or less successfully mitigate the potentially adverse effects of excessive ROS accumulation. For example, a clear correlation was revealed between relatively high endogenous levels of antioxidant activity (enzymes and substrates) in the leaves and roots of a saltresistant wild tomato (Lycopersicon penellii) and lower levels in a much less
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salt-resistant cultivated tomato (Shalata and Tal, 1998; Shalata et al., 2001). Shalata and Neumann (2001) showed that an exogenous supply of ascorbic acid, a water soluble antioxidant substrate found in plants and also known as vitamin C in humans, could mitigate increases in lipid peroxidation caused by highly stressful root exposures of whole, transpiring, tomato seedlings to 300 mM NaCl for up to 9 h. The 9-h salt treatment rapidly and uniformly induced a complete wilting of the shoots, and in the absence of supplementary ascorbic acid, the 9-h treatment was 100% lethal. However, supplementary supplies of ascorbic acid via the roots facilitated a remarkable recovery from wilting and a continuation of apparently normal growth in circa 50% of the wilted seedlings following a return to non-saline root media. This remedial effect was not obtained when roots were supplied equivalent concentrations of other small organic molecules without equivalent antioxidant activity. Subsequently, several studies have provided additional evidence for a potentially protective role of increased levels of either endogenous or exogenous ascorbic acid, in the apoplast as well as the cytoplasm of salinised plants (cf. Athar et al., 2008; Hemavathi et al., 2009; Huang et al., 2005; Yamamoto et al., 2005). However, the practical utility of chemical treatments with exogenous antioxidants, or of using antioxidant traits (among others) in breeding for salinity tolerance, remains to be demonstrated under variable field conditions. Most attempts at improving the resistance of crop plants to salinity stress have rightly focused in recent years on genomic approaches aimed at discovering and utilising genes associated with two vital physiological parameters, that is, salt exclusion and growth maintenance (see reviews by Munns, 1993; Neumann, 1997). The introduction into agricultural practice of genetically engineered crop varieties can, however, be a very prolonged and difficult process even when compared with the time required to introduce new varieties produced by more traditional breeding approaches (cf. Potrykus, 2010; Yang et al., 2010). Fortunately, conventional breeding for enhanced ability to maintain growth and exclude sodium ions can now be accelerated by use of genetic marker technology and appears to be resulting in more saltresistant varieties of major crop species such as maize, rice and wheat (Schubert et al., 2009; personal communications by Rana Munns, CSIRO, Australia and Glen Gregorio, IRRI, Philippines). The process of breeding more resistant varieties may be further accelerated by the recent introduction of industrialised processes for mass characterisation and selection of individuals showing desirable phenotypic traits resulting from phenotypic plasticity in populations of isogenic lines subjected to different environmental challenges (Munns and Tester, 2008; Nicotra and Davidson, 2010). Moreover, the realisation that inheritable epigenetic changes, as well as genomic
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changes are involved in evolutionary selection processes may have important new implications for plant breeding. For example, Hauben et al. (2009) give impressive examples of the potential benefits associated with the exploitation of apparently stable epigenetic variability in breeding programs. They showed that respiratory energy-use efficiency is an important epigenetically regulated factor in determining seed yield in canola (Brassica napus). Thus, individual plants in an isogenic canola population and their selffertilised progenies were recursively selected for respiration intensity and populations with distinct physiological and agronomical characteristics, including increased stress resistance, were isolated. Most importantly, this apparently simple approach appeared to facilitate further improvements in the already high yield potentials of existing commercial hybrids.
III. PLANT GROWTH DURING MODERATE WATER STRESS EPISODES Plant water stress symptoms appear during periods of water deficit when the rate of supply of water from soil to plant falls below the water demands of the combined processes of growth and transpiration. Water deficits can result from drought-induced soil drying, salt-induced osmotic limitations to water uptake, limitations to soil or plant water transport and hot, dry, atmospheric conditions which increase evaporative demands. Drought may take the form of terminal drought that leads progressively to desiccation and death or intermittent drought, such as that occurring between rainfall or supplementary irrigation events. The initial response of whole plants to water deficits involves rapid reductions in leaf (and to a lesser extent root) growth rates followed, sooner or later, by partial or complete closure of stomata. These responses can reduce transpirational water loss at the cost of associated reductions in photosynthetic potential. Moreover, stress-induced root growth inhibition will necessarily limit the capacity of the root system to search for new, untapped water reserves in the soil profile. A practical implication is that breeding or engineering plants for avoidance of growth inhibition during intermittent periods of moderate water stress, or during continued exposure to the osmotic effects of moderate salinity, might offer opportunities for growth and yield stabilisation. Such a goal may benefit from improved understanding of biochemical and genetic mechanisms involved in stress-induced growth inhibition in both roots and leaves. As mentioned in the introductory section, plant growth inhibition can result from hydraulic and/or biomechanical (cell wall) limitations to cell and organ extensibility. This section concerns some recent advances in the
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understanding of mechanisms involved in the biomechanical regulation of growth inhibition during water stress. Bogoslavsky and Neumann (1998) described laboratory studies investigating the relative roles of increased water availability and of apoplastic pH in regulating the recovery of leaf growth from water stress. The responses of the emerging first leaf of whole-maize seedlings with a single primary root were studied. A moderate water deficit, with attendant leaf growth reductions, was first imposed by exposing the roots for 1 h to aerated nutrient solution containing the non-penetrating osmolyte PEG 6000 at a water potential of 0.4 MPa. A micro-syringe fitted with a fine needle was then used to inject 12-mL aliquots of various aqueous solutions into the circa 1-cm long elongation zone at the base of the emerging first leaf of the water-stressed seedlings. Changes in leaf position were determined at 1-s intervals by using electronic position transducers (LVDTs) connected via thread to the leaf tips. The position data was plotted against time and used to simultaneously track instantaneous growth rates in up to 16 individual seedlings. By measuring the effects of gently applying and removing a small force (2 g weight), relative in vivo or in vitro measures of the irreversible component of leaf mechanical extensibility could also be obtained. Injection of water alone into the leaf elongation zone of water-stressed seedlings immediately induced remarkable increases in the (inhibited) rates of leaf elongation. The accelerated growth rate declined gradually to the original low growth rate over about 30 min. Thus, the increased availability of the injected water at 0 MPa appeared to allow increased rates of water uptake for leaf cell expansion. Similar findings were obtained when strong buffer solutions at pH 4.5 were injected in place of water. However, injection of isoosmotic solutions of the same buffers at pH 5.5 largely prevented utilisation of the injected water for growth stimulation. Thus, small upwards changes in the pH of apoplastic solution injected into the elongation zone effectively limited the acceleration of leaf growth normally elicited by local increases in water availability. Injected solutions buffered at pH 5.5 also inhibited the increases in relative leaf tissue extensibility (both in vivo and in vitro) that were induced by injections of water alone or of pH 4.5 buffer. In addition, the acceleration of leaf growth by injected water was also prevented when sodium vanadate or erythrosin B, both inhibitors of proton-pumping ATPases, was included in the injected water. Overall, the findings suggested that the pH 5.5 buffer limited ongoing wall acidification to lower pH values and the associated increases in wall extensibility needed to allow utilisation of the injected water for cell expansion. These findings were consistent with the ‘acid growth hypothesis’, that is, that small changes in apoplastic pH, regulated by outward proton-pumping ATPases in the plasma membranes, can
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affect cell wall proteins such as expansins, XET (xyloglucan endotransglycosylase) and cell wall polysaccharide linkages, thereby effecting wall loosening and increased rates of cell expansion (Cosgrove, 2005; Fry, 1986; Hager et al., 1971; Rayle and Cleland, 1970, 1992). Zo¨rb et al. (2005) and Pitann et al. (2009) provided further evidence for the involvement of protonpumping ATPases in leaf growth inhibition by the osmotic component of salinity stress. Finally, Gevaudant et al. (2007) genetically engineered increased expression in tobacco plants of either a wild-type Hþ ATPase or of a modified Hþ ATPase in which the autoinhibitory domain was removed in order to facilitate constitutive expression. The latter transformant showed aberrant growth but also revealed increases in resistance to growth inhibition by salt stress. Further support for the involvement of the Hþ ATPase and wall acidification in the regulation of cell wall extensibility and growth was provided in several reports in which the more accessible elongation zone at the apex of the maize primary root was investigated (Bassani et al., 2004; Fan and Neumann, 2004; Fan et al., 2006; Zhu et al., 2007). Bassani et al. (2004) used a suppressive subtractive hybridisation technique to reveal that 150 growth-related genes were preferentially expressed in the elongating region of the maize root tip as compared with the subtending fully elongated region. One immediate conclusion is that growth regulation is likely to be a complex, multigenic process (cf. Birnbaum et al., 2003). Interestingly, growth in the accelerating growth region, that is, in the first 3 mm behind the maize primary root tip, is maintained, even under water stress. Bassani et al. used Northern blots to show that transcripts of two candidate genes related to wall metabolism (Hþ ATPase and XET, cf. Wu and Cosgrove, 2000) were highly expressed in this region under both control and water deficit conditions and that transcript expression was relatively down regulated in the more basal decelerating and fully elongated regions. These findings suggested a close spatial association between maintenance of accelerating growth and the expression of these particular genes, as well as several others. Fan and Neumann (2004) more directly investigated the possibility that root growth inhibition during water stress might be related to stress-induced decreases in the ability of proton-pumping ATPases to acidify the expanding cell walls and thereby maintain cell wall extensibility. Again using primary roots of whole-maize seedlings, they revealed clear spatial correlations between the growth-inhibitory effects of a PEG-induced water deficit on segmental elongation rates in the more basal regions of the elongation zone and profiles of proton flux from the root epidermis or of apoplastic pH in epidermal cell walls. They also showed that exogenous acidification of roots
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with succinate buffer at pH 4.5 could partially reverse the stress-induced inhibition of growth. Thus, the more apical zone of accelerating growth responded to acidification with further increases in growth rates while the mid- and basal region of the elongation zone showed limited or zero responses, respectively. This suggested that regulated changes in wall pH were not the only factor involved in regulating root growth inhibition by water deficits and that developmental variations in the responsiveness of the cell walls to pH change were also involved. Fan et al. (2006) provided support for this suggestion. They directly measured the in vitro extensibility of tissues in the three regions of the elongation zone of maize primary roots and confirmed that water deficits did not reduce mechanical extensibility in the region 0–3 mm behind the tip but did do so in more basal regions (cf. Wu and Cosgrove, 2000). More importantly, they used UV fluorescence, Fourier transform IR spectroscopy and a specific stain for lignin to show that an accelerated stelar accumulation of wall-based phenolic compounds and lignin accompanied stress-induced decreases in the extensibility of the basal regions. Moreover, the expression of two gene transcripts involved in lignin synthesis (cinnamyl-CoA reductases 1 and 2) increased within 1 h of initiating water deficits. Water-stressinduced increases in accumulation of UV-fluorescent phenolics in expanding cell walls of soybean roots were also reported by Yamaguchi et al. (2010) suggesting that similar stress-induced processes of cell wall stiffening, by acceleration of phenolic cross linking and lignification, may have evolved in both mono and dicot roots. An obvious question is ‘why have plants evolved and retained a response to water deficits which limits root growth when it is most needed to facilitate the search for additional soil water?’ One possibility suggested by Fan et al. (2006) is that such root growth limitations may increase the relative availability of internal supplies of essential water and solutes to meristematic regions, thereby increasing their chances of surviving drying soil environments and of subsequent root growth recovery, if or when soil water availability is increased by rainfall or irrigation. In conclusion, root and leaf growth regulation by salinity or water stress can involve multiple genetic, biochemical and physiological responses which may help acclimation to the mixed stresses faced by plants in rapidly changing real world environments. Further plant research at all levels of investigation, should facilitate educated trials involving epigenetic trait selection, conventional marker assisted breeding and genetic engineering approaches. These could lead to the introduction of new varieties tailored to specific locales and specific types of stress.
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IV. WHOLE-PLANT WATER AVAILABILITY AND GROWTH CAN ALSO BE LIMITED BY COLLOID STRESS The possibility that the availability of water to higher plants may also be limited by interactions with environmental colloids is generally overlooked. This section briefly introduces recent laboratory findings which indicated the existence of ‘plant colloid stress’. Asli and Neumann (2009) investigated the possibility that colloidal suspensions of inorganic nano-particulate materials of industrial or natural origin, that can be present in soil solutions, could interfere with rhizosphere water transport. They found that external colloidal suspensions of either naturally derived bentonite clay particles or industrially produced TiO2 nanoparticles (30 nm diameter) at 1 gL 1 could significantly reduce water transport through the intact epidermal surfaces of maize primary roots by up to 40%. Moreover, similar additions to the hydroponic solution surrounding the roots of whole-maize seedlings rapidly inhibited leaf growth and transpiration. A subsequent report showed that colloidal suspensions of humic acid at 1 gL 1 had similar inhibitory effects (Asli and Neumann, 2010). Humic acid is an organic colloid that is ubiquitous in soils and soil solutions and may act as a growth stimulant at low concentrations. In addition to reducing root hydraulic conductivity, excessive soil accumulation of humic acid caused an inhibition of shoot growth and reduced plant ability to withstand soil drying. Experiments with model polymers and estimates of cell wall pore sizes suggested that nanosized inorganic or organic particles in soil waters can be transported by mass flow to the root cell wall surfaces of transpiring plants where water flow is reduced by the formation of cake layers and /or by pore blocking. Importantly, the levels of colloids in agricultural soil waters may be further increased when solid wastes and recycled waste waters rich in organic matter are applied to the soil. Recently, the potential for disruption of xylem water transport by physical interactions between endogenous protein colloids in the xylem sap and the cell wall material comprising xylem pit membranes was also reported (Neumann et al., 2010). Thus, ‘colloid stress’ resulting from the inhibitory effects on water transport of physical interactions between plant cell walls and environmental or internal colloids, is a novel stress-factor that may affect plant water relations and further limit plant ability to resist salinity and drought.
REFERENCES Asli, S. and Neumann, P. M. (2009). Colloidal suspensions of clay or titanium dioxide nanoparticles can inhibit leaf growth and transpiration via physical effects on root water transport. Plant, Cell and Environment 32, 577–584.
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Asli, S. and Neumann, P. M. (2010). Rhizosphere humic acid interacts with root cell walls to reduce hydraulic conductivity and plant development. Plant and Soil 336, 313–322. Athar, H. R., Khan, A. and Ashraf, M. (2008). Exogenously applied ascorbic acid alleviates salt-induced oxidative stress in wheat. Environmental and Experimental Botany 63, 224–231. Azaizeh, H. and Steudle, E. (1991). Effects of salinity on water transport of excised maize (Zea mays L.) roots. Plant Physiology 99, 1136–1145. Bassani, M., Neumann, P. M. and Gepstein, S. (2004). Differential expression profiles of growth related genes in the elongation zone of maize primary roots. Plant Molecular Biology 56, 367–380. Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert, G. M., Galbraith, D. W. and Benfey, P. N. (2003). A gene expression map of the Arabidopsis root. Science 302, 1956–1960. Bogoslavsky, L. and Neumann, P. M. (1998). Rapid regulation by acid-pH of cellwall adjustment and leaf growth, in intact maize plants responding to reversal of water stress. Plant Physiology 118, 701–709. Chazen, O. and Neumann, P. M. (1994). Hydraulic signals from the roots and rapid cell wall hardening in growing maize leaves, are primary responses to PEG induced water deficits. Plant Physiology 104, 1385–1392. Chazen, O., Hartung, W. and Neumann, P. M. (1995). The different effects of PEG 6000 and NaCl on leaf development are associated with differential inhibition of root water transport. Plant, Cell and Environment 18, 727–735. Christmann, A., Weiler, E. W., Steudle, E. and Grill, E. (2007). A hydraulic signal in root-to-shoot signalling of water shortage. The Plant Journal 52, 167–174. Cosgrove, D. J. (2005). Growth of the plant cell wall. Nature Reviews Molecular Cell Biology 6, 850–861. Evlagon, D., Ravina, I. and Neumann, P. M. (1990). Interactive effects of salinity and calcium on osmotic adjustment, hydraulic conductivity and growth in primary roots of maize seedlings. Israel Journal of Botany 39, 239–247. Fan, L. and Neumann, P. M. (2004). The spatially variable inhibition by water deficit of maize root growth correlates with altered profiles of proton flux and cell wall pH. Plant Physiology 135, 2291–2300. Fan, L., Linker, R., Gepstein, S., Tanimoto, E., Yamamoto, R. and Neumann, P. M. (2006). Progressive inhibition by water deficit of cell wall extensibility and growth along the elongation zone of maize roots is related to increased lignin metabolism and progressive stelar accumulation of wall phenolics. Plant Physiology 140, 603–612. Fry, S. C. (1986). Cross-linking of matrix polymers in the growing cell walls of angiosperms. Annual Review of Plant Physiology 37, 165–186. Gevaudant, F., Duby, G., von Stedingk, E., Zhao, R., Morsomme, P. and Boutry, M. (2007). Expression of a constitutively activated plasma membrane Hþ ATPase alters plant development and increases salt tolerance. Plant Physiology 144, 1763–1776. Hager, A., Menzel, H. and Krauss, A. (1971). Versuche und hypothese zur primarwirkung des auxins beim shtrekungswachstum. Planta 100, 47–75. Halliwell, B. and Gutteridge, J. M. C. (1989). Protection against oxidants in biological systems: The super oxide theory of oxygen toxicity. In Free Radicals in Biology and Medicine, (B. Halliwell and J. M. Gutteridge, eds.), pp. 86–123. Clarendon Press, Oxford, 0-1985-5294-7.
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Hauben, M., Haesendonckx, B., Standaert, E., Van Der Kelen, K., Azmi, A., Akpo, H., Van Breusegem, F., Guisez, Y., Bots, M., Lambert, B., Laga, B. and De Block, M. (2009). Energy use efficiency is characterized by an epigenetic component that can be directed through artificial selection to increase yield. Proceedings of the National Academy of Sciences 106, 20109–20114. Hemavathi, U. C. P., Ko, E. Y., Nookaraju, A., Reddy, A. C., Chun, S. C., Kim, D. H. and Park, S. W. (2009). Over-expression of strawberry D-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance. Plant Science 177, 659–667. Huang, C., He, W., Guo, J., Chang, X., Su, P. and Zhang, L. (2005). Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant. Journal of Experimental Botany 56, 3041–3049. Lockhart, J. A. (1965). An analysis of irreversible plant cell elongation. Journal of Theoretical Biology 8, 264–275. Lu, Z. and Neumann, P. M. (1998). Water stressed maize, barley and rice seedlings show species specific diversity in mechanisms of leaf growth inhibition. Journal of Experimental Botany 49, 1945–1952. Lu, Z. and Neumann, P. M. (1999). Water stress inhibits hydraulic conductance and leaf growth in rice seedlings but not transport of water via mercury sensitive water channels in the root. Plant Physiology 120, 143–152. McDowell, N., Pockman, W. T. Allen, C. D. et al. (2008). Mechanisms of plant survival and mortality during drought: Why do some plants survive while others succumb to drought? The New Phytologist 178, 719–739. Miller, G., Suzuki, N., Ciftci-Yilmaz, S. and Mittler, R. (2010). Reactive oxygen species homeostasis and signaling during drought and salinity stresses. Plant, Cell & Environment 33, 453–467. Mittler, R. and Blumwald, E. (2010). Genetic engineering for modern agriculture: Challenges and perspectives. Annual Review of Plant Biology 61, 443–462. Munns, R. (1993). Physiological processes limiting growth in saline soils: Some dogmas and hypotheses. Plant, Cell & Environment 16, 15–24. Munns, R. and Tester, M. (2008). Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651–681. Neumann, P. M. (1993). Rapid and reversible modifications of extension capacity of cell walls in elongating maize leaf tissues responding to root addition and removal of NaCl. Plant, Cell & Environment 16, 1107–1111. Neumann, P. (1997). Salinity resistance and plant growth revisited. Plant, Cell & Environment 20, 1193–1198. Neumann, P. M. (2008). Coping mechanisms for crop plants in drought prone environments. Annals of Botany 101, 901–907. Neumann, P. M., Van Volkenburgh, E. and Cleland, R. E. (1988). Salinity stress reduces bean leaf expansion and turgor but not cell wall extensibility. Plant Physiology 88, 233–237. Neumann, P. M., Azaizeh, H. and Leon, D. (1994). Hardening of root cell walls: A growth inhibitory response to salinity stress. Plant, Cell and Environment 17, 303–309. Neumann, P. M., Weissman, R., Stefano, G. and Mancuso, S. (2010). Accumulation of xylem transported protein at pit membranes and associated reductions in hydraulic conductance. Journal of Experimental Botany 61, 1711–1717. Nicotra, A. B. and Davidson, A. (2010). Adaptive phenotypic plasticity and plant water use. Functional Plant Biology 37, 117–127. Pitann, B., Schubert, S. and Muhling, K. H. (2009). decline in leaf growth under salt stress is due to an inhibition of Hþ pumping activity and increase in
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apoplastic pH of maize leaves. Journal of Plant Nutrition and Soil Science 172, 535–543. Postaire, O., Tournaire-Roux, C., Grondin, A., Boursiac, Y., Scha¨ffner, A. R., Morillon, R. and Maurel, C. (2010). A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis. Plant Physiology 152, 1418–1430. Potrykus, I. (2010). Insights from Golden Rice: GE-projects for public good are faced with inhibitive conditions. ASPB annual meeting—Plant Biology 2010, Montreal, Canada, Abs # S042, http://abstracts.aspb.org/pb2010/public/. Rayle, D. L. and Cleland, R. E. (1970). Enhancement of wall loosening and elongation by acid solution. Plant Physiology 46, 250–253. Rayle, D. L. and Cleland, R. E. (1992). The acid growth theory of auxin induced cell elongation is alive and well. Plant Physiology 99, 1271–1274. Schubert, S., Neubert, A., Schierholt, A., Sumer, A. and Zorb, C. (2009). Development of salt-resistant maize hybrids: The combination of physiological strategies using conventional breeding methods. Plant Science 177, 196–202. Shalata, A. and Neumann, P. M. (2001). Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation. Journal of Experimental Botany 52, 2207–2211. Shalata, A. and Tal, M. (1998). The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii. Physiologia Plantarum 104, 169–174. Shalata, A., Mittova, V., Volokita, M., Guy, M. and Tal, M. (2001). Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: The root antioxidative system. Physiologia Plantarum 112, 487–494. Skirycx, A. and Inze, D. (2010). More from less: Plant growth under limited water. Current Opinion in Biotechnology 21, 197–203. Sperry, J. S., Hacke, U. G., Oren, R. and Comstock, J. P. (2002). Water deficits and hydraulic limits to leaf water supply. Plant, Cell and Environment 25, 251–263. Urano, K., Kurihara, Y., Seki, M. and Shinozaki, K. (2010). ‘Omics’ analyses of regulatory networks in plant abiotic stress responses. Current Opinion in Plant Biology 13, 132–138. Uyttewaal, M., Traas, J. and Hamant, O. (2010). Integrating physical stress, growth, and development. Current Opinion in Plant Biology 13, 46–52. Walter, A., Silk, W. K. and Schurr, U. (2009). Environmental effects on spatial and temporal patterns of leaf and root growth. Annual Review of Plant Biology 60, 279–304. Watson, J. D. and Crick, F. H. C. (1953). A structure for deoxyribose nucleic acid. Nature 171, 737–738. Wu, Y. J. and Cosgrove, D. J. (2000). Adaptation of roots to low water potentials by changes in cell wall extensibility and cell wall proteins. Journal of Experimental Botany 51, 1543–1553. Yamaguchi, M., Valliyodan, B., Zhang, J., Lenoble, M. E., Yu, O., Rogers, E. E., Nguyen, H. T. and Sharp, R. E. (2010). Regulation of growth response to water stress in the soybean primary root. I. Proteomic analysis reveals region-specific regulation of phenylpropanoid metabolism and control of free iron in the elongation zone. Plant Cell and Environment 33, 223–243. Yamamoto, A., Bhuiyan, M. N. H., Waditee, R., Tanaoka, Y., Esaka, M., Oba, K., Jagendorf, A. T. and Takabe, T. (2005). Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and arabidopsis plants. Journal of Experimental Botany 56, 1785–1796.
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Recent Advances in Photosynthesis Under Drought and Salinity
MARIA M. CHAVES,*,{,1 J. MIGUEL COSTA*,{ AND NELSON J. MADEIRA SAIBO*
*Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Av. da Repu´blica, Oeiras, Portugal { CBAA, Instituto Superior de Agronomia, Universidade Te´cnica de Lisboa, Tapada da Ajuda, Lisboa, Portugal
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Studying Drought and Salinity Effects on Photosynthesis . . . . . . . . . . . . . . . . A. From Controlled Conditions to the Field ................................. B. The Relevance of Studying Recovery Responses ......................... C. The Use of Model Plants ..................................................... III. Photosynthetic Limitations Under Water and Saline Stress . . . . . . . . . . . . . . . A. Diffusive Limitations (Stomatal and Mesophyll)......................... B. Biochemical and Metabolic Limitations ................................... IV. Is Water Use Optimized by the Leaves Under Water Deficits? . . . . . . . . . . . . V. How are Stomata and Photosynthetic Genes Regulated? . . . . . . . . . . . . . . . . . A. Regulation of Stomatal Aperture ........................................... B. Stomatal Development ....................................................... C. Expression of Photosynthetic Related Genes ............................. VI. Improving Carbon Fixation Under Environmental Stress?. . . . . . . . . . . . . . . . VII. Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding author: E-mail:
[email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00003-5
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ABSTRACT Fast increase in world population, scarcer water resources and climate change are putting pressure on the maximization of crop yield, while optimizing the use of water and soil. Salinity causes tremendous yield losses at world scale, especially in dry areas. We revise the current understanding of the impact of drought and salinity on photosynthesis, a highly sensitive process to these stresses and a major determinant of plant’s growth and yield. The CO2 diffusive limitations (stomatal and mesophyll) to photosynthesis under water deficits and the underlying regulatory mechanisms of stomatal behaviour and photosynthetic metabolism are presented. Recent molecular advances are described, in particular those related to stomatal development and guard cell signalling. Special emphasis is given to the effects of ABA signalling on stomatal regulation under water deficits. The role of transcription factors controlling guard cell movement and photosynthetic activity under drought and high salinity is discussed. Coordination of stomatal conductance with the CO2 requirements of leaf mesophyll that may allow constant water use efficiency (WUE) in different environments is analysed on the basis of recent data from transformed plants. The improvement of WUE by optimizing Rubisco carboxylase capability to increase photosynthetic efficiency has been ineffective. Therefore, we stress the importance of knowledge on leaf gas-exchange limitations caused by drought and high salinity for future breeding strategies. The direct transfer of the knowledge gathered from model plants into crops needs to be carefully considered.
I. INTRODUCTION Soil water deficit and soil salinity are major limiting factors of plant growth and agricultural productivity worldwide. Water scarcity and poor water management increasingly affect large agricultural areas in Asia, Europe, America and Australia (Aldaya et al., 2009; Brown, 2008; Collins et al., 2009; World Economic Forum, 2009). Overall, the occurrence of extreme climate events has been increasing in recent years and is expected to keep rising in the near future (IPCC, 2007; Tin, 2008). Major concern exists in countries like China or India where the combination of increasing domestic/ industrial water consumption and deficient management of soil and water resources endanger food security (Beddington, 2010; Brown, 2008; Costa and Heuvelink, 2004; Morison et al., 2008). In Europe, water scarcity may produce relevant socio-economic and environmental impacts, in particular in the dry South Mediterranean areas or the Sub-Saharan Africa, considered to be amongst the most altered regions in the world by different climate change scenarios (Beddington, 2010; Chaves and Davies, 2010; Tin, 2008). As for salinity, it is known to affect more than 6% of the arable land and about 30–50% of the irrigated area worldwide (Unesco Water Portal, 2007). Salinity risks are larger near coastal areas due to saline water intrusion, which is directly related to the over-exploitation of underground water for
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either domestic or agricultural purposes, causing a decrease in the groundwater levels (Brown, 2008; Carvalho, 2000; Collins et al., 2009; http://www. abc.net.au/learn/silentflood/stats.htm). The ability of plants to adapt and/or acclimate to different environments is directly or indirectly related with the plasticity and resilience of photosynthesis, in combination with other processes, determining plant growth and development, namely reproduction. Evolutionary success depends on integrative and effective regulation of these processes at the whole-plant level (Lawlor, 2009). Regular drought-tolerant plants can withstand moderate tissue dehydration of about 30% water loss. By contrast, desiccation-tolerant plants (generally referred to as resurrection plants) are tolerant to further cell dehydration (around 90% water loss) and keep the ability to rehydrate successfully. Molecular studies suggest that desiccation tolerance in the vegetative tissues of resurrection plants is unlikely to result from the presence of genes that are unique to these plants, since the relevant genes are also present in the genome of non-tolerant plants, but may reside in the expression patterns of those genes, therefore being largely a quantitative characteristic (Ramanjulu and Bartels, 2002). When confronted by salinity, plants can adapt to it by compartmentalizing Naþ and Cl in the vacuole and to keep cellular water status and growth (Blumwald et al., 2000). These mechanisms are particularly developed in halophytes, but over-expression of Naþ/Hþ antiporter has already shown to improve salt and drought tolerance in transgenic Arabidopsis (Shi et al., 2003). Identifying limitations to photosynthesis and the regulatory processes under water deficits and salinity is essential to minimize (or to improve through breeding) the negative impact of such stresses on agricultural crops and to protect ecosystem functioning. Recent reviews have dealt with this subject at various levels of complexity, from the cell to the whole plant (Chaves et al., 2003, 2009; Flexas et al., 2004; Lawlor and Tezara, 2009; Munns, 2002; Munns and Tester, 2008; Zhu, 2001). The reinforcement of research funding in this thematic area was recently proposed by the EU that also recommended increasing research coordination among member states so that plant/crop adaptation to changing climate may be promoted (EU, 2010; The Royal Society, 2009). In this chapter, we aim revising the current status of our understanding of the impact of water scarcity and salinity on photosynthesis. Besides referring to the different systems (model plants, crops) and approaches (physiological and molecular) that are enabling advances in this scientific area, we will revise the controlling factors of carbon uptake in different species and different conditions, with particular emphasis on the regulatory mechanisms that make possible to coordinate carbon assimilation and water loss. Indeed, optimization of water use by plants has and will have major impact in crop
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breeding for arid and semi-arid regions. Major molecular advances related with guard cell signalling and transcriptional regulation of photosynthetic responses will also be reviewed.
II. STUDYING DROUGHT AND SALINITY EFFECTS ON PHOTOSYNTHESIS A. FROM CONTROLLED CONDITIONS TO THE FIELD
In nature, abiotic stress conditions like drought and salinity rarely occur in isolation and combination of different stresses is usually not predictable by single-factor analyses because synergistic, antagonistic, or overlapping effects can occur (Valladares and Pearcy, 1997). An example of an antagonistic response to stresses is the observation that critical temperatures for photosynthesis can increase in leaves of water-stressed plants as compared to wellwatered ones, as it was reported in lupins (Chaves et al., 2002) and in various solanaceae (Havaux, 1992). This suggests that water deficits may provide protection against heat stress, presumably by increasing membrane stability. It is also factual that one stress may originate a second one. This is the case of drought- and salinity-induced stomatal closure that prevents leaf water loss. However, stomatal closure diminishes leaf cooling due to transpiration, generating a superimposed heat stress with leaf temperatures often rising up to 5 or 6 8C in relation to air temperature. Regarding high salinity, the detrimental effects on leaf physiology were shown to depend on exposure to sun light, with more negative impacts being found for sunny than for the shade sites of the canopy, as reported for Olea europea trees (Remorini et al., 2009). Under multiple stresses plants elicit unique and complex responses regarding photosynthesis, respiration (Mittler, 2006; Rizhsky et al., 2002, 2004) and signalling (Okamoto et al., 2009). Therefore, the need to study such interactions under conditions mimicking nature is emphasized (Cimato et al., 2010). At molecular level, co-occurrence of different stresses can generate the co-activation of different response signalling pathways (abscisic acid, ethylene, jasmonic acid, etc.) (Mittler, 2006). For example, in Arabidopsis thaliana plants subjected to both heat and drought about 10% of the regulated genes under such conditions were overlapping with cohort genes regulated by both type of stresses, when they were applied separately (Rizhsky et al., 2004; Voesenek and Pierik, 2008). It is recognized that the velocity of stress imposition dictates plant responses—immediate responses to a short-term stress aiding in plant survival, whereas acclimation responses to slowly imposed stress contribute to
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an improvement of plant performance under adverse conditions. Indeed, these acclimation responses are not only due to direct effects of resource deprivation or hostile conditions, but also to physiological adjustments that minimize disturbances in plant metabolism (Chaves and Oliveira, 2004). Many studies addressing stress effects rely on short-term experiments, which will not be directly applicable to field situation since they are far from the natural growing environment (Cai et al., 2010; Vinocur and Altman, 2005). Although setting up complex design field experiments is a difficult task, the testing under laboratory conditions of newly developed stress-tolerant genotypes to multiple stresses needs to be rechecked with field studies and in different years, to allow the analysis crop performance under a variety of conditions (Mittler, 2006; Richards et al., 2010). In the case of tolerance to salinity, many studies are carried out using hydroponics. However, complex interactions between the soil solution and the soil matrix were shown to affect plant response to salt, as recently shown in Hordeum by Tavakkoli et al. (2010). This implies that field tests are essential, although the confounding effects from several environmental factors changing concurrently have to be taken into account (Cuin et al., 2010; Travers et al., 2010). B. THE RELEVANCE OF STUDYING RECOVERY RESPONSES
Cycles of stress and recovery from stress are prevalent processes occurring under natural conditions during different seasons and under agricultural practices such as irrigation (Vinocur and Altman, 2005). Recovery of photosynthesis following stress relief largely determines plant resilience to water deficits and salinity. For perennial plant species, it is undoubtedly one of the most relevant survival strategies (Hu et al., 2010a). Recovery depends on the intensity of photosynthesis decline under stress (Chaves et al., 2009; Flexas et al., 2006) and is closely linked to plant capacity to avoid or to repair membrane damage when stress intensifies (Chaves and Oliveira, 2004). Moreover, instability of photosynthetic membranes under water deficits, which means becoming transiently permeable, was shown to occur at an early stage of dehydration than in plasma membranes (Speer et al., 1988). In spite of such importance, studies on the capacity of photosynthetic recovery from different stresses have only been conducted in few species, for example, tobacco (Galle´ et al., 2009), beans (Miyashita et al., 2005), grapevine (Flexas et al., 2009; Galme´s et al., 2007a), beech (Galle´ et al., 2007), and several Mediterranean species (Galme´s et al., 2007b). Recovery is generally characterized by a rapid increase (recovery) of leaf water potential (within 2 days or earlier) followed by a later recovery of
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stomatal conductance, which may be associated with hormonal balance being re-established. This will allow the plants to limit transpirational water losses and regain full turgor after rewatering, as shown in citrus plants by Ruiz-Sanchez et al. (1997). The intensity and/or duration of stresses have particular effect on both the velocity and the extent of recovery after stress relief (Chaves et al., 2009). In general, when a severe water stress is imposed, recovery is partial and can reach only 40–60% of the maximum photosynthesis rate during the day after irrigation restarts. Nevertheless, recovery process may continue in the following days but the maximum photosynthetic rates are sometimes never reached again (Bogeat-Triboulot et al., 2007; Galle´ et al., 2007; Grzesiak et al., 2006; Kirschbaum and Pearcy, 1988; Miyashita et al., 2005; Sofo et al., 2004). When a severe water stress is imposed, recovery is much slower and may require de novo synthesis of photosynthetic proteins. It is possible that water stress (and in general all stresses) irreversibly affect photosynthetic capacity and accelerates leaf senescence (Chaves et al., 2011). Slow and/or incomplete photosynthetic recovery has also been linked to sustained oxidative stress (Galme´s et al., 2007a,b). Incomplete recovery of photosynthesis following stress seems to be more frequent in fast growing (e.g. herbaceous) than in slow-growing species. Besides the duration and intensity of the stress, recent findings also suggest a positive effect of a pre-drought treatment on the future recovering after re-watering (Xu et al., 2009). Physiological mechanisms involved in the recovery of plants subjected to high salinity are poorly understood. It is known that the time period required for photosynthesis recovery after salinity stress is generally much longer (up to 15–20 days) than that following drought, even when the average stomatal conductance (gs) before the onset of recovery was threefold that observed in drought studies (Chaves et al., 2011). This likely reflects the metabolic nature of a large proportion of photosynthesis limitations occurring under salinity. Despite the slower recovery, reports on full recovery are more numerous than in plants subjected to severe drought. However, the interaction of salt and water stress strongly reduces plant’s capacity to recover photosynthesis after stress alleviation, as compared with plants subjected to a single stress (Pe´rezPe´rez et al., 2007). C. THE USE OF MODEL PLANTS
1. The model plants Several model plants have been used in stress physiology studies to allow molecular dissection of ‘‘stress-tolerance mechanisms’’ in important crop plants. One of the most well known is A. thaliana, which is considered a
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model for dicotyledonous species. Genetic screens—applied in both forward and reverse modes—have permitted isolation of numerous Arabidopsis mutants, which are important tools to identify genes acting in signalling networks and controlling plant response to environmental stress (Feng and Mundy, 2006; Papdi et al., 2009; Sirichandra et al., 2009a; Vinocur and Altman, 2005). Mutants with abnormal stomatal characteristics (Badger et al., 2009; Hashimoto et al., 2006a,b; Merlot et al., 2002, 2007; Xie et al., 2006) or photosynthetic behaviour (Niyogi et al., 1998; Overmyer et al., 2008; Shikanai et al., 1999; Varotto et al., 2000) have been isolated in large scale screens and used to study different aspects of stomatal regulation and/or photosynthetic mechanisms as well as to identify genes implicated in relevant signalling networks, for example, of abscisic acid (ABA) (Wasilewska et al., 2008). Studies by Koiwa et al. (2006) have shown that T-DNA-tagging followed by phenotypic screens (forward genetics) allow the identification of genes involved in cold, osmotic and salinity stress as well as ABA-mediated gene expression. More recently, Koiwa et al. (2006) isolated mutations causing NaCl hypersensitivity of Arabidopsis seedlings and identified the major components of the SOS pathway which controls ion homeostasis and salt tolerance. Arabidopsis is sensitive to moderate levels of NaCl and has provided much information about both Naþ transport processes and Naþ tolerance (Møller and Tester, 2007). Many of known transcriptional elements of environmental stress-responsive genes in higher plants regulating and controlling stress reactions related to drought or salinity were isolated in Arabidopsis (Ni et al., 2009). More recently, the species Thellungiella halophyla emerged as a new model plant for studies on plant responses to salinity (Amtmann, 2009; Amtmann et al., 2005; Bressan et al., 2001; Zhu et al., 2004). T. halophyla is a close relative of Arabidopsis but tolerates extreme salinity and drought (Amtmann, 2009; Bressan et al., 2001; Inan et al., 2004). An important aspect of salt tolerance is the accumulation of compatible solutes in the cytoplasm to osmotically balance ions accumulation in the vacuole during salt adaptation (Inan et al., 2004). Thellungiella is a dramatic accumulator of proline and can be used to study unique aspects of proline-related signalling pathways in determining fitness under environmental stress conditions (Amtmann, 2009). Rice (Oryza sativa) is probably the most important genetic model species for monocotyledonous (Xu et al., 2009). Research on salt tolerance in legume crops is conducted in model legumes, such as Medicago (Eckardt, 2009; Nunes et al., 2008) or Lotus (Udvardi et al., 2007; Varshney and Koebner, 2006). Regarding woody species, poplar has been used as a model plant (Cronk, 2005; Jansson and Douglas, 2007), including research on stress responses (Bradshaw et al., 2000). Grapevine (Vitis vinifera) also became a
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model system for fruit trees following the fully sequence of its genome (Troggio et al., 2008). The large phenotypic and genetic variation characterizing grapevine is an advantage for comparative physiology and molecular biology studies and/or studies on resistance to water stress (Chaves et al., 2010; Vandeleur et al., 2009). 2. Limitations of model plants The use of Arabidopsis in water deficit stress studies was first limited by methodological difficulties (e.g. gas-exchange measurements). However, specific growing conditions (e.g. short days) and progress in the available equipment for leaf gas-exchange (e.g. development of specific leaf chambers for Arabidopsis plants) or the use of imaging techniques (e.g. IR thermal and Chl a imaging) helped to overcome the limitations regarding physiological characterization of mutants (Badger et al., 2009; Merlot et al., 2002, 2007; Oxborough, 2004). Other limitation of model plants relates to the fact that the knowledge gathered with model species might not be directly transposable to distantly related crop species, namely concerning long-term strategies for improved stress tolerance in the field (e.g. to cereal crops) (Møller and Tester, 2007). For example, the relationship between Naþ tolerance and Naþ accumulation is different in Arabidopsis and cereals, with an inverse relationship often found within cereal species that is not that evident in Arabidopsis ecotypes (Møller and Tester, 2007). Therefore, the results on salinity tolerance obtained in Arabidopsis should be extrapolated to cereal crops with caution. Regarding the root system, root architecture is another trait influencing resistance to water and nutrient stress. The A. thaliana is a Brassica characterized by a tap root system, which represents a limitation for studies of crop root systems that are typically deep (Watt et al., 2008). Therefore, a better understanding of the mechanisms underlying crop species response to environmental stress should include the use of non-model species, grown under field conditions (Travers et al., 2010) and observed over long periods in order to more closely mimic crop’s life span (Vinocur and Altman, 2005).
III. PHOTOSYNTHETIC LIMITATIONS UNDER WATER AND SALINE STRESS A. DIFFUSIVE LIMITATIONS (STOMATAL AND MESOPHYLL)
In steady state photosynthesis, CO2 diffusion from atmosphere to the active site of ribulose 1–5 biphosphate carboxylase/oxygenase (Rubisco) in the chloroplast follows a complex pathway that involves three major conductance
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components: (1) the boundary layer, (2) stomatal conductance and (3) mesophyll conductance (gm) (Farquhar and Sharkey, 1982). Stomata guard cells respond to multiple exogenous and endogenous signals, including light, CO2, leaf to air VPD, ozone, hormones (ABA, auxin) or ions and reactive oxygen species, like hydrogen peroxide (Assmann, 1993; Gray and Hetheringthon, 2004; Schroeder et al., 2001; Wasilewska et al., 2008a,b). This enables stomata to adjust their aperture very fast (within minutes) in response to changes in the surrounding environment and contributes to optimize the balance between water vapour loss and CO2 uptake (Chaves and Oliveira, 2004). By controlling transpiration, stomata also influence leaf temperature and the fluxes of metabolites and long-distance chemical signalling (Brownlee, 2001; Lake et al., 2001). In C3 and C4 plants, stomatal closure is generally the main cause of reduced photosynthesis under mild to moderate drought stress (Chaves et al., 2009; Erismann et al., 2008; Flexas et al., 2004; Grassi and Magnani, 2005) and high salinity (Chaves et al., 2009; Delfine et al., 1999; Ghannoum, 2009; Hura et al., 2006). Because photosynthesis of C4 plants saturates at much lower CO2 concentrations than that of C3 plants, it is unlikely that C4 assimilation is affected by stomatal closure like C3 photosynthesis. However, the limited capacity for photorespiration or for the Water–Water cycle (Mehler reaction) to emerge as alternative sinks of electrons under drought may explain why C4 photosynthesis can be as sensitive or even more sensitive to water stress than C3 photosynthesis, in spite of the larger photosynthetic capacity and water use efficiency (WUE) of C4 plants (Ghannoum, 2009). Once inside the leaf, CO2 has to diffuse from the intercellular air spaces to the chloroplast. However, CO2 diffusion is limited by resistances in both gaseous and liquid phases in the cytosol and by several diffusion barriers like intercellular spaces, the cell wall, the plasmalemma and chloroplast’s envelope. As a whole this represents the so-called mesophyll resistance (or the inverse of the biophysical diffusion resistance, the mesophyll conductance, gm) (Evans and Loreto, 2000; Flexas et al., 2008; Pons et al., 2009). The gm emerged as an important CO2 diffusive limitation of photosynthesis (Barbour et al., 2010; Evans and Loreto, 2000; Flexas et al., 2008; Pons et al., 2009; Warren, 2006). Low mesophyll conductance can reduce the partial pressure of CO2 at the site of carboxylation, limit photosynthesis and affect carbon isotope discrimination () (Niinemets et al., 2009).The relative contribution of stomatal and nonstomatal limitations to photosynthesis depends on the severity, velocity and type of stress being imposed (short term vs. long term). When water deficit is intensified, limitations of non-stomatal processes become more important, in particular, due to lower gm and impaired photobiochemistry (Chaves et al., 2003; Flexas and Medrano, 2002; Flexas et al., 2004, 2008).
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Studies by Flexas et al. (2004, 2008), Niinemets et al. (2005, 2009) and Warren and Adams (2006) showed that both water and salinity stress resulted in decreased gm in many species. Mesophyll conductance also seems to respond very fast to external stimulus such as light, temperature or CO2 (Flexas et al., 2008). This led to the hypothesis that stomatal and mesophyll conductance could be interacting and/or being intrinsically coregulated (Galme´s et al., 2007a,b; Peeva and Cornic, 2009; Warren, 2008a,b). However, recent findings showed that the supposed co-regulation of gs and gm depends to some extent on external signals, such as environmental conditions. Flexas et al. (2009) showed that gm reduction caused by water stress was fully reversed under conditions of cloudy days. In turn, Galle´ et al. (2009) showed that the effects of water stress on gm, and the delayed recovery after re-watering, were dependent on the prevailing irradiance, being more marked under high light. The relevance of leaf structure on gm is also emphasized in the literature (Niinemets et al., 2009). Mesophyll conductance has physical characteristics that relate to surface area of the intercellular spaces, walls and cytosol and dimensions of the intercellular spaces (Lawlor and Tezara, 2009; Niinemets et al., 2009). These physical characteristics change as a function of the shrinking response of tissues to drought (Lawlor and Tezara, 2009). It is known that prolonged water stress or salinity, especially during plant development, may cause profound modifications in leaf anatomy, such as thickened cell walls and smaller and more densely packed leaf cells (Bongi and Loreto, 1989; Qiu et al., 2007). This may explain the long-term reduction of mesophyll conductance in salt stressed plants (Niinemets et al., 2009). In a recent paper, Perez-Martin et al. (2009) showed that leaf structural parameters (mass per unit leaf area—which means mesophyll porosity) influence gm values. Although scattered, the relationship between leaf mass per unit area (MA) and gm revealed that MA imposes a limitation to the maximum values of gm Perez-Martin et al. (2009), an effect that seems to be species dependent. MA decreased under water stress in O. europea but it increased in V. vinifera, resulting in a negative relationship between MA and CO2 between sub-stomatal cavities and chloroplasts in O. europea, and in a positive relationship in grapevine Perez-Martin et al. (2009). We must also have in account the metabolic component of gm, which is possibly related to the activity of proteins like carbonic anhydrase (CA) and aquaporins (as channels for CO2), which together or individually can facilitate CO2 diffusion from the sub-stomatal cavity to the active sites of Rubisco in the chloroplast (Niinemets et al., 2009). The fact that gm is affected by environmental variables and changes rapidly in response to, for example, CO2 concentration, leaf temperature and drought suggests that these
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biochemical factors may indeed be involved in gm response to the environment (Warren, 2008a,b). Mesophyll conductance has also been shown to depend on the genotype (Barbour et al., 2010).
1. The role of carbonic anhydrase Carbonic anhydrase may have a relevant role in CO2 exchange by influencing the metabolic component of gm (Warren, 2008a,b), especially under limiting conditions of CO2 supply, as it happens in severe water and saline stress. It is estimated that CA activity facilitates CO2 diffusion across the chloroplasts by reducing diffusion resistance by about 1/3 (Evans and Von Caemmerer, 1996; Gillon and Yakir, 2000). Although CA is inhibited by progressive water stress, its activity has been considered large enough not to pose limitations on net assimilation (Flexas et al., 2004). In leaves, CA is a Zn-containing enzyme that catalyzes the reversible conversion of CO2 to HCO3. Recently, it was shown that other CA isoforms besides the well-characterized CA1 may also contribute to CO2 transfer in the cell to the catalytic site of Rubisco (Fabre et al., 2007). CA is the second most abundant protein following Rubisco (Fabre et al., 2007; Tiwari et al., 2005) and participates in a broad range of biochemical processes, like carboxylation and decarboxylation reactions, pH regulation, inorganic carbon transport, ion transport, and water and electrolyte balance (Fabre et al., 2007; Moroney et al., 2001; Smith and Ferry, 2000). C4 plants have generally lower amount of CA than C3 plants, which probably explains the low conductance to CO2 across the bundle sheath of C4 plants (Brown and Byrd, 1993; Gillon and Yakir, 2000). Besides the potential role on CO2 diffusion at the leaf mesophyll, CAs were also shown to be upstream regulators of CO2 controlled stomatal movements in guard cells (Hu et al., 2010b). Transcriptome analyses showed that CA genes CA1, CA4, CA6 are highly expressed in both stomatal guard cells and mesophyll cells (Leonhardt et al., 2004; Yang et al., 2008). In rice seedlings, expression of the gene (OsCA1) coding for CA in leaves and roots was induced by salt and osmotic stress (Yu et al., 2007). In Arabidopsis, overexpression of the OsCA1 gene improved growth under NaCl as compared to the wild type, suggesting a beneficial effect of CA activity on growth under saline conditions. Considering the role of CA in CO2 diffusion, future breeding strategies (either of C3 or C4), aiming at crop improvement with regards to both water and saline stress, may involve changes in the expression of CA in guard (and mesophyll) cells.
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2. The role of aquaporins Aquaporins are abundant in the vacuolar and plasma membranes, including mesophyll cells of higher plants (Terashima and Ono, 2002). There is increasing evidence that aquaporins besides facilitating water transport, may also facilitate CO2 diffusion across plasma membrane (Hanba et al., 2004; Katsuhara and Hanba, 2008; Martre et al., 2002; Maurel and Chrispeels, 2001; Terashima and Ono, 2002; Tyerman et al., 2002). This characteristic might be of interest under reduced CO2 availability, for example, under mild to severe water and saline stress. Nevertheless, the relationship between aquaporins and water stress resistance is still elusive (Aharon et al., 2003). In Arabidopsis, when the expression of plasma membrane aquaporins (PIPs) was reduced, there was an increase in the time needed for recovery of hydraulic conductance and transpiration compared to plants normally expressing PIPs (Martre et al., 2002). Aquaporins were also shown to be involved in the recovery of winter embolism in walnut trees (Sakr et al., 2003) and associated to changes in hydraulic conductance of olive tree leaves (Cochard et al., 2007). Several studies show upregulation of aquaporins in response to water stress in species like A. thaliana (Jang et al., 2007), Phaseolus vulgaris (Aroca et al., 2006), V. vinifera (Galme´s et al., 2007a,b; Vandeleur et al., 2009) and tobacco (Aharon et al., 2003; Jang et al., 2007; Siefritz et al., 2002). This is in line with the suggestion by Vandeleur et al. (2009) that increased aquaporin activity is part of an adaptation strategy to water stress. However, and in contrast with this, gene expression studies in various species adapted to desert climate showed a downregulation of aquaporins that reduced water loss. This is the case of Opuntia acanthocarpa (Martre et al., 2001) or Agave deserti (North et al., 2004). Identical behaviour was observed in Populus spp. (Martre et al., 2001; Siemens and Zwiazek, 2003). Although the reduction of gm under water stress may be partly linked to physical changes in the structure of intercellular spaces due to leaf shrinkage (Lawlor and Cornic, 2002), the fine and rapid regulation of gm in response to varying environmental conditions are likely to be explained by alterations in the expression and/or regulation of PIPs (Flexas et al., 2006; Hanba et al., 2004; Uehlein et al., 2003). 3. Stomatal and photosynthetic patchiness under stress Stomatal distribution and/or mean stomatal conductance can vary significantly among adjacent micro-areas, often corresponding to the surface of areoles. Such non-random, spatially arranged variation in gs (and in the dimensions of stomatal aperture) is called ‘‘stomatal patchiness’’ (Mott and Peak, 2007; Pospı´solova´ and Sˆantrucˇek, 1994). The phenomenon occurs in a
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large number of families and species and is induced by multiple factors (Table I). It characterizes by formation of patches of different stomatal opening, with diverse size, shape, movement and is often transient in nature (Mott and Peak, 2007). Stomatal patchiness is relevant for plant physiology because suggests the existence of still unrecognized mechanisms of stomatal functioning (Mott and Buckley, 1998). However, the physiological consequences of the ‘‘patchy’’ stomatal behaviour remain unclear. Patchiness of stomatal conductance is almost always detrimental to instantaneous WUE (Mott and Peak, 2007) and is not consistent with an optimal behaviour of stomata (Buckley et al., 1999). In turn, several authors emphasize that patchiness may have a minor effect on Ci (Buckley et al., 1997) and that it can have a much less important effect on plant assimilation and A/Ci response than once thought (Lawlor and Cornic, 2002; Lawlor and Tezara, 2009). Flexas and Medrano (2002) also concluded that for conductance to water vapour above 30–40 mmol H2O m 2 s 1 the effects of patchy stomatal closure on Ci calculation were small. Patchy stomatal closure is usually more common in pot experiments, where plants experience a rapid dehydration, than under field conditions, where drought is imposed slowly (Grassi and Magnani, 2005). Patchy stomatal conductance cannot be detected unequivocally with standard gasexchange techniques. It has been monitored by using techniques such as starch staining, pressure infiltration and autoradiography and imaging (see Table I). The different interpretation on the impact of patchiness on plant physiology may result not only from the use of different species but also from different methodologies (Table I). Stomatal patchiness can occur in any plant leaf but mostly in heterobaric ones (Beyschlag and Eckstein, 2001). These leaves are characterized by extensions of the vascular bundles that span across the leaf, forming a physical barrier to lateral gaseous diffusion and resulting in the discrete compartmentation of the leaf and no lateral CO2 diffusion between neighbouring areoles. This is more evident in response to water stress (Meyer and Genty, 1999) or ABA treatment (Meyer and Genty, 1998). Homobaric leaves are deprived of those bundle sheath extensions and have large interconnected intercellular air space with few or no barriers for lateral gas fluxes. They should be therefore less prone to patchiness than heterobaric leaves. However, according to Morison et al. (2007), there is no dichotomy between species considered homobaric and heterobaric, but rather a gradation which depends on extension and size of bundle sheath extensions. According to Morison et al. (2007), the relevance of lateral CO2 diffusion depends not only on stomatal conductance and net assimilation but also on lateral permeability. Lateral
TABLE I Non-Exaustive List of Literature Describing the Stomatal/Photosynthetic Patchiness in Leaves of Different Species and Anatomy (Homobaric, H; Heterobaric, Het) in Response to Different Factors
Species
Leaf anatomy
Factor inducing patchiness ABA in xylem No treatment (diurnal variation) Low humidity
Detection method
Vicia Faba Arbutos unedo
H Het
Starch staining Liquid infiltration
Olea europea
H
Quercus coccifera L.
Het
Gossypium hirsutum Xanthium strumarium Rosa rubiginosa L.
Het Het Het
Nicotiana plumbaginifolia Nicotiana plumbaginifolia
Het Het
Rosa rubiginosa
Het
No treatment (diurnal variation) Drought Low humidity Darkness to high PPFD transition Exogenous ABA Changes CO2 concentration, light intensity and leaf detachment Water stress and ABA
Vitis vinifera, Nerium Oleander Rosa rubiginosa
Het Het
Water stress Exogenous ABA
14
Phaseolus vulgaris
Het
Exogenous ABA
IR imaging
14
CO2 autoradiography
Liquid Infiltration
Reference Terashima et al. (1988) Beyschlag and Pfanz (1990) Loreto and Sharkey (1990) Beyschlag et al. (1992)
14
CO2 autoradiography Chl a imaging Chl a imaging
Wise et al. (1992) Mott et al. (1993) Bro et al. (1996)
Chl a imaging Chl a imaging
Eckstein et al. (1996) Eckstein et al. (1998)
Chl a imaging
Meyer and Genty (1999) Downton et al. (1988) Meyer and Genty (1998) Jones (1999)
CO2 autoradiography Chl a imaging
Rosa hybrida Avena sativa Xanthium strumarium L. Gossypium hirsutum L. Tradescantia virginiana Nicotiana tabacum Helianthus annus Phaseolus vulgaris Vicia faba Glycine max Vicia faba
Het Het Het H Het Het H Het H
Leaves from excised cuttings No treatment (attached leaves) Decrease in air humidity No treatment (attached leaves) Rapid desiccation of excised well-watered leaves CO2 concentration Light intensity Light intensity
Chl imaging IR imaging Chl imaging þ IR imaging Chl imaging Chl a imaging
Costa (2002) Prytz et al. (2003) West et al. (2005) Marenco et al. (2006) Nejad et al. (2006)
Chl a imaging Chl a imaging Chl a imaging
Morison et al. (2007) Morison et al. (2007) Pieruschka et al. (2008)
Light intensity and water stress
Chl a imaging
Pieruschka et al. (2010)
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diffusion of CO2, in addition to vertical CO2 diffusion through stomata, has been shown to promote CO2 assimilation in both homo- and heterobaric leaves, in particular when photosynthesis rates were low and stomata were closed (Morison et al., 2007). Pieruschka et al. (2008, 2010) concluded that lateral CO2 diffusion supports photosynthesis in some species whereas in others like Glycine max, which is heterobaric, it did not. Stomatal patchiness has been considered a resultant of heterogeneous water status in different parts of the leaf (Beyschlag and Eckstein, 2001). Hydraulic interactions among stomata were suggested as one of the possible mechanisms involved in formation and movement of stomatal conductance patches. Stomata can interact locally via hydraulic interactions of epidermal cells (Mott and Franks, 2001; Mott et al., 1997) and these hydraulic interactions can serve to coordinate the movements of adjacent stomata (Mott et al., 1999). In a recent paper, Nardini et al. (2008) suggest that spatial stomatal heterogeneity may arise from heterogeneous distribution of local hydraulic resistances, which would maintain local water potential above critical values, possibly by triggering vein cavitation. B. BIOCHEMICAL AND METABOLIC LIMITATIONS
Metabolic potential of photosynthesis is determined by the amount and activity of light harvesting components, electron transport components and energy transduction processes, as well as by carbon metabolism, namely the Calvin cycle enzymes involved in carboxylation (Rubisco) and in the regeneration processes of ribulose 1–5 bisphosphate (RuBP). The photosynthetic apparatus is generally considered very resistant to mild and moderate water stress (Chaves et al., 2002, 2009; Cornic, 2000; Flexas et al., 2004; Warren et al., 2004), with stomata being the main limiting factor to carbon uptake under such conditions (Angelopoulos et al., 1996; Cornic et al., 1992; Kaiser, 1987). However, as water deficit progresses biochemical limitations become apparent. The most frequently reported biochemical mechanisms involved in drought-related downregulation of photosynthesis are: limitations of phosphorylation (Lawlor and Tezara, 2009; Tezara et al., 1999), RuBP regeneration (Medrano et al., 2002) and Rubisco carboxylation (Parry et al., 2002; Zhou et al., 2007). All enzymes related to these main mechanisms may experience a decrease in activity and/or amount under stress. Flexas and Medrano (2002) suggested that in C3 plants alterations in Rubisco activity had a minor role in the drought induced limitation of photosynthesis. However, literature describes discrepant results on the negative effects of drought on Rubisco activity and/or quantity, which can vary
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65
from major reductions (Maroco et al., 2002; Parry et al., 2002) to minor decrease (Flexas et al., 2006) or no effect at all (Delfine et al., 2001; Pankovic et al., 1999). Such conflicting results can be related to the use of different species and/or different experimental conditions (long-term vs. short-term experiments) (Damour et al., 2008; Flexas et al., 2006; Parry et al., 2002). At present atmospheric CO2 conditions (well below saturating values for photosynthesis) the carboxylation rate by Rubisco is the major factor limiting photosynthesis and not the electron transport nor the regeneration of the RuBP (Long et al., 2004). Under drought, stomatal closure will reduce CO2 availability, which leads to low CO2 operating range and the carboxylation by Rubisco will arise as a major rate limiting factor of net assimilation (Flexas et al., 2004; Grassi and Magnani, 2005). Under severe, long-term drought together with high temperatures and irradiance will result in limited net assimilation and an unbalance between PSII photochemical activity and the electron requirement for photosynthesis, leading to an over-excitation and, subsequently, photoinhibitory phenomena (Epron et al., 1992). The damage caused to PSII is associated with lightinduced oxidative stress. Indeed, active oxygen species (H2O2, OH and O2) are produced when photon absorption exceeds the rate of photon utilization (Foyer and Noctor, 2000; Quartacci et al., 2002). Drought stress usually leads to an increase of starch hydrolysis and reduced sucrose translocation, with the maintenance and/or even the buildup of the concentration of reducing sugars in leaf tissue (Bunce, 1982; Chaves, 1991; Campos et al., 1999; de Souza et al., 2005). Such build-up of sugars in leaves may have a protective effect (osmotic protection). The osmotic contribution of sugars may also be essential to the build-up of a driving force for roots to uptake water from the soil (Silveira et al., 2010). Moreover, and although there is no clear evidence for feedback inhibition of photosynthesis by sugars under drought, carbohydrate accumulation may be associated with lower Rubisco activation state and repression of photosynthetic genes expression (Paul and Foyer, 2001; Sheen, 1990). Proline accumulation is another common metabolic response of higher plants to both drought and salinity stress (Taylor, 1996; Hare et al., 1999, for a review). Metabolic limitations occurring under high salinity are related with the high concentrations of Naþ and Cl in leaf tissue (in general above 250 mM) (Munns et al., 2006). The depletion of organic acids accompanies stomatal closure and decreased assimilation. Such a reduction of the organic acids under high salinity may work as a compensatory mechanism for ionic imbalance (Chaves et al., 2009; Sanchez et al., 2007). At higher concentrations, NaCl may directly inhibit photosynthesis due to oxidative stress (Chaves
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et al., 2009). In Sorghum under salinity, Netondo et al. (2004) reported a significant decrease in maximum quantum yield of photosystem II, photochemical quenching coefficient and electron transport rate and an increase in the non-photochemical quenching (qN). However, in studies with different rice cultivars with contrasting salinity tolerance, the potential quantum yield of PSII (Fv/Fm) was almost not affected by salt stress, whereas qN increased in sensitive cultivars with increasing salt stress (Dionisio-Sese and Tobita, 2000). It is possible that sensitivity to salt stress in cereals might be related with a reduction in PSII photochemical efficiency associated with an enhanced qN, as means to dissipate excess energy (Moradi and Ismail, 2007).
IV. IS WATER USE OPTIMIZED BY THE LEAVES UNDER WATER DEFICITS? In leaves under water deficits, caused either by drought or by salinity, the coordinated regulation of gas exchange (water vapour vs. carbon dioxide) takes place. Such a tight coordination between photosynthetic CO2 assimilation and leaf water loss determines land plant survival by preventing desiccation, while allowing some CO2 to enter into the leaf (Nilson and Assmann, 2007). Since the early work by Cowan and co-workers (Wong et al., 1979, 1985), Sharkey and Raschke (1981) and Schulze and Hall (1982) that the parallel response of stomata and photosynthesis observed in the long-term response to different environmental variables has inspired the hypothesis of an ‘‘optimization’’ theory of stomatal opening that may have accompanied plant evolution (Cowan and Farquhar, 1977). In other words, stomatal conductance would be coordinated with the CO2 requirement of the mesophyll, leading to the maintenance of the ratio intercellular CO2 partial pressure/ external CO2 partial pressure (pi/pa) for variable conditions of CO2, light or N nutrition, except when changes were too fast to produce a decrease in photosynthesis with stomata still open (Wong et al., 1979). This assumption had long supported empirical models of photosynthesis (Ball et al., 1987; Leuning, 1995). The question still not fully clarified concerns the mechanism by which guard cells responds to internal CO2 thus creating this correlation between photosynthetic capacity and stomatal conductance. Propositions included several metabolites – ATP, NADPH or RuBP (Farquhar and Wong, 1984) that would act as signals from the mesophyll to stomata or from the guard cells (zeaxanthin) to stomata (Zeiger and Zhu, 1998). Later work using transgenic plants with reduced Rubisco showed that the correlation between photosynthetic capacity and stomatal conductance can
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be broken—indeed stomatal conductance was not affected in the plants with reduced amounts of Rubisco and lower photosynthetic capacity, when they were growing under high light (Krapp et al., 1994; Quick et al., 1991; von Caemmerer et al., 1997). More recently, von Caemmerer et al. (2004), also working with transgenic tobacco plants with reduced Rubisco, confirmed that stomatal conductance is not tightly linked to the photosynthetic capacity of guard cells or the mesophyll and further suggested that either pa (partial pressure of atmospheric CO2) or CO2 in the stomatal pore may be sensed by stomata, instead of pi. Under well-watered conditions and large stomatal conductance, pi is generally around 70–80% of the pa. Under mild to moderate water deficits stomata limit CO2 access to the mesophyll but the photosynthetic demand for CO2 keeps the same, and pi values will decrease to 60–70% of pa (Chaves et al., 2004). This explains why under mild to moderate water deficits an increase in the ratio of carbon assimilated (A) by the leaf and the corresponding water transpired (termed intrinsic water use efficiency— WUEi, for the ratio between A and gs), is commonly observed (Chaves and Oliveira, 2004). This is more evident in C4 plants, because their CO2 uptake is less sensitive to the initial decline in gs than C3 plants (Ghannoum et al., 2002; Long, 1999). In contrast, when leaf water deficits becomes severe an increase in pi is observed due to the decline of net assimilation induced by nondiffusive limitations, with a consequent decrease in WUEi. Summarizing, pi tends to be maintained constant in leaves of plants kept in absence of water or salinity stress. When tissue water deficits is installed, stomata respond to leaf water potential, and both respond to and control the supply and loss of water by leaves (Leuning et al, 2003). Under these circumstances, the balance between the supply of CO2 to the chloroplast (a function of the diffusion from the air to the site of carboxylation) and the demand for CO2 by photosynthesis, governed by chloroplast biochemistry, irradiance or sink strength (Chaves et al., 2004) will dictate the pi and WUEi.
V. HOW ARE STOMATA AND PHOTOSYNTHETIC GENES REGULATED? A. REGULATION OF STOMATAL APERTURE
As discussed above, environmental signals, such as drought and high salinity, regulate stomatal pore opening and closure. By controlling CO2 uptake and transpired water vapour, stomata play a crucial role in abiotic stress tolerance. The phytohormone ABA is the primary signal controlling the reactions
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of guard cells to either drought or high salinity (Chaerle et al., 2005; Wasilewska et al., 2008). In response to these stresses, plant ABA concentration increases, thus modulating the expression of target genes and controlling adaptive physiological responses, both leading to stomata closure (Christmann et al., 2007; Rabbani et al., 2003; Seki et al., 2002a). It is therefore essential to better understand the molecular mechanisms underlying stomata responses to water deficit. During many years, ABA was assigned as the long-distance signal that communicates water stress from the root to the shoot (Wilkinson and Davies, 2002). However, it was recently observed in Arabidopsis that, although the water deficit response does require ABA biosynthesis and signalling in the shoot, it is not affected by the capacity to generate ABA in the root. Instead, water deficit seems to elicit a root-to-shoot communication by a hydraulic signal, which precedes ABA biosynthesis in the shoot and consequent signalling, followed by stomatal closure (Christmann et al., 2007). ABA-induced stomatal closure requires the coordinate control of several cellular processes, such as guard cell turgor, cytoskeleton organization, membrane trafficking and gene expression (Hetherington, 2001) and is mediated by a complex, guard cell signalling network of kinases/phosphatases, secondary messengers, and ion channel regulation (Kim et al., 2010; Wasilewska et al., 2008). An enormous number of signalling components that affect ABA-dependent stomatal closing have been identified by forward and reverse genetic approaches. Most of the Arabidopsis mutants with impaired ABA-mediated stomatal responses correspond to guard cell signalling components (Table II). 1. ABA perception Upon perception of a stress signal ABA biosynthesis is induced primarily in vascular tissues and ABA is exported into other cells by specific ATPdependent transporters (Kang et al., 2010; Kuromori et al., 2010). This mechanism allows the rapid distribution of ABA to the guard cells, where it triggers stomatal closure through changes of ion fluxes (Fig. 1). ABA perception by the guard cells is undertaken by ABA receptors, whose identity has remained under debate until recently (McCourt and Creelman, 2008). Before the discovery of the RCAR/PYR1/PYL-PP2C complexes (Ma et al., 2009; Park et al., 2009), a number of ABA-binding proteins, such as ABAR/ CHLH/GUN5 (Shen et al., 2006), the plasma membrane-localized GCR2 (Liu et al., 2007) and GTG1/GTG2 (Pandey et al., 2009) had already been reported as ABA receptors. They affect ABA responses and are likely involved in the network of ABA responses. However, how these ABAbinding proteins are placed into the molecular events governing the main
69
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TABLE II Non-Exhaustive List of Arabidopsis Mutants Showing Alterations in Stomatal Response to Environmental Cues Mutant abi1 los5/aba3
atmpr4
atmpr5 atmyb60-1 atmyb61
atabcb14 nced3 atabcg40
ost1 ost2 slac1 gork gpa1
gtg1gtg2
Phenotype
Reference
Stomata of abi1-1 are more open than are those of the wild type Reduced accumulation of ABA under drought stress and consequent increased transpirational water loss Stomatal aperture in atmrp4 mutant alleles was larger than in wild-type plants, resulting in increased water loss Limited stomatal opening induced by light Constitutive reduction in stomatal opening and decreased wilting under water stress conditions atmyb61 mutants do not close their stomata to as great an extent as wild-type plants in response to darkness Guard cells close more rapidly in response to high CO2 Stomata are less responsive to drought due to reduced ABA biosynthesis Mutant stomata closed more slowly in response to ABA, resulting in reduced drought tolerance ost1 mutants showed reduced ABA responsiveness in guard cells ost2 responds to CO2 and darkness, but not to ABA The slac1 mutations impair CO2-, ABA- and dark-induced stomatal closure Impaired stomatal closure in response to darkness or the stress hormone abscisic acid Reduced stomatal density and index. More sensitive to lowCO2-induced stomatal opening. Insensitivity in aspects of guard cell ABA responses gtg1 gtg2 mutants are hyposensitive to ABA
Koornneef et al. (1984) Xiong et al. (2001)
Klein et al. (2004)
Klein et al. (2003) Cominelli et al. (2005) Liang et al. (2005)
Lee et al. (2008) Iuchi et al. (2001) Kang et al. (2010)
Merlot et al. (2002) Merlot et al. (2002, 2007) Negi et al. (2008) Hosy et al. (2003) Nilson and Assmann (2010) Pandey and Assmann (2004) Pandey et al. (2009) (continues)
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TABLE II Mutant mpk9-1/12-1
pyr1pyl1pyl2pyl4
dst
AtrbohD AtrbohF era1 abh1 gcr1 pp2ca-2 tpk1 abo1-1
rdc3 ozs1
dri1 hit1
(continued )
Phenotype ABA and calcium failed to activate anion channels in guard cells of mpk9-1/12-1. Mutation in only 1 of these genes did not show any altered phenotype. ABA-induced stomatal closure and ABA-inhibition of stomatal opening are impaired in quadruple mutant plants. Increased stomatal closure and reduced stomatal density, consequently resulting in enhanced drought and salt tolerance in rice Impaired ABA-induced stomatal closure Impaired ABA-induced stomatal closure ABA hypersensitivity of guard cell anion-channel activation and of stomatal closure BA-hypersensitive stomatal closure and reduced wilting during drought Hypersensitivity to ABA in assays of stomatal response Stomatal closure is ABA hypersensitive ABA-induced stomatal closure is slowed The closure of abo1-1 stomata in response to ABA treatment was greatly enhanced compared to that of the wild type gs is significantly higher in control conditions and exhibited no O3-induced closure gs levels and the size of stomatal apertures are greater than in the wild type. Mutant and wild-type plants responded similarly to environmental stimuli Reduced stomatal response to H2O2, CO2 and ABA Impaired CO2 response, but shows functional responses to blue light, fusicoccin and ABA
Reference Jammes et al. (2009)
Nishimura et al. (2010)
Huang et al. (2009)
Kwak et al. (2003) Kwak et al. (2003) Pei et al. (1998) Hugouvieux et al. (2001) Pandey and Assmann (2004) Kuhn et al. (2006) Gobert et al. (2007) Chen et al. (2006)
Overmyer et al. (2008) Saji et al. (2008)
Song et al. (2006) Hashimoto et al. (2006a,b)
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Fig. 1. Guard cell signalling and ion channel regulation leading to stomatal closure in plants under water deficit conditions. Water shortage induces ABA accumulation, which is perceived by the ABA receptor. The receptor is formed by the heteromeric complex of a PP2C such as ABI1 and an ABA-binding RCAR/PYR1 member. ABA receptor is present in both cytosol and nucleus. Without ABA, the phosphatase activity of the PP2C inhibits the activity of the protein kinase OST1 and related SnRKs. In the presence of ABA, the phosphatase activity of the receptor is blocked and the protein kinases released from inhibition. OST1 and related SnRKs, in a Ca2þ-dependent or Ca2þ-independent way, regulate several events leading to stomatal closure. Dashed lines represent regulation by ABA through SnRKs or other proteins (??). Abbreviations: ABA, abscisic acid; ABI1, ABA insensitive 1; PP2C, protein phosphatase 2C; RCAR/PYR1, regulatory component of ABA receptor 1/pyrabactin resistante 1; OST1, open stomata 1; RBOHF, respiratory burst oxidase homologue F; AHA1/OST2, Arabidopsis Hþ ATPase 1/open stomata 2; SLAC1, slow anion channel associated 1; GAP1, G protein alpha 1; KAT1/2, Kþ transporter of Arabidopsis thaliana 1/2; GORK1, guard cell outward rectifying Kþ-channel 1; CDPK, calcium-dependent protein kinase; S-type, slow-type; R-type, rapid-type; VK, vacuolar Kþ selective; TPK1, two pore Kþ channel; CE, cis-element.
ABA responses remains to be clearly determined and their role as ABA receptors is largely controversial. Thus, the recent discovery of RCAR/ PYR1/PYL-PP2C, which was shown to control the main ABA responses (Ma et al., 2009), has paved the way to understand the main ABA signalling events leading to gene regulation and ion channel control. The identification of RCAR/PYR1/PYL was obtained by two independent groups using
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different approaches. Using an Arabidopsis screening to identify pyrabactin (selective ABA agonist)—insensitive mutants, Sean Cutler’s group identified PYRABACTIN RESISTANTE 1 (PYR1) (Park et al., 2009). PYR1 and several PYR1-Like (PYL) homologues were then characterized as ABAdependent inhibitors of group A type 2C protein phosphatases (PP2C), such as ABI1 (ABA INSENSITIVE 1) ABI2, HAB1 (HYPERSENSITIVE TO ABA 1), and HAB2, which are known to negatively regulate the ABA signalling at an early step in the pathway (Merlot et al., 2001; Moes et al., 2008; Saez et al., 2006). On the other hand, Erwin Grill’s group, using a Yeast two Hybrid approach, identified the REGULATORY COMPONENT OF ABA RECEPTOR 1 (RCAR1), identical to PYL9, as an ABI1 and ABI2—interacting proteins (Ma et al., 2009). They also showed that RCAR1 and its homologues bind ABA with strong affinity and with stereoselectivity. In addition, the RCAR1–ABA complex inhibits certain PP2C, such as ABI1, ABI2, HAB1, and HAB2. Given that six PP2C proteins from clade A are related with ABA responses and there are 14 RCARs, an enormous number of combinatorial interactions would be possible if all RCAR members can regulate the same PP2C. It is believed that different RCAR/PP2C complexes may vary in their affinity to the hormone and address different downstream signalling elements, thus allowing the adjustment of the ABA signalling to the broad range of ABA levels (Raghavendra et al., 2010), induced by different intensities of water deficit. Interestingly, it was shown in Arabidopsis that ABA stimulates PYR1/ABI1 interactions within 5 min. (Nishimura et al., 2010). 2. ABA signalling transduction ABA signalling transduction is another important aspect of stomatal regulation. Recently, a series of crystallographic studies have clearly demonstrated that interaction of PP2C with the hydrophobic surface of ABA-bound receptors inhibits PP2C activity (Melcher et al., 2009; Miyazono et al., 2009; Yin et al., 2009). This will then activate the SnRK2 protein kinase SnRK2.6/OST1 (OPEN STOMATA 1), which functions as positive regulator of ABA-induced stomatal closure (Figure 1) (Mustilli et al., 2002). Thus, in non-stress conditions, the PP2C protein ABI1 interacts with OST1 in vitro and negatively regulates ABA-induced OST1 kinase activity (Yoshida et al., 2006). In response to water deficit, ABA increase will avoid inactivation of OST1, which thereby activates the S-type anion channel SLAC1 (SLOW ANION CHANNEL ASSOCIATED 1) (Geiger et al., 2009; Lee et al., 2009) and inhibits the Kþ inward channel KAT1 by phosphorylation (Sato et al., 2009) (Figure 1).
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The SLAC1 is also regulated by calcium-dependent protein kinases (CDPK) (Geiger et al., 2010). Interestingly, the guard cell outward rectifying Kþ-channel GORK is up regulated by drought and salt and its regulation is mediated by ABI1 and ABI2 (Becker et al., 2003); however, whether OST1 (or other protein downstream PP2C) is also involved in GORK regulation is not yet known. OST1 also interacts with and activates the AtRBOHF (RESPIRATORY BURST OXIDASE HOMOLOGUE F), a plasma membrane localized NADPH oxidase that generates H2O2 (Sirichandra et al., 2009a). H2O2 increases is known to mediate stomatal closure through activation of calcium channels (Pei et al., 2000). The ABA-induced signalling transduction mechanisms in guard cells include two pathways, a Ca2þ-dependent and a Ca2þ elevation-independent pathway, which might cross talk to regulate stomata movements (Fig. 1) (Kim et al., 2010). For instance, KAT1 and SLAC1 channels are reciprocally regulated by both pathways (Siegel et al., 2009). Other signals also known to be involved in ABA-induced stomatal closure are: cytoplasmic pH; lipidbased signalling molecules such as inositol 1,4,5-triphosphate (IP3), sphingosine-1-phosphate (SP1), inositol hexakisphosphate (IP6), and phosphatidic acid (PA); and production of signal compounds such as nitric oxide (NO) and malate catabolism (Kim et al., 2010). Besides the regulation of other pumps/channels, these signals have been proposed to inhibit the Hþ pump AHA1/OST2 (ARABIDOPSIS Hþ-ATPASE/OPEN STOMATA 2) activity, in a Ca2þ-dependent way, thus contributing to stomatal closure (Sirichandra et al., 2009b). ABA-induced stomatal closure is also mediated by the vacuolar potassium channel TPK (TWO PORE Kþ CHANNEL1) (Gobert et al., 2007) and the ABC transporter AtMRP5 (MULTIDRUG RESISTANT PROTEIN 5), which is a high affinity IP6 transporter (Nagy et al., 2009). The -subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency. GPA1 regulates stomata density via the control of epidermal cell size and stomata formation (Nilson and Assmann, 2010), but also regulates potassium and anion channels in guard cells (Wang et al., 2001). 3. Transcription factors The control of stomatal aperture by ABA signalling involves the transcriptional regulation of many genes, however, little is known about the transcription factors (TFs) involved. Although the role of the bZIP domain proteins in ABA signalling (through their interaction with gene promoters containing ABRE motifs) is well established, none of these TFs have yet been shown to influence stomatal movements under abiotic stress conditions. On the other hand, three MYB TFs from the subclass R2R3 were already reported to be
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involved in stomatal movements in response to environmental stimuli. AtMYB60 was shown to be specifically expressed in guard cells, and its expression regulated by light conditions, ABA and water stress (Cominelli et al., 2005). Its expression is negatively regulated under drought, concomitantly with stomatal closure. However, elevated CO2 concentrations, which are known to induce stomatal closure, do not modulate AtMYB60 expression. The atmyb60-1 null mutant shows a constitutive reduction in stomatal opening and decreased wilting under water stress conditions. Interestingly, many genes altered in atmyb60-1 (e.g. Aquaporin, ERD10, ERD13 and ERF) are known to be involved in plant response to water stress (Cominelli et al., 2005). AtMYB61 gene is also specifically expressed in guard cells in a manner consistent with its involvement in the regulation of stomatal aperture (Liang et al., 2005). While AtMYB60 gene expression is induced by light, leading to an increased stomatal aperture, AtMYB61 transcription is repressed by light, although it has the same effect on stomatal movement. AtMYB61 gene expression was not altered in plants treated with ABA, salt, and drought, known to induce stomatal closure. Although AtMYB61 seems to act in a mechanism parallel to that responsible for closing stomata in response to water deficit, a post transcriptional/translational regulation of AtMYB61 cannot be ruled out. The AtMYB44 gene is expressed in the vasculature and leaf epidermal guard cells and is rapidly activated (within 30 min) under dehydration, salinity, low temperature and ABA treatment (Jung et al., 2008). When AtMYB44 is over-expressed in Arabidopsis, plants show a higher sensitivity to ABA and a more rapid ABA-induced stomatal closure then wild type. In addition, these transgenic plants exhibit a reduced rate of water loss and an enhanced abiotic stress tolerance, mediated by the reduced activation of PP2C genes, which are known as negative regulators of ABA signalling. DROUGHT AND SALT TOLERANCE (DST) is a zincfinger TF that regulates drought and salt tolerance via stomatal aperture control (Huang et al., 2009). It negatively regulates stomatal closure by direct modulation of genes related to H2O2 homeostasis. Interestingly, loss of DST function, besides increasing stomatal closure, also reduces stomatal density, thus indicating a putative function in the abiotic stress control of stomata development. The control of stomata aperture may also involve the activity of the TF SNAC1. Transgenic rice plants over-expressing SNAC1 are more sensitive to ABA and lose water more slowly by closing more stomata, thus showing an enhanced tolerance to drought and high salinity (Hu et al., 2006). Although we have focused our review on the ABA-mediated stomatal responses, it is likely that other signals can control stomatal movement independently of ABA. This is suggested, for instance, by the ABA-insensitive mutant mrp5, which shows limited stomatal opening induced by light. In
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addition, in the ABA insensitive mutant abi1-1 stomatal closure can be induced by Cd2þ (Perfus-Barbeoch et al., 2002). However, this is an area still largely unknown.
B. STOMATAL DEVELOPMENT
Significant advances have been made in Arabidopsis to understand the basic genetic pathways controlling stomatal development, which requires a strict control of a series of asymmetric and symmetric cell divisions in a specialized epidermal cell lineage, followed by cell differentiation. Stomatal development is initiated by an asymmetric division of a meristemoid mother cell (MMC) producing a small meristemoid and a larger sister cell. This division is regulated by the bHLH TF SPEECHLESS (SPCH) (MacAlister et al., 2007) and requires the novel plant specific protein BASL (Dong et al., 2009). The meristemoid has limited self-renewing capabilities and after one to three divisions differentiates into a guard mother cell (GMC). This transition is controlled by MUTE (MacAlister et al., 2007; Pillitteri et al., 2007), while the final differentiation step is regulated by FAMA (Ohashi-Ito and Bergmann, 2006). Both MUTE and FAMA are also bHLH TFs. It was recently shown that despite the different stomata morphologies in monocots and in dicots, FAMA function is conserved, whereas the roles of MUTE and two SPCH paralogs are somewhat divergent (Liu et al., 2009). In Arabidopsis, two further bHLH TFs, ICE1/SCRM1 and SCRM2, directly interact with and specify the sequential actions of SPCH, MUTE and FAMA (Kanaoka et al., 2008). In addition, the TOO MANY MOUTHS (TMM) leucine rich repeat receptor-like protein (Nadeau and Sack, 2002), which is involved in the spacing mechanism that inhibits the development of adjacent stomata (the one-cell spacing rule), was proposed to associate with members of the ERECTA family of LRR-receptor-like kinases (ER, ERL1 and ERL2) and to negatively regulate several aspects of stomatal differentiation (Shpak et al., 2005). Interestingly, the ERECTA protein is known to regulate plant transpiration efficiency in Arabidopsis through modulation of stomatal density (Masle et al., 2005). The -subunit of the heterodimeric G protein, GPA1, is another regulator of the plant transpiration efficiency and similarly to ERECTA was also shown to have a function in stomatal development (Nilson and Assmann, 2010). Two related secreted peptides, EPIDERMAL PATTERNING FACTOR 1 and 2, were also found to negatively regulate stomatal development and their function is dependent on TMM and ER-family function (Hara et al., 2007; Hunt and Gray, 2009). If EPF1 and/ or EPF2 prove to be peptide ligands for those receptors, and particularly for
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ERECTA, it would be important to investigate whether they also play a role in plant transpiration efficiency. Recently, another secreted peptide STOMAGEN was reported as a mesophyll-derived positive regulator of stomatal density (Sugano et al., 2010). This intercellular signalling factor suggests that inner photosynthetic tissues may optimize their uptake of CO2 by regulating epidermal stomatal density. Whether STOMAGEN expression mediates stomatal development in plants subjected to abiotic stress has not yet been directly addressed. However, in Arabidopsis, the expression of the STOMAGEN (At4g12970) seems to be differentially regulated by ABA, drought, cold, high salinity, and heat treatments (https://www.genevestigator.com). Although the basic genetic mechanisms regulating stomatal development in Arabidopsis have been rather well characterized (as seen above), how environmental cues are influencing stomatal development is still an unresolved question. We know that to optimize leaf gas exchange under prevailing environmental conditions, plants modulate stomata aperture, but they can also regulate stomata number. Changes in environment, such as increased CO2 in the atmosphere, increased temperatures and changes in light quality, all have impact on stomatal density (Casson and Gray, 2008; Casson et al., 2009). It has also been reported that water availability (Wu et al., 2009) as well as exogenous applied ABA (Franks and Farquhar, 2001) can modulate stomatal development. However, only the HIGH CARBON DIOXIDE (HIC) protein has been shown so far to regulate stomatal development in response to CO2 (Gray et al., 2000) and the PHYTOCROME INTERACTING FACTOR 4 (PIF4) has been suggested to regulate stomatal development in response to light (Casson et al., 2009). It is however predictable that environmental stresses, such as water deficit or high salinity, when perceived by plant cells, are transduced through a signalling pathway that will converge on the proteins involved in stomatal development (e.g. SPCH, MUTE, FAMA, ICE1/SCRM1). Curiously, ICE1/SCRM1 was first identified as an abiotic stress-responsive TF (Chinnusamy et al., 2003), thus showing a potential link between environmental adaptation and stomatal development. In Arabidopsis, the MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6, two environmentally responsive mitogenactivated protein kinases (MAPKs), and their upstream MAPK kinases, MKK4 and MKK5, compose the MKK4/MKK5-MPK3/MPK6 module. This module is downstream YODA, a MAPKKK described as key regulator of stomatal development and patterning (Wang et al., 2007). Recently, it was shown that SPCH, which controls entry into the stomatal lineage, is a substrate of AtMPK3 and AtMPK6, suggesting that SPCH activity may be directly affected by adverse environmental conditions thereby enabling
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the plant to modify stomatal development in response to abiotic stress (Lampard et al., 2008). Curiously, the ABA-overly sensitive mutant abo1 shows a drought-resistant phenotype. The abo1 mutation enhances ABAinduced stomatal closing and also influences development of guard cells, resulting in stomata reduced to half the number compared to the wild type (Chen et al., 2006). This suggests that ABA signalling must be also involved in modulation of stomatal development in response to water deficit. C. EXPRESSION OF PHOTOSYNTHETIC RELATED GENES
In addition to stomatal regulation, adverse environmental conditions modulate photosynthetic rate by controlling expression of genes involved in non-stomatal processes associated with photosynthesis and carbohydrate metabolism. Most genes associated with photosynthesis are under control of a transcriptional regulatory network evolved to control plant response to external stimuli. Transcriptional profiling studies have shown that although some are upregulated, most photosynthesis-related genes and the genes for carbohydrate metabolism are downregulated under drought and high salinity (Chaves et al., 2009; Seki et al., 2002b; Wong et al., 2006). 1. Transcription factors regulating photosynthetic related genes TFs are usually defined as proteins that show sequence-specific DNA binding and are capable of activating and/or repressing gene expression. Regulation of gene expression controls many biological processes, such as cell cycle, metabolic and physiological balance, and responses to the environment (Riechmann et al., 2000). In the last decade, many TFs, belonging to different TF families and sub-families, were shown to be involved in plant responses to adverse environmental conditions, such as high salinity, drought, heat, and low temperatures (Saibo et al., 2009; Yamaguchi-Shinozaki and Shinozaki, 2006). Among the TF families present in plants, AP2/EREBP (APETALA2/ ethylene responsive element binding protein), NAC (NAM, ATAF and CUC), ZF-HD (zinc-finger homeodomain), AREB/ABF (ABA-responsive element binding protein/ABA-binding factor), and MYC (myelocytomatosis oncogene)/MYB (myeloblastosis oncogene) have been the most related with abiotic stress responses. Interestingly, AP2/ERF and NAC proteins are widely present in land plant genomes but no homologue has been identified so far in other eukaryotes (Riechmann et al., 2000). Although the differential expression of photosynthesis-related genes in plants subjected to various abiotic stresses has been demonstrated (Chaves et al., 2009; Seki et al., 2002b; Wong et al., 2006), only few TFs have been
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associated with this process. Among the few examples is the regulation of genes encoding CHLOROPHYLL A/B-BINDING (CAB) proteins of PSII. Two MYB-like TFs from barley, HvMCB1 and HvMCB2, bind specifically to defined regions of CAB promoters derived from barley and wheat. These TFs have characteristic features of transcriptional activators and are required for maximal CAB gene expression, but are not necessary for expression related to light and circadian clock. Interestingly, the transcription level of both genes HvMCB1 and HvMCB2 is downregulated by salt, osmotic, and oxidative stress (Churin et al., 2003). GLK1 and GLK2 are MYB TFs known to regulate genes involved in chlorophyll biosynthesis and light harvesting (Fitter et al., 2002; Waters et al., 2009), and their gene expression in Brassica napus is altered by cold stress (Savitch et al., 2005). Whether these TFs also respond to other abiotic stresses, such as drought or high salinity, is yet to be investigated. Remarkably, MYB TFs seem to be involved in regulation of photosynthetic related gene expression upon abiotic stress. The transcript level of Ppcl and Gapl genes encoding a CAM (Crassulacean Acid Metabolism)-specific isozyme of phosphoenolpyruvate carboxylase and NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, respectively, is upregulated under high salinity stress. The promoters of both genes include several common sequence motifs resembling consensus binding sites for the MYB class of TFs (Schaeffer et al., 1995). The regions where these motives are located were shown to be essential for regulation of transcription by salinity, thus suggesting that MYB-type TFs control expression of Ppcl and Gapl in plants under salt stress conditions. CAM is an adaptation of photosynthetic carbon fixation to water-limited environments that results in improved WUE. Upon water-deficit or salt stress CAM adaptation requires that CAM-specific genes are regulated at transcription level. In addition to MYBs, other TF families may be involved in photosynthesis response to stress. The LONG HYPOCOTYL 5 (HY5) is a bZIP-type TF that regulates transcription of several photosynthesis-related genes, such as CAB2 and RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL SUBUNIT (RBCS1A) and also regulates several stress-responsive genes (e.g. CBF1, DREB2A, RD20 and MYB59) (Lee et al., 2007; Maxwell et al., 2003). It has long been known that ABA controls the transcription of CAB and RBCS genes, as observed in tomato (Bartholomew et al., 1991). Hence, although there is no direct evidence showing that expression of photosynthesis-related genes is regulated by HY5 in response to abiotic stress, this is a likely hypothesis worthwhile being investigated. HAHB4 encodes a sunflower TF belonging to the HD-Zip (sub-family I) and is positively regulated by drought and ABA. The over-expression of this TF in sunflower led to the downregulation of a large group of photosynthesis-related genes
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(e.g. genes encoding components of photosystem I (LHCa) and photosystem II (PSBx), related to chlorophyll biosynthesis, and others that comprise the Calvin cycle, such as PRK and Rubisco) (Manavella et al., 2008). Although the interaction between the TF and the promoters of these genes was not investigated, there is strong evidence that HAHB4 regulates expression of numerous photosynthesis-related genes and, besides light signalling, this regulation may also be triggered by ABA/drought signalling. Recently, it was reported in rice that over-expression of TSRF1, which encodes an ethylene response factor (ERF) involved in drought and osmotic stress responses, induces expression of OsRBCS (Quan et al., 2010). The C2/H2-type zinc-finger proteins STZ and AZF2 from Arabidopsis have been shown to function as transcription repressors under drought, cold, and highsalinity stress conditions and expression of both STZ and AZF2 genes is induced mainly in leaves under drought stress (Sakamoto et al., 2004). It has been suggested that they play a role in the regulation of photosynthesisrelated genes. This hypothesis agrees with the fact that transgenic plants over-expressing STZ show growth retardation, which might be explained by STZ repression of genes related with photosynthesis and carbohydrate metabolism. Phytochrome interacting factors (PIFs) are bHLH TFs acting as negative regulators in light responses. In Arabidopsis, various PIFs were shown to negatively regulate chlorophyll and photosynthesis genes in etiolated seedlings (Shin et al., 2009). In rice, there is at least one PIF, OsPIF, regulated by cold and drought (Figueiredo et al., unpublished results). This suggests that PIFs may also be involved in the regulation of photosynthetic responses to different abiotic stresses.
VI. IMPROVING CARBON FIXATION UNDER ENVIRONMENTAL STRESS? Under low water availability or salinity photosynthesis is predominantly limited by the available CO2 to the catalytic site of Rubisco (as a result of stomatal closure) and therefore by the carboxylation rate. Under restricted CO2 and high temperature the use of O2 as substrate for Rubisco (and therefore photorespiration) will increase. This leads to losses of more than 50% of the carbon fixed by photosynthesis, as demonstrated with transgenic antisense plants with reduced Rubisco, where growth under high light and temperature decreased photosynthesis dramatically (Krapp et al., 1994). This means that the activity of Rubisco will play an important role in determining carbon assimilation by the leaves in stress conditions. However, Calvin cycle enzymes involved in RuBP regeneration may also influence the rate of carbon
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uptake, particularly when CO2 is saturating (Raines, 2003), but also under severe drought conditions (Maroco et al., 2002). These limitations have been successfully overcome through evolution with the appearance of C4 plants that are able to concentrate CO2 in the vicinity of Rubisco and therefore to decrease photorespiration, being more efficient under lower stomatal conductance, high light and high temperatures. Indeed, C4 photosynthesis, contrary to C3, is CO2 saturated in the present atmosphere (Ghannoum et al., 2001) and therefore can cope with restricted intercellular CO2 induced by stomatal closure. Interestingly, C4 plants have evolved when atmospheric CO2 was lowest in the history of the planet earth and occurred several times independently, suggesting that this pathway is a strong ‘‘solution’’ to overcome photorespiration in spite of its complexity (Long et al., 2006; Sage, 2004). By growing plants in an enriched CO2 atmosphere limitations by stomata and carboxylation due to water deficits may also be partially overcome, as demonstrated by a large number of experiments in the last two decades (Chaves and Pereira, 1992; Long et al., 2004; Wullschleger et al., 2002). Indeed, by exposing plants to long-term CO2 enrichment a number of growth and physiological alterations do occur, including some that ameliorate the negative impacts of drought. They include (i) an increase in leaf net photosynthetic rates due to higher carbon substrate availability and the competitive inhibition of the oxygenase activity of Rubisco (decreased photorespiration), (ii) stomatal conductance generally reduced and (iii) increased intrinsic WUE, resulting from higher A and lower gs than under present atmospheric CO2 (Chaves et al., 2001; Gunderson and Wullschleger, 1994). Also observed increases in the root system, whole-plant hydraulic conductance and osmotic adjustment may be important in this context. Herbaceous crops and grasslands are reported to be the most responsive to growth at elevated CO2 (Wullschleger et al., 2002). In addition to using CO2 and O2 as substrates, Rubisco is known to have low efficiency. An improvement in the Rubisco specificity factor for CO2 ( ) by genetic modification has been considered by researchers to improve carboxylation efficiency and ultimately plant performance under water deficits and salinity (Long et al., 2006; Zhu et al., 2004). In a survey with 24 species, it was shown that tends to be higher in plants native from drier, hotter and saline environments with respect to those from more mesophytic climates (Galme´s et al., 2005). The goal of increasing the efficiency photosynthesis of plants by improvements on the kinetics of Rubisco has been a long-term goal scoring modest successes (Mueller-Cajar and Whitney, 2008). Attempts to manipulate Rubisco have not been successful so far and resulted in even a less efficient enzyme (Parry et al., 2003; Spreitzer and Salvucci, 2002).
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Reduced photorespiratory rates could be achieved by introducing enzymes of the C4 pathway into C3 plants, through the combined use of genetic engineering and transgenic technologies (Raines, 2006). Indeed, genes encoding for different genes of the C4 pathway have already been successfully transferred to C3 plants such as rice, and tobacco (Hau¨sler et al., 2001; Ku et al., 1999; Leegood, 2002). Although some interesting results have already been reported, such as an increase synthesis of C4 acids or a decrease in CO2 compensation point, still no clear picture of alterations was obtained. As pointed out by Raines (2006), a way forward in this investigation may also be related to exploring C4 native genes in C3 plants, as pioneered by Hibberd and Quick (2002). In a theoretical analysis of C4 photosynthesis in a C3 cell von Caemmerer (2003) concluded that although energetically inefficient it may ameliorate CO2 diffusion limitation in the mesophyll and therefore have positive impact in C3 photosynthetic response to water deficits. In a future atmosphere with saturating CO2, limitation of photosynthesis will be shifted from carboxylation efficiency to the capacity for regenerating RuBP, the CO2 acceptor of the C3 cycle, which depends on the electron transport capacity and the enzymes of the Calvin cycle (Long et al., 2004). Under such circumstances it would be possible to improve photosynthesis in C3 plants by over-expressing the enzymes of the regenerative phase of the C3 cycle, as revised by Raines (2003).
VII. CONCLUSIONS AND FUTURE PROSPECTS Abiotic stresses, such as drought and high salinity, are amongst the primary causes of crops loss worldwide. Fast increase in world population, scarcer soil and water resources, climate change and more environmentally aware consumers are putting pressure on the agricultural sector to maximize crop yield while guaranteeing a more efficient use of resources (Beddington, 2010; Stefanelli et al., 2010). In this context, breeding to improve crop stress tolerance remains a major goal to guarantee food security and sustainable production. Consequently, increased knowledge on the factors limiting relevant plant processes like photosynthesis under abiotic stress emerged as a major topic of research in plant sciences. Under water stress and high salinity, photosynthesis can be restricted by CO2 diffusion (stomatal and mesophyll related) as well as by photochemistry and carbon metabolism. Considering the inefficiency of Rubisco activity and the limitation in its improvements, possible photosynthetic gains under unfavourable environmental conditions may derive preferentially from optimization of leaf gas exchange, via stomatal and mesophyll control.
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Therefore, it is essential to fully understand the mechanisms regulating stomatal aperture and mesophyll conductance under stress conditions. In the plasma membrane of guard cells several ion transporters involved in stomatal movement have been characterized, but it is still unknown how they are globally regulated. In addition, genetic screens may unveil novel ion transporters. The recent identification of the missing ABA receptor, helped to better understand ABA signalling that controls stomatal aperture under water deficits. Transcriptional regulation of genes involved in guard cell movements may also play an important role in stomatal responses to the environment. These findings can be used in breeding programmes to obtain plants with more responsive guard cells to water scarcity or high salinity. Research on the effects of abiotic stress on development of stomata is required. Genetic screens to isolate mutants with abnormal stomatal development (e.g. stomatal index, size) under moderate water deficit, may help to identify new players involved in this regulation. Photosynthetic limitations due to mesophyll conductance are currently under research and, giving the large variation among species, it is expected to find plants with higher gm (Warren, 2008). Stomatal mutants are being used to decouple stomatal from mesophyll response, which could clarify the possible coordination between stomata and mesophyll regarding gasexchange and plant water relations. The role of different CA on stomatal regulation and of different CA isoforms on mesophyll conductance is being investigated (Fabre et al., 2007; Hu et al., 2010a,b). Future research directions may include as well the clarification of the functional role of aquaporins on leaf gas exchange. Many photosynthetic related genes are regulated by either drought or salinity at the transcription level. Given that only few TFs have been reported as being associated with this regulation, further investigation on the transcriptional network underlying photosynthetic responses to abiotic stress should receive attention. A possible approach may include the identification of TFs that bind to promoters of photosynthetic genes transcriptionally regulated by drought or salinity. On the other hand, identification of targets for stress-responsive TFs will make possible to spot photosynthesis-related genes.
ACKNOWLEDGEMENTS Miguel Costa is supported by a fellowship granted by Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT). Our research work is supported by FCT through the projects POCI/AGR/59079/2004, PPCDT/AGR/61980/2004 and PTDC/BIA-BCM/099836/2008.
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Plants in Extreme Environments: Importance of Protective Compounds in Stress Tolerance
´ SZLO ´ CS,* ´ SZABADOS,*,1 HAJNALKA KOVA LA { AVIAH ZILBERSTEIN AND ALAIN BOUCHEREAU{
*Institute of Plant Biology, Biological Research Center, Szeged, Hungary { Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel { Universite´ de Rennes 1, Campus de Beaulieu, Baˆtiment 14A, Rennes Cedex, France
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Global Metabolic Consequences of Osmotic Stress . . . . . . . . . . . . . . . . . . . . . . . . III. Osmoprotective Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Quaternary Amines: Glycine Betaine........................................ B. Sugars: Trehalose ............................................................... C. Polyalcohols: Mannitol, Pinitol, Inositol ................................... D. Amino Acids: Proline .......................................................... IV. Osmoprotective Compounds and Adaptation to Extreme Environments . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding author: E-mail:
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Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00004-7
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ABSTRACT Extreme environmental conditions such as drought, cold or high soil salinity impede plant growth and require specific adaptation capacity. In response to environmental stresses, a number of low-molecular-weight compounds can accumulate in plants: protective amino acids, sugar alcohols, sugars and betaine-type quaternary amines. The function of these compounds includes the stabilisation of cellular structures, photosynthetic complexes, specific enzymes and other macromolecules, the scavenging of reactive oxygen species or acting as metabolic signals in stress conditions. Although a correlation between the accumulation of certain osmoprotective compounds and stress tolerance certainly exists, a causal relationship between osmolyte accumulation and enhanced tolerance could not always be confirmed. Nevertheless, the importance of osmoprotective compounds for the adaptation to extreme environmental conditions is supported by numerous studies obtained with natural variants, mutants or transgenic plants with different capabilities to accumulate these metabolites. Combining genetic analysis with metabolic profiling approaches could considerably increase our understanding of plant stress responses and the importance of the protective metabolites in the adaptation to stress conditions.
I. INTRODUCTION Environmental conditions determine plant growth and development. Abiotic stresses such as drought, heat, cold and soil salinisation restrain the optimal growth of plants and require certain degree of adaptation to such extreme environments. Climate change needs more intense research on reprogramming of physiological, metabolic events during stress, regulation of growth and development under suboptimal conditions and adaptation of plants to extreme environments. Approximately 40% of the arable earth is arid, semi arid or affected by soil salinity, which reduces crop yield leading to increasing ecological, agronomical and economical impact. A correlation between increased frequency of extreme environmental events and global warming requires more efficient, environmentally compatible agricultural practices including the implementation of new crop cultivars with enhanced tolerance to environmental stresses (Ahuja et al., 2010; Boyer, 1982; Etterson and Shaw, 2001; Gregory et al., 2005; Kintisch, 2009). Water shortage, extreme temperatures or high salinity lead to a depletion of cellular water content, enhance the cellular osmotic potential generating osmotic stress in plants. Physiological responses to drought, cold and salt stress are similar and include reduced shoot growth and photosynthetic activity, accumulation of reactive oxygen species (ROS), alterations in ion transport and compartmentalisation, and changes in metabolite profiles (Lugan et al., 2009, 2010; Munns, 2002; Shulaev et al., 2008). One of the metabolic consequences of osmotic stress is the accumulation of low-molecular-weight organic compounds that do not interfere with normal metabolic reactions and
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are considered to have protective functions (Ashraf and Harris, 2004; Bartels and Sunkar, 2005; Bohnert et al., 1995; Hare et al., 1998; Hasegawa et al., 2000; Yancey et al., 1982). This chapter is intended to give an overview of osmoprotective compounds with an emphasis on recent advances describing their function and significance to adaptation in improving stress tolerance.
II. GLOBAL METABOLIC CONSEQUENCES OF OSMOTIC STRESS Metabolism is of essential functional importance to support growth and development by providing building blocks and energy for plant structures and reserves, to generate signalling compounds for coordination and defence or to produce protective substances to cope with adverse environmental conditions. At a systemic level, metabolism can be viewed as the most integrated (and possibly informative) action reflecting both genotypic and environment-dependent regulations and resulting in the phenotype. Control of energy inputs and outputs is mainly driven through metabolic processes to support optimal growth and reproduction, which is a major goal for all organisms (Baena-Gonzalez and Sheen, 2008; Stitt et al., 2010). Metabolic adjustment is a challenging task for plants, which have to resist fluctuating environmental conditions frequently perceived as stress factors. As a matter of fact, plant metabolic networks are under constraint and metabolic reconfigurations are observed under abiotic stress exposition (Bohnert and Sheveleva, 1998; Guy, 1990) Major trends frequently observed in many plant species exposed to high salt, low water availability, and high or low temperatures include the accumulation of primary metabolites like amino acids, non-structural sugars and organic acids (Ahuja et al., 2010; Sanchez et al., 2008). Multiple functions have been proposed for these stress-induced metabolic adjustments like osmotic potential regulation, thermo- and osmoprotection, chaperon-like roles of macromolecules, adjustments in the carbon/nitrogen balance, scavenging of ROS or reactive nitrogen species, redox buffering, pH adjustment, carbon and nitrogen reserves deposition and signal transduction (Bartels and Sunkar, 2005; Noctor, 2006). The impact of stress on metabolic network is often associated with concurrent depression of growth and development depending on the amplitude and the duration of the physico-chemical pressure (Chaves et al., 2003). Exploring correlations between biomass production and metabolite profiles in recombinant inbred line (RIL) populations of Arabidopsis, Meyer and collaborators proposed that growth may drive metabolism and not the opposite and biomass can be described as a function of metabolic composition. Significant correlation
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could be observed between biomass production and specific combination of metabolites (Meyer et al., 2007). Through the analysis of quantitative trait loci (QTL) with RIL and introgression line (IL) populations of Arabidopsis thaliana, it was demonstrated that there was a substantial and significant overlap of at least a subset of the biomass QTL, with metabolic QTL, suggesting a strong link between biomass and primary metabolism (Lisec et al., 2008). In contrast to secondary metabolism, it is proposed that perturbation of the primary metabolic network should have strong detrimental effects on plant performance. Metabolic adjustments in plants under osmotic stress are often observed through accumulation of specific metabolites. Such changes can be interpreted either as regulated adaptive or acclimation events or as biochemical consequences of growth impairment and trophic disruption. Plants that are tolerant to harsh environments (high salt, low water, high temperature) generally display low relative growth rate and often possess high solute concentrations (Alpert, 2006; Lugan et al., 2010). Although much evidence is now available about the adaptive or protective role of specific accumulation of compatible osmoprotectants, antioxidant and ROS scavenging compounds, a clear explanation of functional consequences of symptomatic global metabolic changes is still missing. Studies of metabolic adjustments in plants under stressful conditions have been encouraged in the past years, as technologies available for metabolomic approaches have been significantly improved (Fiehn et al., 2000; Ratcliffe and Shachar-Hill, 2005; Sumner et al., 2003; Weckwerth, 2003). Together with transcriptomic and proteomic approaches, metabolomic analysis gives a more comprehensive view of systems biology studies to investigate biological networks in order to understand plant responses to environmental cues and to develop tolerance improvement strategies. Metabolic profiling has been used recently for the description of plant adaptation to a wide range of biotic and abiotic stresses, creating the emerging field of environmental metabolomics (Bundy et al., 2009; Lugan et al., 2009; Schauer and Fernie, 2006; Shulaev et al., 2008). Complex changes in the metabolome during temperature stress have been characterised by studying the metabolic events associated with cold acclimation or acquired thermotolerance (Browse and Lange, 2004; Guy et al., 2008; Hannah et al., 2006; Kaplan et al., 2007; Korn et al., 2010; Shuman et al., 2006; Stitt and Hurry, 2002). Reconfiguration of central carbohydrate metabolism under stress appears to play a major role in plant response to temperature, where proposed triple function of sugars as nutrients, signalling compounds and putative ROS scavengers complicates the elucidation of the mechanisms involved (Guy et al., 2008; Rolland et al., 2006; Rosa et al., 2009; Stitt and Hurry, 2002). Moreover, the importance of DREB1/CBF
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transcription factors in the regulation of the low-temperature metabolome, associated with cold acclimation has been demonstrated in Arabidopsis (Cook et al., 2004; Maruyama et al., 2009). Changes in lipid molecular profiles during cold acclimation and freezing have also been described in Arabidopsis using ESI-MS/MS-based technologies (Wang et al., 2006; Welti et al., 2002). Few studies have used holistic metabolic fingerprinting or metabolic profiling in plants to highlight biomarkers and understand the molecular basis of tolerance to salt or drought stress (Sanchez et al., 2008). Valuable information has been obtained about global management of metabolic networks under osmotic stress (induced either by exposure to high salt concentrations or water deprivation) in crop species like grapevines (Cramer et al., 2007), tomato (Johnson et al., 2003; Semel et al., 2007), rice (Zuther et al., 2007), pear (Larher et al., 2009) or lupin (Pinheiro et al., 2004). Halophytic extremophiles and desiccation-tolerant species have also been the subject of metabolic profiling, searching for biochemical attributes of salt or dehydration tolerance (Gagneul et al., 2007; Ksouri et al., 2010; Lugan et al., 2010; Moore et al., 2009; Weber et al., 2007). Halophytes belonging to different species collected in the same inland salt marsh habitat have been compared and characterised in terms of nitrogenous compound production, thus highlighting some competitive regulations between betaines and proline accumulation (Tipirdamaz et al., 2006). The importance of antioxidant systems in desiccation tolerance has been supported by a study on Myrothamnus flabellifolia (Kranner et al., 2002). In addition to antioxidant metabolism, carbohydrate metabolism was shown to be reconfigured in resurrection plants undergoing dehydration (Moore et al., 2007; Whittaker et al., 2001). Due to the available genetic and genomic resources most of the recent molecular and integrated studies on metabolic adjustment under salt or drought stress have used Arabidopsis, a typical glycophyte species (Lugan et al., 2009; Tester and Bacic, 2005). Such analysis can combine transcriptomic, proteomic and metabolic profiling approaches, and could markedly increase our understanding of global plant stress response and adaptation to stress conditions such as drought (Mittler and Blumwald, 2010; Seki et al., 2007; Shulaev et al., 2008; Urano et al., 2010). Through multiparallel and iterative combinatorial experiments, the central role of abscisic acid in stressregulated metabolic pathways and redox control has been illustrated (Ghassemian et al., 2008; Kempa et al., 2008; Urano et al., 2010). Genetic variation between naturally occurring populations of Arabidopsis has also provided a valuable source of information to unravel metabolic traits and genetic architecture associated with the complex mechanisms of abiotic stress tolerance (Bouchabke et al., 2008; Brosche et al., 2010; Cross et al., 2006; Hannah et al., 2006; Katori et al., 2010; Wingler and
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Roitsch, 2008). To decipher the metabolic attributes and the genetic bases of extreme stress tolerance, Arabidopsis-relative model systems (ARMS) have recently been developed (Amtmann, 2009; Amtmann et al., 2005; Orsini et al., 2010). Recent comparative metabolic profiling experiences between Arabidopsis and Thellungiella salsuginea (halophila), a relative halophyte, contributed to the detection of relevant variation in metabolic composition and function in response to salt or drought treatments (Gong et al., 2005; Lugan et al., 2010; Pedras and Zheng, 2010). The metabolic composition of the salt tolerant T. salsuginea seems to be more compatible with dehydration, suggesting that this halophyte has more efficient osmoprotection than Arabidopsis (Lugan et al., 2010). It can be predicted that more and more studies will include metabolomics as a comprehensive addition to the systems biology approach to decipher plant stress response (Saito and Matsuda, 2010). The predicted changes in climatic conditions will likely create combinations of various abiotic stresses and make it necessary to adapt novel breeding or genetic engineering approaches to provide new crop varieties for modern agriculture (Mittler and Blumwald, 2010; Shulaev et al., 2008). Such practices will certainly encourage the development of metabolic phenotyping procedures and their adaptation in knowledge-based breeding programmes.
III. OSMOPROTECTIVE COMPOUNDS The most common osmoprotective compounds are amino acids, sugar alcohols, sugars and betaine-type quaternary amines (Table I; Chen and Murata, 2008; Hare and Cress, 1997; Hare et al., 1998; Hasegawa et al., 2000; Kerepesi et al., 1998; Parida and Das, 2005; Shulaev et al., 2008; Szabados and Savoure, 2010; Yancey et al., 1982). Composition and concentration of the solutes in stressed plants can vary considerably, depending on species and are influenced by the environmental conditions (Evers et al., 2010; Kumar, 2009; Lugan et al., 2010; Murakeozy et al., 2003; Sanchez et al., 2008). In contrast to inorganic ions, which can be harmful at high concentrations, osmolytes are considered compatible solutes, which can contribute to cell turgour, protect cellular structures, replace inorganic salts and alleviate ion toxicity. Compatible osmolytes were thought to mediate osmotic adjustment when water supply is limited, to replace water in biochemical reactions, stabilise the internal osmotic potential and to protect macromolecular structures. Therefore, osmotic adjustment has traditionally been accepted to be the primary function of osmolytes in plants (Crowe et al., 1992; Ford, 1984; Hasegawa et al., 2000; Ingram and Bartels, 1996; Parida and Das, 2005;
TABLE I Metabolism of the Most Common Osmoprotective Compounds in Plants
Osmolyte
Type of compound
Glycine betaine Quaternary ammonium compound
Proline
Amino acid
Trehalose
Sugar
Biosynthesis pathway Choline -(CMO)Betaine aldehyde -(BADH)Glycine betaine Glutamate -(P5CS)Pyrroline-5carboxylate -(P5CR)Proline
Enzymes in biosynthesis
Degradation pathway
Enzymes in catabolism
CMO: choline monooxygenase BADH: betaine aldehyde dehydrogenase P5CS: pyrroline5-carboxylate synthase P5CR: pyrroline5-carboxylate reductase
UDP TPS: trehalose phosphate glucose þ glucosesynthase 6-phosphate TPP: trehalose phosphate -(TPS)phosphatase Trehalose-6phosphate -(TPP)Trehalose
References Burnet et al. (1995), Hanson et al. (1994), Weigel et al. (1986)
Proline -(ProDH)Pyrroline-5-carboxylate -P5CDH)Glutamate
ProDH: proline dehydrogenase P5CDH: pyrroline5-carboxylate dehydrogenase
Trehalose -(TRE)2 Glucose
TRE: trehalase
Yoshiba et al. (1995), Hu et al. (1992), Szoke et al. (1992), Delauney and Verma (1993), Verbruggen et al. (1993), Strizhov et al. (1997), Kiyosue et al. (1996), Peng et al. (1996), Deuschle et al. (2004) Blazquez et al. (1998), Frison et al. (2007), Gussin et al. (1969) Lopez et al. (2008), Muller et al. (2001), Pramanik and Imai (2005), Vogel et al. (1998, 2001)
(continues)
Table I
Osmolyte
Type of compound
Mannitol
Polyalcohol
Myo-inositol
Polyalcohol
Pinitol
Polyalcohol
Biosynthesis pathway
(continued )
Enzymes in biosynthesis
Fructose-6P -(M6PI)Mannose-6P -(M6PR)Mannitol-1P -(M1PP)D-Mannitol D-Glucose-6P -MIPSMyo-inositol-1PMyo-inositol
M6PI: mannose-6P isomerase M6PR: mannose-6P reductase M1PP: mannose-1P phosphatase
Myo-inositol -(IMT1)D-ononitol Pinitol
IMT1: inositol-Omethyltransferase ononitol epimerase
MIPS: myo-inositol1-phosphate synthase
Degradation pathway Mannitol -(MTD)Mannose D-Mannose-6P -(M6PI)Fructose-6P
Enzymes in catabolism MTD: mannitol dehydrogenase M6PI: mannose-6P isomerase
References Loescher (1987, 1992), Rumpho et al. (1983)
Majumder et al. (1997), Johnson and Sussex (1995), Yoshida et al. (1999, 2002), Abreu and Aragao (2007), Wei et al. (2010a,b) Rammesmayer et al. (1995), Sengupta et al. (2008)
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Yancey et al., 1982). This model has been challenged by results obtained with transgenic plants and mutants, suggesting that osmolytes could have alternative protective functions. Concentrations of organic osmolytes were found to be lower than inorganic solutes in several halophytes, suggesting that these compounds might not be important for osmotic adjustment in these plants (Gagneul et al., 2007). For example, proline overproducing transgenic tobacco plants were shown to be more tolerant to salt and drought stress, without unequivocal data on osmotic adjustment (Blum et al., 1996; Kishor et al., 1995). Some osmoprotectants might have other protective functions, such as stabilisation of redox balance, maintenance of proper protein folding, mediating sugar or stress signals (Chen and Murata, 2008; Hare et al., 1998; Parida and Das, 2005; Rosgen, 2007; Szabados and Savoure, 2010). One important function of osmoprotective compounds is the stabilisation of proteins under conditions that can lead to protein denaturation. Equilibrium between native and unfolded forms of proteins is influenced by solvent composition. Unfavourable environmental conditions such as extreme temperatures, high salinity or dehydration can alter secondary and tertiary structure of proteins and modify the ratio of active and inactive proteins. Protein denaturation, formation of protein aggregates and accelerated protein degradation can be the cellular consequence of such stress conditions. However, the maintenance of native folding of proteins is essential for their function and can be facilitated by osmoprotective compounds (Bolen and Baskakov, 2001; Burg, 1995; Yancey et al., 1982). Protective osmolytes were shown to stabilise proteins and push the balance of the folding equilibrium towards native, actively folded proteins through raising the free energy of the unfolded proteins (Auton and Bolen, 2005; Street et al., 2006). The protein backbone but not the side chains were shown to be the preferred target for the stabilising function of osmolytes which explains their universal protecting effect (Auton and Bolen, 2004; Liu and Bolen, 1995). Protecting osmolytes improve thermodynamic stability of proteins via hydrogen bonding, which in contrast to hydrophobic interactions, does not affect other cellular functions during stress conditions (Holthauzen and Bolen, 2007; Kumar, 2009). Cyclic polyalcohols such as pinitol and myo-inositol were shown to protect plant and bacterial enzymes against thermally induced inactivation (Jaindl and Popp, 2006). By preserving the native conformation, proteins are protected from aggregation or degradation, which can therefore be considered as principal functions of osmoprotective compounds (Bolen, 2001; Kumar, 2009; Rosgen, 2007; Street et al., 2006). Different osmolytes most probably act independently and have no synergistic or competing effects in their interactions with proteins (Holthauzen and Bolen, 2007).
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Osmolytes can stabilise the structure of large multiprotein complexes as well. Glycine betaine (GB) was shown to support the oxygen-evolving activity of photosystem II (PSII) complex by preventing the dissociation of regulatory proteins from the core complex (Papageorgiou and Murata, 1995) and stabilise the efficiency of PSII photochemistry (Zhang et al., 2008). GB can ameliorate the damage of PSII and thereby stabilise photosynthesis under salt and cold stress (Ohnishi and Murata, 2006). Protection of cellular structures such as membranes and thylakoids against destabilisation during high temperatures or freezing was attributed to some of the protective compounds such as GB (Jolivet et al., 1982; Zhao et al., 1992). Some compounds can function as sinks of reducing power or carbon and nitrogen source after stress is relieved (Greenway and Munns, 1980; Hare et al., 1998). Accumulation of ROS is the result of various abiotic stresses including drought and high salinity. The protective function of osmolytes can include the suppression of radical oxygen production or scavenging of ROS. Accumulation of proline, GB and fructans in transgenic tobacco plants leads to reduced oxidative damage after freezing (Hong et al., 2000; Parvanova et al., 2004). Protection of the photosynthetic apparatus by GB during stress can reduce ROS accumulation and minimise lipid peroxidation during salt stress (Chen and Murata, 2008; Demiral and Turkan, 2004). Activation and protection of the ROS detoxification system is another key component of stress tolerance (Moradi and Ismail, 2007). Osmoprotective compounds can scavenge ROS directly, or contribute to the protection of the enzymes involved in the antioxidant system. Proline was suggested to be a singlet oxygen quencher during osmotic stress as it could reduce ROS damage such as lipid peroxidation in different plants (Hong et al., 2000; Matysik et al., 2002; Mehta and Gaur, 1999; Siripornadulsil et al., 2002; Smirnoff and Cumbes, 1989; Wang et al., 2009). GB increased catalase activity in cold-stressed tomato plants and thereby reduced ROS levels and improved chilling tolerance (Park et al., 2006). Stabilisation of the detoxifying enzymes such as ascorbate peroxidase and glutathione reductase was recently attributed to proline and can be important for the elimination of ROS during salt and drought stress (Sze´kely et al., 2008). Accumulation of ROS can be ameliorated by enhanced rate of proline biosynthesis during stress, which can help to maintain photosynthetic electron flow in the chloroplasts, stabilise redox balance and reduce photoinhibition (Hare and Cress, 1997; Szabados and Savoure, 2010). Osmolytes can control ROS-dependent damage through other, less-known pathways also. ROS was shown to promote Kþ efflux in root epidermal cells, which was significantly reduced by several osmolytes, including proline, GB, mannitol, myo-inositol and trehalose (Cuin and Shabala, 2007).
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Osmolytes can have regulatory functions and modulate metabolic processes and gene activity. Proline and GB were shown to induce the expression of defence genes such as catalase and peroxidase, which can suppress cell death during stress (Banu et al., 2009). GB induced a group of low-temperature-responsive genes, which could elicit cold acclimation and freezing tolerance (Allard et al., 1998). Transcript profiling showed that exogenous GB treatment enhances the expression of numerous Arabidopsis genes, encoding transcription factors, membrane transporters and ROS detoxifying enzymes (Einset et al., 2008). Expression of catalase and an NADPH-dependent ferric reductase (FRO2) was enhanced by GB treatment, which contributed to antioxidant activity and chilling tolerance (Einset et al., 2007; Park et al., 2006). Certain regulatory functions were attributed to proline as well. Transcript profiling revealed that part of the rehydration-inducible Arabidopsis genes can be activated by proline (Oono et al., 2003). bZIP-type transcription factors were suggested to recognise the conserved PRE element in the promoter region of these genes and to activate their transcription (Satoh et al., 2004; Weltmeier et al., 2006). A. QUATERNARY AMINES: GLYCINE BETAINE
The quaternary ammonium compound GB is a methylated derivative of glycine, which accumulates at high concentrations in many halophyte plants (Chen and Murata, 2008; Hanson et al., 1983). GB is synthetised from choline in two steps. Betaine aldehyde hydrate generated from choline by the action of choline monooxygenase (CMO) produces betaine aldehyde hydrate from choline, which is converted spontaneously to betaine aldehyde and subsequently oxidised to GB by NADþ-dependent betaine aldehyde dehydrogenase (BADH; Burnet et al., 1995; Fitzgerald et al., 2009; Fujiwara et al., 2008; Hanson et al., 1983). GB accumulation can be induced by different stress conditions such as osmotic stress (Hanson and Nelsen, 1978), salinity (Hanson et al., 1991), drought (Guo et al., 2009), heat (Jolivet et al., 1982) and cold stresses (Allard et al., 1998; DeRidder and Crafts-Brandner, 2008). The significance of GB accumulation for stress tolerance has been investigated in transgenic plants engineered to produce GB by overexpressing plant-derived CMO and BADH genes (Guo et al., 1997; Nakamura et al., 1997; Shirasawa et al., 2006). Alternatively, bacterial betaine aldehyde dehydrogenase (betB) or choline oxidase (COX or codA) genes were introduced and expressed in transgenic plants, leading to increased GB accumulation (Holmstrom et al., 1994; Mohanty et al., 2002; Park et al., 2007; Sakamoto et al., 1998; Su et al., 2006) .The beneficial effects of GB accumulation
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regarding salt and osmotic stress tolerance have been demonstrated in a number of engineered GB-accumulating plants, including tobacco (McNeil et al., 2001; Nuccio et al., 1998; Zhang et al., 2008), tomato (Park et al., 2004, 2007) and rice (Chen and Murata, 2002, 2008; Guo et al., 1997; Kathuri et al., 2009; Nakamura et al., 1997; Sakamoto et al., 1998; Shirasawa et al., 2006). GB accumulation in BADH-overexpressing transgenic tobacco led to enhanced salt or heat tolerance mainly by protecting photosynthetic activity through the maintenance of Rubisco activity and PSII activity (Yang et al., 2005, 2007, 2008). Targeted accumulation of GB in chloroplasts has been achieved by engineering a plastid-expressed CMO gene, leading to higher PSII activity during salt and drought stress (Zhang et al., 2008). Therefore protection of the PSII by GB-mediated osmoprotection can be a promising strategy to improve drought and salt tolerance in crops. Rice was engineered to produce GB by introducing the chloroplast-targeted choline oxidase (codA9) gene from Arthrobacter globiformis. GB synthesis enhanced the activity of PSII, led to better ROS detoxification and improved physiological and agronomic performance (Kathuria et al., 2009). Plants are usually very sensitive to environmental stress during reproduction. GB was shown to have a particularly important protective effect on reproductive organs, such as inflorescence apices and flowers during drought and cold stress (Chen and Murata, 2008; Sakamoto and Murata, 2000). Engineering of GB accumulation reduced chilling damage on tomato flowers, leading to a 10–30% increase in fruit production (Park et al., 2004). These data confirm that GB has osmoprotective qualities, which can therefore be explored to improve tolerance to salinity and probably to other abiotic stresses such as drought and cold. B. SUGARS: TREHALOSE
Trehalose is a nonreducing disaccharide of glucose, which was first described in desiccation-tolerant organisms capable of surviving dehydration, including resurrection plants (Drennan et al., 1993; Fernandez et al., 2010; Liu et al., 2008; Moore et al., 2009). Later, trehalose accumulation was detected in numerous other plants under different stress conditions such as drought, cold, high salinity (Iordachescu and Imai, 2008; Kaplan et al., 2004; Kosmas et al., 2006; Lopez et al., 2008; Pramanik and Imai, 2005) and during various plant–microbial interactions (Brodmann et al., 2002; Dominguez-Ferreras et al., 2009; Farı´as-Rodrı´guez et al., 1998; Lopez et al., 2008). Trehalose biosynthesis is a two-step pathway. First, trehalose-6-phosphate is produced from UDP glucose and glucose-6-phosphate by trehalose phosphate synthase, which is converted to trehalose by the enzyme trehalose phosphate
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phosphatase (Vogel et al., 1998, 2001). Trehalose is catabolised by trehalase, which converts it to glucose (Brodmann et al., 2002; Goddijn et al., 1997). The importance of trehalose in modulating plant stress responses was demonstrated by engineering the trehalose biosynthetic pathway in transgenic plants. Stress-dependent trehalose accumulation was engineered in transgenic rice by the regulated overexpression of the fused bacterial trehalose biosynthetic genes otsA and otsB. Enhanced trehalose levels resulted in improved salt, drought and cold tolerance, and lower photo-oxidative damage, which was suggested to modulate carbohydrate metabolism (Garg et al., 2002). Several other transgenic plants were produced that accumulated trehalose at high levels, and subsequently improved tolerance to drought, freezing or high salinity (Jang et al., 2003; Romero et al., 1997; Stiller et al., 2008; Yeo et al., 2000). However, constitutive overexpression of the trehalose biosynthetic genes led to pleiotropic growth alterations, dwarfism and abnormal root structure (Garg et al., 2002; Romero et al., 1997; Schluepmann et al., 2004; Yeo et al., 2000). Using stress-induced promoters such as the drought-induced Arabidopsis AtRAB18 or potato StDS2 promoters, the environmentally controlled upregulation of trehalose biosynthesis was important to generate transgenic plants with enhanced stress tolerance, but without morphological abnormalities (Karim et al., 2007; Stiller et al., 2008). The function of trehalose in stress responses is controversial. Trehalose was shown to have the ability to stabilise membranes and protect proteins in desiccated tissues and was suggested to function as chemical chaperon (Crowe, 2007; Crowe et al., 1984). However, trehalose levels in the engineered plants usually remained well below 1 mg/g fresh weight, suggesting that trehalose does not act as a compatible solute, but has an alternative function (Garg et al., 2002). The fact that trehalose-accumulating transgenic plants exhibit abnormal morphology can also indicate that this compound is not a neutral osmolyte. Recently, trehalose-6-phosphate was proposed to function as a metabolic signal involved in the control of the SnRK1 activity, which is a central regulator of sugar and energy homeostasis (Paul et al., 2010; Zhang et al., 2009). Therefore, the signalling function of trehalose and trehalose-6P could be more important than the previously suggested chaperone or osmolyte function, although in some tissues such a protective role cannot be excluded (Fernandez et al., 2010). C. POLYALCOHOLS: MANNITOL, PINITOL, INOSITOL
Accumulation of polyalcohols, such as mannitol and pinitol, has been detected in several water-stressed plants and are considered as important compatible solutes, which serve as ROS scavengers and molecular
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chaperones (Bohnert et al., 1995; Ford, 1984; Sengupta et al., 2008). Mannitol is the most common sugar alcohol and is an important photosynthetic product in a number of plant species (Loescher et al., 1992; Rumpho et al., 1983). In plants, mannitol is synthetised from fructose-6P by subsequent action of mannose-6P isomerase (phosphomannose isomerase), mannose6P reductase (M6PR) and mannose-1P phosphatase (Loescher et al., 1992; Rumpho et al., 1983). Catabolism of mannitol is controlled by mannitol dehydrogenase, which produces mannose. Phosphorylation of mannose results in mannose-6P, which is converted to fructose-6P by mannose-6P isomerase (Loescher, 1987). One of the first demonstrations of the osmoprotective function of an osmolyte was the overexpression of a bacterial mannitol 1-phosphate dehydrogenase (mtlD) in transgenic tobacco plants, which led to increased mannitol accumulation in conjunction with enhanced salt tolerance (Tarczynski et al., 1993). In agreement with these results, engineering of mannitol biosynthesis by overexpression of mtlD led to enhanced mannitol accumulation and improved salt tolerance in various transgenic plants including wheat (Abebe et al., 2003), poplar (Populus tomentosa; Hu et al., 2005) and loblolly pine (Pinus radiata; Tang et al., 2005). Overexpression of celery M6PR is an alternative way to enhance mannitol biosynthesis and was shown to be an efficient way to improve salt tolerance of Arabidopsis (Zhifang and Loescher, 2003). As an alternative use, M6PR was employed as a selectable marker for plant transformation, using mannose tolerance as selection criteria (Song et al., 2010). Myo-inositol is an essential polyalcohol in plants and all eukaryotes. Biosynthesis starts from D-glucose-6P, which is converted to myo-inositol1P by myo-inositol-1P synthase (MIPS; Johnson and Sussex, 1995; Majumder et al., 1997). Myo-inositol is produced from myo-inositol-1P by dephosphorylation and is used for the subsequent biosynthesis of all inositolcontaining compounds, including phospholipids. MIPS genes were shown to be salt-induced, leading to accumulation of myo-inositol in the halophyte ice plant, but not in the glycophyte Arabidopsis (Ishitani et al., 1996). MIPS genes can be regulated by several environmental stress factors such as drought, heat and cold stress, high light and were shown to be controlled by ABA signals (Abreu and Aragao, 2007; Wei et al., 2010a,b; Yoshida et al., 1999, 2002). Myo-inositol serves not only as osmoprotectant compound, but also functions as signal that controls metabolic responses to stress, such as sodium uptake in saline environments (Nelson et al., 1999). Phosphorylated derivatives of myo-inositol are important signalling compounds, which are involved in numerous regulatory pathway and control diverse aspects of plant development, responses to biotic and abiotic stresses.
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Other polyalcohols with osmoprotective features such as D-ononitol and are synthetised from myo-inositol through the extension of the metabolic pathway. Pinitol is a methylated inositol, which is synthetised from myo-inositol by inositol-o-methyltransferase (IMT1) and ononitol epimerase (OEP1) (Bohnert et al., 1995; Rammesmayer et al., 1995; Sengupta et al., 2008). Pinitol accumulation is a characteristic feature of a number of halophytic plants in saline environment and occurs in several glycophytic plants grown under osmotic stress conditions (Ford, 1984; Fougere et al., 1991; Murakeozy et al., 2003; N’Guyen and Lamant, 1988; Sengupta et al., 2008). Salt-induced pinitol hyperaccumulation was found in Porteresia coarctata, a halophytic wild relative of rice, which is missing in domesticated rice. The inositol methyl transferase 1 (PcIMT1) gene is strongly upregulated by salt in wild rice, suggesting that this gene controls the stress-induced pinitol accumulation in this halophytic plant which is an essential metabolic response for salt tolerance (Sengupta et al., 2008). Engineering of complex metabolic pathways by altered expression of single genes has limitations. Simultaneously regulated expression of several genes encoding key enzymes could have more profound effects on the metabolic pools. Biosynthesis of myo-inositol was enhanced by introduction and overexpression of the MIPS gene from P. coarctata in tobacco leading to inositol accumulation and enhanced salt tolerance (Majee et al., 2004). Even higher levels of salt tolerance were reported by the introgression and simultaneous overexpression of the MIPS coding gene from P. coarctata and the IMT1 coding gene from M. crystallinum in transgenic tobacco. By comparison, double transgenics accumulated more inositol and pinitol than plants transformed by single genes, which conferred improved growth, higher photosynthetic activity and lower oxidative damage during salt stress (Patra et al., 2010).
D-pinitol
D. AMINO ACIDS: PROLINE
Proline is an essential amino acid, a common denominator of many stress responses being accumulated during diverse abiotic and biotic stresses such as high salinity (Ben Hassine et al., 2008; Voetberg and Stewart, 1984; Yoshiba et al., 1995), drought (Ben Hassine et al., 2008; Choudhary et al., 2005; Huang and Cavalieri, 1979; Rhodes et al., 1986), UV irradiation (Saradhi et al., 1995), heavy metals (Mehta and Gaur, 1999; Schat et al., 1997; Singh et al., 2010) and oxidative stress (Yang et al., 2009). Moreover, proline was reported to accumulate in plants infected by avirulent bacteria (Fabro et al., 2004) or Agrobacterium (Haudecoeur et al., 2009). In plants, proline is synthesised from glutamate in the cytosol and likely also in the chloroplast by the sequential action of delta-1-pyrroline-5-carboxylate
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Synthesis
synthetase (P5CS) and P5C reductase (P5CR). P5CS produces glutamate semialdehyde, which is unstable and is immediately converted to pyrroline-5carboxylate (P5C). P5CR reduces P5C to proline, a reaction that takes place in the cytosol and according to biochemical data also in the chloroplast (Fig. 1; Delauney and Verma, 1993; Hu et al., 1992; Rayapati et al., 1989; Strizhov et al., 1997; Szabados and Savoure, 2010; Szoke et al., 1992; Verbruggen et al., 1993; Yoshiba et al., 1995). Proline synthesis is considered as an evolutionary conserved process based on the similarity that exists among the prokaryotic and eukaryotic pathways and the high homology of the involved enzymes. The human and plant P5CSs are bifunctional enzymes representing evolved fusion products of the domains responsible for the catalytic activity in the prokaryotic proB and proA genes, encoding glutamate kinase (GK) and g-glutamyl phosphate reductase (GPR), respectively (Csonka, 1989; Hu et al., 1992; Perez-Arellano et al., 2010). These genes are arranged in a single operon in bacteria and their paralogs in lower eukaryotes, such as yeast, still form two separate enzymes (Takagi, 2008). Despite sharing functional homology, the mammalian and plant P5CSs are localised to different cellular compartments. The human P5CS isoforms are functioning in the mitochondria, utilising innermitochondrial glutamate and energy sources (Perez-Arellano et al., 2010),
Chloroplast
Cytosol
Mitochondrion
Glutamate
Glutamate
Arginine
NADPH+ + H+ NADP+ ATP ADP
NADPH+ + H+
P5CS1
P5CS1 & 2
Arginase
NADP+
GSA P5C P5CR
GSA P5C
Ornithine P5CR
NADP+
Proline
Proline
NADPH+ + H+
OAT Accumulation during stress in cytosol, vacuole, and PRPs Transport to the cytosol
Transport into mitochondria
Mitochondrion
Degradation
ProDH 1 & 2
Proline
P5C FAD+
FADH2
P5CDH NAD+/NADP+
eElectron transport chain
NADH+ + H+/ NADPH+ + H+
Glutamate
Fig. 1. Scheme of proline metabolism in plants. Proline synthesis occurs in the cytosol and likely also in the chloroplast. Proline degradation is carried out in the mitochondrion. The dashed arrows indicate the proline cycle. See text for details.
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whereas the two plant isoforms, P5CS1 and P5CS2, are functioning in the cytosol, with a likely abiotic-stress-induced shift of P5CS1 to the chloroplasts (Sze´kely et al., 2008). In yeast, both enzymes (GK and GPR) function in the cytosol (Takagi, 2008). Another source for P5C is the mitochondrial degradation pathway of arginine, whose first step is catalysed by arginase that forms ornithine and urea. In the second step, ornithine amino transferase (OAT) produces P5C by ornithine deamination in the mitochondria (Brownfield et al., 2008; Roosens et al., 1998; Xue et al., 2009). The importance of this pathway in proline biosynthesis has recently been questioned as proline levels were not altered in the oat mutant (Funck et al., 2008). Although P5C transporters have not been identified in plant mitochondrial or chloroplast membranes, biochemical evidence support the movement of P5C from the mitochondria to the cytosol in human and plant cells which enables the reduction of mitochondrial-produced P5C to proline in the cytosol (Miller et al., 2009; Yoon et al., 2004). Proline catabolism occurs in the inner-mitochondrial membrane of all eukaryotes. Proline degradation provides electrons and glutamate for mitochondrial usage. Proline dehydrogenase (ProDH), a FAD-enzyme localised to the inner-mitochondrial membrane, catalyses the first oxidising step of proline to P5C and meanwhile delivers electrons to the mitochondrial electron transport chain (Kiyosue et al., 1996; Peng et al., 1996; Rayapati and Stewart, 1991). P5C is further oxidised to glutamate or transported back to the cytosol for proline re-synthesis by the proline cycle (Fig. 1; Deuschle et al., 2004; Miller et al., 2009). Proline cycle exists in yeast, mammals and plants and contains the cytosolic P5CR and mitochondrial ProDH (Miller et al., 2009; Phang et al., 2010; Takagi, 2008). In human cells, proline cycle acts as a suppressor of carcinogenesis. It oxidises proline and provides an excess of electrons producing ROS that are responsible for initiating programmed cell death that prevents tumour development (Phang et al., 2010). P5CDH is the second enzyme in proline degradation, catalysing the oxidation of P5C to glutamate in the mitochondria and is responsible for maintaining proline-P5C homeostasis (Deuschle et al., 2001, 2004; Forlani et al., 1997). When P5CDH activity is impaired, hyperactivity of proline-P5Cproline cycle provides excess electrons to the mitochondrial electron chain and generates ROS by using O2 as the electron acceptor (Miller et al., 2009). ROS signals can be produced during the first hour of dehydration in Arabidopsis leaves, when a transient increase in ProDH transcription occurs (Kiyosue et al., 1996; Peng et al., 1996). In some plant species, derivatisation of proline occurs following its stress-induced accumulation. In the salt tolerant salt-cider (Tamarix spp.), part of the stress-accumulated proline is
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modified to N-methylproline analogues including N-methyl-L-proline, trans4-hydroxy-N-methyl-L-proline and trans-3-hydroxy-N-methyl-L-proline whose function is still unknown (Jones et al., 2006). Proline accumulation during stress has multiple protective functions. For a long time, proline was considered a neutral osmolyte that protects cellular structures and stabilises enzymes (Delauney and Verma, 1993; Kavi Kishor et al., 2005; Mishra and Dubey, 2006; Sharma and Dubey, 2005; Sharma et al., 1998). Besides osmoprotection, proline was shown to have antioxidant activity, activate detoxification pathways, contribute to cellular homeostasis by protecting the redox balance, function as protein precursor, energy source for the stress-recovery process and even as a signalling molecule (Hoque et al., 2008; Islam et al., 2009; Khedr et al., 2003; Matysik et al., 2002; Szabados and Savoure, 2010; Sze´kely et al., 2008). In plants, maintenance of PSII and PSI activity as well as electron flux through the photosynthetic electron transport chain is very important in stress conditions. Inhibition of Calvin cycle and pentose phosphate pathway can channel NADPH, ATP and glutamate for proline synthesis in the chloroplasts. Thus, proline synthesis in the chloroplast may allow an efficient oxidation of photosynthetically produced NADPH providing the required NADPþ for electron acceptor avoiding the use of O2 that leads to ROS generation (Hare and Cress, 1997; Szabados and Savoure, 2010). Numerous reports describe the importance of proline accumulation in salt and drought tolerance, although a clear relationship between proline accumulation and tolerance could not always be confirmed (Kavi Kishor et al., 2005; Lehmann et al., 2010; Szabados and Savoure, 2010; Verbruggen and Hermans, 2008). Convincing evidence for the protective function of proline was provided by studies of mutants and transgenic plants with proline deficiency or proline hyperaccumulation. Arabidopsis p5cs1 insertion mutants had only 10% proline of the wild type and were hypersensitive to salt stress, produced more ROS and lipid peroxidation products, which confirmed the importance of proline in stress tolerance, in particular, in ROS scavenging (Sze´kely et al., 2008). However, enhanced proline accumulation and improved salt tolerance was achieved by increasing the biosynthetic pathway through constitutive overexpression of the Vigna P5CS cDNA in transgenic tobacco (Kishor et al., 1995) and Clamydomonas (Siripornadulsil et al., 2002). Overexpression of feedback-insensitive P5CS in tobacco or rice could enhance proline levels even more than the wild-type enzyme, showing that post-translational regulation needs to be considered for metabolic engineering (Hong et al., 2000; Kumar et al., 2010). Alternatively the feedback-insensitive proAB gene from Bacillus subtilis was expressed in Arabidopsis, which produced more free proline and improved
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osmotolerance (Chen et al., 2007a,b). While overexpression of Arabidopsis P5CR in transgenic soybean improved drought and heat tolerance, antisense expression of P5CR leads to stress sensitivity (De Ronde et al., 2004).These reports confirm that proline accumulation in dehydrated plants is not only a consequence of stress, but also a part of the metabolic defence system against abiotic stress. 1. Regulation of proline metabolism While proline biosynthesis is augmented and proline catabolism is repressed during stress, the opposite happens during recovery, when synthesis declines rapidly and catabolism is activated. Accumulation of cellular free proline levels has been attributed to increased transcription of P5CS and silencing of ProDH, while reciprocal transcriptional regulation suppress P5CS transcrip´ braha´m et al., 2003; Kiyosue et al., tion and activates ProDH after stress (A 1996; Peng et al., 1996; Ribarits et al., 2007; Strizhov et al., 1997; Verbruggen et al., 1996; Yoshiba et al., 1995). Whereas the stress-induced drastic reduction in ProDH transcription is a general phenomenon in plants, some species lack the concomitant increase in P5CS transcription (Ginzberg et al., 1998; Miller et al., 2005). Stress-dependent P5CS activation and ProDH repression is controlled by both ABA-dependent and independent signalling pathways, ´ braha´m et al., 2003; modulated by light and brassinosteroid signals (A Sharma and Verslues, 2010; Strizhov et al., 1997; Sze´kely et al., 2008; Verslues et al., 2007). During incompatible plant–pathogen interactions P5CS activation is controlled by SA-dependent ROS signals (Fabro et al., 2004). In the ornithine pathway, OAT transcription is upregulated by salt stress in young Arabidopsis seedlings but not in mature plants, suggesting the involvement of developmental and temporal regulation in OAT expression (Roosens et al., 1998). Regulation of key enzymes involved in proline metabolism seems to vary according to environmental conditions and also during plant development (Lehmann et al., 2010; Szabados and Savoure, 2010). Calmodulin and MYB2 are likely involved in P5CS activation under salt stress, as interaction of MYB2 and the salt-induced CaM isoform (GmCaM4) was shown to upregulate P5CS1 transcription and leads to proline accumulation (Yoo et al., 2005). In addition to the MYB2, bZIP transcription factors (bZIP-TF) might also be involved in regulating transcription of the genes encoding enzymes of proline metabolism. Promoter analyses by several servers that identify promoter motifs (e.g., Agris, http://arabidopsis.med.ohio-state.edu) indicate that the promoters of all Arabidopsis genes encoding enzymes involved in proline metabolisms possess bZIP-TF recognition motifs: six sites in P5CS1 (At2g39800), 11 sites in P5CS2 (At3g55610), 14 in P5CR (At5g14800), four in ProDH1 (At3g30775), six in ProDH2 (At5g38710), two
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in P5CDH (At5g62530) and five in OAT (At5g46180). As yet, only the molecular basis of induced ProDH transcription by the drought and salinity stressrecovery process has been unravelled, indicating the importance of bZIP transcription factors. bZIP-TFs from the S and C groups AtbZIP53 and AtbZIP10 (Jakoby et al., 2002) form homo- or heterodimers and promote the recruitment of the transcription complex to the ProDH promoter region. Interaction of these transcription factors with the PRE motif ACTCAT of the ProDH promoter strongly enhances its transcription (Satoh et al., 2002, 2004; Weltmeier et al., 2006). Interactions of bZIP11/ATB2 and other heterodimerforming partners from groups S and C with this PRE motif is also responsible for upregulating ProDH transcription in response to sugar depletion, an effect that also blocks the post-transcriptional inhibition of bZIP11 by sucrose (Hanson et al., 2008). The possible involvement of bZIP-TFs in the regulation of the other genes controlling proline metabolism still has to be confirmed. Upon recovery from stress, a reduction in P5CS and enhancement of ProDH transcription occur, leading to intensive proline degradation that provides energy and glutamate for the surviving cells (Kiyosue et al., 1996; Peng et al., 1996; Verbruggen and Hermans, 2008). In addition to changes in enzyme levels, mostly dictated by regulation at the transcriptional level, proline allosterically inhibits the enzymatic activity of bacterial GK/GPR and the plant P5CS, the initial enzyme in the synthesis pathway (Csonka et al., 1988; Fujita et al., 2003; Hu et al., 1992). The accumulation of proline during various stresses without exerting a feed-back regulation on P5CS activity is not well understood. Although expression of ProDH genes is induced by exogenous proline, ProDH transcription is silenced during stress, despite the high levels of accumulated free proline in the cells (Kiyosue et al., 1996; Miller et al., 2005). Hence, it is likely that a certain compartmentalisation of the stress-accumulated proline exists to avoid interaction with P5CS and cancel the silencing of ProDH transcription. Further studies are required to clearly understand the signalling pathways and define all the regulatory proteins involved. 2. Proline rich proteins and stress response Proline rich proteins (PRP) are plant-specific proteins that are mainly localised to the cell wall, playing different roles in cell wall maintenance and linkage to the plasma membrane. Their synthesis utilises a large portion of the free proline pool and normally occurs in most of the plant cells during development. These proteins are characterised by reiterated proline-rich sequence motifs. Many of the proline residues in these proteins are hydroxylated and thereafter glycosylated through their processing in the ER and Golgi network before being secreted to the outer side of the plasma membrane or linked to the membrane by a GPI-anchore or hydrophobic
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transmembrane domain/s. The hydroxyproline-rich glycoproteins comprise a huge super family in Arabidopsis and other plants, which is divided into three subfamilies; arabinogalactans, extensins and PRP (Showalter et al., 2010). Most of these proteins are involved in cell wall assembly in all plant organs and along the developmental processes or are more specific to certain organs (Fowler et al., 1999). Oxidative cross-linking of tyrosine residues within the subfamily of extensin molecules strengthens the cell wall and increases resistance to invading pathogens. Whereas many hydroxyproline-rich proteins are involved in plant response to wounding and pathogen attacks, only a limited number of these PRP are involved in abiotic stress responses (Deepak et al., 2010). Most of them belong to the HyPRP family. HyPRPs, which are only found in plants, contain a proline-rich N-terminal repetitive domain suggested to protrude from the plasma membrane towards the cell wall and a hydrophobic C-terminal transmembrane domain with a certain conserved arrangement of eight cysteine residues (Jose-Estanyol et al., 2004). MsPRP2 is an HyPRP that is induced by water stress in alfalfa (Medicago sativa) cells under saline conditions (Deutch and Winicov, 1995), whereas another alfalfa HyPRP, MsACIC, reveals enhanced expression in cold-tolerant plants (Castonguay et al., 1994). The Brassica napus HyPRP, BNPRP, is also highly expressed at low temperatures, whereas low level of the BNPRP transcript is also present under normal growth conditions (Goodwin et al., 1996). As yet, the functional role of these proteins and their contribution to stress tolerance are not clear. It is likely that the Arabidopsis cell wall linker protein (CWLP) that shows 85% amino acid sequence homology to BNPRP (Goo et al., 1999), and is induced by cold treatment in Arabidopsis, is involved in the adhesion of the cell wall to the plasma membrane (Ziberstein’s group preliminary results). Since cold-induced synthesis of these proteins is linked to proline accumulation, availability of free proline might support the synthesis of PRP.
IV. OSMOPROTECTIVE COMPOUNDS AND ADAPTATION TO EXTREME ENVIRONMENTS Numerous earlier papers and reviews have discussed the importance of osmoprotective compounds in stress tolerance and the value they might have in the adaptation process to extreme environmental conditions (Hare et al., 1998; Rontein et al., 2002; Table II). While a number of reports described the coincidence of osmolyte accumulation with salt or drought tolerance, the adaptive value of osmoprotective compounds for stress tolerance was unequivocally demonstrated in a few cases only.
TABLE II Plant Species or Genotypes in Which Osmolyte Accumulation is Important for Tolerance to Extreme Environmental Conditions Species
Compound
Environment
Atriplex halimus L (xero-halophyte)
Glycine betaine, sugars, proline
Arid environment, saline soil
Camphorosma annua (halophyte)
Glycine betaine, pinitol
salty–sodic soil
Cicer arietinum (chickpea genotypes with contrasting copper tolerance) Hordeum vulgare L (salt tolerant/ sensitive barley)
Proline
Copper toxicity
Proline, glycine betaine, sugars, hexose phosphates
Saline soil
Lepidium crassifolium (halophyte)
Proline, sugars
salty–sodic soil
Limonium spp. (halophyte, Plumbaginaceae spp.) Limonium gmelini (halophyte)
b-Alanine betaine, choline-Osulphate b-Alanine betaine, choline-Osulphate, pinitol Sugars, chiro-inositol, proline Proline, sugars
Hypoxic saline, sulphate-rich saline soils salty–sodic soil
Limonium latifolium (halophyte) Medicago truncatula, M. laciniata, (contrasting landraces in drought tolerance) Mesembryanthemum crystallinum (ice plant, halophyte) Oryza sativa (drought tolerant/ sensitive rice varieties) Oryza sativa (drought tolerant/ sensitive indica rice variety)
Reference Shen et al. (2002), Wang and Showalter (2004), Martinez et al. (2004) Murakeozy et al. (2003) Singh et al. (2010)
Saline soil Drought, arid environment
Chen et al. (2007b), Widodo et al. (2009) Murakeozy et al. (2003), this study Hanson et al. (1991, 1994) Murakeozy et al. (2003) Gagneul et al. (2007) Yousfi et al. (2010)
Polyols (myo-inositol, D-ononitol, D-pinitol) Proline
Saline soil
Nelson et al. (1999)
Drought, osmotic stress
Reducing sugars, proline, polyols
Saline soil
Choudhary et al. (2005) Zuther et al. (2007), Roychoudhury et al. (2008)
Oryza sativa (proline accumulating mutant) Phaseolus vulgaris (contrasting landraces in drought tolerance) Plumbaginaceae spp. (halophytes) Plumbaginaceae spp. (halophytes) Porteresia coarctata (wild rice, halophyte) Solanum tuberosum L. (drought tolerant/sensitive potato cultivars) Sorghum bicolor (sorghum: salt tolerant/sensitive variety) Thellungiella salsugiana (halophila) (halophyte)
Triticum aestivum (drought tolerant/ sensitive wheat genotypes) Triticum durum Desf. (drought tolerant/sensitive variety) Various contrasting legume species in drought tolerance
Proline
Tolerance to Hg2þ-induced oxidative stress Drought, arid environment
Glycine betaine
Saline soil
Proline betaine Pinitol
Arid environment Saline soil
Hanson et al. (1991, 1994) Hanson et al. (1994) Sengupta et al. (2008)
Proline, inositol, galactose, galactinol Sugars, proline
Drought treatment
Evers et al. (2010)
Drought, arid environment
Premachandra et al. (1995) Lugan et al. (2010), Gong et al. (2005), Inan et al. (2004), Arbona et al. (2010), Taji et al. (2004) Kerepesi et al. (1998)
Proline
Proline, sugars (sucrose, fructose, glucose, raffinose, melibiose), sugar alcohols (inositol, galactinol)
Saline soil
Sugars, fructan
Drought, arid environment
Sugars, proline
Arid environment, osmotic stress Drought, water stress
Pinitol
Such examples are listed where experimental evidence suggests the value of osmolyte accumulation for stress tolerance.
Wang et al. (2009) Tari et al. (2008)
Bajji et al. (2001) Ford (1984)
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Osmoprotective compounds can accumulate to very high concentrations in extremophile plants in saline or arid environments, suggesting that these metabolites contribute considerably to the adaptation of these plants to the harsh environment. Some species produce high levels of sugars or polyols, others preferentially accumulate nitrogenous compounds. Halophytes usually accumulate one dominant compatible solute, which can be proline, GB, sorbitol, b-alanine betaine, choline-O-sulphate or sugar (Arbona et al., 2010; Hanson et al., 1991, 1994; Inan et al., 2004; Lugan et al., 2010; Tipirdamaz et al., 2006). Halophytic Limonium species were shown to accumulate high levels of quaternary ammonium compounds such as choline-O-sulphate, GB and b-alanine betaine, suggesting that these compounds are important for the salt tolerance of these species (Hanson et al., 1991). Sucrose accumulated in Juncus maritima, Phragmites communis and Scirpus maritimus, while maltose and rhamnose were abundant in Atriplex hastata and Plantago maritima, respectively. Polyols were present in Aster tripolium, Juncus maritimus, P. maritima and P. communis (Briens and Larher, 1982). Distribution of osmoprotective ammonium compounds among different species of the Plumbaginaceae family suggested that particular compounds have selective advantage in different environments. GB was dominant in species adapted to dry environments, choline-O-sulphate was advantageous in sulphatecontaining soils, b-alanine betaine is apparently more typical in species growing on hypoxic saline soils, and proline–betaine was detected in plants adapted to arid environments (Hanson et al., 1994). Composition and concentration of compatible osmolytes had species-specificity and showed seasonal fluctuation in three halophyte species in salty–sodic grasslands in Hungary. GB and pinitol was characteristic of Camphorosma annua, b-alanine betaine and pinitol accumulated in Limonium gmelini, while proline was most characteristic of Lepidium crassifolium. The highest osmolyte concentrations were measured in spring, characterised by low temperatures, hypoxic conditions and high salt concentrations in the habitats of the tested species (Murakeozy et al., 2003). Among the extremophile Chenopodiaceae species, Artiplex halimus is a known xero-halophyte plant, characterised by GB and proline accumulation in stressful environments (Shen et al., 2002; Wang and Showalter, 2004). Accumulation of GB and sugars were suggested to be responsible for osmotic adjustment during osmotic stress in this species (Martinez et al., 2004). In a more recent study, P. maritima was found to accumulate sorbitol, proline and GB, while species belonging to the Chenopodiaceae family are typical accumulators of GB in saline environments (Tipirdamaz et al., 2006). In other species, the importance of osmoprotecting compounds in stress tolerance has been studied by comparing genotypes with contrasting
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drought or salt tolerance and osmolyte accumulation. The importance of osmoprotectants for salt and drought stress tolerance of cereals has long been discussed (Garcia et al., 1997). Proline and sugar levels were higher in drought and salt tolerant than sensitive rice varieties, suggesting that these protective compounds can contribute to stress tolerance of rice (Choudhary et al., 2005; Roychoudhury et al., 2008). In durum wheat, main osmolytes that correlated with drought tolerance were sugars, followed by proline and quarternary ammonium compounds (Bajji et al., 2001). A positive correlation between drought tolerance of wheat and glucose, fructose, sucrose and fructan contents was observed, suggesting that soluble carbohydrates have adaptive value for wheat (Kerepesi et al., 1998). However, accumulation of proline and GB did not correlate with salt tolerance in contrasting barley genotypes, while hexoses and TCA intermediates did, suggesting that carbohydrates contribute to salt tolerance of barley (Chen et al., 2007a,b; Widodo et al., 2009). Transcript profiling of drought tolerant and sensitive barley varieties revealed that tolerance correlated with the drought-dependent activation of genes, which are involved in the synthesis, and transport of GB, but not proline (Guo et al., 2009). GB is therefore important for drought but not for salt tolerance in barley, hexoses are significant for salt tolerance, while proline accumulation is rather a symptom of salt susceptibility. In sorghum, drastic increase of both proline and GB levels were recorded upon water deficit (Wood et al., 1996). Accumulation of solutes was higher in a drought-tolerant sorghum line than in a drought sensitive one, with the notable exception of proline, which does not seem to contribute to drought tolerance in this plant (Premachandra et al., 1995). In saline environments, pinitol accumulated in the halophytic wild rice, P. coarctata Roxb., but not in the cultivated rice. Inositol methyl transferase (IMT1) mediates pinitol synthesis in P. coarctata, while this gene is missing in the rice genome (Sengupta et al., 2008). Moreover, a salt tolerant form of L-MIPS was identified in P. coarctata, containing a 37 amino acid stretch, which conferred superior thermodynamic stability and elevated activity to the MIPS enzyme leading to enhanced myo-inositol synthesis in saline environments (Ghosh Dastidar et al., 2006). The capacity to synthesise and accumulate myo-inositol and pinitol can therefore be an important adaptive feature in P. coarctata and probably a number of other extremophile plants (Sengupta and Majumder, 2009). In legumes, drought or salt tolerance correlated with osmolyte accumulation in some but not all species. Drought-induced proline accumulation was not different in contrasting bean landraces or in Medicago truncatula and M. laciniata ecotypes characterised by contrasting drought tolerance. Therefore, proline does not seem to contribute significantly to osmotic stress tolerance in these legumes (Tari et al., 2008; Yousfi et al., 2010). In several
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tropical legumes, water stress induced the accumulation of sugar alcohols and proline, while betaine contents were not changed. Tolerance to low water potential correlated with pinitol accumulation, suggesting that this sugar alcohol contributes to drought tolerance in these species (Ford, 1984). In contrasting, chickpea (Cicer arietinum) genotypes the capacity to accumulate proline correlated with copper tolerance, suggesting that in some species proline can contribute to heavy metal tolerance (Singh et al., 2010). Metabolite analysis of potato cultivars with different drought tolerance showed that enhanced galactose, inositol and proline content in drought-stressed plants correlate with higher degree of tolerance (Evers et al., 2010). The above listed examples confirm the importance of osmoprotectants for stress adaptation not only in extremophiles but in other species as well, including in crops. The elucidation of the importance of osmolyte accumulation in extremophiles became recently possible through comparative analysis of closely related halophyte and glycophyte species, especially those which are related to the model plant Arabidopsis (Orsini et al., 2010).The halophyte T. salsuginea (halophila) and L. crassifolium are close relatives of A. thaliana (a glycophyte), and can grow under extreme saline conditions. Proline contents in T. salsuginea and L. crassifolium are higher than those in Arabidopsis, even in optimal growth conditions, and proline accumulates to higher levels when salt stress is imposed (Fig. 2). Metabolite profiling revealed that in saline environments, proline is the dominant osmoprotective compound in T. salsuginea, followed by sugars, while betaines and polyols do not accumulate in this species (Arbona et al., 2010; Inan et al., 2004; Lugan et al., 2010). Sucrose, galactose and melibiose are among the carbohydrates that also accumulate at high levels in T. salsuginea (Lugan et al., 2010). Transcript analysis showed that the P5CS gene, which controls the glutamate-derived proline biosynthetic pathway, has higher constitutive expression level in T. salsuginea than in Arabidopsis, and is induced more rapidly under stress (Inan et al., 2004; Taji et al., 2004). High proline contents in T. salsuginea could also be the consequence of lower transcription of the ProDH gene, which controls proline catabolism (Kant et al., 2006). Such comparative analysis suggests that proline levels are key for the salt tolerance of T. salsuginea, which is controlled by the activities of its key biosynthetic and catabolic genes.
V. CONCLUSIONS The specific physiological responses elicited by various stresses on higher plants are relatively well described. The accumulation of osmoprotective compounds represents a specific metabolic response that is important to
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A
Arabidopsis
Thellungiella
Lepidium
Salt stress
Control B
Proline (mM/gFW)
400
300 Control
200
Salt 100
0 Arabidopsis
Thellungiella
Lepidium
Plants
Fig. 2. Comparison of salt tolerance and proline accumulation of Arabidopsis thaliana and two close relatives: Thellungiella salsuginea and Lepidium crassifolium. (A) Plants were grown in soil for 4 weeks and then watered twice a week with water or 0.5 M NaCl for 2 weeks. (B) Relative proline accumulation in the three brassicaceae species after 1 week of salt stress. Note that both halophytes contained more free proline in the absence of salt stress and accumulated more proline than Arabidopsis under saline conditions.
withstand harmful conditions. Although osmoprotective compounds belong to various categories of bioactive chemicals, they have similar cellular functions such as the stabilisation of cellular structures, protein complexes or specific enzymes or the control of redox balance and ROS production. Sugars or proline could function as metabolic signals and therefore have broader influence on physiological responses and metabolic adjustment to stress conditions.
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Despite of the enormous amount of information accumulated in the past decades, the exact function of low-molecular-weight protective compounds in the adaptation to extreme environmental conditions is still not completely understood. Combining genomic, proteomic and metabolic profiling approaches could increase our understanding of plant stress responses on a global scale and of the metabolic bases of adaptation to drought, salinity or extreme temperatures. Engineering of model and crop plans via genetic transformation is a promising tool to study the significance of osmoprotective compounds in stress responses and to improve the performance of crop plants under suboptimal conditions. Enhanced accumulation of a metabolite can be achieved via activation of the biosynthetic pathway or inhibition of the catabolic pathway. Alternatively, novel pathways can be established in plants, by introducing genes from other species. Various examples have been published for engineering the synthesis and accumulation of osmoprotective compounds. Although osmoprotectant levels in such transgenic plants are often low, and increase in stress tolerance is small, manipulation of the metabolism of osmoprotective compounds have already produced promising results (Chen and Murata, 2008; Rontein et al., 2002; Szabados and Savoure, 2010; Verbruggen and Hermans, 2008). Adaptation to extreme environmental conditions is a complex trait, controlled by numerous genes. Engineering several traits by simultaneous manipulation of two or more genes is a real alternative to handle complex traits such as stress tolerance. Significant enhancement of drought tolerance have been achieved by pyramiding of transgenes in maize, by combining codA from E. coli and TsVP (V-H(þ)-PPase) from Thellungiella. Expression of the two transgenes led to higher GB accumulation and enhanced H(þ)-PPase activity compared with the parental lines, resulting in lower cell damage and higher yields under drought conditions (Wei et al., 2010a,b). Simultaneous engineering of osmolyte accumulation and various other traits by transgenic technology therefore is a feasible strategy to improve abiotic stress tolerance in crops.
ACKNOWLEDGEMENTS Authors are indebted to Ilse Kranner, Csaba Papdi and Laura Zsigmond for proofreading and correcting the chapter. Research and this publication were supported by OTKA grant K-68226, Cross-Border Cooperation Programme HURO/0801/167 and COST Action FA0901.
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Ion Transport in Halophytes
SERGEY SHABALA1 AND ALEX MACKAY
School of Agricultural Science, University of Tasmania, Hobart, Tas, Australia
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Relevance of Halophytes to Crop Breeding for Salinity Tolerance ............................................................ B. Evolution and Diversity of Halophytes ................................... C. Halophytes as Potential Cash Species for Saltwater Management ...................................................... II. Anatomical and Morphological Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Root Structure ................................................................ B. Succulency ..................................................................... C. Salt Bladders and Glands ................................................... III. Whole-Plant Ionic Relations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Tissue-Specific Compartmentation ........................................ B. Inorganic Ion Accumulation and Osmotic Adjustment ................ C. Organic Osmolytes: Osmotic Adjustment or Osmoprotection? ....... IV. Radial Ion Transport in Halophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plasma membrane transport systems in root epidermis ................ B. Ion Transporters in Root Vacuoles........................................ V. Xylem Ion Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Unloading and Ion Transport in Leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Ion Transport in Leaf Mesophyll .......................................... B. Vacuolar Sequestration in Leaves.......................................... C. Pinocytosis..................................................................... D. Ion Retention in the Vacuole ............................................... VII. Ion Transport in Guard Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Stomata Control in Halophytes ............................................
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Corresponding author: E-mail:
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Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00005-9
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B. Ionic Relations in Guard Cells ............................................. C. Guard Cell Electrophysiology .............................................. VIII. Salt Glands and Bladders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Oxidative Signalling and Damage Repair in Halophytes . . . . . . . . . . . . . . . . . A. Major Antioxidant Systems and Their Control.......................... B. Oxidative Stress Signalling and Tolerance in Halophytes.............. X. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT The increasing problem of global land salinisation and associated multibillion dollar losses in crop production require a better understanding of key physiological mechanisms conferring salinity tolerance in crops. The effective way of gaining such knowledge comes from studying halophytes. Halophytes have always attracted the attention of plant physiologists, due to their remarkable ability to tolerate and even benefit from salt concentrations that kill most other plant species. At the very least, halophytes may provide genes that allow transgenic conference of salinity tolerance to crops. In addition, some halophytes have already been tested as vegetable, forage and oilseed crops in agronomic field trials, whilst others already show good potential to be developed as crops. Surprisingly, our knowledge of fundamental ionic and molecular mechanisms conferring salinity tolerance in halophytes is rather limited and, at best, is restricted to several model species. This chapter summarises the current knowledge of physiological mechanisms regulating ion uptake and sequestration in halophytes. The following topics are covered: specific anatomical and morphological features of halophytes; tissue- and organ-specific ion compartmentation; mechanisms of osmotic adjustment in halophytes; radial ion transport in halophytes roots; mechanisms of Naþ and Kþ loading into the xylem; Naþ sequestration in vacuoles in roots and leaf cells; ion transport in guard cells; control of ion fluxes into salt glands and bladders; and oxidative signalling and damage repair in halophytes. It is concluded that more indepth electrophysiological and molecular studies are needed to reveal the identity of membrane transport systems and better understand what appears to be the complex and highly orchestrated regulation of ion transport and sequestration in halophytes.
I. INTRODUCTION A. RELEVANCE OF HALOPHYTES TO CROP BREEDING FOR SALINITY TOLERANCE
Although most major mechanisms conferring salinity tolerance in plants have been intensively studied over the past few decades, crop breeding for salt tolerance has not met with much success to date (Yamaguchi and Blumwald, 2005). There is little doubt that salt tolerance is a complex multigenic trait showing heterosis, dominance and additive effects (Flowers, 2004; Fooland, 1997). Salt tolerance is also multifaceted physiologically, with numerous tissue- and age-specific components involved
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(Shabala and Cuin, 2008). As such, salt tolerance will be determined by a number of subtraits (specific for each particular species), any of which might, in turn, be determined by any number of genes. Tester and Davenport (2003) estimated that salinity affects the level of transcription of 8% of all genes; however, less than a quarter of these appear to be salt stress specific (Ma et al., 2006). Thus, to genetically manipulate salinity tolerance traits, the specific physiological roles of all these genes must be revealed, and the relative contribution of each of them quantified. The most efficient way of doing this is to use halophytes, both as the model species in physiological studies, and as a potential source of salinity tolerance genes for major agricultural crops (Flowers et al., 2010). B. EVOLUTION AND DIVERSITY OF HALOPHYTES
The Dictionary of Botany defines halophytes as ‘plants that are adapted to live in soil containing a high concentration of salt’. The point at which a plant comes to be called a halophyte, as opposed to glycophyte, is not readily definable. Greenway and Munns (1980) have previously defined halophytes as plants that can tolerate monovalent ion concentrations above 70 mM. More recently, Flowers and Colmer (2008) have classified halophytes as plants that can complete their lifecycle in salinities equivalent to 200 mM NaCl or more. The degree to which plants can tolerate salt varies from species to species. One possible demarcation line could be whether or not plants actually benefit from having some significant (e.g. 100 mM or more) amounts of NaCl in the soil solution. While Naþ is not considered to be an essential nutrient for plants, many halophytes show a peak in growth performance, only when grown in the presence of NaCl. Optimal concentration may vary between species and can range from 150 to 200 mM (e.g. Salicornia biglovii; Neales and Sharkey, 1981; Atriplex spongiosa; Storey and Wyn Jones, 1979) to as high as a seawater, for example, 510 mM NaCl (Sarcocornia fruticose or Arthrocnemum macrostachyum; Redondo-Gomez et al., 2006). To a large extent, the difficulty in agreeing upon what defines a halophyte originates from the fact that halophytes have no clearly defined or unique strategy, structure or mechanism that would be completely absent in glycophyte species. As such, succulence can also occur in glycophytes in response to salinity, and is a feature of xerophytes as well as halophytes. Similarly, epidermal glands and bladders can also be found in non-halophytes, and at least half of the halophytes do not posses these structures. Hence, halophytism appears to be an integration of a large number of adaptive (mainly physiological) mechanisms rather than a distinct morphological or anatomical feature.
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The fossil record suggests that the first land plants arose 470 million years ago (Kenrick and Crane, 2000), and it is thought that these first plants evolved from freshwater algae (Rodriguez-Navarro and Rubio, 2006). The reason this hypothesis is favoured is because all living cells apparently have an absolute requirement for Kþ (Rodriguez-Navarro and Rubio, 2006), so preadaptation for high-affinity Kþ uptake would be critical for the move onto dry land. If this hypothesis is right, then salt tolerance must have evolved later. To speculate on the advent of salt tolerance, it is evident that, in nature, resource rich areas do not remain uncolonised for long; ‘nature abhors a vacuum’. It is known that the earth’s climate is not stable over the long term, fluctuating between glacial and non-glacial periods and the concurrent sea level fluctuations would have resulted in large tracts of land becoming available during various epochs. At the ebb point of these occasions, it would seem highly likely that plants would have colonised the newly available land. Other factors that would create salinised land would be geological uplift, erosion of sedimentary rock containing salts and salt-laden winds depositing salts inland, and these areas would likewise be colonised by plants. The plants that would most likely have been first to take advantage would perhaps be plants in various clades that were to some extent already preadapted, possibly through adaptation to drought stress. If salt tolerance did evolve after plants colonised the land, then it is likely that salt tolerance evolved several times, independently (Flowers et al., 2010). Lines of evidence for this include the low percentage of halophytes (< 1%) in most orders of plants, meaning that the vast majority of the plants in those orders would have to have lost their salt tolerance; the pattern of salt tolerance (or lack thereof) between closely related families; and the use of different morphological and physiological strategies for salt tolerance between and within different lineages (Flowers et al., 2010). It should also be commented on that salt tolerance in plants is not an either– or condition, rather it represents a continuum of degrees of tolerance to salinity. In this context, some salt-tolerant glycophyte species have successfully implemented key physiological mechanisms conferring salinity tolerances in halophytes such as Naþ vacuolar sequestration, or the use of inorganic ions for osmotic adjustment (Flowers and Colmer, 2008). Again, any demarcation line is pretty thin here. It is thought that one fundamental difference between halophytes and glycophytes is the ability to productively utilise Naþ and Cl for osmotic adjustment, and not merely negate the deleterious effects these ions have by sequestering them in the vacuole. However, direct pressure-probe microelectrode experiments have shown that, under hypertonic conditions, Arabidopsis roots were able to regain over 90% of their initial turgor merely by increased uptake of inorganic ions;
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among these, Naþ and Cl have contributed to more than 70% of the observed turgor recovery (Shabala and Lew, 2002). In barley, salt-tolerant varieties loaded much higher amounts of Naþ into the xylem compared with sensitive genotypes (Shabala et al., 2010), presumably for more efficient osmotic adjustment in the shoot. Thus, it appears that there is nothing really unique to halophytes that is not present in glycophytes; the major difference is to how efficiently these mechanisms are controlled in these two plant groups. As mentioned above, the number of halophytic species is relatively low (< 1%) with estimates of how many species there are ranging from 345 to 5000–6000, depending on how one defines salt tolerance (Flowers et al., 2010; Glenn et al., 1999). Halophytes are present in about half the higher plant families (Flowers and Colmer, 2008) and, while some orders contain many species of halophytes (e.g. Caryophyllales, Alismatales, Malpighiales and Poales; Table I), there are many orders that contain only a few halophytic
TABLE I List of Various Orders of Flowering Plants that Contain Halophytic Species (Reproduced From Flowers et al., 2010, with Permission from CSIRO Publishing)
Order
Number of families
Number of families with halophytes
% families with halophytes within the order
Caryophyllales Alismatales Malpighiales Poales Lamiales Myrtales Fabales Malvales Ericales Arecales Gentianales Sapindales Asterales Zygophyllales Apiales Magnoliales Solanales Celastrales Fagales Brassicales Total
34 14 40 16 25 12 4 10 25 1 5 9 11 2 7 6 5 4 8 18 256
9 7 4 4 5 4 1 1 6 1 4 3 2 1 1 1 1 1 1 1 58
26 50 10 25 20 33 25 10 24 100 80 33 18 50 14 17 20 25 13 6 —
Number of halophytic species
% of all halophytic species
74 61 35 28 24 22 21 18 13 11 9 8 8 3 3 2 2 1 1 1 345
21.4 17.7 10.1 8.1 7.0 6.4 6.1 5.2 3.8 3.2 2.6 2.3 2.3 0.9 0.9 0.6 0.6 0.3 0.3 0.3 100
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species (e.g. Zygophyllales and down; Table I). More interestingly, there is no strong correlation between number of families with halophytes within an order and number of species of halophytes within that order. This table is just one interpretation (and the most conservative one) of halophyte phylogeny, yet it may still be possible to say that it further adds to the theory that halophytes have arisen independently within different clades (Table I). C. HALOPHYTES AS POTENTIAL CASH SPECIES FOR SALTWATER MANAGEMENT
Very large volumes of poor-quality water are generated by agriculture, industry and municipal water treatment processes (Glenn et al., 1999). Much of this is saline, containing from 1000 mg/L to above 7000 mg/L of total dissolved salts, with sodium frequently the dominant cation. These wastewaters are commonly discharged into aquifers, municipal sewage systems or surface waters where they represent a potential hazard to downstream water users and the environment (Gerhart et al., 2006). The result is degradation of soil structure and detrimental effects to crops and ecosystems. Although the most environment-friendly way of solving the problem of saline wastewater disposal would be the use of desalination plants, the cost of construction and operating such plants is substantial, and so is its ‘carbon footprint’. It is estimated that the initial cost of construction of one ‘standard size’ desalination plant to dispose of 5 ML/day of saline wastewater is AUD$17.5M, with a further AUD$6M per annum required for operation (G. Haros, personal communication). Thus, economically viable alternatives need to be found. One of these options is to use halophytes as cash crop species. Halophytes have been tested as vegetable, forage and oilseed crops in agronomic field trials. The most productive species yield 10–20 t/ha of biomass on seawater irrigation, equivalent to productivity of conventional crops under non-saline conditions (Glenn et al., 1999; Masters et al., 2007; Jordan et al., 2009). Atriplex species are already used for utilisation of saline water from coal bed methane extraction in Montana, USA where they absorb about 6000 m3 year 1 of effluent saline water (Browning et al., 2006). Some Atriplex species can produce between 12 and 21 t/ha of the fresh biomass when grown at 100–500 mM NaCl salinity (Glenn et al., 1999) and are considered excellent livestock fodder having favourable crude protein levels (10–20% protein; Khan et al., 2000), with the current market value estimated at $800/tonne. Another example is Salicornia bigelovii, an annual C3 halophyte plant from the Salicornioideae subfamily that inhabits salt marshes and estuaries. Salicornia is arguably one of the most salt-tolerant vascular plants and can yield as much biomass and seed as many
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conventional crops under non-saline conditions, even when the soil solution exceeds 1.3 M NaCl (twice seawater salinity; Grattan et al., 2008). It can produce between 12 and 24 t/ha of biomass and between 1.4 and 2.4 t/ha of seed over a 200-day growing cycle (Glenn et al., 1991) and has an estimated market value of $800/tonne. The seed contains 31% protein, 28% oil (mostly polyunsaturated), and only 5% fibre and 5% ash (Glenn et al., 1999). This is similar to the yield and seed quality of soybean grown under non-saline conditions. Salicornia can handle as much as to 3800 mm/annum irrigation and is already used in some parts of USA (e.g. in California) as an environmentally sound method of reusing saline drainage water (San Joaquin Valley Drainage Program; Grattan et al., 2008). It was concluded that halophyte forage and seed products can replace conventional ingredients in animal feeding systems, with some restrictions on their use due to high salt content and antinutritional compounds present in some species (Glenn et al., 1999; Rogers et al., 2005). The recent rapid rise in the number of publications concerning the use of halophytes has suggested that they could play important roles in recycling saline agricultural wastewater and reclaiming salt-affected soil in arid-zone irrigation districts. Commercial-scale field trials have been conducted in Montana (Browning et al., 2006) and California (Grattan et al., 2008).
II. ANATOMICAL AND MORPHOLOGICAL FEATURES As commented above, it appears that halophytes do not possess any specific ‘anatomical hallmarks’ that confer this remarkable salinity tolerance and that will make them strikingly different from glycophytes. However, although the particular structures such as glands or bladders, and tissue succulence are not specifically unique to halophytes, it is apparent that the way the halophytes utilise these features is. It may be that what halophytes have done is to simply improve extant mechanisms of stress tolerance (Bohnert et al., 1995). Some of these mechanisms involve aspects of gross morphology such as variances in the temporal and spatial development of structures within the roots, and development and efficient utilisation of structures associated with both Naþ sequestration and secretion. A. ROOT STRUCTURE
Root suberisation and the presence of the Casparian strip in the root endodermis are two important anatomical features controlling nutrient and water transport patterns. In glycophytes, root suberisation increases with salinity
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stress (Steudle, 2000); this may be functionally important to both prevent apoplastic flow of toxic Naþ ions as well as increase water retention in the root. Some halophytes have made this trait constitutive, by developing an extra endodermis layer (e.g. Mesembryanthemum crystallinum; Inan et al., 2004). Also, the Casparian bands form earlier and are longer and closer to the root apex of Suaeda maritima (Hajibagheri et al., 1985). These authors also reported earlier vacuolisation of cells of the root apex, and much greater volume of cortical cells (e.g. root succulency). However, similar responses were recorded for glycophyte species (e.g. Gossypium hirsutum; Reinhardt and Rost, 1995). Thus, it appears that these adaptive anatomical features cannot be attributed exclusively to halophytes.
B. SUCCULENCY
Leaf succulency is a term used to describe thickening of leaf tissues and the resultant increase in the volume of leaf sap. Increased leaf succulency is a typical adaptive response in glycophytes and may be achieved by increasing the size of mesophyll cells and the relative size of their vacuoles (Gorham et al., 1985; Longstreth and Nobel, 1979). Another option is to increase the number of spongy cell layers (Longstreth and Nobel, 1979). Similar responses were reported for halophyte species. It has long been suggested that succulence increases with salinity as a direct response to having to increase storage area for Naþ and Cl (Jennings 1968; Hajibagheri et al., 1984). Importantly, rapid thickening of the halophyte leaves is often associated with a delay in the development, and longer survival of individual leaves under saline conditions (Black, 1958). Given the existing causal relationship between cytosolic Kþ content and programmed cell death (read ¼ senescence) under saline conditions (discussed in Section IX), it remains to be studied as to whether increased leaf succulency also delays programmed cell death in glycophyte crops.
C. SALT BLADDERS AND GLANDS
Salt bladders/glands are arguably the most remarkable feature found in some halophytes. Both bladders and glands arise from epidermal cells and are modified trichomes (Adams et al., 1998). The distinction between bladders and glands is ill defined in the early literature (1970s). A good distinction would be that glands extrude salts on a regular basis, whilst bladders release salt only once when the bladder ruptures, after a prolonged period of accumulation. Having salt bladders rather than glands may be advantageous for
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species growing in drier climates, by possibly providing a reservoir of useable water (Tester and Davenport, 2003). Glands and bladders can be constructed in several ways, perhaps alluding to their evolutionarily diverse origins. Figure 1 shows three generalised types of bladders/glands found in halophytes (based on Agarie et al., 2007; Oross and Thomson, 1982). These are two-celled excretory structures found in most
Fig. 1. (A) Salt gland typical of those found in the graminoids. The glands are recumbent to the epidermis, and a waxy cuticle covers all epidermal surfaces, broken only by cuticular pores in the cap cell. The postulated increase in the Naþ concentration gradient from low in the mesophyll to highest in the bladders and glands is indicated by the number of Naþ symbols in the vacuoles of the various tissues. (B) Multicellular salt glands, as found in various families of dicots and some graminoids. These glands are thought to consist of layers of specialised cells, namely, collection cells that pass Naþ ions through the intermediary basal cells to the secretory cells. (C) Schematic of an EBC, as found in such clades as Chenopodioideae, Oxalidaceae and Mesembryanthemaceae. The schematic is not to scale, as the bladders can be an order of magnitude larger than epidermal cells. EBC, epidermal bladder cell; CaC, cap cell; SeC, secretory cell; CuP, cuticle pore; BaC, basal cell; ColC, collecting cell; V, vacuole; SC, stalk cell; C, cuticle; ED, epidermis; M, mesophyll; PD, plasmodesmata; AP, apoplast; XY, xylem.
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of the graminoids (Fig. 1A): multicellular structures found in some graminoids and several dicot families (Fig. 1B), and the epidermal bladder cells (EBCs) characteristic of the Chenopodioideae, Oxalidaceae and Mesembryanthemaceae (Fig. 1C). EBCs are thought to be storage sites for excess Naþ, Cl and Kþ (Adams et al., 1998; Agarie et al., 2007; Jeschke and Stelter, 1983; Luttge, 1971). Other functions postulated for EBCs include storage of water and various metabolic compounds such as malate, flavinoids, cysteine, pinitol, inositol and calcium oxalate crystals (Adams et al., 1992; Agarie et al., 2007; Jou et al., 2007). Salt bladders may also play an important role as a secondary epidermis to reduce water loss and prevent excessive UV damage. Salt bladders can be very large structures in relation to epidermal cells, of an order of a few hundred microns in length and diameter (Fig. 2). Bladder densities vary depending upon leaf surface (adaxial vs. abaxial), leaf age and plant species (Fig. 2). Loading of bladder cells is still an area of conjecture. Most of the knowledge on this subject is derived from microscopy studies and lack functional characterisation. Several specialised cell types have been reported (Fig. 1). A clear pronounced concentration gradient for Naþ appears to exist between bladder/gland cells, epidermal cells and leaf mesophyll cells (discussed further in Section III.A.). The pathways for Naþ transport into the bladder cells,
Fig. 2.
(Continued)
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Fig. 2. (A) Micrograph of the salt bladders on the abaxial surface of a young leaf of Chenopodium quinoa. The image was taken using a scanning electron microscope in environmental mode. The bladders shown are a third the size they will ultimately achieve. Upon maturing, the bladders take on the appearance of a greatly distended balloon (as depicted in Fig. 1C). (B) Micrograph of a cross-section through a young leaf of Atriplex lentiformis showing numerous bladders on both adaxial (upper right) and abaxial (lower left) surfaces. The image was taken using a scanning electron microscope in environmental mode. Note that the density of bladders is greater on the abaxial surface and that overall bladder density in young leaves is higher for Atriplex lentiformis and then for Chenopodium quinoa.
however, remain elusive and almost certainly involve both apoplastic and symplastic components. Evidence for a symplastic pathway comes from the abundance of plasmodesmata connections in some of these structures (Fig. 1), while tissue-specific expression of some ion transporters (discussed further in Sections VI and VIII) implies that ions must be released from some of the cells (e.g. mesophyll cells) and then reabsorbed by epidermal or bladder cells. In this context, Tester and Davenport (2003) have suggested that salts are accumulated in a sealed-off region of apoplast resulting in an osmotic gradient that creates sufficient pressure to pump salt-laden solution through glands or into bladders via bulk flow. Another possibility to build this concentration gradient may be through the symplastic pathway by active Naþ removal from the bladder cell cytosol in a process of vacuolar sequestration. This implies that the expression levels and/or activity of tonoplast Naþ/Hþ exchangers must decrease in the sequence inversely proportional to
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the amount of Naþ accumulated in the cell (e.g. EBCs > collecting cell mesophyll; Fig. 1C). Finally, the extent of leaf cutinisation and the presence of cuticular pores (Fig. 1B) may play a pivotal role in water flow into bladder/ gland cells and, hence, affect their ion loading. It should also be stated that at least 50% of halophytes do not utilise glands or external bladders to modulate their tissue ion concentration. For example, Atriplex species contain well-pronounced salt bladders, while (equally salt tolerant) Suaeda species have no mechanisms for extra-foliar salt exclusion (Storey and Wyn Jones, 1979). Salt glands are well developed in black mangroves but are absent in (equally salt-tolerant) red mangroves (Glenn et al., 1999). This poses the following question: how do gland-less halophytes control Naþ transport and sequestration, and is there any difference in the functional expression of Naþ transporters between halophyte species with and without salt glands?
III. WHOLE-PLANT IONIC RELATIONS A. TISSUE-SPECIFIC COMPARTMENTATION
Whole-plant ionic relations and tissue-specific ion compartmentalisation in halophytes have been the subject of extensive reviews over the past two decades (Flowers, 1985; Flowers et al., 1986; Flowers and Colmer, 2008; Glenn et al., 1999) and, thus, are only briefly reiterated here. While dicotyledonous halophytes show optimal growth in 100–250 mM NaCl (Flowers and Colmer, 2008; Flowers et al., 1986), the optima are much lower for monocotyledonous species (Glenn, 1987; Glenn et al., 1999). Dicotyledonous halophytes generally accumulate more NaCl in shoot tissues than their monocotyledonous counterparts (Table II), with total ‘ash’ content in the former often exceeding 50% of the shoot dry mass (Flowers and Colmer, 2008). It is not clear whether this difference is attributed to specific anatomical features, higher transpiration rates or higher activity of Naþ transporters in some plant tissues. Much less (three- to fourfold) Naþ is accumulated in halophyte roots compared with shoots (Table III), while Kþ content in both organs is comparable (Tables II and III). The most obvious ‘physiological hallmark’ distinguishing halophytes from glycophytes is their ability to select Kþ from a mixture dominated by Naþ and yet accumulate sufficient Naþ for the purposes of osmotic adjustment. At the whole-plant level, the selectivity between Kþ and Naþ (SK/Na) in halophytes is within the range of 100–200, even at external salinities exceeding sea water levels (Storey et al., 1983a,b). Specific details beyond this selectivity remain obscure.
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TABLE II Sodium and Potassium Content in Leaves of Selected Halophyte Species Grown at High External Salinities (200–500 mM NaCl Range) [Naþ]
[Kþ]
Reference
Aster tripolium Atriplex nummularia
125–275 330–870
65 65–100
Atriplex prostrata Atriplex spongiosa
660–1205 400–650
160–200
Atriplex portulacoides
220–320
140–160
Atriplex centralasiatica Bolboschoenus maritimus Chenopodium glaucum Crypsis aculeata Jaumea carnosa Juncus gerardii Lepidium crassifolium Mesembryanthemum crystallinum Puccinellia distans Salicornia prostrata Scorzonera parviflora Spergularia media Suaeda salsa Suaeda monoica
325–440 60–400 150–380 100–125 165–500 40–150 90–380 240–690
50–110
50–130 380–620 75–125 240–450 340–1150 750–880
35–80 25–40
Suaeda maritima
380–660
35–160
Thellungiella halophila
200–395
Triglochin maritimum Plantago maritima
170–260 100–300
Ueda et al. (2003) Ramos et al. (2004) Flowers (1985) Flowers (1985) Storey and Wyn Jones (1979) and Storey et al. (1983a,b) Redondo-Gomez et al. (2007) Qiu and Lu (2003) Albert (1975) Albert (1975) Albert (1975) Ramos et al. (2004) Albert (1975) Albert (1975) Agarie et al. (2007) Adams et al. (1998) Albert (1975) Albert (1975) Albert (1975) Albert (1975) Wang et al. (2001) Storey and Wyn Jones (1979) Yeo (1981), Yeo and Flowers (1986), Wang et al. (2007) Vera-Estrella et al. (2005) Albert (1975) Albert (1975)
Species
Data were collated from published sources and expressed as millimoles of leaf sap [Naþ] or [Kþ], for the purposes of consistency.
Tissue-specific compartmentalisation appears to play an important role in controlling the above selectivity. Pronounced Naþ gradients were detected between EBCs, leaf epidermis and mesophyll cells in M. crystallinum (Barkla et al., 2002). Gradients in SK/Na selectivity were also reported between the bundle sheath cells and the bladder cells in A. spongiosa (Storey et al., 1983a). However, over 50% of halophytes do not have glands or external bladders to modulate their tissue ion concentration (Flowers and Colmer, 2008), so the above explanation is not applicable to all situations. It appears that vacuolar
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TABLE III Sodium and Potassium Content in Roots of Selected Halophyte Species Grown at High External Salinities (200–500 mM NaCl Range) Species Atriplex prostrata Atriplex spongiosa Atriplex nummularia Atriplex halimus Atriplex portulacoides Disphyma crassifolium Mesembryanthemum crystallinum Puccinellia maritima Porterisia coarctata Salicornia bigelovii Sarcocornia natalensis Suaeda maritima Sporobolus virginicus Thellungiella halophila Triglochin maritimum
[Naþ] 130–315 80 105–160 70–95 60–100 220–475 110–150 110–140 80–100 220–270 20–150 105–200 39–99 70–210 148
[Kþ] 50–65 120 85–95 50–60
45–60 75–100 65–220 105–215 30–45 70
Reference Anderson et al. (1977) Storey et al. (1983a,b) Ramos et al. (2004) Ben Hassine et al. (2008) Redondo-Gomez et al. (2007) Neales and Sharkey (1981) Barkla et al. (2002) Flowers (1985) Flowers et al. (1989) Ayala and O’leary (1995) Naidoo and Rughunanan (1990) Yeo (1981), Hajibagheri et al. (1984) Marcum and Murdoch (1990) Vera-Estrella et al. (2005) Jefferies (1973)
Data were collated from published sources and expressed as millimoles of root sap [Naþ] or [Kþ], for the purposes of consistency.
sequestration fulfils this role in bladder-less species. Indeed, even within species possessing bladders, Naþ may be sequestered in salt bladders primarily in young leaves, while in mature leaves, the major Naþ storage site are vacuoles (Storey et al., 1983a,b). The above, high SK/Na selectivity in halophytes appear to be intrinsically related to a tissues (and, specifically, roots) ability to retain Kþ. Contrary to glycophytes (Shabala and Cuin, 2008; Tester and Davenport, 2003), increasing salinity stimulated Kþ accumulation in halophyte roots (e.g. Suaeda monoica or Triglochin maritima; Marcum and Murdoch, 1992) and resulted in plants’ ability to maintain fairly constant Kþ concentrations in the shoot. Volkov et al. (2003) have concluded that the main difference between celltype-specific ion distribution in Arabidopsis thaliana and its halophyte relative Thellungiella halophila during salt stress was related to Kþ rather than Naþ. These authors have reported that Thellungiella plants accumulated very high Kþ concentrations in the epidermis under control conditions, although during salt stress, epidermal Kþ concentrations decreased dramatically, whereas bulk Kþ concentrations increased. The authors suggested that the epidermis played the role of storage site for Kþ in this species (Volkov et al., 2003). It should be noted, however, that these findings cannot be extrapolated to all halophytes, with salinity-induced decrease in root Kþ content being reported for some halophyte species (Hajibagheri et al., 1985).
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B. INORGANIC ION ACCUMULATION AND OSMOTIC ADJUSTMENT
Halophytes are adapted to low water potential by their capacity for osmotic adjustment. It is widely accepted that cell turgor is maintained by storage of Naþ and Cl in vacuoles, with the solute potential of the cytosol adjusted by accumulation of Kþ and organic solutes (Flowers et al., 1977; Glenn et al., 1999; Storey, 1995; Storey and Wyn Jones, 1979). According to Glenn et al. (1999), the three major inorganic ions, Naþ, Kþ and Cl, account for 80– 95% of the cell sap osmotic pressure in both halophyte grasses and dicots. As a result, halophytes accumulate substantial amounts (> 10% of dry weight each) of Naþ and Cl in their shoots (Grattan et al., 2008), predominantly in vacuoles (Flowers and Colmer, 2008). At the same time, cytoplasmic Kþ concentrations in halophytes are similar to those of glycophytes (Flowers and Colmer, 2008). As a result, halophytes have a rather high ( 5) vacuole/ cytosol Naþ ratio and, at the same time, a high ( 4) cytosol/vacuole Kþ ratio (Ye and Zhao, 2003).
C. ORGANIC OSMOLYTES: OSMOTIC ADJUSTMENT OR OSMOPROTECTION?
It has long been thought that halotolerance depends on the osmotic adjustment of the cytoplasm, achieved by the accumulation of compatible solutes (Storey and Wyn Jones, 1979). The two major osmolytes are glycine-betaine (GB) and proline, although others such as inositol, pinitol, sorbitol, mannitol and ononitol are also reported (Agarie et al., 2007; Ben Hassine et al., 2008). The expression patterns of compatible solutes vary dramatically in time, as well as between species and tissues. As such, proline accumulated as early as 24 h after stress imposition while GB concentration culminated after 10 days of stress and did not decrease after the stress was relieved, in Atriplex (Ben Hassine et al., 2008). While root betaine concentrations in Armeria maritime increased under saline conditions, no significant increase in betaine in leaves was detected (Kohl, 1997), where concentrations remained between 2 and 4 mM. At the same time, much higher (120 mM) GB levels were reported in the leaves of Sporobolus virginicus (Marcum and Murdoch, 1992). Even assuming that compatible solutes (such as GB or proline) are located exclusively within the cytosol, the reported range of concentrations (typically 20–150 mM; Kohl, 1997; Storey and Wyn Jones, 1979) is hardly sufficient for full osmotic adjustment in the cytosol, given the reported Naþ concentrations in the vacuole (e.g. 500–600 mM; Maathuis et al., 1992) or external media (200–500 mM) found under typical conditions. Hence, it is more likely that the major role of compatible solutes in halophytes may not be in
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conventional water retention but in osmoprotection and/or ROS scavenging (Bohnert and Shen, 1999; Ben Hassine et al., 2008). We have previously reported that compatible solutes are potent regulators of Kþ-permeable ion channels in the plasma membranes of root epidermal cells (Cuin and Shabala, 2007a). In this study, root pretreatment with physiologically relevant concentrations of amino acids resulted in a significant reduction of detrimental salinity effects on cytosolic Kþ homeostasis. Similar results were obtained for physiologically relevant concentrations of glycine– betaine, a representative of another class of compatible solutes (Cuin and Shabala, 2005). Moreover, low (5 mM) concentration of compatible solutes such as glycine–betaine (a quaternary amine), proline (amino acid), and mannitol or myo-inositol (polyoles) were also efficient in reducing disturbance to intracellular Kþ homeostasis caused by oxidative stress (OH. treatment; Cuin and Shabala, 2007b). Taken together, these results suggest that physiologically relevant concentrations of compatible solutes might contribute to plant adaptive responses to salinity by regulating Kþ transport across the plasma membrane, thus enabling maintenance of an optimal Kþ/ Naþ ratio, arguably the most important feature of salinity tolerance in plants (Shabala and Cuin, 2008; Tester and Davenport, 2003). Surprisingly, no such work has been conducted on halophytes. Thus, the question remains to be answered, can plasma membrane transporters in halophyte cells be gated by the stress-induced changes in the osmolyte levels under saline conditions, and whether there is a difference in the gating properties between halophyte and glycophyte species.
IV. RADIAL ION TRANSPORT IN HALOPHYTES A. PLASMA MEMBRANE TRANSPORT SYSTEMS IN ROOT EPIDERMIS
It is generally accepted that weakly voltage-dependent non-selective cation channels (NSCC) represent a major pathway for Naþ uptake by glycophyte roots (Amtmann and Sanders, 1999; Tyerman and Skerrett, 1999; Demidchik et al., 2002). Among other candidates, dual-affinity HKT transporters and low-affinity LCT transporters have been named as potential mediators of Naþ uptake by plant roots under saline conditions (Garciadeblas et al., 2003; Maser et al., 2002; Rubio et al., 1995; Uozumi et al., 2000). However, some other authors deny HKT involvement in root Naþ uptake and instead suggest its involvement in Naþ retrieval from the xylem (Berthomieu et al., 2003; Tester and Davenport, 2003).
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TABLE IV Root Plasma Membrane Transport Systems Reported in Halophytes Transporter þ
þ
Species
Na /H exchanger Naþ/Hþ exchanger Voltage -independent Kþ-selective channel Time-dependent inward-rectifying channel
Thellungiella
Method
Thellungiella
Expression studies Expression studies Patch-clamp
Thellungiella
Patch-clamp
Time-dependent outward-rectifying channel
Thellungiella
Patch-clamp
HAK high-affinity Kþ uptake transporter HAK family transporters HKT transporter
Thellungiella
Molecular
Mesembryanthemum Mesembryanthemum
Hþ-ATPase
Atriplex nummularia
Expression studies TEVC in heterologous expression system Expression studies
Thellungiella
Reference Oh et al. (2007) Vera-Estrella et al. (2005) Volkov et al. (2003)Volkov and Amtmann (2006) Volkov et al., 2003, Volkov and Amtmann 2006 Volkov et al. (2003)Volkov and Amtmann (2006) Aleman et al. (2009) Su et al. (2002) Su et al. (2003)
Niu et al. (1993)
Reports on sodium transport in halophytes are much more limited and usually confined to only a few model species such as Thellungiella halophila or M. crystallinum (Table IV). A significant part of these reports employed various molecular tools to study the effect of salinity on the tissue-specific expression of a particular transporter, or reported its activity after expression in some heterologous system. However, heterologous expression systems have often failed to produce functional channels from transcripts encoding both cyclic nucleotide-gated channels (CNGCs) and glutamate receptor-like channels, which are prime candidates for Naþ uptake channels in A. thaliana (Davenport, 2002; Maathuis and Sanders, 2001). Also, despite the presence of two Naþ/Hþ exchanger isoforms in the plasma membranes of Thellungiella root tissue, no Naþ/Hþ exchange was detected (Vera-Estrella et al.,
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2005), suggesting that some of these transporters may require activation by regulatory molecules that are not present in vitro in transport assays with isolated membrane vesicles. Direct patch-clamp experiments on root plasma membrane transporters mediating ionic homeostasis in halophyte roots are very rare and confined to only one species, T. halophila (Volkov and Amtmann, 2006; Volkov et al., 2003). These studies have identified three distinct types of ion currents in T. halophila root cells: (1) time-dependent inward currents; (2) time-dependent outward currents; and (3) instantaneous currents. The basic properties of these channels are summarised in Table V below. All three current types in T. halophila root cells appear to provide a very limited pathway for Naþ uptake (Volkov and Amtmann, 2006), and at physiological membrane potentials, the instantaneous current constitutes the only pathway for Naþ uptake. These instantaneous currents were fivefold
TABLE V Basic Properties of Major Ion Channels Present at the Plasma Membrane of Thellungiella Halophila Cortical Root Cells (Based on Volkov and Amtmann, 2006) Current Time-dependent inward currents
Time-dependent outward currents
Properties
– – – –
– –
Instantaneous currents
–
– –
Highly selective for Kþ over Naþ Blocked by Csþ PNa/PK < 0.1 Moderately selective for Kþ over Naþ (PNa/PK ¼ 0.07) Inhibited by TEAþ Permeability sequence Kþ> Rbþ> Csþ> NH4þ> Liþ> Naþ Moderately selective for Kþ over Naþ Not inhibited by Csþ or TEAþ Inhibited by Zn2þ, Ba2þ and Ca2þ
Major role
–
–
–
–
–
Allows increasingly large Kþ influx at membrane potentials more negative than 140 mV Provides a pathway for low Kþ influx within a limited range of voltages just below EK Mediates efflux of Kþ at voltages above EK
Facilitates moderate Kþ influx at any voltage more negative than EK Mediates efflux of Kþ at voltages above EK
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more selective for potassium in T. halophila than in A. thaliana (PNa/PK ¼ 0.12 vs. 0.67, respectively; Demidchik and Tester, 2002; Volkov et al., 2003). This is consistent with high SK/Na selectivity ratio observed in halophytes at the whole-plant level (Storey and Wyn Jones, 1979; Storey et al., 1983a,b). Potassium retention in the cytosol has been shown to be one of the key determinants of salinity tolerance in glycophytes (Chen et al., 2005, 2007a,b, 2008; Shabala and Cuin, 2008; Shabala et al., 2006). In this context, the HAK family of genes encoding high-affinity Kþ transport were induced by salt stress in Mesembryanthemum (Su et al., 2002) and Phragmites (Takahashi et al., 2007). However, in the latter species, expression of PhaHAK5 was found in salt-sensitive but not salt-tolerant plants and was attributed to HAK5 permeability to Naþ (Takahashi et al., 2007). At the same time, the down-regulation of McHKT expression in Mesembryanthemum suggests that HKT transporters may have little to do with sodium uptake in this species (Su et al., 2003).Thus, the role of high-affinity Kþ transporters in root Naþ uptake in halophytes need more thorough investigation. It should be kept in mind, however, that Thellungiella is classified as a strong salt excluder (Inan et al., 2004; Taji et al., 2004; Volkov et al., 2003), while the bulk of halophytes show much stronger accumulation of Naþ in their shoots compared with glycophyte species (Flowers and Colmer, 2008). Hence, while the above knowledge on Thellungiella root ion channels is crucial to understanding mechanisms of Naþ exclusion in plants, it cannot be extrapolated to the majority of halophyte species. An interesting attempt has recently been undertaken by Wang and coauthors to study pathways for Naþ uptake in S. maritima, a species which accumulates, without injury, concentrations of the order of 400 mM NaCl in its leaves (Wang et al., 2007). Based on results of pharmacological experiments, these authors have concluded that neither NSCC nor LCT are major pathways for Naþ entry into root cells in Suaeda. Instead, they have suggested that two distinct low-affinity Naþ uptake pathways exist in this species: one mediated by a high-affinity Kþ transporter, and another by an AKT1-type channel inward-rectifying Kþ channel (Wang et al., 2007). Direct patch-clamp experiments are needed to validate these suggestions. Similar to glycophytes (e.g. Chen et al., 2007a,b), voltage gating and roots ability to maintain more negative membrane potential appear to be crucial for salinity tolerance in halophytes. Naþ-induced depolarisation was twofold smaller in T. halophila than in A. thaliana (Volkov and Amtmann, 2006), thus allowing cells to maintain their driving force for potassium uptake. Interestingly, membrane potential values in epidermal root cells in Atriplex hastata ranged between 130 and 140 mV, regardless of variations in external
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NaCl concentrations between 100 and 600 mM NaCl (Anderson et al., 1977). It remains to be tested as to what extent this remarkable ability of Atriplex roots is associated with a greater capacity of the halophyte to induce Hþ-ATPase in response to NaCl (Niu et al., 1993).
B. ION TRANSPORTERS IN ROOT VACUOLES
Given the fact that halophytes use Naþ as a major osmolyte for osmotic adjustment, it is logical to expect that they must possess rather efficient systems for Naþ vacuolar sequestration. Indeed, a tonoplast Naþ/Hþ exchanger gene was isolated and characterised in the roots of Atriplex gmelinii (Hamada et al., 2001). Also, tonoplast Naþ/Hþ antiport activity was induced by NaCl treatment in roots of salt-tolerant Plantago maritima species, but not in salt-sensitive Plantago media (Staal et al., 1991). However, while NHX1 activity was detected in the plasma membrane fractions of Mesembryanthemum roots (Vera-Estrella et al., 2005), tonoplast Naþ/Hþ exchanger activity was not observed in either control or NaCl-treated roots (Barkla et al., 2002). Hence, the relation between salinity and a tonoplast Naþ/Hþ exchanger in halophyte root activity is not as straightforward as one would expect. Naþ vacuolar sequestration in halophyte roots has to be energised; both tonoplast Hþ-ATPases and PP-ases may be involved. However, while some authors have reported NaCl-induced increases in both Hþ transport and HþATPase hydrolytic activity in the tonoplast of Thellungiella root cells (VeraEstrella et al., 2005), specific V-ATPase activities in roots were similar in roots of the halophyte P. maritima and the glycophyte P. media and did not change after exposure to 50 mM NaCl (Staal et al., 1991). Moreover, downregulation of V-ATPase expression was reported in Mesembryanthemum roots (Golldack and Dietz, 2001), suggesting that roots are apparently unable to accumulate Naþ and presumably pass it to the xylem for translocation to the leaves. As for the V-PPase, only a moderate (10%) increase in the hydrolytic activity was detected in salt-treated Thellungiella roots (VeraEstrella et al., 2005), leading to the conclusion that V-PPase plays a minor role in energising the tonoplast. Taken together, it appears that vacuolar sequestration of Naþ in roots is not a key determinant of salinity tolerance in halophytes. To the best of our knowledge, the only attempt to characterise the properties of tonoplast ion channels in halophyte roots was undertaken by Maathuis and Prins (1990). These authors have reported strong similarities
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in tonoplast channel characteristics (conductance, selectivity, gating properties) between P. media (salt-sensitive) and P. maritima (salt-tolerant) species. The major reported channel had a conductance of between 60 and 70 pS, and low selectivity (PK ¼ PNa). The open probabilities of the tonoplast channels dramatically decreased when plants were grown under saline conditions, although single channel conductance and selectivity were not altered (Maathuis and Prins, 1990). Given the fact that the channels’ gating was observed mostly under non-physiological (e.g. high Ca and extreme potentials) conditions, it appears that these channels are closed most of the time in vivo. The biological function of such ‘sleepy’ channels is hard to understand.
V. XYLEM ION LOADING Control of Naþ loading into the xylem has long been considered one of the key determinants of salinity tolerance in glycophytes (Tester and Davenport, 2003). Surprisingly, specific details of this process are lacking. Also controversial are estimates of the amount of sodium taken up by roots and loaded into the xylem to be delivered to the shoot. While Munns et al. (1999) estimated that at 200 mM external Naþ, about 97% of all Naþ presented to the root surface must be excluded, regardless of whether the species is a glycophyte or halophyte, Volkov and Amtmann (2006) were more conservative in their estimates and believe that both T. halophila and A. thaliana (a halophyte and a glycophyte, respectively) export 70% of the Naþ taken, back into the external media. Even more controversial are reports by Balnokin and coauthors who have found that in Suaeda altissima concentration of Naþ in the xylem was 1.3–1.6 times higher than in the bath solution, and reached as high as 350 mM (Balnokin et al., 2005). Such conflicting estimates call for a more thorough comparison between glycophyte and halophyte species, under some sort of standardised conditions. Also highly controversial are reports on the effect of growth conditions on xylem Naþ content. While Clipson and Flowers (1987) have reported about a twofold night-time increase in the xylem Naþ content, other authors believe that xylem sap salt concentration are highest during the day (Scholander et al., 1962). The reason for such discrepancies is yet to be understood. Although the above report of 350 mM Naþ in the xylem sap of S. altissima is clearly outside the typical range found in other halophytes (Table VI), concentrations of around 50 mM may be taken as a reasonable estimate. Halophytes are using Naþ as a cheap osmoticum to maintain cell turgor
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TABLE VI Xylem Naþ Concentration in Some Selected Halophyte Species Grown at High External Salinities (200–500 mM NaCl Range) Species
Xylem [Naþ] (mM)
Suaeda maritima Atriplex litoralis Salicornia virginica Suaeda maritima Avicennia species Suaeda altissima
46 30–48 19–38 50–60 5–122 50–350
Reference Flowers (1985) Rozema et al. (1981) Ownbey and Mahall (1983) Clipson and Flowers (1987) Scholander et al. (1962) Balnokin et al. (2005)
(and, ultimately, shoot growth), and thus, some significant amounts of Naþ is expected to be loaded into the xylem. Also, it was argued that high xylem Naþ concentrations are important for formation of water potential gradients to drive water transport to the shoot (Balnokin et al., 2005). The underlying transport mechanisms involved in both Naþ loading into the xylem and its retrieval from the transpiration stream remains highly controversial. To the best of our knowledge, not a single electrophysiological study has been undertaken to address this issue in halophytes. In glycophytes, both passive (Ko¨hler and Raschke, 2000; Wegner and De Boer, 1997; Wegner and Raschke, 1994) and active (De Boer and Volkov, 2003; Lun’kov et al., 2005; Munns and Tester, 2008) models have been vigorously advocated. This results mainly from the great uncertainty regarding the range of Naþ concentrations in the cytosol of xylem parenchyma cells (originating from the methodological difficulties of such measurements). However, even assuming it being as high as 100 mM (Hajibagheri et al., 1987; Harvey, 1985), a twofold Naþ gradient between the cytosol and the xylem will make passive Naþ loading thermodynamically plausible only if the membrane potential values of xylem parenchyma cells are less negative than 17 mV. This seems not to be the case, with some authors reporting highly negative values for halophyte parenchyma cells (e.g. 130 to 140 mV in Atriplex hastata; Anderson et al., 1977). This leaves active Naþ loading as the only option. The most likely candidate is an SOS1 putative Naþ/Hþ antiporter. In glycophytes, such an exchanger is preferentially expressed at the xylem/symplast boundary of roots, as indicated by promoter-GUS fusions (Shi et al., 2002). Although no direct evidence for halophytes was presented, the observed acidification of the root exudate from 5.5 to 4.5 in response to NaCl treatment in S. altissima (Balnokin et al., 2005) is consistent with this model.
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VI. UNLOADING AND ION TRANSPORT IN LEAVES Maintenance of low cytosolic Naþ levels in photosynthetically active mesophyll cells is considered to be absolutely essential for glycophytes grown under saline conditions. Given the fact that the sensitivity of key cytosolic enzymes to Naþ appears to be similar for both glycophytes and halophytes (Flowers and Colmer, 2008), halophyte species must also possess some effective mechanisms to keep cytosolic Naþ levels under control while accumulating Naþ in leaf tissues for the purposes of osmotic adjustment. This process involves Naþ transport control at both the plasma and tonoplast membranes.
A. ION TRANSPORT IN LEAF MESOPHYLL
With no Naþ pump found in plant systems, a putative SOS1 Naþ/Hþ exchanger remains the only known option for the active Naþ extrusion into apoplast. Indeed, Mesembryanthemum leaves showed constitutive expression of the Naþ/Hþ exchanger that was enhanced by growth of the plants in NaCl (Barkla et al., 2002). However, despite the presence of an Naþ/Hþ exchanger isoform in the plasma membrane of the leaf tissue, no Naþ/Hþ exchange was detected in transport assays using isolated membrane vesicles (Vera-Estrella et al., 2005). This controversy was explained by the fact that plasma membrane Naþ/Hþ exchange activity may require activation by regulatory molecules that are not present in vitro. The activity of an Naþ/Hþ exchanger is energised by the plasma membrane þ H -ATPase pump. Consistent with this, salt treatment increased both the Hþ transport and hydrolytic activity of plasma membrane Hþ-ATPases in salt cress leaves (Vera-Estrella et al., 2005). However, in Aster tripolium NaClinduced stimulation of P-ATPase activity was observed only after 1 day of salt treatment and was followed by the pronounced decline of the P-ATPase activity (Ramani et al., 2006). In S. bigelovii, highest plasma membrane HþATPase activity was observed in plants grown at optimal (200 mM NaCl) salt concentration (Ayala et al., 1996), while in another halophyte, P. maritima, such treatment caused a decrease in the plasma membrane Hþ-ATPase activity in leaves (Bruggemann and Janiesch, 1989). Also, accumulation of NaClinduced Hþ-ATPase mRNA was observed only in fully expanded leaves but not in expanding leaves, or in stems (Niu et al., 1993), and NaCl effects on HþATPase activity showed a clearly pronounced concentration dependence, even within the same species (Ramani et al., 2006). Taken together, these observations may suggest that up-regulation of the plasma membrane HþATPase in leaf mesophyll cells is not a facultative feature of halophytes.
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Very sparse information is available on other transport systems mediating Naþ and Kþ movement across the plasma membrane. Su et al. (2003) have showed a high level of HKT expression in Mesembryanthemum leaves, but no functional analysis has been conducted. Interestingly, however, that McHKT1 transcript amounts increased 2.5-fold during the first 6–10 h of stress and then declined well below pre-stress levels, with kinetics reminiscent of the initial influx of sodium into this halophyte (Su et al., 2003). Surprisingly, to the best of our knowledge, no comprehensive electrophysiological studies have ever been attempted to characterise plasma membrane transport systems in halophyte leaf cells. B. VACUOLAR SEQUESTRATION IN LEAVES
Vacuolar Naþ sequestration is a physiological hallmark of halophytes. This sequestration is achieved via tonoplast Naþ/Hþ antiporters (Barkla et al., 1995; Flowers and Colmer, 2008; Glenn et al., 1999). Tonoplast antiporters are constitutive in halophytes (Barkla et al., 1995; Glenn et al., 1999; Matoh et al., 1989), whereas they must be activated by NaCl in salt-tolerant glycophytes (e.g. Garbarino and Dupont, 1988), while in salt-sensitive plants, their expression levels are extremely low and not salt inducible (Apse et al., 1999; Zhang and Blumwald, 2001). Evidence for both constitutive expression of tonoplast Naþ/Hþ antiporters and stimulation of their activity under saline conditions were reported for at least several halophyte species including Mesembryanthemum, Salicornia and Atriplex (Table VII). Overexpression of an Naþ/Hþ antiporter from Suaeda salsa in Arabidopsis conferred salt tolerance and correlated with Naþ accumulation in the vacuoles of the latter species (Li et al., 2007). The Km values for vacuolar Naþ/Hþ transporter ranges from 2.4 mM in P. maritima (Staal et al., 1991) to 14 mM in A. gmelinii (Matoh et al., 1989). Naþ/Hþ antiporter activity could be energised by both vacuolar HþATPases and Hþ-PPases. Indeed, numerous studies have reported a rather strong increase in both the expression levels and hydrolytic activity of VATPase in a range of halophyte species (Table VII). In general, salinity leads to an increase in V-ATPase activity in halophytes while in glycophytes it remains constant or declines (Wang et al., 2001). It also appears that the effects of salinity on V-ATPase activity are NaCl-specific (Ayala et al., 1996; Vera-Estrella et al., 1999; Wang et al., 2001) and not related to the osmotic component of salt stress. The role of the V-PPase in halophyte salinity tolerance is more controversial (Table VII). While overexpression of V-PPase from Suaeda in Arabidopsis conferred salt tolerance (Guo et al., 2006), V-PPase hydrolytic activity and
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TABLE VII Major Active Transport Systems Reported at Leaf Tonoplast in Halophyte Species Transporter
Species
Findings
Reference
V-ATPase Mesembryanthemum
2.5-fold increase
Mesembryanthemum
Cakile maritima
1.7-fold increase in activity Linear increase in V-ATPase activity with increasing salinity Increased activity, both in vivo and in vitro Increased activity
Mesembryanthemum
Decrease
Mesembryanthemum
1.3 increase in hydrolytic activity Both expression and activity are unaffected Both expression and activity are unaffected Effects were strongly dependent on NaCl concentrations
Suaeda
Salicornia bigelovii
Ratajczak et al. (1994) Barkla et al. (2002) Wang et al. (2001)
Ayala et al. (1996) Debez et al. (2006a,b)
V-PPase
Mesembryanthemum Suaeda Salicornia bigelovii
Rockel et al. (1994) Vera-Estrella et al. (2005) Barkla et al. (2002) Wang et al. (2001) Parks et al. (2002)
NHX exchanger Mesembryanthemum
Strong stimulation
Mesembryanthemum
Increased activity in NaCl-grown plants Twofold increase in activity after 2 weeks of NaCl treatment Increased activity in NaCl-grown plants Increased activity
Mesembryanthemum
Salicornia bigelovii Atriplex gmelinii
Vera-Estrella et al. (2005) Rockel et al. (1994) Barkla et al. (1995)
Parks et al. (2002) Hamada et al. (2001)
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amount of the protein increased in Salicornia plants grown at 200 NaCl but was inhibited by 50 mM NaCl (Parks et al., 2002). This led to the conclusion that V-PPase plays a relatively minor role in energising the tonoplast under the increasing demands imposed by high levels of Naþ/Hþ exchange (VeraEstrella et al., 2005). This may be partially explained by the fact that the activity of the Hþ-PPase is Kþ dependent (Rea and Poole, 1993) and, hence, may be down-regulated by Kþ leak from the cytosol under saline conditions (Shabala and Cuin, 2008). In stark contrast to active transport systems, the properties of vacuolar ion channels in halophyte vacuoles have been studied much less. The only patchclamp study we were able to find was conducted by Maathuis et al. (1992) on S. maritima. These authors reported the presence of a poorly selective SV-like channel with about 170 pS conductance. Its open probability decreased steeply towards positive tonoplast potentials, suggesting that either these channels must be closed most of the time or their number must be really low (Maathuis et al., 1992). The channel also showed a ‘classical’ SV dependence upon cytoplasmic Ca2þ concentrations. It was concluded that Suaeda plants do not possess any special adaptation of the tonoplast ion channels to make them distinctly different from glycophytes. However, it remains to be checked if this conclusion can be extrapolated to other halophyte species, especially in the light of the vital importance of minimising NaCl leak back to the cytosol (as discussed in the following sections). C. PINOCYTOSIS
Another possible pathway for loading of potentially toxic Naþ and Cl ions into vacuoles is via pinocytosis. Pinocytic invaginations have frequently been found in the shoot cells of several salt-accumulating halophytes such as Seidlitzia rosmarinus, Salicornia europaea, Climacoptera lanata, Suaeda arcuata and S. altissima (Balnokin et al., 2007; Kurkova and Balnokin, 1994; Kurkova et al., 2002). Several types of pinocytotic structures were observed. The most typical, defined as ‘type 1’ structures, consisted of pinocytotic invaginations of the PM and the tonoplast. They were surrounded by a double membrane (plasmalemma and tonoplast) and protruded deeply into the vacuole (Balnokin et al., 2007). The ‘floating’ multivesicular bodies were also observed in the vacuoles, which apparently originated from pinocytic invaginations. The pinocytotic structures contained high concentrations of Naþ and Cl (Kurkova et al., 2002), and it was suggested that pinocytosis is instrumental in both transport of toxic ion from the apoplast and their subsequent sequestration in the vacuoles of the shoots cells in salt-accumulating halophytes (Kurkova
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and Balnokin, 1994). Salt-induced pinocytosis was also observed in glycophyte species such as barley and beans (Nassery and Jones, 1976). D. ION RETENTION IN THE VACUOLE
Efficient ion compartmentalisation relies not only on transport across the tonoplast, but also on retention of ions within vacuoles. Indeed, given the four- to fivefold concentration gradient between the vacuole and the cytosol, Naþ may easily leak back, unless some efficient mechanisms are in place to prevent this process. Modified lipid composition of the tonoplast has been mentioned (Glenn et al., 1999; Leach et al., 1990), but the specific details remain obscure. In addition, tonoplast cation channels, through which Naþ might leak back to the cytoplasm, were found to be closed at physiological concentrations of Naþ in patch-clamp experiments on S. maritima vacuoles (Maathuis et al., 1992). However, given the several fold difference in vacuolar Naþ accumulation between different halophyte families (Flowers and Colmer 2008), this finding cannot be extrapolated to all species. Hence, the need to explicitly characterise the functional properties and gating modes of vacuolar channels mediating Naþ and Kþ transport across the tonoplast in different halophyte families remains high on the agenda.
VII. ION TRANSPORT IN GUARD CELLS A. STOMATA CONTROL IN HALOPHYTES
In glycophytes, increasing salinity causes a marked decline in stomatal conductance (gs), reducing both net CO2 assimilation (Pn) and transpiration rates (Munns, 2002). It is believed that halophytes exhibit reduced gs values compared with glycophytes (Lovelock and Ball, 2002) but reports on transpirational studies in halophytes are confusing (Flowers et al., 1986). While most authors agree that halophytes generally show a decline in transpiration rate at salinities above optimal (Debez et al., 2006a,b; Flowers et al., 1986; Geissler et al., 2009; Inan et al., 2004; Redondo-Gomez et al., 2007), some exceptions are reported. For example, no difference in gs values was reported for leaves of S. bigelovii grown at 200 mM (optimal) and 600 mM NaCl levels (Ayala and O’leary, 1995), and no significant effect of salinity on either Pn or gs was found in Spartina densiflora after 90-day treatment with NaCl levels up to 500 mM (Mateos-Naranjo et al., 2010). At suboptimal salinities, some species show a greater rate of transpiration while others show a reduced rate. A perennial halophyte Salvadora persica exposed to 200 mM NaCl had
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slightly higher gas exchange properties relative to non-NaCl controls (Maggio et al., 2000). Similarly, the maximum Pn value in A. macrostachyum was observed at 510 mM NaCl (Redondo-Go´mez et al., 2009). In mangrove species, Avicennia germinans, all gas exchange parameters reached their maximum at a salinity level of about 170 mM (Suarez and Medina, 2006), and Pn rates in Bruguiera parviflora were higher in plants grown at 100 mM NaCl compared with non-saline controls (Parida et al., 2004). However, some studies report much higher gs and transpirational values for suboptimal salinities compared with NaCl levels considered to be optimal for plant growth (e.g. S. bigelovii, Ayala and O’leary, 1995; Atriplex centralasiatica, Qiu and Lu, 2003; Urochondra setulose, Gulzar et al., 2003; Phragmites australis, Pagter et al., 2009). It should be noted that the observed decline in stomatal conductance is not always accompanied by a loss of leaf water content (Redondo-Gomez et al., 2007), and effects of NaCl and water stress on leaf gas exchange in some halophyte species were strikingly different (Ueda et al., 2003). Moreover, the presence of NaCl alleviated detrimental effects of osmotic stress caused by the presence of PEG in the growth media of Atriplex halimus (Martinez et al., 2005). These and similar results led to the conclusion that the observed NaClinduced reduction in gs is likely to be a result of signalling processes rather than a general loss of turgor (Redondo-Gomez et al., 2007). The observed reduction in stomatal conductance in halophyte leaves is assumed to be important for improvements in water use efficiency (Glenn et al., 1999) and may originate from both morphological and physiological adaptive responses to salinity. A comparison between glycophyte species A. thaliana and its halophitic relative T. halophila (salt cress) revealed about twofold higher stomata density in Thellungiella leaves (Inan et al., 2004). At the same time, the overall transpiration rate was higher in Arabidopsis, suggesting more efficient CO2 distribution to mesophyll cells at low stomatal apertures in salt crest. Given the fact that the above paper by Inan et al. (2004) is arguably the only work making such a direct comparison, it remains open as to whether these findings can be generalised to other halophyte species. Also, an increase in leaf thickness (succulency) may affect the number of stomata per unit of leaf volume (Flowers et al., 1986), and a decline in stomatal densities was reported for S. maritima (Flowers, 1985), Distichlis spicata (Kemp and Cunningham, 1981) and Kochia prostrata (Karimi et al., 2005) species under saline conditions. Hence, as commented by Flowers (1985), if changes in gs are paralleled by a change in stomatal frequency, the observed reduction in gs does not necessarily imply any increase in resistance to movement through the stomata themselves. This brings us to the question: how is stomatal aperture controlled in halophyte species?
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B. IONIC RELATIONS IN GUARD CELLS
Growing in highly saline environments, halophytes face the dilemma of choosing between the two following options, as to how to efficiently control their stomata. The first option is to use Naþ instead of Kþ to modulate turgor pressure in the guard cells. The second option is to exclude Naþ enabling Kþ to retain the principle role in guard cell movement. Surprisingly, studies on ionic relations of halophytic stomata are rather rare (Robinson et al., 1997) and often controversial. Several halophyte species were shown to be able to substitute Naþ for Kþ in their stomata. Eshel et al. (1974) reported a dramatic increase in Naþ content in the guard cells of Cakile maritima during stomata opening and concluded that this species has a facultative ability to use either Naþ or Kþ to drive stomatal movement. The apparent ability of Naþ substitution for Kþ in the stomatal mechanism was also reported in S. maritima (Flowers et al., 1989). Here, lower guard cells Naþ concentrations were found in closed stomata, accompanied by a concurrent increase in Naþ in subsidiary epidermal cells. As one would guess, the list is rather short to be able to make a generalisation for all halophytes. At the same time, Naþ content in A. tripolium guard cells was lower than that of other leaf cells when plants were grown at high salinity (Perera et al., 1997). In this species, Kþ remained the dominant cation in the guard cells, and Na/K ratio never exceeded 0.13. Therefore, it was suggested that A. tripolium possesses a very effective mechanism for restricting Naþ entry into guard cells (Perera et al., 1997). Similar results were reported for Aster subcoeruleus species (Robinson et al., 1997). Again, it remains to be answered if this feature is characteristic of the Aster genus only.
C. GUARD CELL ELECTROPHYSIOLOGY
To the best of our knowledge, only one paper so far has attempted a patchclamp investigation of properties of ion channels mediating Naþ and Kþ transport across the plasma membrane of the guard cells in halophyte species. Very et al. (1998) has compared properties of inward- and outward-rectifying cation channels in A. tripolium (halophyte) and Aster smelius (non-halophyte species). In both species, stomatal opening was mediated by inward-rectifying KIR channels with very high (PNa/PK < 0.005) selectivity for Kþ over Naþ. The inward potassium current, IKin, was twofold higher in A. tripolium and insensitive to the addition of Naþ to the external media in both species (Very et al., 1998). The above selectivity of KIR channels was similar to other published reports on non-halophytes (Blatt, 1992; Muller-Rober et al., 1995).
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Potassium efflux in both Aster species was mediated by depolarisationactivated outward-rectifying KOR channels (Very et al., 1998). These channels were also highly selective for Kþ over Naþ (PNa/PK ¼ 0.05), and IKout were twice as large in A. tripolium. Remarkably, outward Kþ currents in this species were inhibited by cytosolic Naþ; this inhibition was absent in A. amelus (Very et al., 1998). Naþ entry into the guard cells occurred via weakly rectifying NSCC. The whole-cell inward current through NSCC was reported to be around 17 pA (Very et al., 1998) which could lead to an accumulation of 500 mM Na in just under 1.5 h. These numbers are consistent with the ones previously reported in the literature (Raschke, 1975). The above results suggest that the fundamental difference in the response of halophyte and non-halophyte Aster species arises from the regulation of cation permeability by cytosolic Naþ but not by the basic properties of ion channels such as their conductances, voltage dependence, selectivity and sensitivity to Ca2þ (Robinson et al., 1997; Very et al., 1998). The specific details of this regulation remain obscure. Indirect effects are most likely, given the fact that the above deactivation of the inward-rectifying Kþ conductance in A. tripolium was absent when cytosolic pH and Ca2þ were buffered (Very et al., 1998). Also, responses were delayed by 20–30 min pointing to the existence of some signal transduction pathway responding to cytosolic Na concentration, most likely mediated by an increase in cytosolic Ca2þ. It should be added that NaCl-induced cytosolic Ca2þ concentrations have been previously reported in guard cells of non-halophyte species (Ranf et al., 2008) but still has to be confirmed in halophytes. The above finding by Very et al. (1998) can explain some earlier experiments on isolated epidermal strips in Aster. In contrast to non-halophylic species (Zeiger, 1983), increasing NaCl concentrations resulted in a decrease in stomatal aperture in A. tripolium and Cochlearia anglica (Perera et al., 1994; Robinson, 1996). Being to some extent counterintuitive (one would expect better stomata ‘protection’ in glycophytes), these observations point to the critical role of stomata in controlling the overall rate of Naþ delivery to the shoot. Also, a clear distinction should be made between Naþ-induced stomatal closure and the osmotic effect of salt stress. Halophytes appear to be rather sensitive to Naþ (if such a conclusion can be generalised from the few available examples) but rather tolerant to osmotic stress at the wholeplant level. Non-halophyte species have these patterns inversed: the overall plant performance is significantly affected by the presence of the NaCl in the growth media while their stomata respond to elevations in apoplastic Naþ by increasing their aperture. The specific mechanisms conferring these traits remain to be revealed (Robinson et al., 1997).
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VIII. SALT GLANDS AND BLADDERS As previously mentioned, some halophytes have evolved specialised epidermal cells, such as glands or bladders, for the elimination or sequestration of excess salt from metabolically active tissues (Agarie et al., 2007; Flowers and Colmer, 2008). Being very large (e.g. 0.5 mm diameter; Fig. 2) and containing a large central fluid-filled vacuole, such cells serve as a peripheral salinity and water storage organ (Adams et al., 1998; Jou et al., 2007; Rygol et al., 1989). Lefevre (2007) demonstrated that over 50% of the accumulated Naþ may be excreted at the leaf surface via such specialised structures, and mutants lacking EBCs showed much poorer growth, reduced leaf succulence and water content (Agarie et al., 2007). Very high (typically between 500 and 1000 mM) Naþ and Cl concentrations are reported for EBCs in salt-stressed plants (Adams et al., 1992, 1998; Barkla et al., 2002). EBCs may also store organic osmolytes such as ononitol, pinitol and proline (Agarie et al., 2007); together with inorganic ions, this generates a turgor gradient that can accelerate the growth of new cells. EBCs also contain a substantial (although much lower) amount of Kþ (Adams et al., 1992). In particular, numerous raphide crystals were found inside EBCs in some species like M. crystallinum (Jou et al., 2007); these crystals contain no Naþ and were suggested to serve as a potassium reservoir to maintain the Naþ/Kþ homeostasis in this halophyte. The latter suggestion was made on a basis that Kþ content in raphide crystals of high-salinity grown Mesembryanthemum plants rapidly disappeared, presumably as a result of Kþ remobilisation to increase cytosolic Kþ concentration of nearby mesophyll cells (Jou et al., 2007). The functional role of EBCs may also differ significantly between species. For example, it appears that, in Atriplex hortensis, bladder hairs removed almost all the Naþ from the young leaf lamina, while they contributed little to the control of Naþ content in a fully expanded leaf (Jeschke and Stelter 1983). At the same time, juvenile leaves in Mesembryanthemum had fewer and smaller EBCs compared with mature leaves (Agarie et al., 2007). Given the limited number of reports on this topic, this controversy is yet to be resolved. Another important issue is the actual mechanism of control of Naþ uptake into gland cells and their ability to discriminate between Naþ and Kþ. Naþ and Cl are the predominantly secreted salts, with plants usually excreting three to four times the amount Naþ present in leaves per daily basis (Marcum and Murdoch, 1992; Marcum et al., 2007). At the same time, Kþ excretion is at least an order of magnitude lower (e.g. 12-fold in D. spicata; Marcum et al., 2007). As a result, the Na/K ratio in the hairs of salt-grown halophytes
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is much higher than that in the mesophyll cells (Flowers et al., 1990). With no electrophysiological work available in the literature, the mechanisms for such selectivity remain a complete mystery. Only a very limited number of papers have attempted to reveal the identity of specific ion transporters in EBCs; nearly all of them are related to the tonoplast Naþ/Hþ exchanger. It is assumed that an increase in the accumulation of sodium ions in the EBCs is achieved by the high tonoplast Naþ/Hþ antiport and V-type ATPase activity (Barkla et al., 2002; Golldack and Dietz, 2001). This may explain the mechanisms by which Naþ is removed from the EBC cytosol but does not address the issue of how Kþ is retained in the EBC cytosol, given the expected dramatic plasma membrane depolarisation following the massive Naþ intake into the EBCs. Also unclear are the identity and gating modes of specific transporters mediating Naþ movement across the EBC plasma membrane. Su et al. (2003) have reported that expression of McHKT1 protein in stressed EBC’s was stronger than in the unstressed leaf, naming this transporter as a potential candidate for the Naþ influx into the cell. The functional characterisation of the role of HKT transporter is yet to be done, so is electrophysiological characterisation of all other potential candidates such as NSCC or LCT transporters.
IX. OXIDATIVE SIGNALLING AND DAMAGE REPAIR IN HALOPHYTES A. MAJOR ANTIOXIDANT SYSTEMS AND THEIR CONTROL
Increased reactive oxygen species (ROS) production has been reported in response to a variety of abiotic and biotic stresses, including salinity (Luna et al., 2000; Mittler, 2002; Miller et al., 2008). Numerous defence mechanisms have evolved to protect cells against oxidative injury (Scandalios, 1993). Such protection relies on the activity of various antioxidant components—enzymes and low molecular weight compounds capable of quenching ROS without themselves undergoing conversion to a destructive radical. The plant antioxidant system consists of both enzymatic and non-enzymatic components and is important both for controlling excessive ROS production during stress and in maintaining the correct levels of ROS for growth and signalling (Mittler et al., 2004). The major ROS scavenging pathways coexisting in plants include (1) superoxide dismutase (SOD; found in all cellular compartments), (2) water– water cycle (in chloroplasts), (3) the ascorbate-glutathione cycle (in chloroplasts, mitochondria, cytosol and apoplastic space), (4) glutathione peroxidase (GPX), and (5) catalase (CAT; both in peroxisomes; Mittler, 2002).
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Among these major antioxidant enzymes, the most important are SOD, ascorbate peroxidase (APX), CAT and GPX (Noctor and Foyer, 1998). The existing balance between these, together with sequestration of metal ions, is important in preventing the formation of highly toxic hydroxyl radicals (Mittler, 2002). The most important non-enzymatic ROS scavenging compounds are ascorbate and glutathione (Colville and Smirnoff, 2008). B. OXIDATIVE STRESS SIGNALLING AND TOLERANCE IN HALOPHYTES
It has been repeatedly stated that the capacity to limit oxidative damage is important for halophytes’ salt tolerance (Ben Amor et al., 2006,2007; Jithesh et al., 2006; Parida and Jha, 2010). It is also believed that halophytes possess higher oxidative stress tolerance than glycophytes (M’Rah et al., 2007; Taji et al., 2004). However, given the fact that these conclusions are derived from comparisons of only a rather limited number of species (e.g. T. halophila and A. thaliana), these reports need to be treated with some caution. Similar to glycophytes, halophytes are capable of up-regulating ROS scavenging systems under high-salinity conditions (Pang et al., 2005). As a rule of thumb, activity of all major antioxidant systems increases noticeably under salt stress (Table VIII). However, for some species, no significant changes, or even a decrease in activity of some antioxidant enzymes have been reported (Table VIII). A rather substantial difference in the kinetics of antioxidant activity appears to exist between roots and leaves, and salinity effects on the activity of major antioxidant enzymes display a clearly pronounced dose- and timedependence (Ben Amor et al., 2007; Ben Hamed et al., 2007; Wang et al., 2008). This complexity most likely reflects the above dual role of ROS in plant-environmental interaction. Of specific importance is an increase in the antioxidant activity in chloroplasts. Chloroplast SOD activity is markedly enhanced with the increase of NaCl or with time, especially in its thylakoid-bound fraction (Wang et al., 2008; Zhang et al., 2005). This is believed to be essential in protecting leaf photosynthetic machinery and enabling its optimal functioning under saline conditions. Interestingly, NaCl salinity and osmotic stress lead to a differential regulation of distinct SOD isoenzymes (Wang et al., 2004). Such differential regulation may play a major role in plant stress tolerance. There also appear to be a few rather specific features characteristic to halophytes only. The facultative halophyte M. crystallinum (ice plant) shifts its mode of carbon assimilation from the C-3 pathway to crassulacean acid metabolism (CAM) in response to many factors that lead to the generation of ROS at the cellular level (Surowka et al., 2007). This is believed to be
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TABLE VIII Selected Examples of Salinity Effects on the Activity of Major Antioxidant Systems in Halophytes Species
Enzyme
Changes
Avicennia marina
SOD
Increase
Crithmum maritimum
CAT, APX, GR
Crithmum maritimum C. maritima
SOD, CAT, peroxidase CAT, APX, DHAR, GR
Reduced at 300 mM NaCl; unaffected at 100 mM NaCl Increase
Mesembryanthemum crystallinum Mesembryanthemum crystallinum Salicornia brachiate
SOD
Increased at moderate salinities; decreased after prolonged exposures Increase
APX
Increase Increase
Suaeda salsa
APX, POX, GR, SOD CAT SOD
Decrease Significant increase
Suaeda salsa
CAT, APX, GR
Significant increase
Suaeda salsa
SOD
Increase
Thellungiella halophila Thellungiella halophila
SOD
30-fold increase
SOD, CAT, peroxidases
Minor changes
Reference Prashanth et al. (2008) Ben Hamed et al. (2007) Ben Amor et al. (2005) Ben Amor et al. (2007)
Miszalski et al. (1998) Slesak et al. (2002) Parida and Jha (2010) Wang et al. (2008) Pang et al. (2005) Zhang et al. (2005) Xu et al. (2009) M’rah et al. (2006)
POX, guaiacol peroxidase; APX, ascorbate peroxidase; SOD, superoxide dismutase; GR, glutathione reductase; CAT, catalase.
important for effectively protecting M. crystallinum against oxidative damage (Hurst et al., 2004) and is attributed to the reduced H2O2 levels in CAM plants under saline conditions compared with C3 photosynthesis. In this context, much lower H2O2 levels were detected in M. crystallinum WT compared with a mutant unable to switch to CAM metabolism (Sunagawa et al., 2010). Extensive studies on another model halophyte species, T. halophila (salt crest), has led to the suggestion that increased proline accumulation may be one of the key features conferring its salinity tolerance (Kant et al., 2006).
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However, proline-accumulating species were found amongst both halophytes (e.g. Artemisia lerchiana and T. halophila) and glycophytes (e.g. P. major and Mycelis muralis; Kartashov et al., 2008), suggesting that proline accumulation cannot be named as a specific hallmark of halophytes. Interestingly, a negative correlation was found between proline content and SOD activity in these species. Thus, it appears that proline’s major role is not in conventional water retention (as assumed for most osmolytes) but in antioxidant defence (Radyukina et al., 2007; Shevyakova et al., 2009). These studies also led to the conclusion that high-SOD activity is not an obligatory trait of salt tolerance. Moreover, plants with high-peroxidase activity and active proline accumulation could acclimate to salt stress independently of SOD activity (Kartashov et al., 2008). Another possible class of ROS scavengers in halophytes are polyamines (Kuznetsov et al., 2007; Shevyakova et al., 2006; Stetsenko et al., 2009). It should be noted that both polyamines and organic osmolytes have previously been shown to be efficient regulators of both non-selective (NSCC; Shabala et al., 2007a,b; Cuin and Shabala, 2007a) and depolarisation-activated outward-rectifying (KOR; Cuin and Shabala, 2005, 2007a; Pandolfi et al., 2010) Kþ-permeable channels in both root and leaf tissues in a range of glycophyte species. At the same time, ROS-induced activation of both NSCC (Demidchik et al., 2003) and KOR (Demidchik et al., 2010) channels was reported in direct electrophysiological experiments. Given the crucial role of both these types of channels for the maintenance of cytosolic K/Na ratio and, hence, the plants ability to perform under saline stress conditions (Shabala and Cuin, 2008), the above results may indicate that stress-induced elevation in free polyamine and organic osmolyte pools may be needed to reduce the detrimental effects of stress-induced ROS production on the permeability of NSCC and KOR channels. Given the fact that all the above data were obtained in glycophyte systems, similar experiments should be conducted on halophytes to validate this suggestion. In animal systems, activation of Kþ-efflux channels by ROS can trigger programmed cell death (Bortner and Cidlowski, 2007; Valencia-Cruz et al., 2009). Similar scenarios have been suggested for plants (Shabala, 2009) and were confirmed in direct experiments using Arabidopsis gork1-1 mutants lacking a major plasma membrane outwardly rectifying Kþ channel (Demidchik et al., 2010). Also, expression of the animal anti-apoptotic CDE-9 gene in tobacco mesophyll has significantly improved plant’s Kþ retention ability and resulted in increased salinity and oxidative stress tolerance (Shabala et al., 2007a,b). In this context, it should be noted that enhanced lipid peroxidation was observed in salt-stressed Hordeum maritimum plants grown under Kþ deficient conditions (Hafsi et al., 2010), most
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likely via increased NADPH-dependent O2. generation in root cells (Cakmak, 2005). Also, suppression of an NADPH oxidase in Arabidopsis rhd2 mutant prevented the up-regulation of genes that are normally induced by Kþ deficiency (Shin and Schachtman, 2004). Taken together, these results suggest a strong causal relationship between intracellular Kþ content, ROS production and scavenging, and programmed cell death under saline conditions. It remains to be tested, as to what extent these mechanisms confer salt tolerance in halophytes.
X. CONCLUSIONS AND PERSPECTIVES Halophytes are highly valuable model species in physiological studies on plant salinity tolerance, and a potential source of salinity tolerance genes in crops. It appears that halophytism is an integration of a large number of physiological adaptive mechanisms rather than a distinct morphological or anatomical feature. Remarkably, very little is known about molecular and cellular aspects of halophytes’ remarkable tolerance to salinity. It appears that halophyte roots operate mainly in the ‘passage mode’, and that xylem Naþ loading is crucial for shoot osmotic adjustment and maintenance of sustained growth under high-salinity conditions. These findings question the existing strategies to improve salinity tolerance in cereals and other crops by maximising plants’ ability to retrieve Naþ from the xylem (Lauchli et al., 2008; Tester and Davenport, 2003). Vacuolar sequestration of Naþ in roots appears not to be a key determinant of salinity tolerance in halophytes (Staal et al., 1991; Vera-Estrella et al., 2005), while such sequestration in leaf vacuoles is absolutely essential to confer the latter trait. Both the process of xylem Naþ loading and its sequestration in leaf cell vacuoles are mediated by active transport systems; the expression of these transporters appears to show high tissue specificity. While only a few direct electrophysiological studies were undertaken to investigate the identity and functional role of key transport systems mediating Naþ and Kþ homeostasis in halophyte cells, all of them point to the importance of regulation of cation permeability, while basic properties of ion channels such as their conductance, voltage dependence and selectivity appear not to be very different between halophytes and glycophytes. Remarkably, this seems to be the case for such diverse systems as root tonoplast channels (Maathuis and Prins, 1990) and plasma membrane channels in stomata guard cells (Very et al., 1998). The specific details of this regulation remain an enigma and calls for more active research in this direction. In this context, the regulatory role of compatible solutes and antioxidant molecules
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should be reexamined, and a causal relationship between oxidative and salinity stress tolerance in halophytes should be studied in greater detail. Also essentially unexplored remains an issue of mechanisms of Naþ loading and regulation of Naþ/Kþ selectivity in epidermal bladder and gland cells. Understanding this and the above issues can be instrumental in developing a better understanding of what appears to be the complex and highly orchestrated regulation of ion transport and sequestration in halophytes, opening up the prospect of improving salinity tolerance in crops.
ACKNOWLEDGEMENT This work was supported by the Australian Research Council Discovery and Linkage grants to Associate Professor Sergey Shabala.
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The Regulatory Networks of Plant Responses to Abscisic Acid
TAISHI UMEZAWA,* TAKASHI HIRAYAMA,{ TAKASHI KUROMORI{ AND KAZUO SHINOZAKI*,{,1
*Gene Discovery Research Group, RIKEN Plant Science Center, Tsukuba, Ibaraki, Japan { Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama, Japan { Gene Discovery Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa, Japan
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. ABA Biosynthesis and Catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. NCED: A Key Enzyme in ABA Biosynthesis.............................. B. ABA 80 -Hydroxylase ........................................................... C. Metabolic Sites of ABA ....................................................... III. ABA Transport and Localisation for Intercellular Signalling . . . . . . . . . . . . . . A. ABA Long-Distance Transport Through the Vasculature ............... B. ABA Short-Distance Transport Through the Apoplast .................. C. ABA Subcellular Transport between Organelles .......................... IV. Intracellular Signal Transduction in ABA Responses . . . . . . . . . . . . . . . . . . . . . . A. Receptors ........................................................................ B. Protein Phosphatases .......................................................... C. Protein Kinases ................................................................. D. ABA-Responsive Gene Expression .......................................... E. Regulation Through Protein Degradation in the ABA Response ...... F. RNA Metabolism and ABA Response...................................... V. Emergence of the Core Signalling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding author: E-mail:
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Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00006-0
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ABSTRACT Abscisic acid (ABA), one of the major plant hormones, is involved in various biological processes in plants, especially in the regulation of seed maturation, dormancy and abiotic stress responses, which have strong relationships with crop yields. For these reasons, ABA has been extensively studied for many years. Very recently, several breakthrough studies were published in ABA research. One was the identification of ABA receptors and the establishment of a major ABA signalling pathway. The other was the discovery of the transport activity of ABA among tissues. These findings open new avenues to understanding hormonal response, not only in cells, but also in the whole organism, and for the improvement of crop yields. In this chapter, we will summarise and discuss our knowledge of ABA and provide a perspective led by those processes.
I. INTRODUCTION As multicellular organisms, plants have developed and established unique and complex systems that allow them to grow by photosynthesis, and survive and produce offspring in harsh environments. For these functions, plants utilise small chemical substances called phytohormones, or plant hormones, to communicate among cells and control total body phenomena. Despite the name ‘‘hormone’’, however, both their structure and their modes of action differ from those of animal hormones. Abscisic acid (ABA) is one of these phytohormones and belongs to a class of terpenoids with a C15 backbone (reviewed by Milborrow, 2001; Nambara and Marion-Poll, 2005). Originally, ABA was identified as an abscission-accelerating compound in young cotton fruit (referred to as ‘‘abscission II’’) and a dormancy-inducing factor from sycamore leaves (referred to as ‘‘dormin’’). Further analysis indicated that the structure of dormin was identical to abscission II, and these names were unified as ABA. ABA can form two chiral enantiomers, the (S)- and (R)-enantiomers, at the 10 carbon position. Both enantiomers have 2-trans isomers, that is, (S)-2-transABA and (R)-2-trans-ABA. Although the (S)-enantiomer is the form found in plants, until recently, a large portion of the commercially available ABA was a racemic mixture. The pure (S)-ABA form, however, can now be obtained, which is what the term ABA refers to in this chapter. The physiological roles of ABA were initially tested in the early 1950s (reviewed in Addicott and Lyon, 1969). The acidic fractions in plant extracts had growth-inhibiting effects in oat coleoptiles (called the -inhibitor complex). These observations of the -inhibitor complex included some ABA actions. The name ‘‘abscisic acid’’ originated from abscission II, but the abscission-inducing ability of ABA turned out to be dependent on ethylene function. ABA is now known to take part in a wide variety of physiological phenomena throughout a
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plant’s life, from germination to reproduction, including establishing seed dormancy, germination, root development, abiotic stress response, defense response to pathogens, and stomatal closure (see recent reviews Cutler et al., 2010; Hirayama and Shinozaki, 2007, 2010; Yamaguchi-Shinozaki and Shinozaki, 2006). Because of the important functions of ABA in plant physiology, numerous studies on the ABA biosynthetic pathway, ABA signalling pathway and degradation mechanism have been conducted, accumulating much data. Recently, several studies succeeded in identifying ABA receptors, allowing us to determine the major ABA signalling pathway. In addition, other studies showed that ABA is actively transferred between tissues. With this knowledge, the view of ABA in plant physiology has changed. In this chapter, we describe the recent progress in our knowledge of ABA biosynthesis/catabolism, intercellular transport and intracellular signalling.
II. ABA BIOSYNTHESIS AND CATABOLISM Endogenous levels of ABA fluctuate in response to environmental conditions, such as drought or salt stress, or to various developmental cues, such as seed maturation. The molecular basis of ABA biosynthesis and catabolism was recently established mainly through genetic and biochemical approaches (reviewed by Nambara and Marion-Poll, 2005; Fig. 1). Briefly, the first half of ABA biosynthesis takes place in chloroplasts. The ABA precursor is derived from a C40 carotenoid, zeaxanthin, which is converted to violaxanthin via antheraxanthin by zeaxanthin epoxidase (ZEP). The ZEP gene encodes a protein with sequence similarities to FAD-binding monooxygenases, which require ferredoxin (Bouvier et al., 1996; Marin et al., 1996). Then violaxanthin is converted to 9-cis-neoxanthin or 9-cis-violaxanthin. A membrane protein, AtABA4, is involved in the conversion to 9-cis-neoxanthin (North et al., 2007). The key point in ABA biosynthesis is the next step, cleavage of 9-cis-epoxycarotenoids to a C15 precursor, xanthoxin, by 9-cis-epoxycarotenoid dioxygenase (NCED). NCED was originally identified from a maize ABA-deficient mutant, vp14 (Schwartz et al., 1997), and its cleavage reaction is critical for dehydration-inducible ABA biosynthesis (Iuchi et al., 2000; Qin and Zeevaart, 1999). Thereafter, xanthoxin is released into the cytosol from chloroplasts and converted to abscisic aldehyde by a short-chain dehydrogenase/reductase (ABA2 in Arabidopsis; Cheng et al., 2002; Gonzalez-Guzman et al., 2002). Finally, the biologically active form of ABA is produced by the oxidation of abscisic aldehyde. Abscisic aldehyde oxidase (AAO) catalyses this step in association with molybdenum cofactors
OH
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Fig. 1. Major pathways of ABA biosynthesis and catabolism in plants. ABA biosynthesis is initiated in chloroplast by cleavage of C40 carotenoid precursors. This step was catalysed by ABA1/ZEP (zeaxanthin epoxydase). Then epoxycarotenoids were cleaved by NCED (9-cisepoxycarotenoid deoxygenase) to produce a C15 precursor, xanthoxin. Xanthoxin is converted to ABA in cytosol by two enzymes: ABA2,
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(MoCo), which are supplied by MoCo sulfurase (FLACCA in tomato; ABA3 in Arabidopsis; Bittner et al., 2001; Sagi et al., 2002; Seo et al., 2000). ABA catabolism or inactivation is largely categorised into two pathways: hydroxylation and conjugation. Hydroxylation is thought to be the major pathway in ABA catabolism. In this pathway, ABA is converted to either phaseic acid or dihydrophaseic acid via multistep hydroxylation. The first hydroxylation mainly occurs at C80 of ABA to produce 80 -hydroxy ABA. The enzyme catalysing this step is important, and a pioneering work strongly suggested that this step depends on ABA-responsive de novo transcription of a cytochrome P450 monooxygenase (Krochko et al., 1998). The cytochrome P450 superfamily in Arabidopsis was surveyed to identify ABA 80 -hydroxylase and the CYP707A subfamily was identified and singled out (Kushiro et al., 2004; Saito et al., 2004). The Arabidopsis genome contains four members of the CYP707A subfamily. In this chapter, two major enzymes of ABA biosynthesis and catabolism, NCED and ABA 80 -hydroxylase, are described as are their functions or upstream regulation under drought or salt stress. A. NCED: A KEY ENZYME IN ABA BIOSYNTHESIS
As described above, NCED is a key enzyme in the ABA biosynthetic pathway. Five members of the NCED family exist in Arabidopsis (Nambara and Marion-Poll, 2005). One of these, AtNCED3, plays a prominent role in ABA accumulation triggered by drought stress (Iuchi et al., 2001). The AtNCED3 gene is strongly induced by drought stress, and the nced3 mutant lacks drought-responsive ABA accumulation. Importantly, stomatal closure can be observed in the nced3 mutants in response to drought stress, but a large part of the stress-responsive gene expression is impaired (Urano et al., 2009). These results suggested that a basal level of endogenous ABA could be maintained even in the nced3 mutant and it was sufficient for stomatal closure, but gene expression requires significant ABA accumulation in response to drought stress. In contrast, AtNCED3 overexpressing plants had a higher content of endogenous ABA and elevated drought tolerance (Iuchi et al., 2001).
ABA-deficient 2 encoding short-chain alcohol dehydrogenase, and AAO3, ABAaldehyde oxygenase. AAO3 activity requires ABA3 (ABA-deficient 3) encoding molybdenum cofactor sulphurase. However, multiple pathways have been proposed for ABA catabolism. Among them, the oxidative pathway seems to be a major one which is initiated by 80 -hydroxylation of ABA. CYP707As encode ABA 80 -hydroxylase which catalyses this step; therefore they are regarded as major enzymes for ABA catabolism in plants.
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Other NCED members are also regulated at the gene expression level. For example, AtNCED6 and AtNCED9 are major enzymes in seeds that are responsible for the endogenous ABA levels in seeds (Lefebvre et al., 2006). Otherwise, comprehensive analysis showed different expression patterns for each NCED member (Tan et al., 2003). Although understanding the regulatory mechanism of NCED genes is important, it is still under investigation. In general, the regulation of gene expression is mainly involved in the promoter region, that is, cis-elements or trans-factors. The promoter–reporter gene system is very useful for such kinds of studies. Indeed, a series of NCED promoter::GUS transgenic plants have already been established (Tan et al., 2003). Thus, one would be able to determine the cis-elements or trans-factors for some NCED genes. However, some problems exist; for example, transgenic plants harbouring AtNCED3 promoter::GUS showed no response to drought stress (Tan et al., 2003). Two possible explanations could be considered; the first is that the promoter used in their study is too short and insufficient for drought-responsive expression. The second possibility is that some posttranscriptional regulation may be involved. Further analysis will be required to clarify the regulatory mechanisms of NCED genes. B. ABA 80 -HYDROXYLASE
As described above, CYP707As encodes ABA 80 -hydroxylase, a key enzyme in the ABA catabolic pathway (Kushiro et al., 2004; Saito et al., 2004). As with NCED, each CYP707A also has distinctive physiological roles. For example, CYP707A2 is a major enzyme in seeds, as indicated in cyp707a2 mutants, which have strong seed dormancy (Kushiro et al., 2004). However, seed dormancy in cyp707a3 seeds was not significantly affected. CYP707A1 is expressed in guard cells and participates in ABA-dependent stomatal movement (Okamoto et al., 2009). CYP707A3 plays a major role in degrading endogenous ABA accumulated in response to dehydration (Umezawa et al., 2006). CYP707A3 expression was strongly induced during rehydration following dehydration in vegetative tissues. Two types of CYP707A3 expression were observed: one is ABA-responsive gene expression, and the other is rehydration-responsive expression. The ABA-responsive expression of CYP707A is expected because CYP707A promoters contain ABA-responsive elements (ABREs). However, the rehydration-responsive expression of CYP707A3 was very strong; it was induced to a maximum level within 30 min after rehydration (Umezawa et al., 2006), and this rapid induction is independent of typical ABA signalling pathways because no differences exist in abi1-1, abi2-1, aba2-2, or nced3-2 mutants
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(Umezawa et al., 2006). Therefore, the rehydration signals in CYP707A3 expression must be separate from ABA, although the mechanism still remains elusive. The regulatory mechanisms of CYP707A3 are important for understanding how the endogenous ABA level is adjusted, as well as those of NCEDs. C. METABOLIC SITES OF ABA
Several studies indicated that the vascular system is an important source of drought-inducible ABA accumulation. For example, immunohistochemical analysis revealed that AtABA2 and AAO3 were located in vascular parenchyma cells, but AtNCED3 was not detected in this area in non-stressed plants (Endo et al., 2008). After drought stress, AtNCED3 was strongly induced in vascular parenchyma cells together with AtABA2 and AAO3. These results suggest that ABA is mainly synthesised in the vascular system both in non-stressed and drought-stressed plants. Guard cells are another site for ABA biosynthesis. Expression and histochemical analyses revealed that some ABA biosynthetic enzymes, AtNCED2, AtNCED3 and AAO3, are expressed in guard cells, suggesting that ABA can be produced inside guard cells (Koiwai et al., 2004; Tan et al., 2003). This is reasonable because guard cells are one of the most sensitive organs to ABA in the rapid stomatal closure response. However, the physiological reliance on internal or transported ABA remains to be determined in stomatal responses. As described in this section, cell-type-specific ABA biosynthesis was clearly demonstrated and reminds us of the important issue of cell-to-cell transport of ABA. In Section III, an emerging ABA transport system will be discussed.
III. ABA TRANSPORT AND LOCALISATION FOR INTERCELLULAR SIGNALLING In ABA physiological studies to date, the translocation and the communication of this phytophormone between cells, organs and tissues in plants play important roles in whole-plant ABA responses (Schachtman and Goodger, 2008; Wilkinson and Davies, 2010). For example, ABA is a key regulator of leaf stomatal conductance: under drought conditions, the ABA concentration increases in the apoplast, resulting in stomatal closure (Schachtman and Goodger, 2008; Wilkinson and Davies, 2010). Also, some studies have reported on the systemic and dynamic changes of gene expression related to ABA or stress responses (Christmann et al., 2007; Endo et al., 2008). Most genes and factors identified so far in ABA signalling are mainly involved in
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ABA intracellular regulation (Cutler et al., 2010; Kim et al., 2010), whereas ABA intercellular regulation has not been studied in any plant species. Thus, the molecular basis of ABA transport and localisation needs to be investigated to understand whole-plant ABA intercellular communication. In this section, our present knowledge of ABA intercellular signalling is summarised and described mainly by three points of view: (A) ABA long-distance transport through the vasculature, (B) ABA short-distance transport through the apoplast and (C) ABA subcellular transport between organelles. A. ABA LONG-DISTANCE TRANSPORT THROUGH THE VASCULATURE
Xylem and phloem constitute the vasculature-based transport systems. They both consist of conductive elements that form continuous tubular columns. Xylem transports and stores water, nutrients and hormones from the roots to the aboveground tissues. Long-distance signalling of ABA was discussed in recent reviews (Jiang and Hartung, 2008; Schachtman and Goodger, 2008; Wilkinson and Davies, 2010). Under conditions of mild stress, as the soil starts to dry, and the water potential of the leaves is not or only slightly affected, ABA accumulates in root tissues; then it is released to the xylem vessels and transported to the shoot, where stomatal and meristematic activities are regulated to help the plant cope with the stress (Jiang and Hartung, 2008). Investigation of the relationship between ABA xylem concentration and leaf conductance in woody and herbaceous species originating from different habitats revealed that stomatal reactions are always much better correlated with ABA xylem concentration than with leaf bulk ABA, pointing to the importance of ABA as a hormonal long-distance signal in the xylem (Heilmeier et al., 2007). Many other studies have reported that soil drying can sensitively increase the ABA level in the roots, and ABA is then transported in the xylem up to the shoot (Davies and Zhang, 1991; Dodd et al., 2008; Wilkinson and Davies, 2010). In contrast, grafting analyses with ABA-deficient Arabidopsis clearly showed a requirement for ABA in response to soil-borne water stress, but not as a long-distance signal (Christmann et al., 2007). ABA biosynthesis in the shoot was necessary and sufficient to mediate stomatal closure of plants water-stressed at the roots, confirming grafting experiments with tomatoes (Holbrook et al., 2002). In other studies, the AAO3 protein, the enzyme that biosynthesises ABA (see above), was predominantly localised in the phloem companion cells next to the phloem sieve element, as well as in the parenchyma cells (Koiwai et al., 2004). Some of the ABA accumulated in the leaves was shown to be transported to the roots, and tracer experiments using
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isotope-labelled ABA indicated that the movement of ABA from leaves to roots is activated by water deficiency in the roots (Ikegami et al., 2009). ABA recirculation, in which ABA is loaded to the phloem and then transported to the roots where it is deposited in the xylem vessels, has also been discussed (Hartung et al., 2002; Sauter et al., 2001). Meanwhile, Christmann et al. (2007) reported that their study did not support ABA as a proposed long-distance signal, but that ABA acted downstream of the hydraulic signal in communicating water stress between roots and shoots. Soil water stress appears to elicit a hydraulic response in the shoot, which precedes ABA signalling and stomatal closure, and additional attenuation of the hydraulic response in various plants prevented longdistance signalling of water stress. Nevertheless, the function of ABA as a long-distance signal communicating water shortage from the root to the shoot is still not clear (Christmann et al., 2007) and needs further investigation. B. ABA SHORT-DISTANCE TRANSPORT THROUGH THE APOPLAST
Auxin is another major phytohormone that coordinates plant development (Benjamins and Scheres, 2008). Recently, the auxin transport system was found to include both cellular efflux and influx carriers (Petra´sek and Friml, 2009; Vanneste and Friml, 2009). In current models, auxin basically migrates to adjacent cells through polar transport to regulate disproportionate cell growth (Friml and Palme, 2002; Tanaka et al., 2006). However, the stress hormone ABA is required for rapid signalling, especially under stress conditions (Kim et al., 2010). For example, under drought, ABA promotes stomatal closure in guard cells to prevent transpiration (Cutler et al., 2010; Kim et al., 2010; Leung and Giraudat, 1998). ABA might spread rapidly to peripheral cells to cope with environmental changes. The ABA transport system may be developed for rapid transmission of ABA molecules and effective distribution of stress signalling between plant cells. Indeed, ABA short-distance transportation is thought to exist in plants: for example, ABA is predominantly biosynthesised and metabolised in vascular tissues, while it acts in stomatal responses of distant guard cells (Cheng et al., 2002; Koiwai et al., 2004; Okamoto et al., 2009). In addition, we can infer the necessity of ABA transporters across plasma membranes from the viewpoint of an anion trap (Hartung et al., 1998; Taiz and Zeiger, 2006). Because ABA is a weak acid (pKa 4.7), it cannot passively cross through the lipid bilayer of plasma membranes, as it is mostly in the ionised form in the cytosol, where the pH is approximately neutral (Hartung et al., 1998; Kang et al., 2010; Taiz and Zeiger, 2006). An ABA exporter would be necessary for ABA diffusion from the cell interior to the cell exterior over the anion trap.
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Similarly, stress conditions elevate the apoplastic pH, so an ABA importer would also be necessary for cellular uptake of ionised ABA, for example, at guard cells, particularly under stress conditions (Kang et al., 2010). Recently, an ATP-binding cassette (ABC) transporter gene, AtABCG25, was reported to encode a protein that is responsible for ABA transport and ABA responses in Arabidopsis (Kuromori et al., 2010; Kuromori and Shinozaki, 2010). atabcg25 mutants were originally isolated by genetic screening for mutants with abnormal ABA sensitivity. AtABCG25 is expressed mainly in vascular tissues, where ABA is predominantly biosynthesised. The fluorescent protein-fused AtABCG25 was localised at the plasma membrane in plant cells. The ABC-type transporter is conserved in many model species from Escherichia coli to humans and is reported to transport various metabolites or signalling molecules in an ATP-dependent manner (Higgins, 1992). In membrane vesicles derived from AtABCG25-expressing insect cells, AtABCG25 exhibited ATP-dependent ABA transport activity. Further, the AtABCG25-overexpressing plants had higher leaf temperatures, implying an influence on stomatal regulation (Kuromori et al., 2010). These results suggest that AtABCG25 is an exporter of ABA through the plasma membrane and is involved in the intercellular ABA signalling pathway (Fig. 2).
Vascular tissues
ABA importer (AtABCG40)
ABA exporter (AtABCG25)
ABA 10 mm
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Fig. 2. Schematic view of hypothetical ABA intercellular transmission. The background photograph is an Arabidopsis leaf section to show two of distinct cells: vascular tissues (circle) including vascular parenchyma cells, and guard cells on leaf epidermis (inset). AtABCG25 could function as an ABA exporter from ABAbiosynthesising cells to extracellular area. ABA would be diffused in apoplastic area. AtABCG40 could function as an ABA importer from outside to inside of guard cells to facilitate stomatal closure.
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At the same time, another ABC transporter in Arabidopsis, AtABCG40, was independently reported to function as an ABA importer in plant cells (Kang et al., 2010). The atabcg40 mutant was selected from knockout mutants of dozens of Arabidopsis ABC transporter genes (atabcg29– atabcg41) testing seed germination and stomatal movements. AtABCG40 is expressed in the leaves of young plantlets and in primary and lateral roots; in leaves, the expression was the highest in guard cells. Plasma membrane localisation was shown by ABCG40::sGFP expression driven by the native promoter in Arabidopsis guard cells. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 increased, whereas ABA uptake into protoplasts of atabcg40 plants decreased compared to control cells. In loss-of-function atabcg40 mutants, stomata closed more slowly in response to ABA, resulting in reduced drought tolerance, and the upregulation of ABA-inducible genes was strongly delayed, indicating that ABCG40 is necessary for timely responses to ABA. These results suggest that AtABCG40 is an importer of ABA through the plasma membrane, and integrates ABA-dependent signalling and transport processes. In both cases, the stereospecificity of ABA in transport was shown by experiments using ABA stereoisomers. Note that the Km saturation kinetics of ATP-dependent ABA transport are not much different (260 nM and 1 M for AtABCG25 and AtABCG40, respectively), although different assay systems were used to calculate activity (Kang et al., 2010; Kuromori et al., 2010). These findings strongly suggest the existence of an active control of ABA transport between plant cells. From two reports (Kang et al., 2010; Kuromori et al., 2010), a simple model can be proposed: ABA is exported from ABA-biosynthesising cells to the apoplastic area; then, ABA is imported from the apoplast to the interior of guard cells. This model is consistent with recent reports that some ABA receptors triggering ABA signalling in cells are soluble and localised to the cytosol (Ma et al., 2009; Park et al., 2009), which suggests the potential importance of an ABA transporter that could deliver ABA in a regulated fashion to initiate rapid and controlled responses to the various stress conditions that are perceived by ABA (Kang et al., 2010). Investigation of the ABA transport mechanism has just started, and it provides a novel impetus for examining ABA intercellular regulation. C. ABA SUBCELLULAR TRANSPORT BETWEEN ORGANELLES
Very little information exists on the subcellular localisation of ABA molecules and transportation between cellular compartments and/or the cytoplasm. De novo ABA synthesis is initiated in plastids via the oxidative
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cleavage of xanthophylls (see Section II). The cleaved product xanthoxin is further modified in the cytoplasm to produce ABA. According to this model, xanthoxin is presumed to migrate from the plastid to the cytosol so that some unknown transporters for ABA precursors exist at the plastid membrane (Floss and Walter, 2009; Nambara and Marion-Poll, 2005). In another point related to ABA storage and movement, ABA is conjugated with glucose, resulting in the formation of an ABA glucose ester (ABAGE). ABA-GE is an inactive form of ABA, and is widespread in the plant kingdom (Hartung et al., 2002). Lee et al. (2006) revealed that ABA-GE is hydrolysed in response to stress by the -glucosidase AtBG1, thus leading to an increase in the active ABA concentration. AtBG1-deficient Arabidopsis plants exhibit lower ABA levels in leaves and produce stress-sensitive phenotypes (Lee et al., 2006). While ABA-GE is located in the vacuoles, the xylem sap, and probably in the cytosol and cell wall as well (Dietz et al., 2000), AtBG1 -glucosidase is located in the endoplasmic reticulum (ER) and remains in the ER during stress responses, suggesting that an important aspect of stress responses may be the activation of transporter proteins that shuttle ABA-GE into the ER (Lee et al., 2006; Schroeder and Nambara, 2006). While ABA-GE was also suggested to be a long-distance signal in the vascular system, especially under stress conditions (Jiang and Hartung, 2008), a mechanism for ABA-GE transport has not yet been reported.
IV. INTRACELLULAR SIGNAL TRANSDUCTION IN ABA RESPONSES ABA strikingly affects various aspects of plant life, for example, seed dormancy, maturation and plant growth and development, as well as stress responses to water deficits. Of these, stomatal closure is well characterised as a model system of ABA response. This requires control of the ion balance in guard cells by a series of ion channels or transporters, largely regulated by ABA signalling pathways (Kim et al., 2010). At the whole-plant level, ABA induces a wide variety of gene expression, and those gene products range from regulatory proteins, such as transcription factors and other signalling factors, to functional proteins, such as metabolic enzymes, hydrophilic proteins, and scavengers (Yamaguchi-Shinozaki and Shinozaki, 2006). They have central roles in adaptation or tolerance to water deficit conditions. In addition, ABA is essential for seed development and dormancy, which also requires ABA-responsive expression (Nambara et al., 2010). Several transcription factors have been identified in ABA responses that are strictly regulated by intracellular ABA signalling.
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Such ABA responses as just described may be controlled by well organised intracellular signal transduction pathways. Many signalling factors have been reported for ABA responses in plants (Cutler et al., 2010; Hirayama and Shinozaki, 2007, 2010; Hubbard et al., 2010; Klingler et al., 2010; Raghavendra et al., 2010). They include receptors, protein kinases/phosphatases, and transcription factors, among others. In this chapter, we outline ABA signalling factors and recent advances in our knowledge. A. RECEPTORS
In general, intracellular signal transduction is initiated with the perception of stimuli or chemicals. The molecule for signal perception is called a ‘‘receptor’’ and is regarded as one of the most important components for deciphering a complex network of signal transduction pathways. Phytohormones are critical regulators of plant life, and their receptors have been extensively studied. Recently, receptors of the major phytohormones, auxin, gibberellin, ethylene, cytokinin and jasmonic acid, were determined and well characterised (Lumba et al., 2010; Santner and Estelle, 2009). In most cases, receptor identification triggered the quick resolution of an unexpectedly simple signalling mechanism, for example, the TIR1-Aux/IAA-ARF pathway in auxin signalling. In contrast, the identification of an ABA receptor has encountered much difficulty (McCourt and Creelman, 2008). To date, several receptor candidates, for example, G-protein-coupled receptors (GPCRs) and the Mgchelatase H subunit (ChlH), have been reported, mainly based on their ABA-binding ability (Liu et al., 2007; Pandey et al., 2009; Shen et al., 2006). Although such receptor candidates were suggested to be involved in regulatory pathways in ABA responses, they did not make a breakthrough in our understanding of ABA signalling. Under this situation, two research groups identified a novel candidate for the ABA receptor, the PYR/PYL/ RCAR family in 2009 (Ma et al., 2009; Park et al., 2009). Importantly, the PYR/PYL/RCAR family was connected to a major ABA signalling factor, protein phosphatase 2C (see Section IV.B). This study shed new light on ABA signalling and led to the uncovering of the ‘‘core signalling pathway’’ of ABA (see Section V). Presently, other receptor candidates are still waiting to be connected in ABA signalling pathways. In this section, a brief history of the search for ABA receptors and their molecular functions is reviewed. 1. PYR/PYL/RCAR The PYR/PYL/RCAR family was identified by two different approaches. Ma et al. (2009) screened interacting proteins with a PP2C, ABI1 (see Section IV.B’’) and identified an unknown protein that selectively bound
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ABA. Further, the protein interacted with and inhibited the PP2C activity of ABI1; thus they named this protein ‘‘regulatory components of ABA receptor 1’’ (RCAR1). The other was a chemical genetic approach by Park et al. (2009). They screened chemical libraries and found a selective agonist of ABA, pyrabactin. The Pyrabactin-resistance 1 (PYR1) or PYR1-like (PYL) genes were identified from a genetic screen of pyrabactin-insensitive mutants. Although 14 members of PYR/PYL/RCAR exist in Arabidopsis, Park et al.’s (2009) chemical genetic approach successfully bypassed the barrier of functional redundancy. PYR/PYL/RCAR proteins belong to START-domain/Bet v 1 allergen proteins. They contain a ligand pocket in which ABA can bind with high affinity, as demonstrated by an isothermal titration assay and several structural analyses (Ma et al., 2009). Some differences in sensitivity or selectivity of (þ)- or ()-ABA exist among PYR/PYL/RCAR members. Studies proposed that PYR/PYL/RCARs form dimers in the absence of ABA (Nishimura et al., 2009; Yin et al., 2009). Structural analyses confirmed the mode of ABA binding in a ligand pocket of PYR/PYL/RCARs (Melcher et al., 2009; Miyazono et al., 2009; Nishimura et al., 2009; Santiago et al., 2009; Shibata et al., 2010; Yin et al., 2009). Further, recent studies presented more details about the mechanistic basis of PYR/PYL/RCAR receptors, and discriminated each of these receptors by their selectivity against ABA chirality or pyrabactin (Hao et al., 2010; Melcher et al., 2010; Peterson et al., 2010). As RCAR1 was identified as an ABI1-interacting protein (Ma et al., 2009), PYR/PYL/RCARs are major in vivo interactors with group A PP2Cs (Nishimura et al., 2010). ABA affects the interaction of PYR/PYL/RCAR– PP2C to some extent. Group A PP2Cs are known as global negative regulators of ABA signalling, and PYR/PYL/RCARs inhibit PP2C activity in an ABA-dependent manner (Ma et al., 2009; Park et al., 2009). This means that PYR/PYL/RCARs form a double-negative regulation system in ABA signalling. The mechanistic basis of their interaction and inhibition was clearly established by structural analysis (Melcher et al., 2009; Miyazono et al., 2009). Briefly, ABA binding to an internal cavity of PYR/PYL/RCAR induces conformational changes, especially on two ‘‘gate’’ and ‘‘latch’’ loops. This gate and latch system closes on the cavity to shape an interface with PP2C. The interface makes direct contact with a catalytic domain of PP2C, resulting in the inhibition of phosphatase activity. These studies strongly connected newly identified ABA receptors and a well-known ABA signalling factor, PP2C. The identification of PYR/PYL/ RCAR was a real breakthrough in ABA signalling and triggered the establishment of a core regulatory system for ABA signalling in subsequent studies on SnRK2 protein kinases, as described below.
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2. G-protein-coupled receptors Generally, heterotrimeric GTP-binding proteins (G-proteins) mediate various signals in association with GPCRs in mammals. Although plants have only a limited number of canonical G-proteins or GPCRs, they have been implicated in developmental processes and stress responses, especially ABA responses (Temple and Jones, 2007). In Arabidopsis, a series of G-protein components, for example, GPA1, AGB1 and GCR1, were identified and their roles in ABA signalling were characterised (Perfus-Barbeoch et al., 2004). First, a GPCR-type protein, GCR2, was identified as a plasma membranetype ABA receptor (Liu et al., 2007). Biochemical analysis demonstrated that GCR2 binds ABA with high affinity and interacts with GPA1, the sole Gprotein -subunit in Arabidopsis. However, several questions remain open concerning GCR2, for example, whether GCR2 is a typical GPCR and whether GCR2 is involved in major ABA signalling (Chen and Ellis, 2008; Gao et al., 2007; Guo et al., 2008). Further analysis will be required to determine GCR2 function in plants. In contrast, the second G-protein-related ABA receptors, GTG1 and 2, were reported by Pandey et al. (2009). GTG1 and 2 encode typical GPCRs, which show homology to noncanonical GPCRs, and bind ABA with high affinity, physically interacting with GPA1. GTG1 and 2 are localised to the plasma membrane, and genetic analysis using double-knockout mutants demonstrated that GTG1 and 2 positively regulate ABA responses. These results suggested that GTG1 and 2 comprise a new-type of ABA receptor. However, how GTG1 and 2 regulate the downstream factors involved in ABA signalling remains to be determined. 3. Mg-chelatase H subunit An affinity purification approach identified a 42-kDa ABA-binding protein, termed ABAR, from bean epidermis (Zhang et al., 2002). ABAR encodes the Mg-ChlH (Shen et al., 2006), which is a known component of a multisubunit Mg-chelatase supplying Mg2þ to a chlorophyll precursor. This protein was suggested to be an ABA-receptor candidate and functionally characterised in Arabidopsis. ABAR specifically binds S-ABA with a Kd of 32 nM, and it acts as a positive regulator of multiple ABA responses in Arabidopsis (Shen et al., 2006). ABAR/ChlH is identical to GUN5 (GENOMES UNCOUPLED 5), which is involved in plastid-to-nucleus retrograde signalling (Mochizuki et al., 2001). However, ABAR functions in ABA signalling were distinguished from retrograde signalling (Shen et al., 2006). A recent study revealed that ABAR/GUN5/ChlH transmits ABA signals from plastid to nucleus through
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WRKY transcription factors, which repress the gene expression of some ABA-related transcription factors. For example, ABAR interacts with WRKY40 in an ABA-dependent manner, resulting in the release of negative regulation by WRKY40 in the nucleus (Shang et al., 2010). Moreover, ABAR functions could be regulated by circadian oscillation rhythms. A study reported that TOC1, a central component of the circadian clock, regulates ABAR expression by direct binding to its promoter region (Legnaioli et al., 2009). B. PROTEIN PHOSPHATASES
Protein phosphorylation is involved in countless regulatory systems controlling various biological phenomena. Therefore, protein kinases have attracted much attention in biological research over the past several decades. The enzymes that reverse the phosphorylation status of a protein, protein phosphatases, are as important as kinases and have also been investigated. Eukaryotes have many types of phosphatases, such as protein phosphatase 1 (PP1), PP2A, PP2B and PP2C for phospho-serine/threonine, and PTP for phospho-tyrosine (for more details, see Schweighofer and Meskiene, 2008). Among them, PP2C is unique in requiring metal ions such as Mg2þ or Mn2þ. Plants have many PP2C genes. According to the genome sequence of Arabidopsis, this plant has at least 76 PP2C genes (Schweighofer et al., 2004). The functions of most of them are not yet elucidated. At least six clade A PP2Cs (ABI1, ABI2, HAB1, HAB2, AHG1 and AHG3/AtPP2CA) were shown to regulate the ABA signalling pathway (see below and Hirayama and Shinozaki, 2007 for a review). These six PP2Cs can be separated into two groups, one consisting of ABI1, ABI2, HAB1 and HAB2, and the other of AHG1 and AHG3 based on amino acid sequence similarity. These two group PP2Cs behave differently. AHG1 and AHG3 are strongly expressed in seeds and localised in the nucleus, while the other four PP2Cs are more strongly expressed in adult tissues and localised in both the nucleus and cytoplasm (Moes et al., 2008; Umezawa et al., 2009). Therefore, besides biochemical characteristics, the PP2Cs of these two groups might have different functions in plant physiology. Another three Arabidopsis group A PP2Cs, namely, At1g07430, At2g29380 and At5g59220, have the ability to interact with ABA-activated SnRK2s (see below) and the genes for these PP2Cs are induced by exogenous ABA treatment, demonstrating their function in the ABA response (Fujita et al., 2009; Umezawa et al., 2009; Xue et al., 2008). The important link between PP2C and ABA response was first provided by the analysis of the well-known Arabidopsis ABA-insensitive mutant, ABA insensitive (abi) 1-1 (Leung et al., 1994). A point mutation that causes an
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amino acid conversion from Gly to Asp at amino acid 180 confers a dominant, strong ABA insensitivity for most ABA responses. Recombinant abi1-1 proteins had significantly reduced PP2C activities to artificial substrates, such as casein phosphorylated by animal protein kinase A. No clear explanation exists for the molecular mechanisms by which the abi1-1 mutation causes dominant ABA insensitivity. Several intragenic suppressor mutants for abi1-1 were isolated and all of them were loss-of-function-type mutations for PP2C, such as amino acid conversions in the active site (Gosti et al., 1999), implying that the abi1-1 phenotype requires PP2C activity. Thus, abi1-1 was thought to be a dominant-negative type mutation for years, although this idea remains controversial. Similarly, another PP2C, ABI2, was identified. The abi2-1 mutant has a similar mutation in the ABI2 gene at the corresponding amino acid residue to abi1-1 and exhibits a similar ABA-insensitive phenotype. However, detailed analysis of ABA response in abi1-1 and abi2-1 mutants showed some differences. For example, the effects of these two ABA-insensitive mutations on guard cell responses to ABA are different (Murata et al., 2001). Thus, PP2Cs were thought to have distinct targets and physiological functions regardless of the high similarity in amino acid sequence. Among the 76 PP2Cs in Arabidopsis, 9 have a similar amino acid sequence to ABI1. Overexpression or gene disruption of Homology to ABA1 (HAB1), which was isolated based on a sequence similarity to ABI1, affects ABA sensitivity in transgenic plants (Saez et al., 2004), leading us to the idea that other PP2Cs similar to ABI1 have some functions in the ABA response. Eventually, two independent gene screening studies identified a PP2C, AtPP2CA/AHG3, involved in the ABA response in germination. Kuhn et al. (2006) screened for cDNA clones conferring an ABA-insensitive phenotype when overexpressed in plants. Yoshida et al. (2006b) isolated an ABA-hypersensitive mutant ABA-hypersensitive germination (ahg) 3, using germination efficiency in the presence of ABA or ABA analogues as a physiological marker, and identified the same gene as an ABA-hypersensitive locus. The ahg3-1 mutation is a point mutation that affects an amino acid residue required for PP2C activity. Thus, the clear ABA-hypersensitive phenotype of this mutation indicates that a single loss-of-function-type mutation does not cause a dominant-negative effect but a recessive ABA hypersensitivity. Nishimura et al. (2007) also identified AHG1 as another ABA-hypersensitive locus. In total, six PP2Cs were shown to be involved in the ABA response. The loss-off-function mutations of these genes showed ABA hypersensitivity in germination (Nishimura et al., 2007; Yoshida et al., 2006b). The strength of the phenotype seems to correlate with the expression level of the gene. The multiple mutations had a more pronounced phenotype,
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indicating that these PP2Cs have redundant functions (Nishimura et al., 2007; Rubio et al., 2009). Consequently, six PP2Cs are known to negatively regulate the ABA response. No known regulatory system has been reported for PP2C. Therefore, the molecular mechanisms by which these PP2Cs mediate the ABA response in plants were unclear until very recently. As mentioned above, discovery of soluble ABA receptors gave a clear and impressive answer. PYR1/PYL/RCAR-type ABA receptors interact with ABA-related PP2Cs in an ABA-dependent manner and inhibit PP2C activity (Ma et al., 2009; Park et al., 2009). In addition, the abi1-1 type mutation interferes with this interaction and partially explains the paradox of the abi1-1 type mutants. Recent studies showed that clade A PP2Cs dephosphorylate ABA-activated SnRK2s (see Section IV.C.1). However, how many substrates these ABA-related PP2Cs have other than ABA-activated SnRK2s is not yet clear. Many studies have attempted to identify the targets of these PP2Cs. Most of them focused on the identification of interacting factors. These studies have identified several proteins, such as potassium transporter AKT2/AKT3, homeodomain protein ATHB6, several calcineurin-B-like interacting protein kinases and chromatin remodelling protein SWI3 (Himmelbach et al., 2002; Ohta et al., 2003; Saez et al., 2008; Vranova et al., 2001). These predicted targets more or less take part in the cellular response to ABA, but the physiological relevance of these targets has not yet been established. Vlad et al. (2009) systematically searched phosphopeptides as a potential target for PP2C HAB1. The proteins identified include GUN5, another ABA receptor (see Section IV.A), and ICE1, a transcription factor involved in stomatal development, in addition to transcription factors, metabolic enzymes, and proton ATPases (Kanaoka et al., 2008; Shen et al., 2006; Vlad et al., 2009). Whether HAB1 or other ABA-related PP2Cs actually dephosphorylate these candidate proteins has not been reported. Why plants need more than six PP2Cs to regulate ABA responses is unclear. These PP2Cs have slightly different tissue or organ distributions and have different subcellular localisations. Therefore, their biochemical properties are very similar but their physiological roles might be quite different. The multiple ABA-related PP2Cs may reflect the complexity and/ or diversity of the ABA response of the cell. To regulate or fine-tune such ABA responses, multiple PP2Cs with different properties are required. These PP2Cs were shown to also be targets of various second messengers with important roles in the ABA response. Phosphatidic acid, produced by phospholipase D, inhibits the phosphatase activity of ABI1 and recruits ABI1 from the cytoplasm to the plasma membrane (Zhang et al., 2004). Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), reduce
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the PP2C activity of ABI1 and ABI2 in vitro (Meinhard and Grill, 2001; Meinhard et al., 2002). An Arabidopsis glutathione peroxidase, ATGPX3, inhibits the activity of ABI2 and/or ABI1 in a H2O2-dependent manner (Miao et al., 2006). The existence of multiple regulators implies a complex regulatory system for PP2Cs in modulating ABA signalling flow to integrate other signals. The relationship of these effectors and cytoplasmic ABA receptors remains to be clarified. 2A-type phosphatases (PP2A) were also implicated in ABA signalling. Kwak et al. (2002) showed that a regulatory subunit of an Arabidopsis PP2A, RCN1, functions as a positive regulator of the ABA response in germination and guard cells. In contrast, loss-of-function mutations in PP2Ac-2, encoding a catalytic subunit of PP2A of Arabidopsis, had ABAhypersensitive phenotypes, and the overexpression of this gene resulted in ABA-insensitive phenotypes, suggesting a negative regulatory function in the ABA response (Pernas et al., 2007). The target of PP2A has not been identified. RCN1 is involved in several biological processes, such as auxin transport, ethylene response, and blue light response, implying broad substrate specificity.
C. PROTEIN KINASES
Given that protein phosphatases, especially PP2Cs, have central roles in ABA signalling, the importance of protein phosphorylation was recognised in understanding signalling pathways in response to ABA. In general, protein kinases function as a counterpart to protein phosphatases, and they are known as major signalling factors in eukaryotes. To date, many protein kinases have been identified as signalling factors in ABA responses, and some of them are well characterised (Hirayama and Shinozaki, 2007, 2010). In this chapter, we mention several protein kinases implicated in ABA signalling, for example, SnRK2, CDPK, CIPK/PKS/SnRK3 and MAPK, among others. 1. SnRK2 Yeast sucrose-nonfermenting 1 (SNF1) protein kinase is a central regulator of sugar metabolism, and the mammalian SNF1 homolog, AMP-activated protein kinase (AMPK), functions as an energy sensor in mammalian cells (Hardie, 2007). Plants also have an SNF1-type protein kinase, SNF1-related protein kinase 1 (SnRK1), which seems to be functionally conserved with SNF1/AMPK. In addition, two other types of SNF1-related protein kinases are known in plants, that is, SnRK2 and SnRK3 (see the following sections).
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SnRK2 belongs to a plant-specific protein kinase family that is highly conserved in higher plants. Ten members exist in Arabidopsis and rice, and they are classified into three subclasses, subclasses I–III (Kobayashi et al., 2004). Among them, subclass II and III members are weakly and strongly activated by ABA, respectively. In contrast, all subclass members are strongly activated by osmotic stress (Boudsocq et al., 2004; Kobayashi et al., 2004; Umezawa et al., 2004). Studies have proposed that the activation mechanisms of SnRK2 should differ between ABA and osmotic stress (Kobayashi et al., 2004; Yoshida et al., 2006a). To date, numerous SnRK2s have been analysed and their in planta functions characterised. Wheat PKABA1 is a member of subclass II and was identified as an ABA-inducible gene (Anderberg and Walker-Simmons, 1992). Constitutive expression of PKABA1 drastically suppressed GA-inducible gene expression in barley aleurone cells, but it had only a small effect on ABA-inducible gene expression (Go´mez-Cadenas et al., 1999). SRK2C/ SnRK2.8 is a member of subclass II in Arabidopsis, and it is strongly activated by osmotic stress. SRK2C-overexpressing plants had elevated drought tolerance in concert with the upregulation of several drought-inducible genes, suggesting that SRK2C/SnRK2.8 is a positive regulator involved in drought stress-responsible gene expression (Umezawa et al., 2004). Fava bean AAPK is a member of subclass III, and it was isolated as an ABAactivated protein kinase in guard cells (Li and Assmann, 1996). Transient expression of AAPK in fava bean guard cells revealed that it positively regulates stomatal closure induced by ABA (Li et al., 2000), suggesting that subclass III SnRK2s are important in ABA signalling in guard cells. Consistent with this finding, an Arabidopsis AAPK homolog, SRK2E/OST1/ SnRK2.6, is an ABA-activated protein kinase, and genetic evidence supports its central role in ABA-responsive stomatal closure in Arabidopsis (Mustilli et al., 2002; Yoshida et al., 2002). Three members of Arabidopsis subclass III SnRK2s exist, and another two members, SRK2D/SnRK2.2 and SRK2I/SnRK2.3, are also important in the ABA response (Fujii et al., 2007). A double-knockout mutant snrk2.2 snrk2.3 showed significant ABA-insensitive seed germination and impaired ABAresponsive gene expression, but the stomata were unaffected. This suggests that subclass III SnRK2s have distinctive roles, SRK2E/OST1/SnRK2.6 in guard cells and SRK2D/SnRK2.2 and SRK2I/SnRK2.3 in other tissues. However, other studies demonstrated that all three subclass III members fundamentally share a functional redundancy. A triple-knockout mutant srk2dei (snrk2.2 snrk2.3 snrk2.6) had an extremely ABA-insensitive phenotype displayed in tissues ranging from seeds to guard cells. Remarkably, this triple mutant can germinate normally even in the presence of irregularly high
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concentration of ABA, and ABA-responsive gene expression is impaired in hundreds of genes (Fujii and Zhu, 2009; Fujita et al., 2009; Nakashima et al., 2009; Umezawa et al., 2009). This indicates that the triple mutant lacks a large part of the ABA response. Although subclass II SnRK2s, SRK2C/ SnRK2.8 and SRK2F/SnRK2.7, are also implicated in ABA signalling and their functions partly overlap with subclass III members, the strong phenotype of srk2dei tells us that the three subclass III SnRK2s function globally and are essential positive regulators of ABA signalling in plants. Given the importance of subclass III SnRK2s, the activation mechanism of SnRK2 should be a key to understanding the main regulatory system of ABA signalling. Recently, two research groups identified a negative regulatory mechanism of SnRK2s mediated by group A PP2Cs (Umezawa et al., 2009; Vlad et al., 2009). Yeast two-hybrid analysis revealed that all subclass III SnRK2s interact with group A PP2Cs in various combinations, and some interactions were confirmed in vivo (Umezawa et al., 2009). This provides biochemical evidence that some group A PP2Cs, ABI1, AHG1 or HAB1, can inactivate SnRK2s by direct dephosphorylation. When SnRK2s are activated by ABA, it is accompanied by phosphorylation of the kinase activation loop. The phosphorylation sites are Ser175 and some other Ser/Thr residues (at least one more), and this same region is dephosphorylated by PP2C. Thus, PP2C is an upstream factor of SnRK2 and maintains the inactive state of SnRK2 under normal conditions. 2. Ca2þ-dependent protein kinase Many studies have suggested that Ca2þ acts as second messenger in ABA signalling (Hubbard et al., 2010). Several reports proposed that Ca2þ-dependent protein kinase (CDPK) is a strong candidate for Ca2þ-dependent ABA signalling. CDPKs form a large family of plant protein kinases that are involved in various signalling pathways (Harper et al., 2004). CDPKs consist of three major domains: the kinase catalytic domain, autoinhibitory domain, and calmodulin (CaM)-like Ca2þ-binding domain. Ca2þ-binding to the CaM-like domain unlocks the autoinhibition of CDPK, allowing CDPK to fulfil its protein kinase activity. The role of Ca2þ in ABA signalling has been extensively studied in guard cells. For example, an elegant approach using pharmacological techniques demonstrated that Ca2þ oscillation and its specific pattern are required for stomatal movement in response to ABA (Allen et al., 2001). Therefore, the existence of Ca2þ-dependent signalling mediators in guard cells was expected. Mori et al. (2006) identified two CDPKs, CPK3 and CPK6, as positive regulators of ABA signalling in guard cells. These CDPKs were identified based on their significant expression in guard cells. Genetic analysis
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revealed that they cooperatively regulate ABA signalling, as shown in the double cpk3 cpk6 mutant. In this mutant, activation of slow anion channels or Ca2þ-permeable channels in response to ABA was impaired, resulting in partial inhibition of stomatal closure (Mori et al., 2006). In contrast, a recent study demonstrated that other CDPK members, CPK21 and CPK23, are major interacting partners with the slow anion channel, SLAC1, in Arabidopsis guard cells (Geiger et al., 2010). CPK21 and 23 are closely related CDPKs, and they can phosphorylate SLAC1 in vitro. Further analysis is required to understand whether CPK3/6 and CPK21/23 are functionally redundant in guard cells. Other CDPKs have also been implicated in ABA signalling, mainly in transcriptional regulation. Previously, some evidence was shown for the importance of Ca2þ in ABA-responsive gene expression (Harper et al., 2004). Consistent with this finding, a recent study revealed that CPK4 and CPK11, closely related CDPKs in Arabidopsis, positively regulate multiple ABA responses in seeds and guard cells (Zhu et al., 2007). Further, these CDPKs are capable of phosphorylating the ABA-responsive transcription factors, ABF1 and AREB2/ABF4, in vitro, suggesting their regulatory roles in ABA-responsive gene expression. Multiple CDPKs are likely to be involved in the regulation of AREB/ABF transcription factors and CPK32 was reported to also interact with and phosphorylate AREB2/ABF4 (Choi et al., 2005). Presently, we do not expect any evolutionary relationship between CDPKs involved in ABA signalling (i.e., CPK3, 4, 6, 11, 21, 23 and 32), because each member is widely distributed in the plant CDPK superfamily. Additionally, understanding how plants orchestrate various ABA responses using different types of CDPKs is important. 3. CIPK/PKS/SnRK3 In addition to CDPK, plants have several groups of Ca2þ-regulated protein kinases (Harper et al., 2004). One of these, CBL-interacting protein kinase** (CIPK)/protein kinase SOS2-like (PKS) is a plant-specific protein kinase family that is involved in multiple stress responses, as well as ABA responses (Gong et al., 2004; Luan, 2009; Weinl and Kudla, 2009). CIPK/PKS is also called SNF1-related protein kinase 3 (SnRK3) because the N-terminal region contains a protein kinase catalytic domain, which is highly conserved when compared to yeast SNF1 protein kinase (Hrabak et al., 2003). CIPK/PKS is autoregulated through an autoinhibitory domain in the C-terminal stretch. CIPK/PKS functions as a pair with a Ca2þ-binding protein, calcineurin-Blike protein (CBL)/SOS3-like Ca2þ-binding protein (SCaBP), which interacts with a specific domain (NAF/FISL) of CIPK/PKS in a calcium-dependent
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manner, which releases the CIPK/PKS from autoinhibition (Gong et al., 2004; Luan, 2009; Weinl and Kudla, 2009). Internal phosphorylation of a Thr residue in the kinase activation loop is required for CIPK/PKS activity (Gong et al., 2002a,b). A study suggested that group A PP2Cs, such as ABI1 or ABI2, might be involved in the regulation of CIPK/PKS via physical interaction, but the molecular interaction between PP2C and CIPK/PKS remains unclear. Several CIPK/PKSs and CBL/SCaBPs function in ABA signalling. For example, PKS3/CIPK15 and its interactor SCaBP5/CBL1 were identified as a global negative regulator of ABA responses in seed germination and stomata (Guo et al., 2002). Likewise, CIPK23/PKS17, which interacts with CBL1 and CBL9, is involved in a negative regulatory pathway in ABAdependent stomatal closure (Cheong et al., 2007). CIPK1/PKS13 also interacts with CBL1/9 and negatively regulates seed germination and gene expression in response to ABA (D’Angelo et al., 2006). Moreover, a study proposed that CBL9 has a major role in CIPK1-mediated ABA signalling (D’Angelo et al., 2006). In contrast, CIPK3/PKS12 positively regulates gene expression in response to ABA, although it interacts with CBL9 (Kim et al., 2003; Pandey et al., 2008). As compared to other protein kinases, for example, SnRK2 and CDPK, as positive regulators of ABA signalling, the mode of action of the CIPK/PKS/SnRK3 family seems to be diverse and complicated. The role of CIPK/PKS in ABA signalling could be conserved in higher plants because a rice CIPK/PKS, OsCIPK31, negatively regulates ABA responses in seed germination, seedling growth, and gene expression (Piao et al., 2010). Although some differences exist in tissue specificity and signalling mode between each ABA-related CIPK/PKS, all members are characterised as Ca2þ-dependent signalling factors in ABA responses. To better understand their functions, how each CIPK/PKS regulates ABA signalling should be clarified. Identification of downstream factors is key to understanding CIPK/PKS-mediated signalling. An ABA-related CIPK/PKS member, CIPK23, directly regulates a potassium channel, AKT1, suggesting that CIPK23 affects ABA signalling via cellular potassium adjustment (Lee et al., 2007; Xu et al., 2006). Other downstream targets of CIPK/PKS were already reported in other signalling pathways. Salt-overly-sensitive 2 (SOS2), one of the best characterised CIPK/PKS in salt tolerance, directly interacts with a plasma membrane Naþ/Hþ antiporter SOS1, in association with SOS3 or some other CBL/SCaBPs (Qiu et al., 2002). PKS5/CIPK11 was identified as a negative regulator of the plasma membrane proton pump, and it phosphorylates AHA2 in vitro (Fuglsang et al., 2007). These results strongly suggest that membrane-bound ion channels are major targets of CIPK/PKS.
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4. MAPK mitogen-activated protein kinase (MAPK) is known to regulate various signals in eukaryotes. MAPK is regulated by a phosphorylation cascade that consists of MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). In this cascade, MAPKKK phosphorylates and activates MAPKK, and then MAPKK phosphorylates and activates MAPK. Arabidopsis is known to have 80 MAPKKKs, 10 MAPKKs and 20 MAPKs (MAPK Group, 2002). They form a large number of specific combinations in plant cells, and various MAPK cascades are responsible for different signals. To date, several MAPKs are implicated in ABA signalling. AtMPK3 and AtMPK6 were first identified as ABA-activated MAPKs by an in-gel kinase assay (Ichimura et al., 2000; Lu et al., 2002). Also, AtMPK1 and AtMPK2 are significantly activated by ABA (Hwa and Yang, 2008). Such ABAdependent activation of MAPKs demonstrates their roles in ABA signalling. Recently, a study reported that AtMKK1 and AtMPK6 are involved in ABA and glucose signalling, as shown by single- and double-knockout mutants (Xing et al., 2009). In another case, AtPP2C5, a protein phosphatase interacting with AtMPK3, 4, and 6, was recently identified (Brock et al., 2010). A knockout mutant of AtPP2C5 showed enhanced ABA-dependent activation of AtMPK3 and 6, resulting in ABA insensitivity, suggesting that the MAPK cascade negatively regulates ABA signalling in plants. In contrast, two MAPKs functioning in guard cells were reported (Jammes et al., 2009). MPK9 and MPK12 were screened by microarray analysis of guard cell transcripts. Genetic evidence revealed that MPK9 and MPK12 cooperatively and positively regulate stomatal responses to ABA, and they act upstream of anion channels but downstream of ROS signalling.
5. RLK In addition to SnRK2, CDPK, CIPK/PKS and MAPK (mitogen-activated protein kinase), several protein kinases have been implicated in ABA signalling. For example, RPK1 is a member of the receptor-like protein kinase superfamily and was isolated based on ABA-responsive gene expression (Osakabe et al., 2005). Although genetic analysis suggested that RPK1 is a membrane-bound protein involved in early ABA signalling, the ligand of RPK1 is still unknown. Studies reported that RPK1 is involved in developmental control, such as embryonic pattern formation, and the establishment of the cotyledon primordial, in association with another receptor-like protein kinase (RLK), TOAD1 (Nodine and Tax, 2008; Nodine et al., 2007). The next step is to determine how RPK1 participates in ABA signalling, that is, identify RPK1 targets or its connections to other ABA signalling pathways.
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D. ABA-RESPONSIVE GENE EXPRESSION
ABA rapidly induces a series of gene expressions, and those gene products are essential for various responses. ABA-responsive gene expression is strictly regulated in concert with several transcriptional factors and cis-elements in the promoter region. Among them, a subgroup of basic leucine-zipper (bZIP)-type proteins has critical roles in ABA-responsive gene expression in plants (Yamaguchi-Shinozaki and Shinozaki, 2006). In Arabidopsis, nine bZIP proteins are related to ABA (Fujita et al., 2005; Jakoby et al., 2002). The importance of bZIP-type transcription factors was genetically identified in a screening of ABA-insensitive mutants using an Arabidopsis background. The isolated abi5 mutants exhibit a moderate ABA-insensitive phenotype specifically in seeds (Finkelstein and Lynch, 2000; Lopez-Molina and Chua, 2000). The expression of Em genes was impaired in this mutant. ABI5 interacts with an ABRE in the promoter region of these target genes and upregulates their transcription. bZIP-type transcription factors were also identified with biochemical approaches, for example, yeast one-hybrid screening using ABRE-containing promoters (Choi et al., 2000; Uno et al., 2000). They are designated as ABRE-binding protein (AREB) and ABRE-binding factor (ABF). AREB1/ ABF2, AREB2 and ABF3 regulate gene expression via ABRE, as well as ABI5, but are mainly involved in stress responses in vegetative tissues. These types of proteins require posttranscriptional/translational modifications for their activity. Phosphorylation state is a major factor determining activity, and SnRK2 and CDPK were identified as upstream protein kinases (Choi et al., 2005; Furihata et al., 2006; Kagaya et al., 2002; Kobayashi et al., 2005). Mutational analysis showed that multiple phosphorylation sites are required for full activation of bZIPs (Furihata et al., 2006). Which phosphorylation sites are mediated by SnRK2s and CDPKs is not clear. In contrast, other types of regulations were reported, mainly for ABI5. One is proteasomal degradation, which is described in another section E in this chapter. Another possible regulation is sumoylation of ABI5 mediated by a SUMO E3 ligase AtSIZ1 (Miura et al., 2009). ABI5 interacting protein (AFP) was identified by a yeast two-hybrid screen using ABI5 as bait, and it affects protein degradation of ABI5 during the post-germination stage (LopezMolina et al., 2003). In addition, AFP-type proteins, NINJA, can form a complex with JAZ, AtMYC2 and TOPLESS-type repressor proteins in jasmonic acid signalling (Pauwels et al., 2010). TPL proteins repress the transcriptional activity of AtMYC2, and this negative regulation can be released after COI1 degrades JAZ, suggesting that some similar mechanisms are involved in ABA-related bZIP proteins.
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As described above, several transcription factors have been implicated in ABA signalling, for example, bHLH, NAC, AP2 and WRKY. See recent reviews in which other transcription factors are well documented (Hirayama and Shinozaki, 2010; Yamaguchi-Shinozaki and Shinozaki, 2006). E. REGULATION THROUGH PROTEIN DEGRADATION IN THE ABA RESPONSE
The ubiquitin-26S proteasome-dependent protein degradation system is a prominent regulatory mechanism in various eukaryotic biological processes (Vierstra, 2009). ABA response is not an exception. Lopez-Molina et al. (2001) showed that ABI5 undergoes proteolysis via the ubiquitin-26S proteasome system. They also identified a protein called AFP that is required for ABI5 degradation (Lopez-Molina et al., 2003). Another forward genetic study revealed an E3 ligase, KEEP ON GOING (KEG), whose loss-offfunction mutants are ABA-hypersensitive and abnormally accumulate ABI5 protein (Liu and Stone, 2010; Stone et al., 2006). Another ABA-related transcription factor, ABI3, is also regulated at the protein level. Ubiquitination of ABI3 is regulated by AIP2 E3 ligase (Zhang et al., 2005). Consistent with these findings, impairment of RPN10, a subunit of the 19S regulatory complex of proteasomes, caused pleiotropic phenotypes, including ABA hypersensitivity (Smalle et al., 2003). In addition, mutations in the genes encoding E3 ligases, SDIR1 and RHA2a, confer abnormal responses to exogenous applied ABA, implying that these E3 ligases function in the regulation of the ABA response (Bu et al., 2009; Zhang et al., 2007). That some of the ABA-related transcription factors, ABI3 and ABI5, are regulated by proteolysis is becoming clearer. However, the molecular mechanisms that initiate the degradation process of these transcription factors are not known. In other hormone response pathways, ligand binding of hormone receptors triggers the degradation of transcription repressors that interact with hormone receptors in a ligand-dependent manner (Santner and Estelle, 2009). As mentioned earlier, a recent study on the transcriptional regulation of the jasmonic acid response revealed a transcriptional complex including the TOPLESS transcriptional repressor (Pauwels et al., 2010). A similar complex containing AFP is expected to regulate ABA-responsive genes. Very recently, Liu and Stone (2010) showed that phosphorylation of AFP might trigger its proteolysis and in turn stabilise ABI5. As discussed above, the ABA signal is converted by a protein phosphorylation/dephosphorylation switching relay. Therefore, phosphorylation of the components of such a transcription repression complex might be a key step in the degradation process of transcription factors.
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F. RNA METABOLISM AND ABA RESPONSE
Recent reports have shown that defects in the components of RNA processing, including splicing, passive mRNA export from the nucleus to the cytoplasm and degradation, affect ABA response (Fedoroff, 2002; Hirayama and Shinozaki, 2007). For example, defects in each subunit of the cap-binding complex (CBC) causes ABA-hypersensitive phenotypes in Arabidopsis (Hugouvieux et al., 2001; Papp et al., 2004). One of these mutants, ABA hypersneitive1 (abh1), which is defective in CBC80, actually has abnormal mRNA levels of several genes (Bezerra et al., 2004; Hugouvieux et al., 2001; Verslues et al., 2006). Similarly, supersensitive to ABA and drought1 with an enhanced stress response phenotype turned out to have a defect in an Lsm protein, which is involved in RNA splicing and/or mRNA decapping (Xiong et al., 2001). DEAD-box RNA helicase is also involved in various RNA metabolic processes (Linder and Owttrim, 2009). Analysis of the Arabidopsis mutant low expression of osmotically responsive genes4 showed that a DEAD-box RNA helicase gene is required for cold stress and ABA responses (Gong et al., 2005). Some DEAD-box RNA helicase genes are upregulated under stress conditions and their disruptant mutations weakly affect the stress response (Kant et al., 2007; Kim et al., 2008). A link also exists between miRNA-mediated gene regulation and ABA response. HYPONASTIC LEAVES1 (HYL1), which is involved in miRNA processing, is required for several phenomena, including the ABA response (Han et al., 2004; Lu and Fedoroff, 2000). In addition, the RNA degradation process also appears to be involved in the ABA response. An impaired function of polyA-specific ribonuclease (PARN) ABA HYPERSENSITIVE GERMINATION2 (AHG2)/AtPARN, which is one of three eukaryotic deadenylases involved in the first step of the mRNA degradation process, causes an abnormal ABA response (Nishimura et al., 2005). A defect in a component for nonsense-mediated mRNA decay LOW BETA-AMYLASE1 (LBA1)/UPF1 affects ABA and sugar responses (Yoine et al., 2006). These observations indicate that the ABA response is tightly linked with the regulatory systems for mRNA processing. With regard to RNA, one of the most important discoveries in the past decade is the regulatory network of small RNAs, such as microRNA (miRNA) and small interfering RNA. Small RNAs play pivotal roles in various biological phenomena of plants by regulating target mRNA stability, translation and the epigenetic status of genes. ABA is affected by the small RNA regulatory network. The hyl1 mutant, which is defective in a miRNA processing factor, shows abnormal responses to various hormones, including ABA Lu and Fedoroff (2000). Moreover, malfunction of SERRATE,
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a Zn-finger binding protein and ABH1 (a subunit of the cap-binding complex) plays important roles in miRNA processing with HYL1, resulting in abnormal responses to ABA (Laubinger et al., 2008). Consistent with these findings, application of ABA changes the level of some miRNAs (Reyes and Chua, 2007; Sunkar and Zhu, 2004). miR159 is upregulated by ABA through the ABA-related transcription factors ABI3 and ABI5, and this miRNA inhibits the expression of MYB genes that positively regulate the ABA response, constituting a negative feedback loop regulating germination (Reyes and Chua, 2007). Abiotic stresses also change the composition of the miRNA network (Liu et al., 2008; Sunkar and Zhu, 2004). Therefore, ABA might regulate abiotic stress responses through reorganisation of the miRNA network. As mentioned above, small RNAs are implicated in epigenetic regulation and deeply involved in the developmental regulation of organisms. ABA was shown to take part in phase transitions, such as flowering. Recently, a study on post-translational gene silencing (PTGS) in rice showed that ABA regulates PTGS by modulating the expression of OsRDR6, which encodes an RNA-dependent RNA polymerase (Yang et al., 2008). These findings might be the tip of the iceberg. More studies on the relation between ABA action and the small RNA network will open a new avenue on ABA function in plant life. RNA-binding proteins were implicated in various phenomena, including developmental regulation and stress response (Lorkovic, 2009). Many types of RNA-binding proteins exist, categorised by their RNA-binding motifs and overall structures. However, their function cannot be easily deduced from their structures. They are thought to be involved in all RNA processes. The expression levels of genes for several Arabidopsis glycine-rich RNAbinding proteins fluctuate under environmental stress conditions and ABA treatment (Kim et al., 2005; Kwak et al., 2005). Recently, the alternative splicing pattern of ABI3 transcripts was shown to be regulated by a RNAbinding protein that interacts with a splicing factor (Sugliani et al., 2010). Some, but not all, of the stress up- or downregulated RNA-binding protein genes were shown to affect stress responses when overexpressed or malfunctioning (Kim et al., 2007). UBP1-associated RNA-binding proteins, UBA1 and UBA2 of Arabidopsis, were shown to regulate mRNA stability (Lambermon et al., 2002). Notably, AKIP1, the UBA2 ortholog of Vicia faba, interacts with a SnRK2 kinase, AAPK. ABA treatment activates the RNA-binding activity of AKIP1 and induces its relocation into nuclear speckles (Li et al., 2002), presumably through phosphorylation by AAPK. Similar observations were reported for an Arabidopsis UBA2 (Riera et al., 2006). Although the role of nuclear speckles is unclear, they appear to function as storage areas for transcription or splicing factors (Lamond and
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Spector, 2003). Thus, ABA presumably induces dynamic changes in nuclear events not only in gene activation but also in the splicing mode by changing the composition of splicing factors (Tillemans et al., 2006). A recent highly sensitive transcriptome analysis indicated that ABA treatment modulates the expression of the more abundant genes (Nemhauser et al., 2006). To exert such a dramatic change in gene expression, the ABA response might require the fine regulation of mRNA processing. Therefore, any malfunction in gene expression processes and posttranscriptional regulation might affect the execution of the ABA response. Related to this assumption, note that protein degradation events, especially those related to transcriptional factors, are also deeply involved in the ABA response (Dreher and Callis, 2007; Smalle and Vierstra, 2004). Such sophisticated systems might be required to regulate gene products to achieve fine regulation of the ABA response. Much evidence suggests that alternative splicing regulates gene expression and the function of gene products in responding to various stimuli in eukaryotes. Environmental stresses affect the splicing pattern in Arabidopsis (Iida et al., 2004; Zhang et al., 2010). Therefore, stress stimuli may affect RNA processing and metabolism to cope with stress by rapidly and dramatically changing gene expression profiles. As a stress regulator, ABA should play important roles in the regulatory process.
V. EMERGENCE OF THE CORE SIGNALLING PATHWAY As described in Section IV.A, PYR/PYL/RCAR proteins were identified as major ABA receptors in 2009, providing a major breakthrough in establishing the ‘‘core component system’’ in ABA signalling. In the first step, PYR/ PYL/RCARs negatively regulate group A PP2Cs, which are also well-known global negative regulators in ABA signalling, suggesting that PYR/PYL/ RCARs and PP2Cs form a double-negative regulation system (Ma et al., 2009; Park et al., 2009). PYR/PYL/RCARs directly interact with PP2Cs in an ABA-dependent manner, and their interactions were firmly confirmed by several structural analyses. This means that PP2Cs dephosphorylate downstream factors, for example, some positive regulators, in the absence of ABA. In the same year, another breakthrough was made by two research groups. Umezawa et al. (2009) and Vlad et al. (2009) demonstrated that group A PP2Cs negatively regulate a group of protein kinases, subclass III SnRK2s. As described in Section IV.C, subclass III SnRK2s were characterised as being major positive regulators of global ABA responses. They are strongly and rapidly activated by ABA in association with internal phosphorylation
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(Boudsocq et al., 2007; Kobayashi et al., 2004). LC–MS/MS analysis identified multiple residues in the kinase activation loop involving Ser175 in SRK2E/OST1 as ABA-responsive phosphorylation sites (Umezawa et al., 2009; Vlad et al., 2010), and the same sites were dephosphorylated by group A PP2Cs (Umezawa et al., 2009). The core pathway of PYR/PYL/RCAR–PP2C–SnRK2 was rapidly established as shown in Fig. 3 (Park et al., 2009; Umezawa et al., 2009). In the absence of ABA, group A PP2Cs dephosphorylate some specific residues in the activation loop of SnRK2. In other words, PP2Cs keep SnRK2s in the inactive state by direct dephosphorylation. When the ABA level is induced by environmental stresses or developmental cues, ABA-bound PYR/PYL/ RCARs inhibit PP2C activity, resulting in SnRK2 release from PP2C-dependent negative regulation. Further, the PYR1–ABI1–SnRK2 signalling system was successfully reconstituted in vitro. (Fujii et al., 2009; Umezawa et al., 2009). Moreover, Umezawa et al. (2009) found that an abi1-1 mutant PP2C had strong phosphatase activity for SnRK2s but almost no activity for artificial substrates, suggesting that the abi1-1 mutation affects the interaction with ABA receptors and PP2C activity for artificial substrates but does not affect the activity for natural substrates. These results provided a clear answer to the long-standing question of how the abi1-1 mutation causes a dominant ABA-insensitive phenotype (Umezawa et al., 2009; Vlad et al., 2009). Notably, a co-immunoprecipitation assay suggested that PP2Cs constantly interact with SnRK2 in vivo (Nishimura et al., 2010; Umezawa et al., 2009). Conversely, another possibility is that PP2Cs dissociate from SnRK2s in response to ABA, as shown in a yeast three-hybrid analysis (Fujii et al., 2009). The activation mechanism of SnRK2s is still open to question, but two possibilities were already proposed. One is autophosphorylation (Belin et al., 2006), but in vivo evidence is lacking. The other is that some unknown upstream kinases activate SnRK2s as suggested by a pharmacological assay (Boudsocq et al., 2007). To date, several downstream factors of SnRK2s have been reported. One is a group of transcription factors, the bZIP family, which is well known as a major regulator of ABA-responsive gene expression (see Section IV.D). Previous reports demonstrated that subclass III SnRK2s directly phosphorylate and activate bZIP transcription factors, such as ABI5 or AREB1/ABF2 in Arabidopsis, TRAB1 in rice and TaABF in wheat (Furihata et al., 2006; Johnson et al., 2002; Kobayashi et al., 2005). Therefore, ABA-responsive gene expression is regulated by four components, PYR/PYL/RCARs, PP2Cs, SnRK2s and bZIPs. These components seem to function mainly in the nucleus.
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A Receptors
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Fig. 3. The core signalling pathway of ABA responses in plants. (A) A schematic view of the core ABA signalling pathway. An upper scheme shows a silent state of this pathway. Under a normal condition (in the absence of ABA), clade A PP2C inactivates SnRK2 by direct dephosphorylation to shut off ABA signals. A lower scheme shows an active state of this pathway. ABA is induced by various environmental or developmental cues, and it binds to PYR/PYL/RCAR receptors. ABA-bound PYR/ PYL/RCAR inhibits clade A PP2C by physical interaction, and SnRK2 is released from PP2C-dependent negative regulation. Then downstream factors, including transcription factors or ion channels, etc., are phosphorylated by SnRK2 to turn on ABA signals. (B) Given differences in gene expression pattern, ligand specificity, protein-interaction specificity and substrate specificity, combinations of 14 cytosolic ABA receptors, 6 (or 9) clade A PP2Cs and three ABA-activated SnRK2s offer a wide variety of signalling cascade, which are supposed to cover a broad spectrum of ABA signals produced in different tissues/organs under various environmental stress conditions. Circles indicate signalling factors (receptors, PP2Cs and SnRK2s). They are distributed diversely in a three-dimensional coordinate according to their characters (such as expression pattern, ligand affinity or selectivity, etc.). State of a signal from ABA is shown as a yellow arrow.
Alternatively, subclass III SnRK2s can access membrane proteins. SLAC1 is a slow anion channel with a critical role in guard cells (Negi et al., 2008; Vahisalu et al., 2008), and its activity is regulated by SnRK2s (Geiger et al., 2009; Lee et al., 2009). Other membrane proteins, such as a potassium channel, KAT1, or an NADPH oxidase, AtrbohF, were suggested to be targets of SnRK2s (Sato et al., 2009; Sirichandra et al., 2009). Notably, these SnRK2 targets are also phosphorylated by CDPKs (Geiger et al.,
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2010; Ogasawara et al., 2008; Sato et al., 2010), suggesting that multiple pathways could be involved in ABA signalling. Further analysis will be required to identify other SnRK2 targets to fully understand the signal transduction network in ABA responses. In addition, studies have suggested that PP2C not only regulates SnRK2 but also other substrates, for example, transcription factors, chromatin remodelling factors, and ion channels (see Section IV.B; Hirayama and Shinozaki, 2007,2010). Our knowledge will be expanded when the downstream network of the core signalling pathway is clarified.
VI. PERSPECTIVES As reviewed in this chapter, recent progress has led to the understanding of the regulatory networks in ABA responses in plants, for example, ABA biosynthesis and catabolism, intercellular ABA transport and intracellular ABA signalling. Although our knowledge was recently greatly expanded, many new questions are emerging. For example, we now have a complete set of ABA biosynthetic and catabolic enzymes (see Section II), but their upstream regulators are still unclear. Likewise, two ABC transporters were characterised as ABA transporters as described in Section III, but no information exists about the regulation of their activity. Whether other ABC transporters are also involved in ABA transport poses another interesting question. Establishment of the core component system of intracellular ABA signalling was certainly an important milestone in 2009. However, it leads to many questions about ABA signalling (Fig. 3): How is the core component system integrated into other ABA signalling pathways? What combinations of the receptor complex are functional in planta? What other substrates or downstream factors of, for example, SnRK2 or PP2C exist? Further studies are needed to answer these questions. Recent accumulation of genomic sequences in various plants, including bryophytes or lycophytes, tells us the evolutionary process of ABA signalling, that is, the core regulatory pathway evolved around bryophytes (Umezawa et al., 2010). This suggests that the development of ABA signalling could be correlated with the evolution of land plants. Comparative analysis between bryophytes and angiosperms will be important, because such studies can highlight an ancient and primary ABA signal transduction mechanisms. Since the regulatory networks of ABA responses must be quite complex, understanding ABA responses at the systems level will be important. Therefore, an approach based on systems biology will be necessary to elucidate the whole picture of ABA responses in plants.
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Molecular Mechanisms of Abscisic Acid Action in Plants and Its Potential Applications to Human Health
ARCHANA JOSHI-SAHA, CHRISTIANE VALON AND JEFFREY LEUNG1
Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, UPR 2355, Gif-sur-Yvette 91198 Cedex, France
I. II. III. IV. V.
VI. VII.
VIII. IX. X.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ABA as a Positive and Negative Regulator in Plant Growth. . . . . . . . . . . . . ABA Circulation in the Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Transport in Guard Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The ABA Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. A Family of Soluble Receptors PYR/PYL/RCAR..................... B. ABA Receptor in the Chloroplast Membrane .......................... C. Plasma Membrane-Localised ABA Receptors: The Link to G Proteins ................................................................. The Soluble PYR Signalling Complex is Part of a Short Phospho-Relay Cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ABA Controls Rapid Drought Adaptive Responses by Modification of Selective Transport Across the Plasma Membrane. . . . . . A. The Potassium Channels ................................................... B. The Anion Channels ........................................................ C. The P-Type Proton Pumps................................................. D. The Control of Downstream Targets by the Core ABA Signalling Complex in the Guard Cell ................................... Targets of SnRK2s in medium-term aba responses-gene expression and chromatin modelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetics in ABA Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitogen-Activated Protein Kinases in ABA Signalling . . . . . . . . . . . . . . . . . .
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Corresponding author: E-mail:
[email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00007-2
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XI. Root Growth in Response to Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. ABA is Conserved in Evolution and has Potential to Improve Human Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT Drought tolerance actually embodies several protective mechanisms deployed by plants commensurate with the stress severity and duration. Against mild drought, one of the most rapid defensive measures is the closing of the stomatal pore, caused by shrinking of the flanking guard cells, in order to reduce transpiration that accounts for 90% of the water loss from plants. In contrast, the widening of the pore caused by expansion and bowing out of the guard cells is stimulated by light, permitting CO2 entry for carbon fixation. It is clear, therefore, that the two most decisive factors in plant growth—photosynthesis and water consumption—are directly influenced by guard cell regulation. Drought induces the synthesis of the ‘‘stress’’ hormone abscisic acid (ABA). Work within the past two decades has outlined the signalling pathway consisting of phospho-relay and ion transport across membranes that link ABA reception to stomatal closure. The threshold of the stomatal response to stress may also be set by more long-term processes that include gene expression and epigenetic regulation (‘‘stress memory’’), implying a feedback integration between rapid and slower protective mechanisms. In this chapter, some of the most recent, insightful, and exciting findings in the signalling network that orchestrate these ABA-dependent adaptive processes will be related. We will also extend our discussion in applying ABA research—not on the more obvious agricultural benefits—but as a novel and potent modulator of the immune system in human.
I. INTRODUCTION The seventeenth century psalmist George Herbert mused in his poem, ‘‘Man’’, after being inspired by his perception of congruity between man and his surroundings, that ‘‘Herbes gladly cure our flesh; because that they finde their acquaintance there’’. Indeed, biologists typing in a search for a string of codons from Arabidopsis will find hits in the mammals, flies, sculpin, fungus, and much more. With high-throughput technologies getting speedier by the day, and centralised databases easily accessible, large-scale discoveries are routinely being reduced to the effortless click of a button. We are already taking for granted that hundreds of thousands of genes in the plant genome have homologs in many other species, including humans (Jones et al., 2008). Perhaps with some reluctance, advances in science have also forced us to grapple with the sobering reality that, despite being the most evolutionary complex organisms, we are not even endowed with much more proteincoding capacity that the ‘‘lowly’’ Arabidopsis, whose life is surely less eventful
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than ours. The congruity in all life forms would have pleased Darwin, but these modern day insights seem to have already been an old truth for poets. The interconnectivity among all life forms is no less exemplified by adaptative responses to stress, an innate ability well known in both plants and animals. In the past years, many independent lines of anecdotal and experimental evidence converge on the startling conclusion that the ‘‘phytohormone’’ abscisic acid (ABA) is in fact a conserved signalling molecule that triggers protective functions in both the plant and animal kingdoms. We will return to this fascinating point later in the chapter. Land plants are subjected to continuously fluctuating microclimatic changes, particularly humidity, temperature and light quality. Being sedentary, they have evolved many elaborate mechanisms with built-in ‘‘redundancy’’ to optimise productivity in spite of adverse conditions. These adaptive responses have been operationally categorised as short term (e.g., stomatal closure in seconds to minutes), medium term (e.g., reprogramming of transcriptome in minutes to hours) and long term (morphological changes following days or weeks of stress, most notably, the root structure). A wealth of knowledge on the mechanistic nature of drought adaptation has already been distilled from imposed conditions in the laboratory, but still, all such controlled environments entail inadvertent biases to facilitate clear interpretations of the outcome. In nature, the climatic changes affect plant growth in more complicated ways. For example, temperature affects photosynthesis, but plants have considerable adaptive capacities enabling them to grow even at high temperatures providing that adequate water is available. Beyond a certain temperature, vapour pressure deficits of the air will be severe enough to heighten the transpiration rate from plant canopies, triggering stomatal closing and thereby suppressing growth. Increasing atmospheric CO2 (330–360 ppm) can also increase photosynthetic rates; but high CO2 will also stimulate stomata to close. It is not surprising, therefore, why most of the genes, when knocked out, do not necessarily lead to obvious visible phenotypes in the simplified and highly controlled laboratory conditions. Because of these apparent experimental limits, there has been a resurgent interest in exploring natural polymorphisms in plants, including Arabidopsis, to identify allelic variations across entire genomes that confer particular selective (dis)advantages (trade-offs). These complementary population approaches will enlighten us on quantitative traits, the nature of the genes across species or kingdoms that eventually fashion the developmental patterns of these plants for those environmental niches (‘‘Evolutionary Developmental Biology’’, or ‘‘evo-devo’’). There is a solid experimental proof that ABA is critical, in a dose-dependent manner (the underlying reason for which is still scarcely explored), for both normal plant development and stress
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adaptative capacity, especially to drought. The subject of how the ABA signal is transduced is a passionate scientific enquiry in itself, and with its obvious added value for enhancing agriculture for societal benefits, makes this topic truly vast as well as topical in view of climate threat, all of which cannot be dealt within the scope of a single chapter. At the time of writing, there are two recent and comprehensive reviews that provide excellent background on the initial steps of ABA signalling (Cutler et al., 2010; Raghaendra et al., 2010). This chapter will therefore focus on the more global advances made in the molecular aspects of the so-called short-, medium- and long-term adaptive responses in plants, with most of the mechanistic knowledge of the role of ABA derived from Arabidopsis thaliana. We hope that this chapter will be useful as a summary of a body of important work on molecular mechanisms of ABA signalling, but more so, a seemingly odd observation here and there would also ignite some latent ideas for further exploration, to perhaps even beyond plants. This is because an added intrigue that we put forth here concerns the origin of ABA, which seems ancient, as it also exists in model animals spanning the evolutionary scale from the most primitive to the most advanced—including humans. There is also tantalising clinical evidence that ABA is bioactive against certain ailments, most notably, type II diabetes, and could act as a powerful modulator of the immune system. This is ‘‘evo-devo’’ on a grand scale.
II. ABA AS A POSITIVE AND NEGATIVE REGULATOR IN PLANT GROWTH ABA is often viewed as a negative regulator because reduced growth under stress conditions is correlated with increased cellular ABA content, and that exogenously applied ABA (usually in micromolar) arrests seed germination and seedling growth. Several observations clearly indicate that this view is too simplistic, and that low levels of ABA can promote vegetative growth. ABA, while low in concentrations, can still be detected in extracts from aerial parts of wild-type plants grown even in well-watered conditions (Merlot et al., 2002). The basal level of ABA does not seem to be in passive storage, but studies carried out in tomato and Arabidopsis suggest that it is required to stem ethylene production (LeNoble et al., 2004; Sharp et al., 2000). Indirect in vivo imaging of ABA using reporter genes driven by ABA-sensitive promoters could also detect above background levels of ABA in some unstressed tissues, for example, the guard cells (Christmann et al., 2005). The concentration-dependent nature of ABA action is evident in root growth. Root elongation in Arabidopsis can in fact be stimulated significantly
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by exogenous ABA at 0.1 mM, while the hormone delivered at above 1.0 mM becomes inhibitory (Ghassemian et al., 2000). It seems that even in severe water stress, ABA is required to sustain growth in the root apex (3 mm-region) of maize, but inhibitory to cells (3–7 mm) proximal to the apex (Sharp et al., 2004), suggesting tissue-specific or developmental stage-dependent sensitivity to ABA. Indeed, suppressing ABA production in mutants or in transgenic plants was shown to result in developmental defects such as altered organisation of the mesophyll and stomatal morphogenesis (Barrero et al., 2005; Wigger et al., 2002). Thus ABA can act as both a promoter and an inhibitor of growth and development depending on its concentration and site of accumulation.
III. ABA CIRCULATION IN THE PLANT ABA is predominantly synthesised in bundle sheath cells (Marion-Poll and Leung, 2006) and then rerouted from there to all other tissues. These being the primary site of ABA synthesis is coincidental with expression of the biosynthetic genes AtNCED3, AtABA2 and AAO3 (Cheng et al., 2002; Koiwai et al., 2004; Tan et al., 2003) and the indirect detection of in vivo pools of ABA in these cells (Christmann et al., 2005; Wachter et al., 2003). The mechanisms by which ABA is rerouted from cell-to-cell is not known with precision, but it might circulate as an inactive glucose ester conjugate. The chemical properties of ABA glucose ester are well suited for its longdistance translocation in the xylem as it has low biomembrane permeability (Jang and Hartung, 2007). The ABA conjugate is stored in vacuoles or apoplastic space (Dietz et al., 2000), which is then released into the active form by apoplastic and endoplasmic reticulum b-glucosidases (Lee et al., 2006) in response to dehydration.
IV. MEMBRANE TRANSPORT IN GUARD CELLS How does ABA enter and exit cells? Previous pharmacological assays have hinted that ATP-binding cassette (ABC) transporters might have important roles in guard cell functions linked to environmental changes or hormone signalling (Leonhardt et al., 1997,1999). The ABC transporters are constituents of one of the largest gene families and are present in all taxa and assume diverse cellular functions, including detoxification of organic toxins, heavy metals and resistance against pathogens. More recent and direct evidence has now revealed the identities of some of these members in ABA transport,
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although there might still be others. In its acidic form, ABA can diffuse passively across biological membranes. This, of course, had begged the obvious question of whether ABA required at all active and vectorial transport, or by simple random diffusion coupled to other types of regulation such as controlled modification and/or cleavage of the hormone. Indeed, one essential component that facilitates active ABA expulsion into intercellular transport has now been identified by reverse genetic screens of mutants altered in ABA sensitivity (Kuromori et al., 2010). The gene corresponds to AtABCG25 (also known as AtWBC26), predicted to encode a so-called halfsize ABC transporter. One structural hallmark of ABC transporters is the presence of two conserved Walker A and B motifs within the nucleotidebinding-folds (NBF) which drive transport by ATP hydrolysis. Another diagnostic feature is the transmembrane domains (TMDs), each with 6–10 membrane-spanning a-helices with divergent sequences that determine substrate specificities. A full-size ABC transporter requires the concerted action of two NBFs and two TMDs, typically arranged in one contiguous polypeptide chain. AtABCG25 has a single copy of each TMD and NBF motifs, and thus qualified as a half-size ABC transporter. Insertion allelic mutants of abcg25 all display accentuated sensitivity to ABA. The protein is restricted to the plasma membrane, and it is expressed in cells close to the vascular tissues, presumably poised to export ABA into the intercellular space. Isotopically labelled 3H-ABA efflux activity can be detected in vesicles derived from Sf9 insect cell line expressing ABCG25. Overexpression of this gene in Arabidopsis conferred ABA resistance on seed germination, which is consistent with ABCG25 being an exporter or efflux factor of ABA. Stomatal response to rapid drought onset triggered by detaching leaves from these transgenic plants was found to be slower than that in the wild type, presumably because ABA was efficiently expelled from the guard cells (Kuromori et al., 2010), limiting their access to cytosolic targets that include receptors (see below). Simultaneously, a full-size ABC transporter functioning as an ABA importer was also identified. This latter discovery is particularly relevant in the context of stress response, whereby the increase in apoplastic pH would hinder the passive diffusion of the nonprotonated form of ABA across the plasma membrane; this situation logically necessitates active transport mechanisms to facilitate its delivery to the cytosolic receptors as primary targets. The plasma membrane-resident PDR12/AtABCG40, originally proposed to be a pump excluding lead or lead-containing toxic compounds (Lee et al., 2005), turned out to import ABA (Kang et al., 2010). 3H-ABA uptake showed time-dependent enhancement in the yeast mutant YMM12 or tobacco BY-2 cells expressing the AtABCG40. Uptake of 3H-ABA was sensitive to competition by the biologically active (S)-ABA, but not (R)-ABA, and also
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to inhibitors of ABC transporters such as glibenclamide, verapamil and vanadate. Conversely, Arabidopsis mutants disrupted in AtABCG40 (abcg40-1 and abcg40-2) showed reduced ABA uptake. It is noteworthy that, as compared to the wild type, the reduction of ABA uptake was greater for the knock-out mutants at higher pH, in which passive ABAH diffusion would be limited. The mutants also showed delayed expression of several ABA-inducible marker genes. Not surprisingly, these mutants are impaired in drought tolerance, and in a number of elementary developmental processes, including seed germination, that are known to be influenced by ABA. AtABCB14 (also known as AtPGP14 or AtMDR12) is expressed in several tissues as visualised by promoter fusion using the reporter uidA gene encoding b-glucuronidase. The stronger expression signals come from the guard cells (Lee et al., 2008). This transporter has a role in malate import from the apoplastic space into guard cells in response to CO2 (800 ppm), neither to high Ca2þ, ABA, nor to transition from light to darkness (Lee et al., 2008). Mutants disrupted for the AtABCB14 gene show significantly more reduced stomatal aperture than that of the wild-type guard cells exposed to malate and high CO2. Conversely, overexpression of the AtABCB14 transgene blocks stomatal closure to both of the above stimuli (Lee et al., 2008). The supposition that AtABCB14 is a malate importer comes from the fact that when the bathing solution contained only malate as the anion, stomatal opening was observed to be much faster in the AtABCB14 overexpressing lines than for the two mutants. This is consistent with malate acting as an osmoticum in guard cells to maintain stomatal opening. Another line of compelling evidence is that expression of AtABCB14 can restore growth of the Escherichia coli mutant defective in dicarboxylate transport on malate-containing medium. The combined results would thus link this particular ABC transporter to malate uptake in different cell types in response specifically to high CO2. However, in guard cells, high CO2 privileges stomatal closure by, at least in part, stimulating anions extrusion (and a decrease in osmotic pressure; Negi et al., 2008), the malate uptake by AtABCB14 may thus reflect a recycling mechanism. There are additional transporters whose mutations also lead to impaired ABA responses, but the underlying mechanisms are less obvious. Only some of the recent and more insightful studies are summarised here. Anion transporters that confer resistance to aluminium were first reported in wheat. These transporters were presumed to share a generalised function in excreting organic anions such as citrate or malate into the soil to chelate aluminium, thereby neutralising its toxicity. However, members of this transporter family may have functions more diverse than originally suspected. In Arabidopsis, the 13 homologous members of this gene family have been targets of
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physiological as well as molecular genetic studies. It seems that at least AtAML12 (At4g17970) is an anion transporter permeable to nitrate and chloride, and the corresponding mutant is impaired in guard cell regulation (Sasaki et al., 2010). The Arabidopsis mutant Atmrp5 is impaired in Ca2þ signalling and partial ABA-induced anion current activity as part of its pleiotropic phenotype (Klein et al., 2003; Suh et al., 2007). AtMRP5 (AtABCC5) turned out to transport inositol hexakisphosphate (IP6), a ubiquitous signalling molecule and the principle storage form of phosphorus in many plant species. Expression of the wild-type transgene restores the mutant’s sensitivity to ABAmediated inhibition of stomatal opening (Nagy et al., 2009).
V. THE ABA RECEPTORS A. A FAMILY OF SOLUBLE RECEPTORS PYR/PYL/RCAR
The passionate hunt for the ABA receptor(s) was ignited 25 years ago that began with three ABA-binding proteins detected biochemically in the plasmalemma of Vicia guard cells (Hornberg and Weiler, 1984). Subsequently, several other proteins with affinity to ABA were also reported, but either their molecular characteristics were not known, or they do not seem to work in the conventional pathways outlined by genetics and physiology. Owing to its accessibility to experimental manipulations, and most importantly, a simple physiological output (stomatal closure or opening), the guard cell dominated as the hunting ground over the next 15 years for ABA perception sites, whose presence were detected in the mid-1990s by various physiological means to be on both the cell surface as well as the ‘‘inside’’ of the cell (Leung and Giraudat, 1998). In 2009, the hunt culminated in the two independent molecular studies that converged simultaneously on the same candidates, which turned out to be the soluble ABA receptors. They are encoded by a gene family of 14 members sharing homologies with the steroidogenic acute regulatory related lipid transfer (START) proteins (Ma et al., 2009; Park et al., 2009; Fig. 1). The study headed by S. Cutler in the United States successfully exploited chemical genetics by selecting Arabidopsis mutants that can germinate on pyrabactin, a synthetic growth inhibitor of seed germination that can act as a selective agonist of ABA (Park et al., 2009). Pyrabactin and ABA trigger highly correlated transcriptional responses in seeds (although only moderately so in seedlings), suggesting that these two compounds share common targets. Even so, the mechanisms of pyrabactin and ABA in growth inhibition also seemed to implicate distinct
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Fig. 1. The core complexes of the two better established ABA signalling pathways. The PYR/PYL/RCAR receptor is cytosolic/nuclear, whereas the ABAR is in the chloroplast. In the ground state (in the absence of ABA), PP2Cs in the clade A (ABI1, ABI2, HAB1, PP2CA, etc.) restrain the SnRK2 kinase activities (OST1, SnRK2.2, SnRK2.3) by dephosphorylation. The binding of ABA to PYR induces a structural change in the latter, creating a binding surface that sequesters ABI1, allowing OST1 and other SnRK2 kinases to phosphorylate downstream targets, which include at least three major b-ZIP transcriptional activators, and in the guard cell, plasma membrane transporters that are either activated (SLAC1) or repressed (KAT1), contributing to an overall decrease in osmotic pressure to bias stomatal closure. SLAC1 can also be directly dephosphorylated (and inactivated) by PP2CA independent of OST1. The second parallel ABA signalling pathway is highly unusual in that it starts in the chloroplast with the receptor ABAR (same as GUN5), a large 120-kDa protein that spans the envelopes. The binding of ABA allows ABAR to recruit, by as yet unknown mechanisms, at least three transcriptional repressors WRKY from the nucleus, relieving the suppression of gene expression. The differential sensitivity of the anion channel and the proton pump to Ca2þ is likely due to the action of Ca2þ-sensitive kinases (CPK21 and PSK5, respectively).
properties, because the founding member of the pyrabactin-insensitive mutants, pyr1 (also named as rcar11 in the other study headed by Grill in Germany), was isolated based on its resistance to pyrabactin and yet it was still wild type in sensitivity to ABA. It was in combining triple or quadruple knock-outs of the PYR, and some of its homologs (PYL), reduction in ABA sensitivity was finally revealed (Park et al., 2009). Conversely, overexpressing one of the members, PYL9/RCAR1 (for Regulatory Component of ABA Receptor), enhanced ABA sensitivity in virtually all elementary criteria such as root elongation, stomatal closure and seed germination (Ma et al., 2009). This suggests that PYR family members assume overlapping functions in
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ABA perception and that chemical genetics can by-pass functional redundancy that can sometimes hinder traditional genetic screens. It remains a curious observation that despite the fact that ABA and pyrabactin share very little similarity in their chemical structure, they both bind to PYR. Results from yeast two-hybrid screens, done independently by the two laboratories cited above, revealed that PYR interacted with several PP2Cs, including ABI1, ABI2, HAB1, and other closely related homologs, in the presence of ABA or pyrabactin, but not inactive ABA analogues or other hormones. Further, mutations in either PYR1 (S152L and P88S), or in other close homologs of HAB1, such as ABI2 (abi2-1) (Ma et al., 2009; Park et al., 2009) and ABI1 (abi1-1) (Ma et al., 2009), disrupted PYR–PP2C interactions in yeast. Importantly, the interaction was recapitulated in plant cells when selected PYR and PP2C members were coexpressed by transfection into Nicotiana benthamiana epidermal cells (Park et al., 2009), in Arabidopsis protoplasts (Ma et al., 2009; Szostkiewicz et al., 2010) or by in vitro pulldown assays using components expressed in E. coli (Park et al., 2009). Moreover, the protein PYR9/RCAR1 interacted with the PP2C in the cytosol and nucleus, the same subcellular compartments at the RCAR1 alone (Ma et al., 2009). Isotopically labelled ABA directly binds to PYR/PYL/RCAR, as ascertained by 15N-labelled PYR1 and PYR1P88S in heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments, which probe chemical shifts of protein amide-NH bonds in response to ligands (Park et al., 2009). The addition of (S)-ABA in the nanomolar range, and in the presence of PYR/PYL/RCAR, inhibits efficiently PP2C activities (Ma et al., 2009; Park et al., 2009; Szostkiewicz et al., 2010). No inhibition was observed if the PYR/PYL/RCAR was disrupted physically (Ma et al., 2009) or by mutations (Park et al., 2009). Thus, PYR/PYL/RCAR negatively regulates particular PP2Cs in response to ABA, which defines an unprecedented mechanism for ligand-mediated regulation of PP2C activity. Isothermal titration calorimetry revealed binding of (S)-ABA to PYL9/RCAR1 and ABI2 with an apparent binding affinity (Kd) of 64 8 nM ABA and a single binding site. The analysis for the binding of (S)-ABA to PYL9/RCAR1 yielded lower energy changes and a higher apparent Kd of 0.66 0.08 nM ABA (Ma et al., 2009). The considerably lower Kd value of the heteromeric protein (PP2C–RCAR1)-ABA complex was argued to reflect a ligand-induced complex stabilisation, similar to FLS2 and BAK1 receptor complex stabilisation by flagellin (Chinchilla et al., 2007). Because both the ABA receptors (14 members) and PP2Cs (nine members in clade A) are encoded by multigenes, combinatorial interactions between these two components could promulgate subtly distinct downstream
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messages. Although the picture is far from complete, the different receptors that have been tested display variable affinities to ABA isomers (Park et al., 2009; Szostkiewicz et al., 2010). Further, the selectivity observed between receptor and members of the clade A PP2C could constitute a second layer of control to fine-tune the ABA signal input. For example, PYL5/RCAR8 can bind HAB1, ABI1, ABI2, but not AHG3 (Santiago et al., 2009). PYL8/ RCAR3 was also shown to repress ABI1 and ABI2 in vitro and to stimulate ABA signalling in protoplasts (Szostkiewicz et al., 2010). The efficiency of ABA-mediated phosphatase inhibition was higher with ABI1 than ABI2, and higher with RCAR3 than RCAR1. To put this into perspective, halfmaximal inhibition of RCAR3/ABI1 was observed at 23 nM ABA, whereas RCAR1/ABI2 revealed a more than fourfold higher IC50 value of 95 nM ABA. This finding reflects major differences in the heteromeric receptor complexes with respect to ABA-mediated inhibition. Although these studies using different combinations of the PYR/PYL/ RCAR and PP2C hinted that these two components may function as coreceptor complexes (Szostkiewicz et al., 2010), structural studies (using models PYR1/RCAR11, PYL1/RCAR12, PYL2/RCAR13) revealed that ABA is actually bound deep inside an occluded protein cavity, rather than at the interface between receptors and the PP2Cs (Miyazono et al., 2009; Nishimura et al., 2009; Yin et al., 2009). This indicates that the PYR/PYL/ RCAR are direct ABA receptors and signal transduction partners. On the mechanistic level, ABA receptors therefore function more like the gibberellin receptor GID1, rather than like the auxin-linked TIR1 and AUX/IAA coreceptor complex in which both components cradle the hormone. PP2Cs are monomeric enzymes, and those implicated in ABA signalling interact with PYR/PYL/RCAR in equal molar ratio (Leube et al., 1998; Ma et al., 2009; Yin et al., 2009); nevertheless, the receptors themselves can form dimers through interaction between the so-called CL2 loop (or the proline gate) from each of the subunits, even in the absence of ABA (Nishimura et al., 2009; Yin et al., 2009). PYR1 in the presence of nonsaturating concentrations of ABA yield an ABA-bound and an ABA-free subunit, related by a 1708 rotation around a pseudo twofold axis. The PYR1 is thus an unusual symmetric homodimer. Moreover, a specific leucine residue (Miyazono et al., 2009; Nishimura et al., 2009; Yin et al., 2009) from the ABA-free subunit reaches across the PYR1 dimer to block the remaining cavity access, thus sequestering the ABA. In excess ABA, when both subunits are bound by the ligand, the dimer assumes an exact twofold symmetry, consisting of a flattened biconcave disc that has been described to resemble a red blood cell (Nishimura et al., 2009). The CL2 loop also forms the lid, to which PP2C binding apparently favours lid closure and decreases the ABA
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off rate. This could also explain the observation of tighter binding of ABA to PYR in the presence of a compatible PP2C (Ma et al., 2009; Nishimura et al., 2009; Yin et al., 2009). It has also been speculated that PP2C docking onto the CL2 loop of the receptor–ABA complex might require the prior dissociation of the dimer into monomers (Yin et al., 2009). These structural studies also clarified the nature of the dominant mutations abi1-1 and abi2-1 which were the first to establish the important role of these PP2C in ABA signalling (Koornneef et al., 1984; Leung et al., 1994, 1997; Meyer et al., 1994; Rodriguez et al., 1998). The corresponding mutant proteins are genetically defined as suppressors of ABA signalling, exactly the same role as the wild-type protein, suggesting that these dominant mutations might have rendered the phosphatases constitutively active (without them necessarily being hyperactive on a per enzyme basis). The interaction of PYL1/RCAR12 with ABI1 is mediated by the CL loops (especially the CL2 mentioned above and helix a2) through a network of van der Waals contacts, water-mediated contacts, and a few direct hydrogen bonds. Ser112 of PYL1/RCAR12 donates one hydrogen bond to Glu412 of ABI1 and accepts one from the amide of Gly180 which makes a hydrogen bond to the backbone carbonyl oxygen of Ser112. The mutation abi1-1, which converts Gly180 to Asp, or the equivalent Gly168 to Asp in abi2-1 would disrupt this amide bond with Ser112 of PYL, and the Asp substitution may cause additional steric hindrance into the interface between PYL1 and ABI1 (Miyazono et al., 2009; Yin et al., 2009). Hence, these dominant mutations impart immunity to these PP2C from recruitment by the ABA-receptor complex (Ma et al., 2009; Park et al., 2009). The Trp300 of ABI1 (presumably equivalent amino acids in the other close homologs) is accorded particular importance, as it is the only amino acid in simultaneous contact with a hydrophobic pocket of the receptor and the ABA molecule via a water molecule (Miyazono et al., 2009; Yin et al., 2009). B. ABA RECEPTOR IN THE CHLOROPLAST MEMBRANE
Another potential ABA receptor is ABAR, corresponding to the H subunit of the magnesium-chelatase (CHLH; Shen et al., 2006). The identity of this being a potential ABA receptor was based on homology with a 42-kDa protein (or a fragment of a larger protein) that has been affinity purified from the abaxial epidermis of Vicia leaves earlier by the same group (Zhang et al., 2002). Scatchard plot analysis of this protein showed an equilibrium dissociation constant of 21 nM and stereospecificity in that ()ABA and trans-ABA were incapable of displacing 3H-() ABA bound to the protein and () ABA was less effective than (þ) ABA in the competition. As a
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candidate ABA receptor, the Arabidopsis homolog ABAR is surprising in that it is a chloroplast membrane-localised protein of over 120 kDa, and moreover, it was identified previously as genome uncoupled (or gun)5, that disrupted a component of plastid-to-nucleus communication to coordinate expression of both nuclear- and chloroplast-localised genes encoding photosynthesis-related proteins (Susek et al., 1993). The wild-type protein GUN5 is one of three subunits of the Mg2þ-chelatase required for Mg2þ-protoporphyrin IX synthesis and has been postulated to be a monitor of porphyrin levels by binding excess proto and/or Mg2þProto, and sends a signal to the nucleus via a hypothetical downstream effector. This function is separable from that of ABA signalling. It has been proposed that, after direct ABA reception, GUN5/CHLH/ABAR binds via its C-terminal to at least three WRKY proteins that are negative regulators of ABA signalling in seed germination and postgermination growth (Shang et al., 2010; Fig. 1). Overexpression and RNA-interference transgenic Arabidopsis lines led to altered ABA responses in seed germination, postgermination growth, stomatal movement and expression of certain ABA-regulated genes (Shen et al., 2006). C. PLASMA MEMBRANE-LOCALISED ABA RECEPTORS: THE LINK TO G PROTEINS
Heterotrimeric G proteins are fundamental in transmembrane signalling by relaying a large variety of receptors to channels, enzymes and myriads of effector molecules (Wettschureck and Offermanns, 2005). In the mammalian genomes, multiple subforms of G proteins together with receptors, effectors and teams of regulatory proteins make up the components of a highly versatile signal transduction network (Wettschureck and Offermanns, 2005). G protein-mediated signalling is employed by virtually all cells in the organism and is centrally involved in diverse physiological functions, notably sensory perception, synaptic transmission, hormone release and cell contraction and mobility, just to name a few. The key members are the heterotrimeric G proteins—comprising the Ga, Gb, Gg subunits—as well as the G protein-coupled receptors (GCPRs). The Ga subunit, with both GTP-binding and GTPase activity, acts as a bimodal switch, typically a GDP-bound ‘‘off’’ state and a GTP-bound ‘‘on’’ state. The GPCRs classically act as guanine nucleotide exchange factors (GEF), and a change in GPCR conformation upon signal perception triggers an exchange of GDP for GTP at the Ga subunit. This triggers the dissociation of Ga from the Gbg dimer, both of which can interact with an array of downstream signalling elements. Note, however, plant genomes are notorious for their seemingly poor endowment of G proteins, at least based on the criterion of overt sequence homologies
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with their counterparts in other species. A. thaliana, for example, has a rather modest repertoire of one Regulator of G protein Signalling (RGS), one prototypical Ga (GPA1), one Gb (AGB1), two Gg (AGG1 and AGG2) subunits (Jones and Assmann, 2004). Disruption of GPA1 was previously shown to disturb normal ABA functions (Pandey and Assmann, 2004), a first hint suggesting the involvement of GCPRs. In silico analysis of the Arabidopsis has, however, recently revealed two plasma membrane-localised proteins with 45–68% sequence identity to the human protein GPR89, which was initially annotated as an orphan GCPR (Pandey et al., 2009), but functionally it is a pH-sensitive chloride channel resident in the Golgi (Maeda et al., 2008). These Arabidopsis homologs, which are conserved across phyla, are named GTG1 and GTG2 (GPCRtype G protein), and both have been shown to display GTPase activity in vitro. As compared to the wild type (accession Wassilewskija) or each of the single gtg mutants, the double mutant is hyposensitive to ABA at seed germination, seedling growth and stomatal closure. The two GTG proteins interact with GPA1, the sole canonical Ga subunit (see above), in the yeast split-ubiquitin two-hybrid system (SUS) designed to assess membrane proteins, and they coimmunoprecipitate from plant extracts. GPA1 binding stimulated GTP-binding activity of the GTGs but remarkably the latter’s GTPase activities are inhibited. The purified recombinant GTGs show saturable 3H-ABA binding with apparent Kd of between 30 and 40 nM. The dissociation constant could be even halved, considering that binding was completed by ()ABA, but not the biologically inactive ()ABA, and ABA binding was improved by GDP (Pandey et al., 2009). The stoichiometry of binding was low in these experiments, being 0.01 mol ABA/mol of GTG, perhaps due to the fact that the receptors require a membranous environment for optimal ABA binding (Pandey et al., 2009; Risk et al., 2009).
VI. THE SOLUBLE PYR SIGNALLING COMPLEX IS PART OF A SHORT PHOSPHO-RELAY CASCADE In the ABA signalling core complex, members of the protein phosphatases 2C belonging to clade A—as epitomised by the two closely homologous founding members ABI1 and ABI2 (Gosti et al., 1999; Merlot et al., 2001)—are negative regulators of ABA signalling. In an idealised environment devoid of stress (presumably no or low levels of ABA), these clade A PP2Cs would antagonise the pathway by inactivating specific downstream protein kinases, which function as positive regulators of the ABA signal transduction pathway (Umezawa et al., 2009; Vlad et al., 2009). Indeed,
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these PP2Cs can physically dock via a particular motif called domain II that is found in the noncatalytic C-termini of SnRK2.2, SnRK2.3 and OST1/ SnRK2.6/SRK2E (Fujita et al., 2009; Umezawa et al., 2009; Yoshida et al., 2006). The PP2Cs probably interact transiently with these SnRK2s because coimmunoprecipitation of the two proteins was found to be inefficient unless chemical cross-linkers were added (Vlad et al., 2009). These PP2Cs can efficiently dephosphorylate the multiple Ser/Thr residues in the activation loops of these kinases (Umezawa et al., 2009; Vlad et al., 2009). In response to stress, ABA binds to PYR/PYL/RCAR. As mentioned above, the change of the receptor protein conformation caused by the binding of ABA in turn leads to the creation of contact surfaces specific for at least some of the clade A PP2Cs, thereby restraining physically these negative regulators. In the absence of these free PP2Cs, the equilibrium of the SnRK2s is shifted towards their active states, permitting them to phosphorylate downstream targets. These core signalling steps have been successfully and elegantly recapitulated in vitro using a peptide derived from the ABA-responsive transcription factor ABF2 as the model target of phosphorylation (Fujii et al., 2009). The in vitro reconstruction of the signalling core is largely consistent with a number of in vivo observations. As alluded to above, the dominant nature of the mutation equivalent to abi1-1 in various PP2Cs owes this to the loss of a contact amino acid, allowing them to escape recruitment by the soluble receptors. In addition, the lower in vivo SnRK2 kinase activity in the dominant PP2C mutant genetic background, and inversely, their elevated activity in the PP2C knock-outs (Mustilli et al., 2002; Vlad et al., 2009) are in accordance with the direct binding and inactivation of SnRK2s by PP2Cs in vitro or in yeast (Umezawa et al., 2009; Vlad et al., 2009). The homologous SnRK2.2, SnRK2.3 and SnRK2.6/OST1/SRK2E are likely the major and direct targets of negative regulation by at least some of the clade A PP2Cs related to ABA signalling. Of these three kinase-encoding loci, SnRK2.6/OST1/SRK2E was independently identified in forward genetic screens for mutants that transpire excessively in conditions of low humidity (Merlot et al., 2002; Xie et al., 2006) and by reverse genetics in search of kinases activated by exogenous ABA (Yoshida et al., 2002). Earlier, the ortholog AAPK was purified from Vicia faba guard cells by virtue of its activation by ABA and was shown to be functionally important in mediating stomatal closure by ABA (Li et al., 2000). Thus, OST1 and AAPK seem to be the predominant SnRK2s in relaying the ABA signal in the guard cell. No forward genetic screen has identified SNRK2.2 and SNRK2.3, and one reason is that these two kinases may be functionally very similar (in those defined experimental conditions). This is supported by the fact that knock-out mutants for either SNRK2.2 or SNRK2.3 are phenotypically
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indistinguishable from the wild type, but the simultaneous loss of both loci causes altered root growth and seed germination behaviour on ABA (Fujii et al., 2007). Despite their apparent tissue-specific phenotypes that imply functional distinction, OST1 may still share a subset of common downstream targets with SNRK2.2 and SNRK2.3. In comparison to the double mutant, the triple knock-out snrk2.2 snrk2.3 snrk2.6/ost1/srk2e shows further reduction by several fold in ABA sensitivity, remains stunted in development, produces few seeds, and is prone to wilting when ambient humidity is not high (Fujii and Zhu, 2009; Umezawa et al., 2009). The severity of the phenotype throughout plant growth and a pronounced decrease in ABA sensitivity suggest that these three kinases act as key regulators of most of the elementary downstream ABA responses.
VII. ABA CONTROLS RAPID DROUGHT ADAPTIVE RESPONSES BY MODIFICATION OF SELECTIVE TRANSPORT ACROSS THE PLASMA MEMBRANE A. THE POTASSIUM CHANNELS
As an overview, most of the so-called rapid ABA responses are derived from studies using the guard cell as the model (Fig.1). These rapid responses are most likely conserved, at least at the qualitative level, as they have also been observed in cell cultures (Meimoun et al., 2010). One of the earliest detectable responses to drought onset is the reduced turgor pressure and volume of the pair of flanking guard cells leading to stomatal closure. The closing stimulus is mediated, at least in part, by the production of H2O2, and through increases in cytosolic [Ca2þ]cyt as well as enhancement of its sensitivity as a form of positive feedback (coined as ‘‘priming’’; Kim et al., 2010; Siegel et al., 2009). The Ca2þ signals prevent turgor increase by downregulating both the P-type Hþ-ATPases required for plasma membrane hyperpolarisation (Kinoshita et al., 1995) and channels for Kþ influx. In parallel, Ca2þ stimulates anion extrusion (Hedrich et al., 1990; Schroeder and Hagiwara, 1989), which contributes to the depolarisation of the plasma membrane. As well, in parallel to the Ca2þ signal, the associated cytoplasmic alkalinisation evoked by ABA promotes Kþ extrusion via efflux channels (Blatt and Armstrong, 1993). In plant cells, Kþ is the most abundant cation (which can make up to 10% of the dry weight if Kþ is unlimited in availability) and serves as a charge carrier, enzyme cofactor and an osmoticum, as in the case of the guard cell. There are six Shaker-type (named after the Drosophila founding member) Kþ
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inward-rectifying channels expressed in the guard cells, with the homologs KAT1 and KAT2 being responsible for majority of the Kþ influx activity that drives stomatal opening. Each of these two major Shaker channels, when expressed alone, can form homotetramers which constitute the functional unit of the Kþ channel. However, in plants, they preferentially form heterotetramers consisting of two subunits each of KAT1 and KAT2 (Lebaudy et al., 2010). KAT1–KAT2 heterotetramers generate synergistic enhancement in Kþ transport in that the current is much larger as compared to the equivalent amounts of either KAT1 or KAT2 homotetramers. In comparison to the large number of inward rectifiers, there is only one Kþ outward-rectifying channel, GORK, responsible for Kþ efflux in the guard cell requisite for stomatal closing. The voltage-gated Kþ channels presumably have evolved from a common Shaker-type ancestor because they share the characteristic framework of six TMDs, designated S1—S6, with the pore region between transmembrane segments S5 and S6 that form the major constriction and lining of the pore. Despite the sequence conservation among these Kþ transporters, they are impressive in their remarkable functional diversity. Previous work on the sole Kþ outward rectifier expressed in the stellar cells of the root, SKOR, has shown that its gating mechanism is achieved by certain amino acids deep in the S6 domain that opposes and contacts with the base of the helix pore (Johansson et al., 2006; Liu et al., 2006). However, the precise regulatory mechanism remains intriguing: it has been reported that the activity of SKOR is stimulated by internal [Kþ] (Liu et al., 2006), or alternatively, a more unusual mode that depends rather on external Kþ (Johansson et al., 2006) even though the function of SKOR is to extrude Kþ. It also appears that both intracellular and extracellular acidification inhibit SKOR (Lacombe et al., 2000). As described above, SKOR bears a ‘‘S6 gating domain’’, including the key residues D-M-I within the last transmembrane segment that opposes and interacts with the base of the pore helix, transmitting information about pore occupancy to the channel gate. Altering this interaction through residue exchange—either in the S6 gating domain or at the base of the pore helix— affects the Kþ sensitivity as well as the voltage-dependence of SKOR gating and, in the extreme, also renders the channel nonrectifying. These results raised the question of whether the S6 regions of Kin and Kout channels are key to understanding the divergence in their function. B. THE ANION CHANNELS
The long-sought after channel that is critical for slow sustained anion efflux, and the subsequent decrease in osmotic pressure, needed to drive stomatal closure was identified by independent genetic screens for mutants with
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altered sensitivity to ozone (Saji et al., 2008; Vahisalu et al., 2008) and to CO2 (Negi et al., 2008). The mutation slow anion channel-associated 1 (slac1, also variously known as rcd1, rcd3, or cdi3; Fig. 1) results in larger stomatal aperture, and the corresponding guard cells contain higher Kþ content, possible as a counter ion to the accumulation of malate and fumarate (Negi et al., 2008). As well, the mutant turns out to be reduced in sensitivity to a variety of other stomatal closing-inducing signals, but several details differ in the independent mutant characterisation in terms of its responses to H2O2, ABA and high CO2. For example, the mutant guard cells were said to be insensitive to ABA by two groups (Negi et al., 2008; Vahisalu et al., 2008) but were described as wild type by another (Saji et al., 2008). Both rapid transients and long-term O3-induced decreases in stomatal conductance were abolished in the mutant. Notably, only the S-type (but not the R-type) anion current was impaired by the mutation. This observation is important because it provides strong evidence that the S- and the R-type anion currents are mediated by distinct entities, rather than due to the same channel being modified differently, for example, by phosphorylation, as previously proposed. Whole-cell patch clamp techniques detected a permeability ratio for malate to chloride anions of 0.128 consistent with previous anion selectivity analyses of S-type anion channel currents. The corresponding protein (At1g12480) has low homology with the C4-dicarboxylate transporter/ malic acid transport protein domain defined from the E. coli TehA and Schizosaccharomyces pombe Mae 1 protein. C. THE P-TYPE PROTON PUMPS
Activated proton pump is required for hyperpolarisation of the plasma membrane that eventually drives stomatal opening. The pumping activity is substantially reduced in the phot1 phot2 mutant background, thus placing the Hþ-ATPases, such as OST2 (Fig. 1), in the phototropin-mediated pathway (Shimazaki et al., 2007). Plant proton pumps can functionally complement yeast mutants disrupted for the major Hþ-ATPase PMA1 (which is lethal for yeast), which has facilitated detailed studies into the structure–function relationship of these plant proton pumps. The activity of the proton pump is known to be influenced by protein phosphorylation and structural rearrangements. All P-type Hþ-ATPases have a cytosolic C-terminal domain of 100 amino acids which act as an autoinhibitory domain (Palmgren et al., 1991). The current model proposes that this domain suppresses proton pumping activity by folding back onto the rest of the protein. Note that the inactive form of the P-type Hþ-ATPase is dimeric and it is not clear how the individual folded proteins are arranged relative to each other. This presumed
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closed protein conformation, nonetheless, is consistent with the interaction between the C-terminus and the rest of the OST2 protein in trans by using the yeast split-ubiquitin (Merlot et al., 2007) and bimolecular fluorescence complementation (BiFC) assays in transfected tobacco epidermal cells (Sirichandra et al., 2009a). Recently, the N-terminal soluble portion of the Actuator domain has also been shown to possess autoinhibitory functions as well; in particular, an extrapolation from the available data suggests that deleting amino acids 4–10 in this domain of AHA2 led to enhanced yeast growth in acidic medium (Ekberg et al., 2010). This was further shown to correlate with an increased phosphorylation of a specific Thr947 and its subsequent ability to bind to 14-3-3 proteins in the C-terminus of the pump. Further deletions up to 20 amino acids negate this enhancement of yeast growth on acidic medium, and in a majority of the cases, without apparent effect on the stability these truncated AHA2 (Ekberg et al., 2010). There might be other subtle regulatory effects exerted by the N-terminal portion of the Actuator domain that are difficult to decipher by increasingly larger deletions which might disrupt the protein structure, as hinted by the existence of several point mutations further up to the first TMD (M1) capable of increasing the proton pumping activities as well (Merlot et al., 2007; Morsomme et al., 1996). Activation of the pump requires phosphorylation of the penultimate Thr, which then binds 14-3-3 proteins to assume the active conformation. This is also accompanied by the conversion from the dimeric to a hexameric form joint by six 14-3-3 proteins at the C-termini of the Hþ-ATPases (Kanczewska et al., 2005; Ottmann et al., 2007). At least for two of the most studied tobacco Hþ-ATPases, PMA2 and PMA4, do not heterodimerise. Moreover, the hexamer assumes a true sixfold symmetry rather than a threefold symmetry, consistent with a lack of heterodimeric forms between PMA2 and PMA4. This has also been interpreted that regions other than the C-termini might be engaged in the formation of this hexameric configuration (Ge´vaudant et al., 2007). Besides the bilateral autoinhibitory domains, the proton pumping activity is also reduced by phosphorylation by the calcium-dependent kinase PKS5 on a specific and conserved serine residue in the C-terminal domain that impedes subsequent 14-3-3 binding (Fuglsang et al., 2007). This might explain the sensitivity to Ca2þ inhibition of Hþ-ATPases as described above. PKS5 is itself inactivated by the chaperone J3, homologous to the E. coli DnaJ/hsp40 protein (Yang et al., 2010). The inactivation mechanisms are not clear; it could be that J3 decreases the affinity between Hþ-ATPase and PKS5, or J3 could directly inhibit the PKS5 kinase activity.
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Treatment with ABA is invariably associated with an increase in H2O2 production, as shown in Arabidopsis and in V. faba guard cells (Kwak et al., 2003; Pei et al., 2000; Zhang et al., 2001). In Arabidopsis guard cells, two of the 10 NADPH oxidases, AtrbohD and AtrbohF, are responsible for ABAinduced ROS production and subsequent stomatal closure. Until recently, it remained unknown how ABA activated these two NADPH oxidases. The ost1 mutant was observed to lack detectable H2O2 rise in the guard cells after ABA stimulation (Mustilli et al., 2002). This kinase has now been shown to activate H2O2 production by direct phosphorylation of AtrbohF (Ser13, Ser174; Sirichandra et al., 2009b; Fig. 1). Another H2O2 production pathway exists, independent of the core complex, but that requires phospholipase Da1 and phosphatidic acid (Zhang et al., 2008). Using Xenopus oocyte as the main functional assay system, OST1 has also been shown to be one of the kinases which directly phosphorylate (Thr303) and inactivate KAT1 (Sato et al., 2009; Fig. 1). However, the anion channel SLAC1 is activated by OST1 (Geiger et al., 2009; Lee et al., 2009) as well as the Ca2þ-dependent CPK21 (Geiger et al., 2010), while the channel’s inactivation is mediated by direct interaction with PP2CA (Lee et al., 2009), and indirectly by this same PP2C and ABI1 via interactions with OST1 (Geiger et al., 2009). Note that the physical interaction of these PP2Cs with their targets has not been directly shown to be dephosphorylation step, as this is only suggested by the coexpression of these components in Xenopus. H2O2 is an intriguing choice for a signalling molecule because it is induced by a variety of stresses and, being a reactive free-radical, can inflict severe cell damage if uncontrolled. The H2O2 signal must therefore be tamed if it were to serve as a signalling molecule in order to activate the proper physiological responses, especially the coreceptor ABA complex with ABI1/ABI2 (see below). Excess free radicals are, at least in part, removed by scavenger enzymes such as superoxide dismutase, catalase, peroxidases and enzymes involved in the ascorbate–glutathione cycle (Noctor and Foyer, 1998). It has been shown that glutathione peroxidase3 (ATGPX3) can interact with ABI2 and to a lesser extent, ABI1 (Miao et al., 2006). The redox state of both ATGPX3 and ABI2 was found to be regulated by H2O2. The in vitro phosphatase activity of ABI2 was reduced by fivefold in the presence of added ATGPX3. The reduced form of ABI2 was converted to the oxidised form by the addition of oxidised ATGPX3, suggesting that this latter protein plays a dual role in mediating ABA and oxidative signalling by relaying H2O2. Consistent with this is that T-DNA mutants of GLUTATHIONE
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PEROXIDASE3 show higher rate of water loss under drought, higher sensitivity to H2O2 treatment during seed germination and subsequent early development. The mutant also produces higher amount of H2O2 in the guard cells. In contrast, lines overexpressing AtGPX3 were less sensitive to drought stress than the wild type and displayed lesser transpirational water loss. The Atgpx3 mutation also disrupts ABA activation of Ca2þ channels and expression of ABA-/stress-responsive genes. Thus, besides being sequestered by PYR during ABA signalling, ABI2 (and perhaps ABI1 to a lesser extent) could also be inactivated by the oxidised ATGPX3. In fact, both ABI1 and ABI2 are sensitive to inactivation directly by H2O2 without ATGPX3, as demonstrated in vitro (Meinhard and Grill, 2001; Meinhard et al., 2002). So, both PP2Cs could also be direct targets of H2O2 in vivo.
VIII. TARGETS OF SnRK2s IN MEDIUM-TERM ABA RESPONSES-GENE EXPRESSION AND CHROMATIN MODELLING It has been estimated that about 5% of the plant transcriptome is under the influence of ABA. The direct class of downstream target genes is probably represented by those controlled by the characteristic ABA-responsive element (ABRE) motifs in the promoters (see below), which are binding sites of the b-ZIP class of transcription factors. In turn, some of these b-ZIP proteins are also in vivo substrates of SnRK2. A fragment corresponding to ABF2/ AREB1 (ABA Response Element Binding Factor) had been indeed used successfully in the reconstitution of a functional ABA core signalling complex in vitro (Fujii et al., 2009). In fact, a b-ZIP transcription factor (TaABF) was the first plant target identified for this kind of kinases in a yeast twohybrid screen by using the wheat homolog PKABA1 as the bait (Johnson et al., 2002). Further, PKABA1 was able to phosphorylate in vitro, among six test peptides derived from TaABF, one was described as relatively efficient (containing the sequence RMIKNRESAARSRARK). Independent work in rice cell cultures also showed that three representative members of the SnRK2 family, SAPK8, 9 and 10, were particularly effective in mediating expression of reporter genes containing the motif ABREs, which are recognition sites of b-ZIP proteins (Kobayashi et al., 2005). When SAPK10 was used as the model kinase, it was shown that it can indeed phosphorylate in vitro peptide fragments derived from the b-ZIP transcription factor TRAB1 and coimmunoprecipitated from rice cells transiently expressing the two proteins. Mapping the phosphorylation sites by MALDI-TOF mass spectrometry revealed two phosphoserines in a peptide with a sequence
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composition (RGQGSLTLPRTLSVKTVDEVW) that differs significantly from that for PKABA1 above. Detailed investigations in Arabidopsis using peptides derived from several shared domains in b-ZIP proteins (AREB1/ABF2, AREB2/ABF4 and ABF3) and test SnRK2s (SnRK2.2/SRK2D, SnRK2.3/SRK2I, SnRK2.6/OST1/ SRK2E, SnRK2.7/SRK2F and SnRK2.8/SRK2C). These studies allowed the deduction of phosphorylated (S/T) in a ‘‘consensus’’ motif R 3–X 2–X 1– (S/T) in the ABFs, which also first revealed the highly conserved arginine residue at the 3 position (Furihata et al., 2006). Phosphorylation at these particular Ser or Thr in the b-ZIP proteins was proven important for their function, as their replacement to Ala compromised severely their capacity to transactivate a reporter gene. Using SnRK2.8/SRK2C as a representative SnRK2 to phosphorylate in vitro 200 pools of semidegenerate peptides that represented 1012 unique sequences and then quantifying the influence by the neighbouring amino acids on phosphorylation of a fixed Ser or Thr, it was confirmed that Arg is indeed preferred at the 3 position, and further, this method also predicted that SnRK2s may have a general selectivity for hydrophobic amino acids (LIMVF) at the 5 position, in particularly Leu (Vlad et al., 2008). Indeed, there is a preponderance of Arg at 3 and Leu at 5 relatively to the phosphorylated serine in the peptides derived from AREBs/ ABFs that supported phosphorylation by the SnRK2s (Furihata et al., 2006). Promoter analyses carried out on a number of ABA-responsive genes led to the identification of a conserved motif, named the ABRE (with the consensus PyACGTGG/TC) enriched in their presumptive promoters. The core ACGTG also resembles the G-box (CACGTG) prevalent in promoters of genes whose expression is sensitive to light. As mentioned above, the ABRE is the binding site of b-ZIP transcription factors, some of which are targets of SnRK2s. Three of the b-ZIP transcription factors—AREB1, AREB2 and ABF3—together seem to assume a particular important status in the adaptive responses to high salinity, drought and exogenously applied ABA. As compared to the wild type, or to lower order mutants, the triple mutant disrupted in these genes was found to be highly resistant to inhibitory concentrations (up to 50 mM) of exogenous ABA in seed germination tests and root growth (Yoshida et al., 2010). More than 80% of the downregulated genes in the triple mutant contain two or more copies of ABRE in their promoters, consistent with the idea that these three AREBs control a major portion of the transcriptome triggered by water stress. It is striking though the transpiration remained nearly normal in this triple mutant, suggesting that there are as yet additional b-ZIP transcription factors (or other unrelated proteins) that regulate the guard cell responses to drought and ABA transcriptionally. When coexpressed in the same cell, these three b-ZIP
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transcription can form heterodimers and homodimers. Whether this might be the case as well in intact plants is not known, since whether all three proteins are simultaneously expressed in time and in space has not been investigated. Many of the downstream genes transcribed either directly, or perhaps indirectly, by these AREBs encode dehydrins, whose appearance has been known to coincide with water deficit. It should be noted that, besides being unstructured proteins, the diverse biochemical functions of these dehydrins in protecting the cells against water penury are still not fully elucidated.
IX. EPIGENETICS IN ABA REGULATION In addition to changes in specific genes brought about by interaction between b-ZIP transcription factors and promoters bearing target binding sites, it appears that ABA also mediate large-scale changes in gene expression through modification of chromatin changes. Epigenetics was coined by C. H. Waddington in 1942—prior to any knowledge concerning the physical nature of the gene and its role in heredity—as a conceptual model of how genes might interact with their surroundings to produce a phenotype. ‘‘Epi-’’ in epigenetics implies ‘‘above’’ genetics; thus epigenetic traits are addressing regulatory mechanisms that are more than those explainable by the traditional Mendelian basis of inheritance. The definition of epigenetics has evolved over time, and the modern usage now refers to heritable traits, stable over cell division and generations that do not entail changes in the DNA sequences. In molecular parlance, these epigenetic modifications are equivalent to chromosomal marks or imprinting, including cytosine 50 methylation, posttranslational modification of histones (generating the so-called histone code) and changes in compositions or positions of chromatin complexes (nonhistone proteins) along the DNA in response to some environmental or developmental signal. There are suggestions that modifying chromatin structure may serve to impose a sort of ‘‘stress memory’’ in plants (Chinnusamy and Zhu, 2009). A histone H1 variant induced by drought through an ABA-dependent pathway has been reported for tomato, as transgenic plants expressing an antisense directed at this histone variant resulted in higher stomatal conductance (Scippa et al., 2004). Some of the proteins with homologies to the polycomb group (PcG), whose role is to condense chromatin as a means to coordinately repress genes within a chromosomal region, are responsive to ABA in barley (Kapazoglou et al., 2010). As well, in Arabidopsis, the histone deacetylase HDA19 is associated with a chromatin complex that is required for suppressing stomatal conductance and ABA sensitivity (Song et al., 2005). ABA also represses the expression of the
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histone deacetylase gene AtHD2C (Sridha and Wu, 2006), the overexpression of which was shown to result in lower stomatal conductance. These results imply that chromatin modification underpins even the so-called rapid drought adaptive responses that include stomatal regulation. Further evidence for histone deacetylase in ABA and abiotic response emerged from the identification of the hos15 mutation (Zhu et al., 2008), whose affected protein shows similarities to the human WD-40 repeat TRANSDUCIN BETALIKE PROTEIN-1 (TBL1), which is a component of the chromatin repressor complex in histone deacetylation. HOS15 is induced not only by ABA, but also by cold and high salinity, and the protein is associated with histone H4. It might thus be possible that HOS15 modulates ABA and other abiotic stress responses by H4 deacetylation-dependent chromatin remodelling. The Arabidopsis PP2C, HAB1, besides functioning as a suppressor of SnRK2 activity to abrogate ABA transmission, may also directly regulate large-scale gene expression by modifying chromatin structure or nucleosome positioning. HAB1 interacts with SWI3B, an A. thaliana homolog of the yeast SWI3 subunit of SWI/SNF (SWITCH/SUGAR NONFERMENTING) chromatin-remodelling complexes (Saez et al., 2006). Based on BiFC and immunoprecipitation assays, this interaction is confirmed to take place in the nucleus of plant cells. This nuclear-confined action of HAB1 (representing less than 10% of the total HAB1) is reminiscent of another PP2C, ABI1, which has also been described to exert its negative regulatory effects in the nucleus, even for the socalled rapid plasma membrane transport events associated with stomatal closure, although the reason for this is not clear (Moes et al., 2008). In this respect, ABI1 (ABI2, PP2CA/AHG3) can also interact with SWI3B in plant cells (Saez et al., 2006) and suggests that ABA may reprogram gene expression by epigenetic modification. In the absence of ABA, HAB1 is enriched in the vicinity of ABRE and TATA elements upstream of classical ABA-inducible marker genes such as RAB18 and RD29B, but it is then evicted from these chromatin regions during ABA response. SWI3B also interacts with FCA (Sarnowski et al., 2005), suggesting a molecular link between stress, ABA and flowering time through the remodelling of an SWI/SNF complex.
X. MITOGEN-ACTIVATED PROTEIN KINASES IN ABA SIGNALLING Mitogen-activated protein kinases (MAPKs) have been described to be components in many signalling pathways mediating abiotic and biotic stress responses as well as during normal development such as cell division. Indeed, activities of MAPK associated with hormone actions, pathogen invasion, or
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harsh environmental treatments have often been reported. The most studied in Arabidopsis are MPK3, MPK4 and MPK6 which respond to myriads of stimuli. In particular, disruption of MPK6 also impaired downstream H2O2 production stimulated by ABA (Xing et al., 2008). Moreover, both ABA- and H2O2-induced stomatal closure was severely reduced in the presence of the MAPKK inhibitor PD98059 suggesting important functions for these kinases in reducing transpiration (Jammes et al., 2009). In silico examination of gene expression data revealed that the transcripts of MPK9 and MPK12 are highly enriched in guard cell protoplasts than those derived from mesophyll. The double, but not each of the single, TILLING mutants of these two MAPKs was impaired in stomatal closure induced by ABA, cold and H2O2 (Jammes et al., 2009). The activities of both kinases, as ascertained by immunoprecipitation and in vitro tests, were found to be enhanced by ABA and H2O2. Further, studies by electrophysiology found that in the double mutant, the anion channels are refractory to activation by either ABA or by Ca2þ. These two MAPK are likely functionally redundant, most likely downstream of the Ca2þ signal, as expression of MPK12 (fused to the epitopes, YFP and HA) alone was able to rescue the phenotypes of the double mutant. In animals, MAPKs can be deactivated by both dual-specificity and type 2C protein phosphatases. Although there is evidence that certain dual-specificity protein phosphatases may control ABA sensing (Monroe-Augustus et al., 2003; Quettier et al., 2006), the more solid evidence of MAPK inactivation in plant has come from the studies of PP2C. The plant PP2C shown to negatively regulate MAPK was the alfalfa MP2C, which directly interacts with the salt-stress-inducible SIMK and inactivates it by dephosphorylation of the Thr in the conserved pTEpY motif (Meskiene et al., 1998,2003). Similarly, the Arabidopsis AP2C1, which is the closest MP2C homolog, was also shown to interact with and dephosphorylate MPK4 and MPK6 (Schweighofer et al., 2007). These two MAPKs as well as MPK3 are also negatively regulated by a nuclear-localised phosphatase PP2C5, phylogenetically belonging to clade B (Brock et al., 2010). Depletion of PP2C5 and its closest homolog AP2C1 results in plants with increased stomatal aperture, partial ABA sensitivity during seed germination and reduced responsiveness of some ABA-inducible genes after ABA application (Brock et al., 2010).
XI. ROOT GROWTH IN RESPONSE TO ENVIRONMENT ‘‘Can science feed the world?’’ printed on the front cover of the leading journal Nature in the 29 July 2010 issue. Whether this is an open question or rather a desperate plea is hard to tell, but it is no doubt a blunt reminder of
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the momentous achievement by the Green Revolution between 1940 and 1970, and for which the founder member Norman Borlaug was recognised by the Nobel prize. One of the current efforts to increase yield without causing environmental damage is to enhance root development. Root delivers nutrients and water, two of the most essential and often limiting factors that a plant needs. Designer roots could thus be part of the future strategy to diminish fertiliser and/or water input while still maintaining optimal output. Obviously, this strategy, if successful, would also tender a plausible solution to avoid encroaching increasingly on more land to feed the growing world population. But we need to first get down to the basics with respect to the regulatory mechanisms of how a root normally develops, with its attendant complex tissue specialisation and how a root sense environmental stresses such as drought? Finally, how are the genetic programme and environmental variations integrated? Previous physiological studies have provided evidence that ABA might be required for root growth, even in conditions of the environmental constraints (Sharp et al., 2000; Spollen et al., 2000). There are other corroborating, but indirect, evidence such as high concentrations of ABA in the columella cells and the quiescent centre (Christmann et al., 2005). Root is also the active site of ABA biosynthesis and precursor conversion. The biosynthetic genes AtNCED2 and AtNCED3 are expressed in the pericycle at the site of lateral root initiation (Tan et al., 2003). The ABSCISIC ALDEHYDE OXIDASE (AAO)3 gene is highly expressed in root tips and vascular bundles and ABA2 is detectable in the branching points of lateral and mature roots (Cheng et al., 2002; Koiwai et al., 2004; Tan et al., 2003). Mutations affecting root patterning and development exist (e.g., cobra, werewolf, myb23, caprice, tryptychron), but exploiting them to understanding the link between development and environmental constraints has been limited. Roots, being underground, create more of a technical challenge for agricultural researchers to observe their growth patterns in their natural milieu. Their inherent growth flexibility could also confound the relative contributions from the tremendous genetic variations and the heterogeneity of the soil environment with a given phenotypic criterion or a physiological response. Nevertheless, when osmotic stress treatment goes on for extended period of time, notable changes in root architecture occurs. There had been a couple of reports describing bulbous and shortened root hairs in drought conditions that implicate the role of hormones, particularly auxin, GA and ABA (Schnall and Quatrano, 1992; Vartanian et al., 1994). Root hair development is also transiently inhibited by salt stress, but in time-course experiments, it was noted that development resumed after a few hours, suggesting some sort of physiological adaptation. This is correlated with fluctuation of
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certain hair-cell developmental marker genes and many of the repressed genes encode structural components of the cell wall, or in tricoblast differentiation (Dinneny et al., 2008; see below). It has also been observed that when plants were treated in mannitol (50–75 mM), lateral roots did not develop or delayed (Xiong et al., 2006). Lateral root growth is also very sensitive to inhibition by ABA. All ABAdeficient mutants have more lateral roots under normal conditions, but these same mutants are also less sensitive to the inhibition by mannitol. A forward mutant screen was thus conducted using inhibition of lateral root development as a typical response to mannitol. This screen has identified a mutant, dig3, that showed lateral root growth in experimental drought conditions, although the nature of the gene product is not yet known. The slight increase in lateral root primordia in slight decrease in osmotic potential in an artificial medium has also been exploited to identify another mutant, lateral root development(lrd)2, which displays a constitutive increased lateral root system in the repressive osmotic condition as well as in the absence of stress (Deak and Malamy, 2005). Again the nature of the LRD2 gene product is not yet known. In addition to the above mutant screens, recent whole-genome technologies have provided a glimpse of some of the molecular aspects of root adaptability to stress. A high-resolution spatial expression atlas of stressinduced genes in various Arabidopsis root tissues was constructed by using whole-genome RNA arrays (Dinneny et al., 2008). Radial patterns of gene expression maps were generated by GFP-reporters expressed in particular cell layers whereas longitudinal sections of the roots were used as a proxy of different developmental stages. These GFP-labelled cells were recovered by fluorescence activated cell-sorting and the RNAs were profiled by Affymetrix gene-chips. These whole-genome results revealed that, along the longitudinal axis, an increase in salt responsiveness was found in those cells at the elongation zone. This suggests that cells that are the most developmentally competent to respond to high salinity are those undergoing differentiation. In examining the radial pattern of gene expression in response to high salt, many of the induced genes are found to express in only one cell layer. This suggests that there is both developmental and tissue-specific competence (or constraints) in conferring salt responsiveness. In contrast, genes responding to ABA are not cell-type specific. In those genes that were induced by salt along the longitudinal zones, their corresponding promoters were enriched by many known cis-regulatory elements such as drought-responsive elements (DRE) and ABRE. These cis-elements were also found in genes that are classified as semiubiquitous based on the fact that they are expressed in at least three radial zones. Thus, although canonical stress-responsive pathways
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appear to be active in all cell layers, the authors argued that cell-type-specific responses are distinguishable at the promoter level and probably controlled by other cis-elements. ABA insensitive mutants are also partly resistant to salt (Achard et al., 2006), and the affected ABA-regulated genes are also saltresponsive in all cell layers of the root. This apparent widespread activity has been taken to mean that ABA might primarily mediate semiubiquitous transcriptional responses to salt. This picture is certainly more complex. In ABA-deficient mutants, salt-induced expression is diminished for many ABA-responsive cell-type-specific markers. This would also indicate that ABA regulates cell-type-specific responses to salt stress in a manner independent of characterised ABRE.
XII. ABA IS CONSERVED IN EVOLUTION AND HAS POTENTIAL TO IMPROVE HUMAN HEALTH Laying down the basic knowledge concerning the mechanisms of ABA action has obvious application in the field to enhance plant resilience to a variety of environmental constraints. For interested readers, there are many excellent reviews and primary publications that point in this direction, especially in view of climate change (Schroeder et al., 2001; Wang et al., 2005; Zhang et al., 2004). There are, however, tantalising and less-publicised advances in the medical field in using ABA as a experimental treatment for certain ailments. This comes in the wake of our increasing awareness of the general benefits of stress-protective compounds derived from plants for human health, a concept known as ‘‘xenohormesis’’ (Hooper et al., 2010). As we have said in the opening to this book chapter, biological sciences are inextricably tied to the theory of Evolution, which embodies the continuity in all life forms as its unifying concept. For the molecular biologists, this concept of continuity is inscribed by the modular nature of protein structures, and these composite functional domains can be found rethreaded together in myriads of different combinations across plant and animal kingdoms. Learning the potential function of an unknown gene from one organism by comparing its sequence against public data bases by ‘‘blast’’ is reaffirming faith in evolution. Despite all our open-mindedness, it still comes as a scientific cultural shock that this continuity actually goes beyond genes, but encompasses ABA, which also implies that large portions of pathways of biosynthesis and signal transduction must be somehow conserved or reinvented by convergent evolution. ABA was first isolated from plants in the 1960s as a substance that can induce bud dormancy and fruit abscission, and thus has logically inherited
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the designation of phytohormone. However, this tacit acceptance has been jolted by its rediscovery in marine sponges, fresh-water hydra, parasites, and even in humans (Wasilewska et al., 2008). Although the pathways by which ABA is synthesised in organisms other than plants is not known, its discovery in animals has naturally sparked interests in the possible parallels between its role in plant and animal stress response systems. In sea sponges, ABA acts as a trigger to increase water filtration to cool the body in response to high sea temperature (Zocchi et al., 2001). This requires cyclic ADP-ribose as the second messenger, and thus the stimulus is thought to pass through Ca2þ release by means of ryanodine receptors in the sarcoplasmic reticulum. The ABA–cyclic ADP-ribose–Ca2þ connection reappears in Toxoplasma gondii (Nagamune et al., 2007), a worldwide parasitic protozoan (found in faeces in domestic animals such as cats) that causes congenital retinitis and brain damage. T. gondii can remain dormant in the cystic form throughout the host’s lifespan, but can be reactivated to lytic growth (egress) in immunocompromised hosts, such as patients suffering from AIDS. A series of elegant experiments conducted by Sibley and colleagues (Nagamune et al., 2007) have helped to track down the origin of the ABA signal. Stress causes ABA synthesis in the apicoplast, a remnant organelle of an algal endosymbiont. The presence of ABA was confirmed by high-performance liquid and gas chromatography, conjugated to mass spectrometry. In a cell culture model, the addition of the classical ABA synthesis inhibitor fluoridone can block specifically egress of the pathogen, but not its replication and host invasion. Moreover, exogenous reapplication of ABA rescues egress, by Ca2þ release through cADP ribose to promote secretion of the marker parasite adhesion MIC2 (microneme protein2). Importantly, in whole animal studies, fluridone inhibition of the ABA-dependent lytic parasite cycle protected mice against toxoplasmosis. In humans, ABA was first detected in the brain (Le Page-Degivry et al., 1986), then rediscovered in granulocytes (Bruzzone et al., 2007), in pancreatic islets (Bruzzone et al., 2008), in monocytes (Magnone et al., 2009) and in the plasma of mice fed with an AIN-93-G-based rodent diet (Bassaganya-Riera et al., 2010). The biosynthetic origins of ABA in mammals have still to be worked out, but it has hormone-like effects as nanomolar concentrations are sufficient to modulate the immune system (Magnone et al., 2009). The value of the public Arabidopsis resources for these new medical investigations are unquestionable, as they provided leads on over 1000 human orthologs that are related to those implicated in ABA signalling (Bassaganya-Riera et al., 2010). By predicting interactions among all proteins by informatics treatment, combined with available data on tissue-specific expression of these proteins, four human homologs of plant ABA-related genes that would anchor in a peroxisome proliferator–activator receptor g (PPARg) network were
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identified. PPARg is the target of the popular though controversial insulinsensitising drugs, thiazolidinediones (TZDs) for the treatment of type II diabetes. Based on studies using cell lines (3T3-L1 preadipocytes), ABA was shown to activate the expression of PPARg and that ABA can prevent the onset of type II diabetes in obese mice (Guri et al., 2007). Importantly, the antidiabetic effect of ABA, based on glucose homeostasis and macrophage infiltration, was reduced in mice deleted for PPARg, thereby providing in vivo genetic data that PPARg is a key player in the mechanisms of ABA antidiabetic action. There is no evidence that PPARg is the direct-binding site for ABA, however. Binding of ABA to the cell surface was observed in granulocytes, monocytes and aortic smooth muscle cells (Magnone et al., 2009; Sturla et al., 2009). In human granulocytes, ABA-induced responses including Ca2þ rise and ROS production were observed to require LANCL2, a plasma membrane-localised protein through N-terminal myristoylation with homology to the prokaryotic lanthionine synthetase (Sturla et al., 2009). Moreover, transfection of a transgene encoding LANCL2 into HeLa cells created ABA-binding sites (Sturla et al., 2009). It should be emphasised that, in contrast, a previous report for a cell-surface ABA receptor with homology to lanthionine synthetase (named GCR2) in Arabidopsis has remained controversial (Gao et al., 2007; Illingworth et al., 2008), and there is no evidence for direct ABA binding to LANCL2. In human monocytes, ABA induces nuclear translocation of NF-kB which regulates the expression of several inflammatory proteins (Magnone et al., 2009). Remarkably, ABA acts as a chemoattractant for granulocytes (Sturla et al., 2009), as well as monocyte or aortic smooth muscle cell migration and positively stimulate MCP-1 (believed to be the primary chemoattractant for monocytes to the antherosclerotic plaque) secretion from monocyte to constitute a positive feedback loop; these cellular behaviour forms part of the important repertoire for the development of antherosclerotic lesions. Both this study with human monocytes and those using the mouse model (Guri et al., 2007) converge on the potency of ABA as an immune modulator.
XIII. CONCLUDING REMARKS Transpiration and photosynthesis are intrinsically linked in gas-exchange processes. Biomass accumulation requires light interception by leaves and stomatal opening (Tardieu, 2003). Light, especially in the blue spectral range, stimulates stomatal opening by hyperpolarisation of the plasma membrane. In contrast, drought and high CO2 stimulate stomatal closing. As the stomatal pore is the only channel allowing communications between plant and
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environment, the trade-off of these two processes is critical for biomass, with important implications for agricultural productivity in view of the climate change. Within the past 2 years, one of the core complexes in ABA signalling has been solved. It consists of three essential components: the soluble receptor, a PP2C and an SnRK2. The binding of ABA promotes sequestration of the PP2C by the receptor, thereby releasing the SnRK2 to phosphorylate downstream targets. Because each of the above components are members of rather large families, their combinatorial actions could fine-tune downstream signalling intensity, although it remains unclear how this is related to the positive role played by low concentrations of ABA in plant development. This is also the second core signal complex that begins with the unusual receptor of 120 kDa resident in the chloroplast membranes, ABAR (GUN5), which was first identified as an essential component in retrograde signalling between the nucleus and chloroplast. It turned out that once bound to ABA, ABAR then becomes competent to interact with several nuclear repressors of the WRKY family, presumably to relieve transcriptional repression of target genes. Although the PYR and the ABAR pathways affect some common reporter genes, it will be exciting to see how they are coordinated (cross-talk) to control the repertoire of ABA responses. Electrophysiological and cell biological approaches have also indicated the presence of cell-surface ABA receptors, and two of these (GTG1 and GTG2) with homology to chloride channels in mammalian cells have been proposed as candidates. However, their affinity to ABA has been questioned (Risk et al., 2009). The genome of Arabidopsis has 129 annotated ABC transporters (Verrier et al., 2008). At least two have been assigned functions in ABA export (AtABCG25) and import (AtABCG40). The genetic and physiological evidence available suggests that they modulate the cellular content of ABA, and hence, perhaps the intensity of the ABA response (much like the combination of soluble PYR receptor and the different PP2Cs). With the discovery of ABAR, one could expect ABA transporters in the chloroplast envelope as well. AtABCB14 and SLAC1 are an interesting combination, because they have counter directions in malate transport, as would be expected, for example, when stomatas are exposed transiently to high atmospheric CO2. The long-distance transport of ABA, and whether this also involves active mechanisms, is unclear. As some of the ABA biosynthetic genes are strongly expressed in cells juxtaposing the vascular tissues these cells might represent sites of maturation and delivery of ABA to the rest of the plant. The SLAC1 and the potassium inward-rectifying channel KAT1 are direct targets of OST1. This same kinase is able to phosphorylate members of the b-ZIP transcriptional activators which control a substantial portion of the ABA
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transcriptome. Both lines of evidence indicate that OST1 (or the AAPK homolog of Vicia) is a key element in this core signalling pathway. Three b-ZIP transcription factors in Arabidopsis seem to control up to 80% of the ABA-inducible transcriptome. These proteins can form heterodimers, at least if they happen to be coexpressed in the same cell, but whether this is accompanied by changes in binding affinity to target promoters as another level of fine-tuning regulatory mechanism is not known. Another key regulator in the core ABA complex is the PP2C (represented by members ABI1, ABI2, HAB1, PP2CA, etc.) that acts as a coreceptor of ABA along with PYR, and the phosphatase is also the upstream suppressor of the OST1 kinase. ABI2 and, to a lesser extent, ABI1 are also rapidly inactivated by H2O2, which can be produced by OST1 phosphorylation of AtrbohF, which altogether, suggests a mechanism of mutual regulation between these two proteins. Moreover, HAB1 has a role in chromatin modification by binding to SWI3 that predicts alteration of the ABA transcriptome by epigenetics, besides those genes directly regulated by ABRE-binding transcriptional activators. The mRNA levels of ABI1, ABI2 and PYR/PYL/RCAR are also either induced or repressed by ABA treatments, but the current available data are somewhat contradictory, probably of the cryptic influence of the different experimental conditions (Shang et al., 2010; Szostkiewicz et al., 2010). It will be fascinating to see the extension of the nascent ABAR signalling pathway, as it implicates completely unexpected elements so far. Even the recognition and binding to ABA by ABAR seem to involve distinct mechanisms than those of the PYR. For example, the active group of ABA that is buried deeply within PYR was actually used to chemically couple ABA to an affinity column used to purify the 42-kDa protein from Vicia and ABAR from Arabidopsis. This predicts that ABAR will recognise a different part of the ABA molecule. The mechanisms of delivering WRKY from the nucleus to ABAR in the chloroplast are not known. Another obvious question, no less fascinating, will be potential points of cross-talk between ABAR and PYR to coordinate downstream events. One of the connecting point to ABAR could be somehow through the APETELA-related nuclear transcription factor ABI4, which has been shown to work downstream of the chloroplast protein GUN1 in retrograde signalling between the chloroplast and the nucleus (Koussevitzky et al., 2007). Root response to drought (and high salinity) remains a difficult subject for forward genetic approach. Roots alter their growth patterns when exposed to prolonged dehydration. Although there are several mutants with altered root development, how the activities of their corresponding genes might be linked to environmental constraints are not yet clear. The high-resolution transcriptomic atlas that recorded gene activities along the longitudinal and radial
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axes, indicate very complex and intertwined gene networks that are influenced by salt, ABA, developmental and tissue-specificity signals. In general, genes responding to exogenous ABA are not confined to any cell layers, nor to particular developmental stages, but are widespread. The most astounding findings in the last years is the rediscovery of ABA in a wide range of nonplant organisms that span several kingdoms. Thus, the existence of ABA is probably universal. The ability of fluridone to inhibit egress of T. gondii may open up development of novel antiparasitic compounds with minimal side effects on mammalian hosts. Extending this logic, if the entire pathway is worked out, from ABA perception to secretion of the microneme proteins, a wider range of targets will be available to combat this type of parasitic infections. However, results in studies using human monocytes (Magnone et al., 2009) contradict somewhat the interpretation of ABA as an anti-inflammatory agent in stromal vascular cells extracted from murine white adipose tissue, in which MCP-1 expression was inhibited instead (Guri et al., 2008). In both of these cases, the results converge on the fact that ABA is produced in mammals, and acts a powerful modulator of the immune system. Not surprisingly, some of the cellular components that are important for ABA signal transduction in plant have homologs in humans, but it is too early to pronounce whether they assume the equivalent functional roles.
ACKNOWLEDGEMENTS A. J. S. is supported by a postdoctoral fellowship from the French Agence Nationale de la Recherche ANR-08-BLAN-0123-01. JL and CV are grateful for the support from the Centre National de la Recherche Scientifique. JL thanks Dr. Philip Hooper (MD) at the University of Colorado, USA for the inspiring exchanges, reprints, preprints, and the philosophical waxing on xenohormesis.
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Signalling Strategies During Drought and Salinity, Recent News
TIJEN DEMIRAL, ISMAIL TURKAN1 AND A. HEDIYE SEKMEN
Department of Biology, Faculty of Science and Arts, Harran University, Sanlıurfa, Turkey
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Osmosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Signalling Components Involved During Salt and Drought Stress . . . . . . . . . A. ROS Signalling.................................................................. B. ABA and Stress Signalling Through ROS .................................. C. Antioxidative Signalling ....................................................... IV. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT Agricultural production has been adversely affected worldwide by environmental restraints, especially by drought and salinity because of their high scale of impact and wide distribution. Conventional breeding programmes seeking improvement of stress tolerance are a long-term endeavour as the trait is multigenic, and genetic variability among crop plants is scarce. Many effective protection systems exist in plants that allow them to perceive, respond to and appropriately adapt to a range of stress signals, and a variety of genes and gene products have been identified that involve responses to drought and high-salinity stress. In the past decade, a genetic model plant, Arabidopsis thaliana, has been widely used for unravelling the molecular
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Corresponding author: E-mail:
[email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00008-4
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basis of stress tolerance. The availability of knockout mutants and its suitability to allow genetic transformation proved the vital importance of Arabidopsis for assessing functions for individual stress-associated genes. In this review, the responses of plants to salt and water stress are described, the regulatory circuits, which allow plants to cope with stress, are presented and how the present knowledge can be applied to obtain tolerant plants is discussed.
I. INTRODUCTION Salinity and drought are responsible for much of the yield decline in agricultural lands throughout the world. Moreover, persistent salinization of arable land is becoming more widespread because of poor local irrigation practices, thus decreasing the yield from formerly productive land (Kaya et al., 2010). Not only anthropogenic factors but also some natural sources, such as parent material, entrance of seawater along the coast, salt-laden sands blown by sea winds, shallow groundwater and capillary rise, decay and release of salts, absence of natural drainage, result in accumulation of salts in soil. Approximately 6% of the world’s land and 30% of the world’s irrigated areas are already estimated to suffer from salinity problems (Unesco Water Portal, 2007). Further, the rapid change in global climate which is more than estimated (Intergovernmental Panel on Climate Change, 2007) seems to increase dryness for the semiarid regions of the world (Bates et al., 2008; Lehner et al., 2005). Therefore, drought in concert with overpopulation will lead to an overexploitation of water resources for agriculture purposes, increase restraints to plant growth and survival and thus reduce crop yield potential (Chaves et al., 2002, 2003; Passioura, 2007) as much as salinity does. The basic physiological responses developed against salinity stress and drought stress overlie with each other as both these stresses eventually lead to dehydration of the cell and osmotic imbalance. However, recent molecular, genomic and transcriptome analyses have shown that many genes and various signalling factors with diverse functions are induced by drought and high-salinity stresses (Seki et al., 2007). Although there has been a remarkable progress in revealing the molecular mechanisms of stress tolerance and responses of higher plants through the development of microarray-based expression profiling methods, together with the availability of genomic and/ or cDNA sequence data, and gene-knockout mutants (Alonso et al., 2003; Cheong et al., 2002; Chinnusamy et al., 2004; Edgar et al., 2002; Seki et al., 2007; Shinozaki and Yamaguchi-Shinozaki, 2007), the understanding how to employ this knowledge to engineer plants with improved stress tolerance is still in the developmental stages.
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In recent years, several hundred genes have been identified that are induced or repressed at the transcriptional level when plants or plant parts are subjected to drought and salinity (Mahajan and Tuteja, 2005; Miller et al., 2010; Saibo et al., 2009; Shinozaki and Yamaguchi-Shinozaki, 2000, 2007; Xiong et al., 2002; Zhu, 2002). The mechanism in which genes are regulated during the stress conditions brings out an important question as well. After the perception of the stress factor by the receptors, the signal is transduced downstream, which induces the generation of second messengers such as calcium, reactive oxygen species (ROS) and inositol phosphates (Mahajan and Tuteja, 2005) to switch on the stress-responsive genes for mediating stress tolerance. One such signal is the plant hormone abscisic acid (ABA) that plays a critical role in response to drought/salinity stresses. ABA treatment imitates the effects of a stress condition, and the concentration of ABA shows increment during stress. Therefore, the expression pattern of stressrelated genes after cold, drought, salinity stresses and ABA application overlaps, suggesting that diverse stress signals and ABA share common aspects in their signalling pathways and these common elements cross-talk with each other, to be able to maintain cellular homeostasis (Finkelstein et al., 2002; Leung and Giraudat, 1998; Shinozaki and YamaguchiShinozaki, 2000). The introduction of many stress-inducible genes through gene transfer significantly improved stress tolerance of transgenic plants (Chen et al., 2009; Hasegawa et al., 2000; Shinozaki and Yamaguchi-Shinozaki, 2000; Zhang, 2003). However, recent molecular and genetic analyses have unravelled that newly identified small RNAs as stress modulators, in addition to small RNAs, RNA processing and chromatin regulation, are also involved in the drought and salinity stress responses (Phillips et al., 2007; Seki et al., 2007; Sunkar and Zhu, 2004) besides the regulation of stress-induced gene expression at both transcriptional and posttranscriptional level by various types of molecules. Regulation by small RNA can cause both transcriptional and posttranscriptional suppression of gene expression (Phillips et al., 2007). Further, in a recent work, the key role of epigenetic regulation in ABAmediated plant mechanisms has been highlighted (Chinnusamy et al., 2008). Chromatin regulators, such as histone deacetylase and linker histone gene, have been shown to be involved in abiotic stress responses of higher plants, and ABA has been shown to induce chromatin remodelling which regulates stress-responsive genes and stress tolerance (Meyer, 2001, Seki et al., 2007). Therefore, it is apparent that for the success of the transgenic plants to exert increased resistance to salt/drought stress, the regulatory role of transcriptomic factors needs to be considered (Chinnusamy et al., 2007).
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Drought and salinity entail oxidative stress accompanied by the formation of ROS due to stomatal closure that restricts CO2 influx through the leaves. ROS such as superoxide (O2.), hydroxyl (OH.) radical, hydrogen peroxide (H2O2) and alkoxyl radical (RO) are produced by enhanced leakage of electrons to molecular oxygen. Chloroplasts, mitochondria and peroxisomes are the major sources of ROS in plant cells (Asada, 1999). Toxic concentrations of ROS disturb normal metabolism through peroxidation of lipid membranes and consequently lead to membrane injury, protein degradation, enzyme inactivation, pigment bleaching and disruption of DNA strands (Fridovich, 1986; McCord, 2000). Nonetheless, oxidative damage in the plant tissue is alleviated by a concerted action of both enzymatic and non-enzymatic antioxidant mechanisms. These mechanisms include carotenoids, -tocopherol, ascorbate, glutathione and enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX) and glutathione reductase (GR) (Miller et al., 2010; Mittler et al., 2004; Smirnoff, 1993). There are many reports in the literature that emphasize the close relationship between enhanced or constitutive antioxidant enzyme activities and increased resistance to drought or salinity stress (Acar et al., 2001; Bor et al., 2003; Demiral and Turkan, 2005; Koca et al., 2007; Ozkur et al., 2009; Seckin et al., 2009, 2010; Sekmen et al., 2007; Turkan et al., 2005; Yazici et al., 2007). Although ROS have the potential to cause oxidative damage to cells during environmental stresses, recent studies have shown that ROS play a key role in plants as signal transduction molecules involved in mediating responses to environmental stresses, pathogen infection, programmed cell death and different developmental stimulus (Mittler et al., 2004; Torres and Dangl, 2005; Verslues et al., 2007). The rapid increase in ROS production, referred to as ‘‘the oxidative burst,’’ has been shown to be crucial for many of these processes. Genetic studies have shown that respiratory burst oxidase homolog (Rboh) genes, encoding plasma membrane-associated NADPH oxidases, are the key producers of signal transduction-associated ROS in cells during these processes (Kwak et al., 2003; Mittler et al., 2004; Torres and Dangl, 2005). In this review, the most recent advances in revealing ROS-mediated signalling under salinity and drought stresses were focused, and the regulatory circuits that allow plants to cope with stress are presented. Some examples were also cited of how osmotic change is sensed and relayed, and the role of some signalling components covering of ROS and ABA has been discussed.
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II. OSMOSENSORS Despite remarkable advances in revealing signalling components during osmotic stress, how plants sense osmotic stress is still an open question. As no plant molecule has actually been identified as an osmosensor so far, the studies were focused on how yeast and microorganisms sense osmotic stress instead. Cellular adaptation to hyperosmotic stress in yeast is mediated by HOG (high osmolarity glycerol) response pathway (Hohmann, 2009). Phosphorylation and hence activity of the MAP kinase Hog1, the yeast orthologue of mammalian p38, are controlled by two branches, the Sln1 (synthetic lethal of N-end rule1) branch and the Sho1 (SH3 domain osmosensor1) branch, which congregate on the MAP kinase kinase (MAPKK) Pbs2. Sho1 branch has been suggested to play a role to direct signalling between the HOG and other MAPK pathways. (SLN1), a two-component histidine kinase, is more sensitive to osmotic changes around and activates full pathway even in the absence of Sho1 branch (O’Rourke and Herskowitz, 2004). Hyperosmotic stress stimulates loss of turgor that leads concurrently to shrinkage of cell volume and an increase in the space between plasma membrane and cell wall. SLN1 possibly senses the change in turgor pressure (Reiser et al., 2003). The two-component regulatory system consists of a phosphorelay among three proteins, Sln1, Ypd1 and Ssk1. Although SLN1 is active under optimal conditions, it is inactivated upon hyperosmotic shock. Active Sln1 is a dimer that autophosphorylates a histidine residue in the N-terminal sensor domain and then transfers the phosphate group to an aspartate residue in the C-terminal-located response regulator domain. The phosphate is then transferred to receiver domain in YPD1, and eventually to the receiver domain in Ssk1. The inactive form of Ssk1, phosphor-Ssk1, is dephosphorylated upon hyperosmotic shock and thus increased concentration of unphosphorylated Ssk1 binds to the regulatory domain of the Ssk2 and Ssk22 MAPKKKs (Posas and Saito, 1998), which allows Ssk2 and Ssk22 to autophosphorylate and stimulate themselves. Active Ssk2 and Ssk22 then phosphorylate and activate Pbs2, which in turn phosphorylates (on Thr174 and Tyr176) and activates Hog1 (Hohmann 2002, 2009). Consequently, activation of HOG pathway up-regulates osmolyte (glycerol) accumulation that play a role in osmoregulation and redox balancing (Hohmann, 2009; Posas et al., 1996; Wurgler-Murphy and Saito, 1997). Hog1 has been suggested to control glycerol accumulation by four ways (Hohmann, 2009): (i) by partly up-regulating the expression of the biosynthetic genes including glycerol-3phosphate dehydrogenase (Gpd1) and glycerol-3-phosphatases (Gpp1 and Gpp2) (Remize et al., 2001; Rep et al., 2000), (ii) enhancing the expression of
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the Stl1 active glycerol uptake system (Rep et al., 2000), which allows glycerol uptake from the surrounding medium (Ferreira et al., 2005); (iii) increasing the activity of phosphofructo-2-kinase to increase the rate of glycerol production and (iv) by controlling the activity of the glycerol export channel Fps1 (Thorsen et al., 2006). Yeast osmoregulating HOG pathway seems very suitable model to study system-level properties of signalling pathways in higher plants. Regarding this, an SLN1 homologue, AtHK1, was identified in Arabidopsis, which also complements SLN1-deficient yeast mutants (Urao et al., 1999). Osmosensing role of AtHK1 has further been shown in transgenic lines of Lycium barbarum plants (Chen et al., 2009). Transgenic L. barbarum plants ectopically expressing AtHK1 exhibited improved tolerance against drought and salinity and faster recovery with subsequent rewatering in comparison to wild-type plants. Further, AtHK1 remarkably prevented oxidative damage caused by ROS through enhancing the activities of antioxidative enzymes such as SOD, CAT and POX (Chen et al., 2009). Chefdor et al. (2006) presented the first proof of osmotic stress sensing multi-step phosphorelay system in a woody species, Populus, by showing the up-regulation of histidine-aspartate kinase (HK1) during osmotic stress and detecting a specific interaction between HK1 and histidine-containing phosphotransfer protein2 (HPt2) through yeast two-hybrid system. HK1 shares the same characteristics as those reported for the yeast (SLN1) and Arabidopsis (ATHK1) osmosensors, which suggest that HK1 in Populus might have the same function as Arabidopsis AtHK1 during osmotic stress. Similarly, a high-affinity Kþ transporter has been cloned in Eucalyptus camaldulensis (EcHKT) showing some similarities to AtHK1 in sensing osmotic changes in external medium (Liu et al., 2001). In accordance with this, Urao et al. (2000) cloned three potential phosphorelay intermediates (ATHP1-1) and four response regulators (ATRR1-4) that might be involved in the step after osmosensing. Further, the results of Reiser et al. (2003) implicated the regulation of plant histidine kinase cytokinin response 1 (Cre1) by changes in turgor pressure, in a similar manner to that of Sln1, in the presence of cytokinin. Tamura et al. (2003) investigated the responses of transgenic tobacco seeds overexpressing NtC7 and found out that overexpression of NtC7 provided osmotic stress tolerance induced by mannitol but not by NaCl. Therefore, NtC7 might be involved in sensing specifically osmotic stress (Bartels and Sunkar, 2005). The interaction of cationic and anionic amphiphilic components with plasma membranes can change the physical status or protein–lipid interactions of membranes that relay osmosensing, and such a mechanism has been described in Lactococcus lactis, where activation of the osmoregulated ABC
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transporter OpuA was mediated by changes in membrane properties and protein–lipid interactions (Heide and Poolman, 2000). More importantly, the activity of major integral membrane proteins involving aquaporins may also be regulated by the changes in the physical state of membranes (Tyerman et al., 2002), gene expression of which is affected by water and osmotic stresses (Kawasaki et al., 2001; Morillon and Chrispeels, 2001). Integrins in mammalian cells are involved in the perception of mechanical stimuli (Shyy and Chien, 1997). Concomitant with this, the important role of the interaction between the cell wall and plasma membrane to sense osmotic stress has recently been highlighted, and the fundamental position of integrin-like protein to mediate this interaction and thus to induce osmotic stress-induced ABA biosynthesis has been revealed in Arabidopsis thaliana (Lu¨ et al., 2007a) and maize (Lu¨ et al., 2007b). The knowledge about the role of integrins in stress responses of higher plants is limited to few studies (Trewavas and Knight, 1994; Zhu et al., 1993). Therefore, the indispensable roles of integrin-like proteins in mediating osmotic stress perception and transmission in higher plants are awaiting further research.
III. SIGNALLING COMPONENTS INVOLVED DURING SALT AND DROUGHT STRESS The early responses of plants to stress are the perception and consequent signal transduction leading to stress-responsive gene expression. As a response to osmotic stress induced by water deficit or high salt, the expressions of a set of genes are altered (Seki et al., 2007). Stress-responsive genes have been identified in many plant species including Arabidopsis (Kreps et al., 2002; Matsui et al., 2008; Seki et al., 2002), Thellungiella halophila (Taji et al., 2004; Wong et al., 2006), sunflower (Hewezi et al., 2008), barley (Oztur et al., 2002), maize (Wang et al., 2003; Yu and Setter, 2003), rice (Gorantla et al., 2007; Kawasaki et al., 2001; Lan et al., 2005; Rabbani et al., 2003), wheat (Gulick et al., 2005), poplar (Brosche et al., 2005), pine (Watkinson et al., 2003), hot pepper (Hwang et al., 2005), potato (Rensink et al., 2005) and sorghum (Buchanan et al., 2005). Although the interaction and exact positions of transduction components in the intricate signalling network are largely unknown, various components of the signal transduction have been characterized. These signalling pathways include a network of protein–protein reactions and signalling molecules that generally increase or decrease in a transient mode (e.g. hormones, ROS, Ca2þ, sugars, etc.). Organisms also regulate cellular processes in response to environmental cues through reversible phosphorylation of proteins. Consequent systemic signals generated
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conduct the management and implementation of plant stress responses with respect to metabolic and developmental adjustments. After the first sensing of osmotic changes during osmotic stress conditions by osmosensors, signal transduction cascade is transduced by protein phosphorylation and dephosphorylation events through various protein kinases and phosphatases, the genes of which have already been shown to be up-regulated by initial perception of dehydration (Lee et al., 1999; Luan, 1998; Zhang et al., 2006). Osmotic stress imposed by drought or high salt is transmitted through at least two pathways; one is ABA-dependent and the other is ABA independent (Mahajan and Tuteja, 2005; Shinozaki and Yamaguchi-Shinozaki, 1996). As the components involved in ABAdependent and ABA-independent pathways often cross-talk or even converge on the signalling pathway, there is no clear-cut discrepancy between two pathways (Kizis et al., 2001; Knight and Knight, 2001; Xiong and Zhu, 2001). Calcium also serves as a second messenger under various stress conditions and mediates cross-talk. Numerous studies have shown that ABA, drought and high salt rapidly increase calcium levels in plant cells (Pardo, 2010; Sanders et al., 1999). The resulting signalling pathway activates various genes that play crucial role to maintain cellular homeostasis. A. ROS SIGNALLING
ROS such as hydrogen peroxide (H2O2), superoxide (O2.), hydroxyl (OH.) radicals and the singlet oxygen (1O2) are produced inevitably as by-products of aerobic metabolism in a cell. Abiotic stresses including drought and salinity result in the enhanced formation of these toxic species by disturbing the metabolic balance of the cells. These compounds indeed have long been considered toxic to the organisms. Recently, however, increasing number of evidence suggests that they play significant role in stress signal transduction (Foyer and Noctor, 2003; Hong-bo et al., 2008; Jaspers and Kangasja¨rvi, 2010; Kacperska, 2004; Miller et al., 2008, 2010; Pardo, 2010). Hence, ROS play a dual role both as toxic compounds and key regulators of biological processes such as growth, cell cycle, programmed cell death, hormone signalling, biotic and abiotic cell responses and development (Foyer and Noctor, 2005; Fujita et al., 2006; Miller et al., 2008). During their long evolutionary history, plants have developed an elaborate and efficient network of ROS-scavenging mechanisms composed of enzymatic and non-enzymatic molecules. Antioxidative enzymes such as SOD, CAT, POX, GR and APX are produced in subcellular organelles with a highly oxidizing metabolic activity such as chloroplasts, mitochondria, peroxisomes or microbodies to overcome ROS toxicity (Bailey-Serres and
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Mittler, 2006; Foyer and Noctor, 2003; Miller et al., 2008). Chloroplast and peroxisomes are considered the two major contributors to the oxidative load in plant cells during abiotic stresses (Miller et al., 2008). It has been shown that ABA accumulation induced by drought triggers the increased generation of ROS, which, in turn, leads to the up-regulation of the antioxidant defence system in plants (Hu et al., 2005; Jiang and Zhang, 2002a,b). ABA has been reported to induce the expression of antioxidant genes encoding SOD, CAT and APX (Guan and Scandalios, 1998; Guan et al., 2000; Park et al., 2004) and enhance the activities of these antioxidant enzymes in plant tissues (Bellaire et al., 2000; Bueno et al., 1998; Jiang and Zhang, 2001, 2002a, b, 2003). A rapid increase in the production of H2O2, OH. and O2. was observed in Arabidopsis guard cells after they were induced to close by ABA (Pei et al., 2000), and the effect was concentration dependent. These results bear a question about the source of ROS in the cell induced by ABA. Among potential sources of ROS in the cell, chloroplasts, mitochondria and peroxisomes, plasma membrane NADPH oxidases (Rbohs for respiratory burst oxidase homologues), cell wall POX, apoplastic oxalate oxidases and amine oxidases are remarkable (Ming-Yi and Jian-Hua, 2004; Mittler, 2002; Neill et al., 2002). Saline soils are often recognized by the presence of white salt encrustation on the surface and have predominance of chloride and sulphate of sodium, calcium and magnesium in quantities sufficient to interfere with growth of most crop plants. Regulation of NaCl responses are controlled by ROS involvement. Rapid increase in cytosolic calcium induced by Naþ within seconds is sensed by calcineurin B-like (CBL) protein CBL4/SOS3 and its interacting protein kinase CIPK24/SOS2 (Luan, 2009; Munns and Tester, 2008; Zhu et al., 2007). The resulting SOS3/SOS2 complex phosphorylates and activates SOS1, a plasma membrane Naþ/Hþ antiporter. Naþ/Hþ antiport activity of SOS1 promotes efflux of excess Naþ ions and hence contributes to Naþ ion homeostasis (Fig. 1; reviewed comprehensively in Munns and Tester, 2008; Turkan and Demiral, 2009). Recent evidence has indicated that Naþ-induced stability of SOS1 transcripts upon exposure to NaCl stress is mediated by ROS generated by NADPH oxidase (Chung et al., 2008). The ROS generated by NADPH oxidase were then suggested to stabilize SOS1 mRNA, thus greatly increasing its activity and consequently NADPH oxidase activity (Chung et al., 2008). NaCl-induced Ca2þ spikes and consequent SOS1 activation cause apoplastic alkalization, which consequently activate Rboh (Chung et al., 2008). These results indicate the potential role of SOS1 at early signalling step of a signal transduction pathway that is common to several abiotic stresses including drought and salinity.
Salt stress Osmotic stress
[Na Salt ? ? sensor
SOS3
P
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+
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Fig. 1. SOS signalling pathway for salt stress adaptation in higher plants. The interaction among SOS1, SOS2 and SOS3 was explained in the text. SOS4 gene encodes a pyridoxal (PL) kinase that play a role in the biosynthesis of PL-5-phosphate (PLP), which contributes Naþ and
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SOS2 kinase also plays a special role in signalling process through its interaction with triphosphate kinase 2 (NDP kinase2), CAT2 and CAT3 (Verslues et al., 2007). Moon et al. (2003) revealed previously uncharacterized regulatory role of NDP kinase2 in ROS signalling by showing its involvement in H2O2-induced activation of MPK3 and MPK6. Further, mutants lacking AtNDPKinase2 had increased sensitivity to salinity stress (Moon et al., 2003), and sos2-2 ndpkinase2 double mutants further deteriorated the sensitivity of sos2 mutants to salinity (Verslues et al., 2007). A sos2-2 ndpkinase2 double mutant did not accumulate H2O2 in response to salt stress, suggesting that it is a change in signalling rather than H2O2 toxicity alone that is responsible for the increased salt sensitivity of the sos2-2 ndpkinase2 double mutant in comparison to single mutants. Very recently, an additional sos mutant SOS6, encoding a cellulose synthaselike protein, AtCSLD5, has been identified by Zhu et al. (2010). sos6-1 mutant plants have been shown to accumulate higher levels of ROS after exposure to osmotic stress than the wild-type plants and are hypersensitive to the oxidative stress reagent methyl viologen (MV). These results suggested that SOS6 and SOS6-dependent cell wall components might control osmotic stress tolerance partly by regulating and maintaining stress-induced ROS levels in plant cells (Zhu et al., 2010). High accumulation of ROS in sos6-1 mutant upon exposure to osmotic stress might imply its specific role in osmotic sensing as well. Plasma membrane NADPH oxidase, which transfers electrons from cytoplasmic NADPH to O2 to form O2., has been shown one of the major sources of ROS generation induced by ABA and drought stress (Zhu et al., 2006). Jiang and Zhang (2002a), using two-phase fractionated plasma membrane extracts and several widely used NADPH oxidase inhibitors, such as DPI, imidazole and pyridine, have demonstrated that NADPH oxidase is involved in ABA and drought stress-induced ROS production, and drought stress-induced ABA accumulation plays important role in the regulation of
Kþ homeostasis by regulating ion channels and transporters. SOS5 is involved in the maintenance of cell expansion. Dashed arrow shows SOS3-independent and SOS2dependent pathway. Formation of self-amplifying loop through the activity of plasma membrane NADPH oxidase (Rboh) is modulated by ABA-induced activation of Ca2þ channels. Ca2þ is involved in Rboh activation as well as serving as a target for the Rboh product (ROS) (Jaspers and Kangasja¨rvi, 2010; Mittler et al., 2004; Sagi and Fluhr, 2006). (The figure was modified from Chinnusamy et al., 2005; Jaspers and Kangasja¨rvi, 2010; Mahajan and Tuteja, 2005; Mahajan et al., 2008; Shi and Zhu, 2002; Turkan and Demiral, 2008, 2009; Zhang et al., 2004; Zhu, 2003).
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NADPH oxidase activity in maize leaves. Similarly, Hu et al. (2005) pointed out the importance of apoplastic H2O2 accumulation induced by ABA to induce the cytosolic antioxidant enzyme activities. These results suggest that NADPH oxidase contributes to early ABA signalling. NaCl stress comprising both ionic and osmotic stresses has recently been shown to induce formation of endosomes containing high concentrations of H2O2 in Arabidopsis cells (Leshem et al., 2006, 2007). Phosphatidylinositol 3-kinase (PI3K)-dependent plasma membrane internalization and ROS production have been triggered within endosomes of Arabidopsis root cells (Leshem et al., 2007). Endosomal membrane in root cells has rolled up ROS which is possibly the product of NADPH oxidase in response to salinity stress. pi3k mutants exhibited reduced oxidative stress but enhanced salt sensitivity due to suppression of the salt-specific induction of NADPH oxidase-mediated ROS production within endosomes (Leshem et al., 2007). Further, level of ROS in endosomes of root hair cells was reduced after treatment with a PI3K-specific inhibitor LY294002, suggesting that PI3K is essential for ROS generation inside endosomes and thus for the final stage of endocytosis in tip-growing root hair cells (Lee et al., 2008). These results suggest new vital regulatory roles of ROS in intracellular trafficking trough vesicles for developmental control of organelle biogenesis in addition to their role in retrograde stress signalling (Miller et al., 2010). B. ABA AND STRESS SIGNALLING THROUGH ROS
ABA can be produced both in shoots and roots as a response to salinity and drought stresses. ABA produced in the roots is transported to the shoots, thus causing stomatal closure and eventually restricts cellular growth (Wilkinson and Davies, 2002). Xylem/apoplastic pH has been shown to affect ABA compartmentation and accordingly the amount of ABA arriving at the stomata. Drought, high light and salinity conditions might lead to a higher xylem sap pH (Jia and Davies, 2007) facilitating the modulation of stomatal aperture in response to a variety of environmental variables. The resulting more alkaline pH in xylem/apoplast decreases the removal of ABA from xylem and leaf apoplast to the symplast thus allowing more ABA to reach the guard cells. Control of stomatal aperture is strongly regulated by ROS–ABA signalling as ABA has been shown to enhance the expression of the genes encoding CAT1, APX1, GR1 and their activities (Cho et al., 2009) and also the activities of cytosolic Cu/ZnSOD, APX and GR in leaves of maize plants (Hu et al., 2005). ABA-induced stomatal closure is partially dependant on NADPH oxidase activity (Kwak et al., 2003; Pei et al., 2000; Torres and Dangl, 2005). ABA and drought stress induced the activities of
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cytosolic aldehyde oxidase (AO) and xanthine dehyrogenase (XDH) that produce, respectively, H2O2 and O2. (Guan and Scandalios, 2000; Hu et al., 2005; Yesbergenova et al., 2005; Zhang et al., 2001), which suggested that drought can enhance ROS accumulation through XDH and AO in an ABA-dependent manner (Yesbergenova et al., 2005). Confirming this, ABAdeficient mutants of Arabidopsis, tomato and tobacco plants did not show AO and XDH activities (Leydecker et al., 1995; Sagi et al., 1999; Schwartz et al., 1997). Further, stomatal conductance is regulated by the signal transduction initiated by photorespiratory glycolate oxidase reaction in peroxisomes under certain stresses including drought, osmotic stress and salinity, highlighting the importance of redox homeostasis as suggested by Foyer and Noctor (2005). However, stomatal closure by ABA is also controlled through ion channels such as SLAC1 and KAT1 which are activated by phosphorylation via an ABA-activated protein kinase OST1 (open stomata1) and Ca2þ (Mustilli et al., 2002; Siegel et al., 2009). One of the targets of OST1 is NADPH oxidase that generates H2O2 (Pei et al., 2000; Sirichandra et al., 2009). Enhanced production of H2O2 intercedes stomatal closure by inactivating ABI1 and ABI2, which were shown to be very sensitive to H2O2 and oxidation (Meinhard and Grill, 2001; Meinhard et al., 2002). OST1-dependent H2O2 production could start release of further active OST1 by PP2C inactivation in a positive feedback loop (Raghavendra et al., 2010). C. ANTIOXIDATIVE SIGNALLING
ROS-scavenging enzymes have been shown to be involved in signalling as well as their more customary role in protection from oxidative stress in recent years (Chen et al., 2005; Miller et al., 2010). Although overproduction of antioxidant enzymes in transgenic plants has been resulted in enhanced tolerance to drought and salinity stresses (Eltayeb et al., 2007; Lu et al., 2007; Tseng et al., 2007; Yan et al., 2003), in some cases their reduced expression unexpectedly resulted in tolerant plants. For example, tobacco was engineered to repress APX and CAT expression levels, individually and together by Rizhsky et al. (2002). Double antisense plants exhibited more tolerance than in plants that lacked only APX or CAT against oxidative damage by suppressing photosynthetic activity, up-regulating the pentose phosphate pathway, enhancing monodehydroascorbate reductase activity and inducing a chloroplast homologue of the mitochondrial alternative oxidase (AOX) (Rizhsky et al., 2002). Likewise, each of antisense APX1 and antisense CAT1 tobacco plants was constantly subjected to oxidative damage, but the double antisense lines exerted more tolerance (Rizhsky et al., 2002). Consistent with this, double mutant Arabidopsis plants apx1/tylapx
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showed enhanced sensitivity to sorbitol treatment while sustaining salinity tolerance (Miller et al., 2007). Even though apx1 plants show increased sensitivity to photo-oxidative as well as paraquat-induced oxidative stress (Davletova et al., 2005; Miller et al., 2007), they grew better than wild-type plants under hyperosmotic or salinity condition (Ciftci-Yilmaz et al., 2007; Miller et al., 2007). Similarly, reduced expression of tylAPX in Arabidopsis led to increased tolerance to both osmotic and salt stresses but did not affect growth under oxidative stress conditions. Arabidopsis plants lacking cytosolic APX1 had constitutively higher levels of H2O2 than wild-type plants and induced the expression of many stress-responsive genes when exposed to a modest level of light stress (Davletova et al., 2005). Likewise, antisense repression of AOX also leads to considerably higher ROS production, whereas AOX overexpression has the opposite effect (Maxwell et al., 1999; Parsons et al., 1999). Moreover, mutant Arabidopsis plants aox1a, deficient in mitochondrial AOX1a, showed higher sensitivity to a combination of drought and moderate light stress (Giraud et al., 2008). It appears that integrated signalling networks are responsible for the activation of acclimation pathways. Moreover, while some changes in ROS metabolism cause enhanced tolerance to stress, other changes cause enhanced sensitivity (Miller et al., 2010).
IV. CONCLUDING REMARKS Numerous types of plant stress conditions enhance the production of ROS along with ABA accumulation, which has been suggested to be key constituents of ‘cross tolerance’ to multiple types of stresses. Previously, the oxidative stress was used to be considered as a harmful event to be avoided or alleviated but is now viewed as an advantage for the plant to appropriately respond and induce adequate acclimation mechanisms. Increasing body of evidence suggests the vital importance of ROS in signal transduction. Links between ROS and hormone signalling have already been suggested (Cho et al., 2009; Guan et al., 2000; Kwak et al., 2003; Raghavendra et al., 2010; Zhu et al., 2006). Further, one of the emerging issues that must be kept up with the twenty-first century is to increase water use efficiency (WUE) of cultivated plants facing an environment with increasing CO2 concentrations and global temperature. With regard to this, getting the hang of stomatal response to osmotic stress is rather vital, and recently considerable progress has been made in this respect by researchers. Therefore, understanding the perception and transmission of osmotic stress signal from plasma membrane to the nucleus and then regulation of gene expression to give a better response is crucial to enhance WUE in plants. Thus, future issues to be
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addressed concern the questions of how ROS are incorporated into the general signalling network of a cell, in which metabolic processes are the source of ROS and could lead to the breakthrough of new signalling functions for well-known metabolic enzymes, and what factors determine the specificity of the biological activities of ROS. Filling these gaps in our knowledge is an urgent need to avert a looming crisis of global warming and its side effects on plants.
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An Overview of the Current Understanding of Desiccation Tolerance in the Vegetative Tissues of Higher Plants
MONIQUE MORSE, MOHAMED S. RAFUDEEN AND JILL M. FARRANT1
Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Global Stresses Caused by Desiccation and Associated Protective Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mechanical Stress and Mechanisms used to Minimize such Damage .. B. Metabolic Stresses and Associated Protection Mechanisms ............. III. Proteomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Whole Proteomic Approaches ................................................ B. Subcellular Proteomics Approaches ......................................... IV. Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT In this chapter, we review the current understanding of desiccation tolerance in the vegetative tissues of resurrection plants. We present an overview of the stresses associated with desiccation and the physiological and biochemical protection reported to result in amelioration of these stresses and discuss the contribution of the genomics era in furthering our understanding of these protection systems in the
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Corresponding author: E-mail:
[email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00009-6
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attainment of desiccation tolerance. We discuss the advances made in proteomics and give a brief overview of recent contributions in the field of metabolomics that have contributed to the understanding of desiccation tolerance.
I. INTRODUCTION The phenomenon of desiccation tolerance (DT) is found throughout the microbial, fungal, animal and plant kingdoms (Alpert, 2006; Farrant, 2007; Ricci and Caprioli, 2005) and is the ability of an organism to survive the loss of most (> 95%) of its cellular water for extended periods and to recover full metabolic competence upon rehydration (Farrant et al., 2007). The commonly held definition of DT is the ability to survive drying to the air-dry state at relative humidities 65%, this usually bringing the absolute water content of the tissue to or below 0.1 g H2O g–1 dry mass (g g–1) and corresponding to a water potential of 100 MPa (Vertucci and Farrant, 1995; Walters et al., 2005). In the plant kingdom, it is relatively common in reproductive tissues such as spores, seeds and pollen (Berjak et al., 2007) and in vegetative tissues of non-tracheophytes, such as bryophytes and lichens (Kappen and Valladares, 1999; Oliver et al., 2000). However, DT in vegetative tissue is rare in pteridophytes and angiosperms and non-existent in extant gymnosperms (Alpert and Oliver, 2002; Farrant, 2007; Gaff, 1989). The mechanisms of vegetative DT differ between the lower and higher orders. In the former, desiccation occurs very rapidly, and protection prior to drying is minimal and constitutive. Survival is thought to be based largely on rehydrationinduced repair processes (Alpert and Oliver, 2002; Oliver et al., 1998). In angiosperms, while some repair is probably inevitable, considerable and complex protection mechanisms are laid down during drying (Bartels, 2005; Blomstedt et al., 2010; Farrant, 2007; Gaff, 1989; Moore et al., 2009), and increasingly, it is becoming evident that many of the protection systems instituted in vegetative tissues of these plants, commonly called ‘resurrection plants’, are similar to those described for DT (orthodox) seeds (Illing et al., 2005; Leprince and Buitink, 2010). While the acquisition of DT in seeds is part of a maturation programme in seed development, it is possible that resurrection plants enlist expression of these genes within vegetative tissues to survive desiccation. Understanding of the regulatory processes of how this is achieved in vegetative tissues of resurrection plants may well ultimately enable induction of an appropriate selection of these genes in crops for drought tolerance. In this chapter, we will review the current understanding of vegetative DT in resurrection plants, and where appropriate draw parallels with DT described in orthodox seeds. In the first part of this work, we will present an
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overview of the stresses associated with desiccation and the physiological and biochemical protection reported to date to result in some amelioration of these stresses. We will also discuss the contribution of the genomics era in furthering our understanding of these protection systems and its role in discovery of other putative protectants and the mechanisms of control of these in the acquisition of DT. The second part of this work will focus on the post-genomic era, specifically the advances made in proteomics towards understanding DT in the vegetative tissues of higher plants. Finally, we will conclude with the recent advances in metabolomics studies in resurrection plants to date.
II. GLOBAL STRESSES CAUSED BY DESICCATION AND ASSOCIATED PROTECTIVE MECHANISMS In plant tissues, the role of water is complex and varied. It fills intra- and intercellular spaces providing turgor pressure and structural support, termed mechanical stabilization (Iljin, 1957; Levitt, 1980). It is involved in metabolism as both a reactant and a product of many processes, and it is the medium in which the intracellular milieu is suspended. By providing hydrophobic and hydrophilic interactions, it determines conformation of macromolecules and membranes and controls and maintains intracellular distances between them (Buitink et al., 2002; Hoekstra et al., 2001; Vertucci and Farrant, 1995; Walters et al., 2002). Even slight water loss can cause disruption of the mechanical and metabolic stability, and resurrection plants appear to have unique mechanisms to minimize such disruption in the face of near total water loss. While there are some obvious differences among the various angiosperm resurrection plants in their mechanisms of protection against such stresses (reviewed below and also in Farrant, 2007; Vicre´ et al., 2003), there are considerable similarities in putative protection mechanisms among them and, as indicated above, orthodox seeds. A. MECHANICAL STRESS AND MECHANISMS USED TO MINIMIZE SUCH DAMAGE
During desiccation, cells lose most of the protoplasmic ‘free or bulk’ water, and only the ‘bound water’—water associated with the cell matrix—is available for cell survival. This loss of water causes mechanical stress as decreased cell volume places tension on the plasmalemma as it shrinks from plasmadesmatal attachments to the cell wall, the ultimate rupture of which allows entry of extracellular hydrolases and cell death. In many species, wall
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collapse occurs which is equally lethal (Walters et al., 2002). Angiosperm resurrection plants are able to survive these changes by active induction of protection mechanisms that allow avoidance of plasmalemma rupture and wall collapse. This is achieved by active and reversible wall folding and/or replacement of water in the vacuoles with compatible solutes (reviewed in Farrant, 2007). Desiccation-induced cell wall folding is essential for structural preservation of tissue (Webb and Arnott, 1982), and the extent and manner of folding is species-specific and dependent upon the chemical composition and molecular architecture of the cell wall. While the overall wall composition of the resurrection species is similar to other related desiccation-sensitive species, the resurrection species appear to utilize inherent wall characteristics, with only slight modifications during drying, to achieve stable and reversible conformational changes (Farrant et al., 2007). In Craterostigma wilmsii, where wall folding is almost exclusively used as a form of mechanical stabilization, the mechanism of folding appears to involve more complex structural and biochemical changes (Vicre´ et al., 1999, 2004b). On drying, there is a reduction in glucose and an increase in galactose substitutions to the xyloglucans (XG) and it has been proposed that cleavage, or partial cleavage of the long-chained XG units into shorter, more flexible ones, allows for wall folding. During the final stages of drying, an increase in wall-associated Ca2þ occurs, and as this ion plays an important role in cross-linking wall polymers, such as acid pectins, it has been proposed that this serves to stabilize walls in the dry state and, more importantly, prevent mechanical stress of rehydration. C. wilmsii is a small plant, and rehydration is rapid and is initially mainly apoplastic (Sherwin and Farrant, 1996). If walls hydrate and unfold before cell volume is regained, plasmalemma tearing and further subcellular damage could occur (reviewed in Vicre´ et al., 2003, 2004a). In species where wall folding is accompanied by vacuole filling as a mechanism of mechanical stabilization, such as in Myrothamnus flabellifolia and Eragrostis nindensis, there are no notable biochemical changes on drying, but these species have constitutively high proportions of arabinose, associated with pectins in the former (Moore et al., 2006) and xyloglucans in the latter (Plancot et al., 2009). Interestingly, the desiccation-sensitive Eragrostis tef, while having similar chemical wall constituents as E. nindensis, has significantly lower arabinose content. As arabinose polymers are highly mobile and allow wall flexibility (Foster et al., 1996; Renard and Jarvis, 1999) and have a high water absorbing capacity (Belton, 1997; Goldberg et al., 1989) which would be important for rehydration, we have proposed that such constitutively high levels allow constant preparedness for dehydration/rehydration in these resurrection plants (Moore et al., 2009).
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In addition to these biochemical and ultrastructural studies, transcriptomic studies on at least three species of resurrection plant have reported on genes potentially involved in wall changes upon desiccation and recovery. Genes encoding glycine-rich proteins (GRP) have been shown to be upregulated upon drying in Sporobolus stapfianus (Neale et al., 2000) and Boea hygrometrica (Wang et al., 2008). These include a GRP and proline-rich protein (SDG137c) and a small GRP (SDG137c) in the former species, while a GRP (BhGRP1) was upregulated in the latter species. A BhGRP1GFP fusion protein was located to cell walls of B. hygrometrica and the desiccation-sensitive control species Nicotiana benthamiana (Wang et al., 2008). While the function of these genes was not elaborated upon by the authors of those papers, GRPs can facilitate structural flexibility in developing walls of most plants, and their synthesis is stimulated under stress conditions (Mousavi and Hotta, 2005). It is thus possible that upregulation of such proteins in resurrection plants enables wall folding upon dehydration and unfolding upon rehydration. Three -expansin genes (CplExp1/2/3) have been implicated in desiccation-associated wall changes of Craterostigma plantagineum (Jones and McQueen-Mason, 2004). All three genes were upregulated upon drying, but upon rehydration of this species, CplExp3 expression remained largely unchanged, while expression of CplExp1 increased as the leaf regained full turgor (Jones and McQueen-Mason, 2004). Expansin proteins are proposed to increase wall elasticity and thus may facilitate wall folding observed to occur in Craterostigma spp. during desiccation (Vicre´ et al., 1999). Replacement of water in vacuoles within dry tissues of resurrection plants was first suggested based on ultrastructural observations that vacuoles continued to take up a large proportion of the cytoplasmic space despite the fact that there was no longer bulk water available in tissues, the remaining water being purely structure associated (Farrant, 2000; Farrant et al., 2007; Moore et al., 2007a,b; van der Willigen et al., 2001). The content of vacuoles from desiccated leaves of Eragrostis nindensis was analyzed after non-aqueous extraction and was shown to contain proline, sucrose and protein in equal proportions (van der Willigen et al., 2004a,b). Vacuoles from both hydrated and dry leaves of Myrothamnus flabellifolia contain 3,4,5-tri-O-galloylquinic acid, but this chemical increases to entirely fill the vacuoles in dry leaves (Moore et al., 2005b, 2007a,b). Use of highthroughput GC–MS to analyse metabolome changes in Mohria caffrorum showed 10- and 12-fold increases in glycerol and monohexadecanoglycerol, respectively, during drying, and as these chemicals are believed to be cytotoxic in large quantities (Fahy, 1986), we proposed that they accumulated in vacuoles within the dry leaves (Farrant et al., 2009). Studies using
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metabolomic protocols are currently underway in which vacuolar content of a variety of resurrection plants is being analyzed.
B. METABOLIC STRESSES AND ASSOCIATED PROTECTION MECHANISMS
Loss of water interferes with metabolic processes and, at the extreme level, results in loss of membrane structure and causes metabolic destabilization within the cell. There is increased aggregation of essential macromolecules which results in the disintegration of organelles due to increased macromolecule concentration (Hoekstra et al., 2001; Vertucci and Farrant, 1995). A commonly noted consequence of water deficit-related disruption of metabolic processes is the increasing formation of reactive oxygen species (ROS) (Berjak, 2006; Hendry, 1993; Kranner et al., 2006; Smirnoff, 1993; Walters et al., 2002). Overproduction of ROS causes damage to macromolecules and subcellular components by reacting with proteins and lipids. In all plants, ROS form as a natural consequence of metabolic processes involving electron transport (Apel and Hirt, 2004; Bailly, 2004; Halliwell and Gutteridge, 1999). Thus, mitochondria and chloroplasts are major sites of ROS production. Photosynthesis, in particular, is very sensitive to water deficit. Electron leakage during photosynthetic electron transport and the formation of singlet oxygen are significantly increased when cells of photosynthetic tissues suffer water loss, and this has frequently been cited as a primary cause of damage and resultant plant death in most species (Kranner and Birtic´, 2005; Seel et al., 1992a,b; Smirnoff, 1993). Under mild deficit stress, ROS are effectively quenched by what are termed ‘classical’ (Kranner and Birtic´, 2005) or ‘housekeeping’ antioxidants (Illing et al., 2005), so called because they are present in all plants and are crucial to maintenance of cellular homeostasis under day-to-day conditions and in protection against a myriad of abiotic and biotic stresses (for an overview, see Elstner and Osswald, 1994). However, with more severe water loss, such antioxidants are themselves compromised, and ROS damage is exacerbated (Farrant et al., 2007; Kranner et al., 2006). Even in the desiccated state when metabolic activity has ceased, ROS can still be generated through auto-oxidation processes, for example, of lipids, and damage to cellular macromolecules continues to occur as a result of Maillard reactions (Bailly, 2004; Wettlaufer and Leopold, 1991). Resurrection plants appear to protect against the potential damage of desiccation-induced ROS production by: (1) minimizing the formation of photosynthesis-associated ROS; (2) more effective use of housekeeping antioxidants and (3) production of novel antioxidants in response to drying. These will be reviewed below.
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1. Regulation of photosynthesis-associated ROS production The extent of ROS production is effectively minimized by downregulation of photosynthesis relatively early in the dehydration time course, usually between 80% and 65% relative water content (RWC) depending on the species (Farrant, 2000; Farrant et al., 2003; Illing et al., 2005; Sherwin and Farrant, 1998; Tuba et al., 1998; van der Willigen, et al., 2001). Downregulation of photosynthesis is achieved by one of two mechanisms: termed poikilochlorophylly and homoiochlorophylly (Farrant, 2000; Gaff, 1989; Sherwin and Farrant, 1998; Smirnoff, 1993; Tuba et al., 1998). Poikilochlorophyllous types, many of which are monocots, break down chlorophyll and dismantle thylakoid membranes during dehydration (Farrant, 2000; Hambler, 1961; Tuba et al., 1993a,b, 1998). Breakdown of photosystem II (PSII), which is responsible for the water-splitting, oxygen evolving and thus oxidizing reactions of photosynthesis, is a highly effective strategy to minimize damaging levels of ROS formation, and indeed it has been shown that poikilochlorophyllous species are able to retain viability in the dry state for far longer than homoiochlorophyllous ones (Proctor and Tuba, 2002; Tuba et al., 1998). However, as the photosynthetic apparatus has to be reassembled on rehydration, recovery time is generally longer in these species (Farrant et al., 2003; Sherwin and Farrant, 1996). Molecular studies have shown that partial recovery of photosystem II function is independent of transcription and that some transcripts such as PsbA and PsbD are stably stored in the dry state (Collett et al., 2003; Dace et al., 1998), while others are transcribed de novo on rehydration with water being the primary cue for induction of transcription although presence of light is ultimately essential for synthesis of chlorophyll-binding proteins and assembly of the grana (Ingle et al., 2008). Homoiochlorophyllous species retain most of their chlorophyll (the amount retained depending on the light levels under which the plants are dried) and thylakoid membranes in the dry state but use various mechanisms to prevent the light-chlorophyll interactions that might cause ROS production during drying and rehydration (Farrant, 2000; Farrant et al., 2003, 2009; Sherwin and Farrant, 1998). This is achieved by leaf folding and shading of inner leaves (e.g. the Craterostigma spp.) or adaxial surfaces (e.g. M. flabellifolius, M. caffrorum) from light. Surfaces that remain exposed to light have reflective hairs and/or waxes, and there is accumulation of anthocyanin and xanthophylls pigments and polyphenols all of which act as ‘sunscreens’ reflecting back photosynthetically active light, masking chlorophyll and acting as antioxidants (Farrant, 2000; Farrant et al., 2003, 2009; Georgieva et al., 2007, 2009; Moore et al., 2007a,b; Sherwin and Farrant, 1998; Smirnoff, 1993).
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2. Antioxidant systems As outlined above, resurrection plants use housekeeping antioxidants to minimize ROS damage during water loss but appear to have the additional capacity to maintain function of their such antioxidant capacity in the desiccated state or quickly resynthesize antioxidants upon rehydration (Bresler, 2010; Farrant et al., 2007; Kranner and Birtic´, 2005; Pukacka and Ratajczak, 2007). They also use ‘novel’ antioxidants in that they are newly described as having antioxidant properties and/or have not to date been associated with vegetative tissues but reported to occur only in seeds (Moore et al., 2005a,b; Mowla et al., 2002; Mulako et al., 2008). Housekeeping antioxidants are essential for maintenance of redox homeostasis by scavenging excess ROS under normal and mildly stressful conditions. These include the water-soluble glutathione (g-glutamyl-cysteinyl glycine; GSH) and ascorbic acid (Asc) (Noctor and Foyer, 1998), the lipid soluble tocopherols and -carotene (Munne-Bosch and Alegre, 2002) and enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (AP) and other peroxidases, mono- and dehydroascorbate reductases and glutathione reductase (GR). There have been numerous reports on the activities of such antioxidants during drying and recovery in various resurrection plants. From this literature, there appears to be considerable variation between DT species with respect to the extent of upregulation of the various housekeeping antioxidants, and the water contents during a dehydration/rehydration time course that the observed changes occur (e.g., reviewed in Farrant, 2000; Farrant et al., 2003; Illing et al., 2005). It is difficult to know whether this variation is real, as reports in the literature are controversial. The conditions under which plants are dried vary, frequently the water content to which the tissues are dried is not presented and/or the activity on rehydration is not recorded, or the manner of quantification differs. Further, use of antioxidant concentrations alone has limitations, as they often show a Gaussian response to stress (Kranner et al., 2006) making interpretation of a single measurement ambiguous. However, what appears to be a distinguishing feature of the functioning of these antioxidants in resurrection plants is the ability to maintain their antioxidant potential in the dry state such that the same antioxidants can be utilized during the early stages of rehydration thus protecting against the ROS stress associated with reconstitution of full metabolism (reviewed in Farrant, 2007; Moore et al., 2009). Farrant et al. (2007) have shown that the enzymes AP, GR, CAT and SOD retain the ability (in vitro assays) to detoxify ROS even at RWC of < 10%, suggesting that there is some protection of these proteins that prevents their denaturation and maintains the native state in dry conditions. This was not the case in DS species and it
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has been proposed that this ability in resurrection plants is a unique DT mechanism (Farrant et al., 2007; Illing et al., 2005). The Smirnoff–Wheeler pathway of ascorbate synthesis in plants has recently been established by the discovery of VTC2 as being the gene responsible for the first committed step to ASC synthesis (Linster and Clarke, 2008; Linster et al., 2007). Bresler (2010) has shown that transcription of VTC2 in the resurrection plant Xerophyta viscosa is upregulated when the plants are dried below 60% RWC, and that mRNA levels remain high in the desiccated plant and during early stages of rehydration. Ascorbate levels in roots and leaves of this plant follow the same trend (Kamies et al., 2010), and we propose that elevated ascorbate levels are maintained during drying and early rehydration by both a combination of de novo synthesis and regeneration of ascorbate by AP, which itself retains the ability to remain active (Farrant et al., 2007; Kamies et al., 2010). This compares well with data by Suarez-Rodriguez et al. (2010) who show the AP transcript to be more abundant in the desiccated leaves of C. plantagineum with increasing levels in rehydrated leaves. In addition to the protection afforded by housekeeping antioxidants, resurrection plants have the ability to induce, de novo, antioxidants such as 1- and 2-cys-peroxiredoxins, glyoxylase I family proteins, zinc metallothionine, metallothionine-like antioxidants and several members of the aldehyde dehydrogenases in response to desiccation (Blomstedt et al., 1998; Chen et al., 2002; Collett et al., 2004; Farrant et al., 2007; Illing et al., 2005; Kirch et al., 2001a,b; Mowla et al., 2002; Mulako et al., 2008; Velasco et al., 1994). While these have been reported to be important in the acquisition of DT of orthodox seeds, they are never found to be upregulated in the DS vegetative tissues of such plants (Aalen, 1999; Stacy et al., 1999). Increasingly, transcriptome and proteome studies are reporting the upregulation of genes and proteins annotated as potential antioxidants in response to drying in resurrection plants (Collett et al., 2004; Deng et al., 2006; Ingle et al., 2007; Jiang et al., 2007; Le et al., 2007; Suarez-Rodriguez et al., 2010). Polyphenols are widely believed to have antioxidant potential (Kahkonen et al., 1999; Smirnoff, 1993; Wang et al., 1996) and may well play such a role in resurrection plants. In a relatively comprehensive study on polyphenols in M. flabellifolia, it has been shown that there is extensive accumulation (up to 50% of the leaf dry weight) of 3,4,5-tri-O-galloylquinic acid upon desiccation and that this compound acts as a potent antioxidant in vitro (Moore et al., 2005a,b). Although this polyphenol is predominantly located in the vacuole and cell wall, it has been proposed that these reservoirs act to absorb electrons from the cytoplasmically located antioxidants and act in a redox buffering capacity (Moore et al., 2007a,b). Support for the antioxidant role of polyphenols and phenolic antioxidant enzymes has been gained from
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studies on Ramonda serbica, in which polyphenol content and activity of polyphenol oxidase (PPO) were shown to be enhanced on drying (Sgherri et al., 2004; Veljovic-Jovanovic et al., 2008). 3. Stabilization of the subcellular milieu Upon water loss to 10% RWC, the hydrophobic effect of water that is essential for the maintenance of macromolecular and membrane structure is lost and irreversible subcellular denaturation occurs. Theories on mechanisms whereby this is achieved are thought to be due to the ability to substitute water with molecules that form hydrogen bonds that are able to stabilize macromolecular interactions in their native configuration (Crowe et al., 1986, 1987, 1998). In addition to water replacement, stabilization of the subcellular milieu is thought to be brought about by vitrification of the cytoplasm by the same candidates achieving macromolecular stabilization (Hoekstra et al., 2001; Leopold, 1986; Leopold et al., 1994; Vertucci and Farrant, 1995; Walters, 1998). The candidates for such replacement/stabilisation reactions are given as (a) sugars, particularly sucrose together with oligosaccharides (reviewed in Berjak, 2006; Illing et al., 2005; Scott, 2000); (b) proteins, particularly late embryogenesis abundant (LEA) proteins (reviewed by Illing et al., 2005; Mtwisha et al., 2006) and (c) small heat shock proteins (Almoguera and Jordano, 1992; Mtwisha et al., 2006; Wehmeyer et al., 1996). Physiological and biochemical studies have gone some way in showing the importance of sugars, and to some extent, LEA proteins in attainment of DT, but understanding of the role and contribution of proteins and metabolites is still in its infancy. a. Sugars. In virtually all resurrection plants studied to date, there is accumulation of sucrose during drying (Bartels and Salamini, 2001; Bianchi et al., 1991; Farrant, 2007; Ghasempour et al., 1998; Illing et al., 2005; Norwood et al., 2000, 2003; Peters et al., 2007; Whittaker et al., 2001, 2004). Sucrose is also universally accumulated in orthodox seeds (Amuti and Pollard, 1977; Berjak, 2006; Koster and Leopold, 1988; Pammenter and Berjak, 1999; Vertucci and Farrant, 1995), suggesting that sucrose plays an important role in DT in general. In both systems, the accumulation of raffinose family oligosaccharides (RFOs), particularly raffinose and stachyose, also occurs (see e.g., Blackman et al., 1992; Ghasempour et al., 1998; Horbowicz and Obendorf, 1994; Koster and Leopold, 1988; Leprince et al., 1990; Obendorf, 1997; Peters et al., 2007), and it has been suggested that together with sucrose, they play an important role in formation of an intracellular glass phase (vitrification) in dry tissues. Vitrification is thought
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to limit the damaging effects of ROS through the slowing down of chemical reaction rates and molecular diffusion in the cytoplasm and prevent damaging compaction of macromolecules and organelles (Berjak et al., 2007; Hoekstra, 2005; Vertucci and Farrant, 1995; Walters et al., 2002). Transcriptomic studies have contributed towards identification of candidates that might contribute to sugar accumulation and their roles in stabilization of the cellular milieu. Sucrose and galactinol synthase transcripts were found to be upregulated during early dehydration in leaves of C. plantagineum (Suarez-Rodriguez et al., 2010), and Collett et al. (2004) have reported upregulation of cDNAs annotated as enzymes that synthesize osmoprotectants such as aldose reductase and galactinol synthase during desiccation of Xerophyta humilis. b. LEA proteins. As the name suggests, LEA proteins were first identified due to their abundant (4% of total cellular protein, Roberts et al., 1993) accumulation during the late stages of seed development coincident with the onset of DT (Baker et al., 1995; Blackman et al., 1992, 1995; Galau et al., 1986; Manfre et al., 2006; Russouw et al., 1995 inter alia). They have been reported to occur in vegetative tissues in response to various abiotic stresses such as cold, drought, salt, osmotic stress (Bray, 1993; Ditzer et al., 2001) and recently desiccation stress (Collett et al., 2004; Ingle et al., 2007). A common feature of LEA proteins is that they are extremely hydrophilic and are soluble at high temperatures. They do not possess any apparent catalytic activity or structural domains, and most of them lack cysteine and tryptophan residues (Close, 1996). As they are largely unfolded in the hydrated state, it is experimentally difficult to assign structure and determine potential function. Thus far, predicted functions of LEA proteins include the unwinding or repair of DNA, forming cytoskeletal filaments to counteract the physical stresses imposed by desiccation, acting as molecular chaperones, stabilization of membrane (Wise and Tunnacliffe, 2004, 2007), maintenance of hydration shells of proteins (Bartels, 2005) to prevent protein aggregation (Chakrabortee et al., 2007; Goyal et al., 2005) and together with sugars facilitate subcellular vitrification (reviewed in Berjak et al., 2007). Transcriptome studies on DT organisms have shown that LEA genes are among the most differentially expressed and highly upregulated genes in DT organisms (Leprince and Buitink, 2010). In resurrection plant studies, Le et al. (2007) reported an LEA transcript from a total of four cDNA clones (specifically expressed in S. stapfianus desiccation-tolerant leaf tissue) upregulated on drying in S. stapfianus, and Collett et al. (2004) reported 16 cDNA clones, from a total of 55 upregulated genes, that were annotated as LEA proteins. A study in which Group 4 LEA genes from the resurrection plant
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B. hygrometrica were overexpressed in transgenic tobacco plants showed increased drought tolerance and an increase in peroxidase and SOD activity when compared to wild-type tobacco plants (Liu et al., 2009). The increased antioxidant activities were also associated with enhanced stability of photosynthesis-related proteins and membranes in the transgenic plants following drought stress (Liu et al., 2009). Overexpression of a dehydrin or group 3 LEA gene in tobacco and Arabidopsis resulted in an increase in production of osmolytes such as proline, polyamines and sugars and significant increases in growth rates under stress conditions (Figueras et al., 2004; Roychoudhury et al., 2007). These data suggest that the accumulation of LEA proteins can, in addition to the stabilization roles suggested above, have an indirect effect on the accumulation of other protective molecules, either altering osmotic adjustment or by the induction of signalling pathways.
III. PROTEOMICS While a number of studies have investigated the transcriptome of resurrection plants (Blomstedt et al., 1998; Collett et al., 2004; Le et al., 2007) during dehydration and rehydration, there have been few studies corresponding to the proteome of resurrection plants. Transcriptome studies offer insight into gene expression profiles, but a major advantage of proteomics over transcriptomics is that it focuses on the actively translated portion of the genome. The importance of post-transcriptional regulation has been demonstrated by several studies revealing a weak or moderate correlation between mRNA and protein levels, except for very abundant proteins in yeast (Gygi et al., 1999; Ideker et al., 2001). In the case of resurrection plants, a further consideration is that many mRNAs appear to be stored during drying and only translated during rehydration (Collett et al., 2003; Dace et al., 1998), thus there may be significant differences between mRNA and protein levels during dehydration. In contrast to transcriptomics, proteomics provides a more physiologically accurate snapshot of biochemical processes by revealing the actual protein constituents performing the enzymatic, regulatory and structural functions encoded by the genome and transcriptome at a given point in time. Further, proteomics approaches provide additional information on gene regulation, especially important when mRNAs may be present but not translated, or when changes in protein level occur without any detectable change in transcript abundance due to translational or other levels of control (Gygi et al., 1999). To date, there have been only a few studies reported on proteomes of angiosperm resurrection plants, which are typically aimed at identifying protein changes in leaf tissues during dehydration to identify proteins that might
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facilitate the acquisition of DT. All these studies have utilized the approach of 2DGE with protein identification by mass spectrometry. There have been no reports on the proteomes of root tissues in any resurrection plant. A. WHOLE PROTEOMIC APPROACHES
Ingle et al. (2007) have reported changes in the proteome of leaves of X. viscosa when whole plants were subject to dehydration. Among the proteins that were upregulated or produced, de novo proteins were involved in antioxidant metabolism, PSII stabilizers, chaperonins and RNA-binding proteins, and downregulated proteins were predominantly those involved in photosynthesis. As this plant is poikilochlorophyllous, reduced expression of such proteins is expected. Table I shows proteins induced during dehydration stress and identified by mass spectrometry. The de novo proteins are those proteins present at 65% and/or 35% RWC which could not be detected in soluble protein extracts from fully hydrated plants (Ingle et al., 2007). They found that marked changes in protein expression appeared to occur in two phases; the first occurring upon drying to 65% RWC and the second more extensive change occurring when leaves were dried to 35% RWC, and those authors have proposed an ‘early’ and ‘late’ stage of preparation for induction of protection. Interestingly, it is at these water contents that changes in transcription have been noted and where marked changes in physiological and biochemical parameters occur in Xerophyta species. For example, Northern blot analysis shows the de novo appearance of inter alia metallothionines I and II, ferredoxin III, an RNA-binding protein, galactinol synthase and oleosin (Collett et al., 2004) and 16 LEA-like proteins (Illing et al., 2005) at or just below 63% RWC. Shutdown of photosynthesis is initiated around this water content and there is increased activity in several antioxidant enzymes (Farrant et al., 2007; Illing et al., 2005). Below 35%, RWC mechanisms that putatively facilitate preservation of structural integrity, for example, accumulation of sucrose, are evident (Farrant et al., 2007). Proteins associated with drying and rehydration of detached leaves of the dicotyledonous homoiochlorophyllous resurrection plant B. hygrometrica have been identified by Jiang et al. (2007). While use of detached leaves might preclude observation of changes that are a consequence of rootderived signalling, such leaves recovered from desiccation and in situ stabilization obviously occur. The rapid drying associated with use of detached leaves possibly also precluded identification of late stage (ca. 35% RWC) changes in protein abundance reported by Ingle et al. (2008), as leaves were sampled at 79%, 6.7% and 2.4% RWC only. The authors report that of the differentially expressed proteins, 19% showed decreased abundance, 35%
TABLE I Table Showing Dehydration-Induced Proteins Identified from B. hygrometrica and X. viscosa Response of protein
RWC (%)
Boea hygrometrica (detached) Induced within 0.5 h Induced within 0.5 h Induced within 0.5 h Induced at 8–48 h Induced at 8–48 h
79.3 79.3 79.3 6.7 and 2.4 6.7 and 2.4
Induced at 8–48 h Induced at 8–48 h Induced at 8–48 h Xerophyta viscosa (whole) Decreased abundance Decreased abundance Decreased abundance Decreased abundance Decreased abundance Decreased abundance Decreased abundance Increased abundance Increased abundance Increased abundance Increased abundance Increased abundance Increased abundance De novo proteins De novo proteins De novo proteins De novo proteins
Homologue/putative identification
pI/MW
Accession number
5.2/62 5.1/17 5.6/57 5.3/20 6/24
NP_220204 AJ238744 gi|22729 P28440 CF079724
6.7 and 2.4 6.7 and 2.4 6.7 and 2.4
ABC Transporter ATPase (Chlamydia trachomatis) Glutathione peroxidase-like protein from barley Polyphenol oxidase precursor from tomato Rubisco large subunit (Pinguicula caerulea) QHK7H18, oxygen-evolving complex of photosystem II from sunflower Vacuolar HþATPase A subunit (Citrus unshiu) Unknown protein (Arabidopsis thaliana) ATGSTU6 (glutathione S-transferase 24) (A. thaliana)
5.6/70 6.1/44 6/29
AB036926 NP_567979 gi|15227084
65 and 35 35 35 35 35 35 35 65 and 35 65 and 35 65 and 35 35 35 35 65 and 35 65 and 35 35 35
PSII stability factor HCF136 (Oryza sativa) PsbO (A. thaliana) PsbP (Xerophyta humilis) Transketalose (Solanum tuberosum) F-ATPase ( subunit) (Ranunculus macranthus) Glu:glyoxylate aminotransferase I (A. thaliana) Ascorbate peroxidase (A. thaliana) Chloroplast FtsH protease (A. thaliana) GDP-mannose-3 0 ,50 -epimerase (O. sativa) Protein phosphatase type 2C (A. thaliana) Alcohol dehydrogenase (Citrus paradise) VDAC1.1 (Lotus corniculatus) 2-Cys peroxiredoxin (O. sativa) 14 dnaK-type molecular chaperone (O. sativa) Phosphopyruvate hydratase (Zea mays) RNA-binding protein (Daucus carota) Desiccation-related protein (Craterostigma plantagineum)
5.2/40 5.6/30 7.0/26 5.4/78 5.2/59 6.5/52 5.0/32 5.2/72 6.2/47 6.0/33 6.4/46 6.5/28 5.0/26 5.3/75 6.0/50 6.3/76 5.0/33
BAD62115 CAA36675 AAN77240 CAA90427 AAZ03784 AAN62332 CAA66925 CAA68141 Q2R1V8 CAB79642 AAY86033 AAQ87019 CAJ01693 NP_001048274 P26301 AAK30205 AAA63616
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were transiently induced during dehydration and 5% were upregulated during rehydration. Among the upregulated proteins identified, at least three are associated with antioxidant metabolism, two with photosynthesis and two with energy metabolism (Table I). The relatively small number of proteins that were upregulated specifically during the rehydration phase supports the hypothesis that most changes in the gene expression occur during the dehydration phase in desiccation tolerant higher plants (Phillips et al., 2002). Some of the proteins identified by Ingle et al. (2007) and Jiang et al. (2007) are discussed briefly below. 1. Photosynthesis-related genes Ingle et al. (2007) showed that the abundance of five chloroplast proteins involved in photosynthesis was significantly decreased at 35% RWC: psbO and psbP, two components of the luminal oxygen-evolving complex (OEC) of PSII, the PSII stability factor HCF136, the -subunit of the F-ATPase and the Calvin cycle enzyme transketalose (Table I). Of these, only HCF136 (a thylakoid luminal protein required for PSII stability and assembly) was also significantly lower at 65% RWC. The reduced levels of HCF136 protein in X. viscosa during drying could be one of the components regulating shutdown of photosynthesis possibly by reducing the rate of stable PSII formation. The decrease in psbO and psbP protein levels in X. viscosa during drying is most likely a consequence of the dismantling of thylakoids observed to occur during the late stages of drying in poikilochlorophyllous resurrection plants (Farrant, 2000; Farrant et al., 2003). Collett et al. (2003, 2004) observed that psbP and psbO mRNA levels also decline in the related species X. humilis at RWCs below 50%. This suggests that at least some of the observed decrease in protein levels is likely caused by downregulation of gene expression. In contrast to the observations of photosynthesis-related proteins in X. viscosa (Ingle et al., 2007), one of the chloroplast-located proteins (identified as a precursor of the OEC of PSII) by Jiang et al. (2007) was found to accumulate in dehydrated and partially dehydrated B. hygrometrica tissue. This observation that OEC PSII accumulates in dehydrated tissue (Jiang et al., 2007) contrasts with the report by Collett et al. (2004, 2005) who reported that many of the genes related to photosynthesis (including the genes encoding for OEC fragments and PSII reaction centre proteins) were downregulated in X. humilis. While B. hygrometrica is homoiochlorophyllous and X. humilis is poikilochlorophyllous, this difference in expression patterns of the photosynthesis-related genes in these two resurrection plants may reflect the different approaches these plants employ to minimize the damage of excess light energy during dehydration, or it may imply that in some cases, the steady state mRNA levels do not accurately reflect the
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changes observed at the protein level (highlighting the necessity of proteomic approaches; (Jiang et al., 2007). 2. Energy metabolism Jiang et al. (2007) found two ATPases induced during dehydration. An ATPbinding cassette (ABC) transporter ATPase was induced during early dehydration (after 0.5 h at RWC of 79%), and protein levels increased when leaves were dried to < 7%. High expression levels were maintained during rehydration. The expression pattern observed suggests that the protein might play a role in providing energy for protective and repair reactions in both dehydration and rehydration phases. The second ATPase, a vacuolar HþATPase A subunit, was apparent only at low water contents, but as there was no measure of protein levels at intermediate water contents in this study, it could have been upregulated at an earlier stage. This protein could well be involved in regulating water replacement in vacuoles, so providing a mechanical stabilization role (Farrant, 2007). Similar genes have been identified in desiccated leaf tissue from S. stapfianus and X. viscosa (Ingle et al., 2007). 3. Detoxification and protection All the studies identified proteins with potential for protection against oxidative damage during drying. These include AP (X. viscosa), glutathione peroxidase and glutathione S-transferase (B. hygrometrica). These data correlate with enhanced ascorbate and glutathione levels in dehydrated leaves of these and other resurrection plants (Farrant et al., 2007; Jiang et al., 2007; Kranner et al., 2006). An increase in PPO protein levels and enzyme activity was observed in drying leaf tissues of B. hygrometrica (Jiang et al., 2007). It has been proposed that PPO may play an indispensable role in chloroplast function with a possible involvement in a Mehler-like reaction detoxifying oxygen species (Sherman et al., 1995), while polyphenols possess ideal structural chemistry for free radical scavenging activities (Rice-Evans et al., 1997). PPO levels were shown to be enhanced on drying in R. serbica (Sgherri, et al., 2004; Veljovic-Jovanovic et al., 2008), and the polyphenol 3,4,5-triO-galloylquinic acid is present in large quantities in desiccated leaves of M. flabellifolius (Moore et al., 2005a,b); these compounds having demonstrable antioxidant properties, possibly also acting in a redox buffering capacity. B. SUBCELLULAR PROTEOMICS APPROACHES
Whole proteomics approaches such as undertaken by Ingle et al. (2007) and Jiang et al. (2007), while useful for an overall survey of changes occurring in a particular tissue, have the disadvantage of not being able to identify changes
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associated with particular organelles or cytoplasmic domains. Studying the proteomes of such sub-compartments allows for more specific localization of proteins which can in turn be related to function with respect to DT. Further, the reduction in protein complexity and increased technical resolution (Dreger, 2003; Jiang et al., 2005; Jung et al., 2000; Pandey et al., 2008) allows for identification of low abundance proteins such as signal or regulatory proteins (Bae et al., 2003; Molloy et al., 1998; Pandey et al., 2006, 2008). To this end, we have provided the first detailed report on the upregulated proteins in the nucleus of a resurrection plant in response to dehydration stress at 35% RWC (Abdalla et al., 2010). Whole plants were dehydrated, and nuclei were extracted from leaf tissues. Eighteen proteins (Table II) were found to be significantly upregulated on drying to this water content of which four proteins involved in gene regulation and four associated with translation were identified. There were two proteins with molecular chaperone type activities and one with a role in energy metabolism and seven with no assigned function. Three of these were identified as having the same protein identity (Abdalla et al., 2010). This is not unusual, as in 2D electrophoresis, the same protein identity can be obtained for different spots due to protein degradation, protein isoforms, heterodimer formation or the proteins in question may belong to an extensive protein family (Colvis and Garland, 2002; Pandey et al., 2008). This drawback, together associated with working
TABLE II Dehydration-Induced Nuclear Proteins from X. viscosa Homologue/putative identification
Accession number
Non-LTR retro element reverse transcriptase Novel GAG-pol Orf100f protein Novel Novel Novel Unknown protein F2P9.19 Chaperonin EF-Tu precursor EF-Tu precursor Chaperonin ATP sythase chain Intron maturase Novel Zinc-finger helicase Ribosomal protein L28 EF-Tu precursor
Q9LGM1 Not available Q93Y69 Q9ASH2 Not available Not available Not available A96767 Q943P8 S21567 S21567 E86388 ATP_ARATH Q9BAA0 Not available Q8LHZ4 D84580 S21567
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on non-model organisms for which the genome has not yet been sequenced, places limitations on correct identification of proteins and thus denies detailed interpretation of the role of many proteins in the acquisition of DT. However, given that the nucleus is an important regulator of many subcellular activities, including gene expression, manufacture and transport of regulatory factors and in stress response signalling (Fink et al., 2008; Komatsu and Tanaka, 2005; Moriguchi et al., 2005; Repetto et al., 2008), this chapter, albeit with giving only a small number of positively identified protein changes, at least confirms that the X. viscosa nucleus responds to dehydration stress and that DT is controlled by multiple genes within the plant nucleus. While these studies on the proteome changes associated with drying of resurrection plants have confirmed the importance of protection mechanisms such as minimization of photosynthetically produced ROS and associated antioxidant systems and have given some insight to other potential pathways that might afford protection against extreme water deficit, they also have highlighted some of the problems associated with use of this technology in relation to the study of DT. An overarching one is the limitations of current databases with respect to identifying proteins from plant species that are phylogenetically distant from model plants. While this is true for all plants in which the genome has not yet been sequenced, it is possible that, as vegetative DT is such a rare phenomenon, that at least some of the genes and/or regulatory aspects of these genes are unique to resurrection plants and will never be identified, given the current status of the databases. It is important that the genome of at least one monocot and one dicot be sequenced. The 2D SDS-PAGE approach used in the studies discussed above has limitations, as it allows the detection and analysis of only a subset of relatively abundant and soluble proteins. To increase the number of proteins that can be analyzed, fractionation of the proteome into subcellular fractions could be employed (as done by Abdalla et al., 2010) or investigators could use the gel-free iTRAQ MS system. The iTRAQ MS system has the additional benefit that hydrophobic proteins lost during isoelectric focusing (IEF) can be studied (Suzuki et al., 2006). Finally, studies on the proteomes of roots in response to desiccation must be initiated. The profiling of the protein constituents together with performing correlative enzymatic, metabolic and physiological assays at given RWCs is designed to complement transcriptome studies in resurrection plants (Jiang et al., 2007). The studies discussed above have reported the differential expression of proteins and the programmed regulation of protein expression in response to dehydration (Abdalla et al., 2010; Ingle et al., 2007; Jiang et al., 2007) and rehydration (Jiang et al., 2007) to be consistent with the accompanying findings at the structural and metabolic levels in resurrection plants studied to date.
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IV. METABOLOMICS Metabolic profiling, correlated with changes in transcriptome and proteome expression, is an important tool used in identifying the early compounds that signal the perception of stress (Shulaev et al., 2008). The approaches currently used in plant metabolomics research include metabolic fingerprinting, where only signatures associated with a stress are identified, metabolic profiling for identification of global metabolites associated with a sample, and targeted analysis used to determine the precise concentration of a limited number of known metabolites (Fiehn, 2002; Halket et al., 2005; Shulaev, 2006; Shulaev et al., 2008). As outlined above, biochemical and physiological studies have shown that resurrection plants produce a number of metabolites such as sugars, amino acids, water and lipid soluble antioxidants, anthocyanins and many small signalling molecules in response to dehydration stress (reviewed in Farrant, 2007; Moore et al., 2009). However, with the exception of a limited profiling study on the resurrection fern M. caffrorum (Farrant et al., 2009), there has as yet no bona fide metabolomic studies reported on angiosperm resurrection plants. Bioinformatics tools have allowed for the in silico metabolic profiling of C. plantagineum (Suarez-Rodriguez et al., 2010). This study characterized the transcriptomes of C. plantagineum leaves at four stages of dehydration and rehydration using deep sequencing, and data was compared with previously reported transcript profiles of orthodox seeds and pollen as well as with transcript profiles of desiccation-sensitive plants. The comparisons indicated that vegetative DT may be the result of differential regulation of pre-existing, non-vegetative DT mechanisms, and that most water-stress related genes are shared by tolerant and non-tolerant species but that changes in their expression patterns ultimately provide tolerant plants with more effective protective mechanisms. Gene ontology enrichment identified metabolic pathways which may be important for DT. These included those involved in the biosynthesis of ubiquinone and other terpenoids; phenylpropanoid, geranylgeranyl diphosphate II, photosynthetic carbon fixation and methane metabolism (Suarez-Rodriguez et al., 2010).
V. CONCLUDING REMARKS There is no doubt that a combination of transcriptomics, proteomics and metabolomics approaches will provide us greater insight into how plants respond to dehydration stress. There is a need to do such studies at the subcellular as well as the whole plant level to gain a comprehensive
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understanding of DT. Insights gained from such a systems-biology approach will ultimately allow informed biotechnological approaches for the production of drought tolerant crops.
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Walters, C. (1998). Understanding the mechanisms and kinetics of seed ageing. Seed Science Research 7, 223–244. Walters, C., Farrant, J. M., Pammenter, N. W. and Berjak, P. (2002). Desiccation and damage. In Desiccation and Survival in Plants—Drying without Drying, (M. Black and H. W. Pritchard, eds.), pp. 263–291. CABI Publishing, Wallingford. Walters, C., Hill, L. M. and Wheeler, L. M. (2005). Dying while dry: Kinetics and mechanisms of deterioration in desiccated organisms. Integrative and Comparative Biology 45, 751–758. Wang, H., Cao, G. H. and Prior, R. L. (1996). Total antioxidant capacity of fruits. Journal of Agricultural and Food Chemistry 44, 701–705. Wang, L., Shang, H., Liu, Y., Zheng, M., Wu, R., Phillips, J., Bartels, D. and Deng, X. (2008). A role for a cell wall localized glycine-rich protein in dehydration and rehydration of the resurrection plant Boea hygrometrica. Plant Biology 11, 837–848. Webb, M. A. and Arnott, H. J. (1982). A survey of calcium oxalate crystals and other mineral inclusions in seeds. Scanning Electron Microscopy 3, 1109–1131. Wehmeyer, N., Hernandez, L. D., Finkelstein, R. R. and Vierling, E. (1996). Synthesis of small heat-shock proteins is part of the developmental program of late seed maturation. Plant Physiology 112(2), 747–757. Wettlaufer, S. and Leopold, C. (1991). Relevance of Amadori and Maillard products to seed deterioration. Plant Physiology 97(165–161), 69. Whittaker, A., Bochicchio, A., Vazzana, C., Lindsey, G. and Farrant, J. M. (2001). Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa. Journal of Experimental Botany 352, 961–969. Whittaker, A., Martinelli, T., Bochicchio, A., Vazzana, C. and Farrant, J. (2004). Comparison of sucrose metabolism during the rehydration of desiccationtolerant and desiccation-sensitive leaf material of Sporobolus stapfianus. Physiologia Plantarum 122, 11–20. Wise, M. J. and Tunnacliffe, A. (2004). POPP the question: what do LEA proteins do? Trends Plant Science 9, 13–17. Wise, M. J. and Tunnacliffe, A. (2007). The continuing conundrum of the LEA proteins. Die Naturwissenschaften 94, 791–812.
Root Tropism: Its Mechanism and Possible Functions in Drought Avoidance
YUTAKA MIYAZAWA,1 TOMOKAZU YAMAZAKI TEPPEI MORIWAKI AND HIDEYUKI TAKAHASHI
Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai, Japan
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Plant Responses to Water Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Water Stress and Osmotic Adjustment Cause Growth Inhibition ...... B. Severe Drought Conditions Damage Plant Cells .......................... C. Avoidance and Tolerance of Severe Drought Conditions................ III. Mechanisms for Root Hydrotropism and its Possible Functions in Drought Avoidance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mechanism for Sensing Hydrostimulation ................................. B. Mechanism for Hydrostimulation Signal Transmission .................. C. Mechanism for Hydrotropic Root Bending ................................ D. Molecular Identification of Genes Responsible for Hydrotropism in Arabidopsis Roots .............................................................. IV. Mechanisms for Other Root Tropisms Related to Drought Avoidance. . . . A. Root Gravitropism ............................................................. B. Regulation of Gravitropism by Water Stress .............................. C. Root Phototropism............................................................. D. Ecological Function of Phototropism ....................................... V. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding author: E-mail:
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Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00010-2
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ABSTRACT Land plants have evolved various mechanisms for responding to unfavourable environmental signals, which allows them to tolerate or avoid environmental stresses such as water deficit. To date, physiological and molecular mechanisms that contribute to drought tolerance have been intensely studied, however the mechanisms that confer drought avoidance have been less understood. To avoid drought conditions roots must sense environmental stimuli and respond by regulating growth away from water scarce areas or toward wet areas. Indeed, roots respond to numerous environmental stimuli, such as gravity, light and moisture gradient, and exhibit gravitropism, phototropism and hydrotropism, respectively. Of these root tropisms, hydrotropism can be considered to contribute directly to drought avoidance. As soil water status is affected by gravity or intense light, positive gravitropism and negative phototropism are assumed to contribute to drought avoidance. In this chapter, we describe what happens to cells faced with a water deficit and then outline the molecular mechanisms underlying different tropisms, with particular emphasis on the molecular mechanism contributing to root hydrotropism.
I. INTRODUCTION All organisms depend upon water availability. Unlike mobile organisms, land plants are sessile in nature and must complete their life cycles where they germinate. Accordingly, land plants have evolved various mechanisms for responding to unfavourable environmental signals, allowing them to tolerate or avoid environmental stresses such as water scarcity. The physiological and molecular mechanisms contributing to drought tolerance have been studied extensively. Such mechanisms include stomatal closure, the synthesis and activity of the phytohormone abscisic acid (ABA), the synthesis of compatible solutes and the functions of regulatory genetic elements. These drought tolerance mechanisms are detailed in other chapters. In contrast, the mechanisms underlying drought avoidance are much less well understood. The root is the primary organ for water absorption. To avoid drought conditions, roots must sense environmental stimuli and respond by regulating growth away from dry areas or towards wet areas. To regulate growth in a directional manner, land plants have evolved tropisms, whereby organ growth is redirected in response to an environmental stimulus, such as gravity, light, a moisture gradient or touch. Some of these environmental stimuli, namely, gravity, light and a moisture gradient, are closely related to soil water status. As gravity affects water, positive gravitropism is assumed to be one mechanism for drought avoidance. Similarly, negative phototropism also plays a role in drought avoidance, as soil is dried by exposure to intense light. Further, plant roots can sense a moisture gradient and grow towards the wet area, a process termed positive hydrotropism. In this chapter, we will
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describe what happens to cells faced with a water deficit and then outline the molecular mechanisms underlying different tropisms, with particular emphasis on the molecular mechanism contributing to root hydrotropism.
II. PLANT RESPONSES TO WATER STRESS Environmental factors, such as water, light and temperature, will change across a day, season and year. Plants use these changes as cues for controlling the timing of developmental transitions such as germination and flowering. These environmental factors also provide plant cells with stresses, which result in the inhibition of cellular processes. Water is the most important environmental factor for plants. It is integrally linked to biophysical and biochemical process such as cell elongation and enzyme activity, respectively. Consequently, water shortages cause water stress in plants. A. WATER STRESS AND OSMOTIC ADJUSTMENT CAUSE GROWTH INHIBITION
Water stress can be estimated by measuring water potential. Water moves according to a water potential gradient within a plant and between the plant and its environment. Water is taken up from the environment and then absorbed into cells, which results in increased cell volume. This increase in cell volume is also driven by a water potential gradient between the outside and inside of the cell. Changes to cell volume are thought to be determined by several factors, namely differences between osmotic potential and turgor pressure, water permeability across the plasma membrane and cellular surface area. The growth responses of terrestrial plants are particularly sensitive to water shortages (Mullet and Whitsitt, 1996), as these decrease the environmental water potential and reduce water movement into plants. However, water shortage does not result in a large difference between osmotic potential and turgor pressure ( 0.1–0.2 MPa). Thus, sensitivity to changes in cell volume may explain drought-induced inhibition of the growth response. Following a decrease in environmental water potential, cell growth is inhibited via adjustments to the osmotic potential, that is, solute concentration. Sugars and amino acids are thought to be instrumental in osmotic adjustment, as their distribution is unlikely to affect cellular components such as enzymes and membranes. Sugars and amino acids can be taken up by cells or obtained via the degradation of osmotically inactive compounds. High molecular mass solutes such as proteins and starch do not contribute to osmotic potential. Until recently, ions have not been considered suitable osmotic
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components because alterations to their distribution could cause charge effects on enzymatic processes. However, comprehensive analyses of plant growth, metabolite composition, enzyme activity and gene expression in Arabidopsis exposed to different drought conditions have identified potassium and organic acids as the primary components for osmotic adjustment (Hummel et al., 2010). In addition, Arabidopsis cells use enzymes to perform these osmotic adjustments without altering the primary metabolic system. This lack of metabolic change is in sharp contrast to the drought-induced alterations to expression of genes involved in metabolism, transport, signalling and transcription, as well as genes encoding hydrophilic proteins (Bray, 2004; Kawaguchi et al., 2004; Kreps et al., 2002; Seki et al., 2002). Water permeability can be important for cell volume increases. Water molecules may pass passively through the lipid bilayers of the plasma membrane or they may be transported via water channels. Aquaporins are the most well-known water channels. These integral membrane proteins contain six transmembrane domains, with both the C- and N-termini found on the cytoplasmic side of the membrane. In plants, important water uptake roles are performed by aquaporin family members that localize to the plasma membrane and vacuole (Kjellbom et al., 1999). While overexpression of a plasma membrane-localized aquaporin improves the growth of transgenic tobacco plants under well-watered conditions, these plants wilt easily under drought conditions (Aharon et al., 2003). Recently, drought conditions were shown to decrease aquaporin levels in the plasma membrane of Arabidopsis (Lee et al., 2009). This decrease resulted from aquaporin degradation in the endoplasmic reticulum via a ubiquitin-mediated protein degradation pathway. The observations described above suggest that cells may control water permeability to regulate water uptake during times of drought. B. SEVERE DROUGHT CONDITIONS DAMAGE PLANT CELLS
Under severe drought conditions, the water potential outside of a plant may be lower than inside, resulting in the dehydration of cells and wilting from loss of turgor pressure. Excessive water loss can irreversibly damage plant cells, and at the macro level, increased ion leakage can be detected (Blum and Ebercon, 1981; Whitlow et al., 1992). Electron microscopy of barley leaf cells under drought conditions shows that volume reduction is achieved by deformation and folding of the cell surface (Pearce and Beckett, 1987). Although ultrastructural particles are usually present in biomembranes such as the plasma membrane, tonoplast and chloroplast envelopes, freezefracture observations reveal patches lacking intramembranous particles (free IMP) in well-watered wheat plants that are rapidly exposed to drought
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conditions (Pearce, 1985). The presence of IMP-free patches or patches with few IMPs is associated with the formation of lamellae between membranes. These patches appear similar to the plasma membrane lesions that occur following exposure to subzero temperatures, which freeze extracellular water (Steponkus et al., 1993). Lamellar and hexagonal II structures are wellknown lipid formations. For example, the lipid bilayer of the plasma membrane represents a unit of lamellar structure, whereas lipids form tube-like units in the hexagonal II structure, and these tubes are packed into a regular pattern. On the plasma membrane, freeze-induced lesions result during the transition from a lamellar to a hexagonal II structure. Lamellae are thought to appear during the loss of cytoplasmic water, when the plasma membrane closes to other membranes in the endomembrane system. Under drought conditions, free-IMP patches are thought to be induced by ion leakage from the cytoplasm. Loss of these ions results in irreversible lesions on the plasma membrane, and accumulation of such lesions will eventually kill the plants. Severe drought conditions also cause loss of available water from cellular macromolecules such as enzymes, the lipid membrane and polysaccharide components of the cell wall (Hoekstra et al., 2001; Moore et al., 2008). These macromolecules are conjugated to water molecules via hydrogen bonds, and this association is necessary for basic molecular function. The loss of water molecules results in significant structural and functional changes in cellular macromolecules. For instance, the loss of water molecules from polysaccharides (e.g. cellulose) causes the cell wall to tighten, reducing elasticity (Moore et al., 2008). Irreversible structural changes result from the complete loss of water molecules from macromolecules, a process referred to as desiccation. C. AVOIDANCE AND TOLERANCE OF SEVERE DROUGHT CONDITIONS
In arid regions, terrestrial plants have evolved several strategies for resisting water stress under severe drought conditions. In angiosperms, the most common mechanism for avoiding a water shortage is the control of germination timing. Succulents such as cacti have tissue modifications for water storage, and Eucalyptus roots penetrate deeply into soil to reach the available water. These examples are part of a plant’s water loss avoidance strategy, which also includes modifications to vegetative organs such as the leaf and stem. The other type of strategy is to generate increased tolerance to the water deficit by protection of cellular components. The seed is a major developmental phase that can be used to withstand water deficit. By sensing the presence of water, seeds are able to time the onset of germination appropriately. An extreme example of desiccation tolerance is found in the resurrection plant Ramonda serbica, which can
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recover from almost complete water loss (Farrant, 2000). When exposed to moderate drought conditions, many plants, including Arabidopsis, can acclimate by increasing drought tolerance (Mullet and Whitsitt, 1996; Whitlow et al., 1992). When rapid exposure to severe drought conditions follows a period of moderate drought conditions, plants will show reduced ion leakage (Blum and Ebercon, 1981; Mullet and Whitsitt, 1996; Whitlow et al., 1992). In barley, fewer IMP-free patches are formed in the leaves of plants that have experienced mild drought than in well-watered plants (Pearce, 1985). During drying, unsaturated fatty acids increase in the plasma membranes of Arabidopsis leaf cells (Gigon et al., 2004). A similar increase in unsaturated fatty acid species occurs under non-freezing cold stress and represents the acquisition of freezing tolerance (Uemura et al., 1995). The relationship between changes in fatty acid composition and freezing tolerance is also observed in other plants such as rye and oat (Steponkus et al., 1993). Interestingly, resurrection plants can survive more than 90% water loss by increasing the unsaturated fatty acids in the plasma membrane during drying (Quartacci et al., 2002). Freezing and drought cause similar stresses, as both induce dehydration of cells. Therefore, the physiological responses observed in the membrane under dehydrating conditions may relate to the molecular mechanisms for tolerance to dehydration.
III. MECHANISMS FOR ROOT HYDROTROPISM AND ITS POSSIBLE FUNCTIONS IN DROUGHT AVOIDANCE As far as we are aware, root hydrotropism was first described by Knight (1811). Knight wrote as follows: ‘‘When a tree, which requires much moisture, has sprung up or been planted, in a dry soil, in the vicinity of water, it has been observed, that much the largest portion of its roots has been directed towards the water.’’ Since then, root hydrotropism has fascinated many plant physiologists. Water acquisition is critical for plant survival on land and it is easy enough to imagine the importance of root hydrotropism. However, root hydrotropism was poorly understood until its ‘‘rediscovery’’. Its obscurity occurred because roots also display gravitropism (directed growth towards the centre of gravity; see next section), and researchers were unable to differentiate between the two tropisms. In addition, it was difficult to establish an experimental system that could maintain a stable moisture gradient. For these reasons, the existence of root hydrotropism remained in doubt for many years. It was in 1985 that Jaffe et al. rediscovered root hydrotropism using an agravitropic mutant of pea (Jaffe et al., 1985). When this pea mutant, ageotropum, was grown under moisture-saturated
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conditions, its roots emerged from the soil and grew into the humid air; however, when the atmosphere was dried, its roots would bend towards the moistened soil. This unequivocal demonstration of the existence of hydrotropism prompted a few researchers to begin to elucidate the nature of hydrotropism. More importantly, this demonstration showed that gravitropism often interferes with hydrotropism. Hence, in the early days, physiological studies were performed using the roots of agravitropic mutants or clinorotation (continuous rotation of samples to nullify the effects of gravity vector) of the seedlings. Following development of these experimental systems, root hydrotropism has been observed in pea, cucumber, maize, wheat and Arabidopsis (Mizuno et al., 2002; Oyanagi et al., 1995; Takahashi and Scott. 1991, 1993; Takahashi et al., 2002). From these experiments, many researchers have concluded that the ability to express root hydrotropism might be universal among monocots and dicots; however, arguments for and against the existence of root hydrotropism continue (Coutts and Nicoll, 1993; Plaut et al., 1996). Very recently, our group identified MIZUKUSSEI1, a gene responsible for root hydrotropism (Kobayashi et al., 2007). Since homologues of MIZU-KUSSEI1 are found across terrestrial plant species, it is likely that the capability to express root hydrotropism has been strongly conserved. In this section, we will describe physiological and genetic studies of root hydrotropism, as understanding the molecular mechanisms underlying this phenomenon could assist with the improvement of plant growth in arid areas. A. MECHANISM FOR SENSING HYDROSTIMULATION
Generally, tropism comprises three steps: sensing, signal transmission and response. The Cholodny-Went hypothesis suggests that the detection of environmental stimuli leads to lateral auxin redistribution (Went and Thimann, 1937), and recent molecular genetic studies have demonstrated that, for the most part, this hypothesis holds for the gravitropic response in roots. The Cholodny-Went hypothesis will be described in detail in the Section IV.A. As root hydrotropism is often masked by gravitropism, early investigations focused primarily on interactions between these two tropisms in various plant species. Several lines of evidence have suggested that the sensing apparatus for hydrostimulation resides in the root tip. In pea and maize, microsurgical removal of the root tip caused diminishment of root hydrotropism (Takahashi and Scott, 1993; Takahashi and Suge, 1991), and this tropism was lost completely following laser ablation of columella cells in the roots of Arabidopsis seedling (Miyazawa et al., 2008). Further, when agar blocks
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containing different concentrations of sorbitol are applied bilaterally to the root cap of an agravitropic pea mutant, the roots respond to gradients in water potential of 0.5–1.5 MPa by bending away from the sorbitol agar block (Takano et al., 1995). It is likely that plants respond to hydrostimulation at the cellular level, reacting to differences or gradients in water potential. As mentioned previously, a water potential gradient is formed across the root cap between the dry and wet sides. Although the molecules that form the sensory apparatus remain unknown, a mechano-sensitive ion channel responsive to osmotic stresses has been reported (Kung, 2005). Such a channel could be responsible for inducing differences in water potential between extracellular and intracellular regions. Recent studies using homologues of such genes have led to the hypothesis that these channels might be involved in the perception of hydrotropic stimuli (Haswell and Meyerowitz, 2006; Nakagawa et al., 2007). However, no experimental evidence on the involvement of these channels is demonstrated, rather it has been shown that a range of single and multiple knockout mutants for genes encoding such proteins showed no alteration in hydrotropism (Fujii et al., unpublished). In yeast, osmotic stress leads to the activation of a mitogen-activated protein kinase, Hog1p, via Sln1p and Sho1p (Reiser et al., 2003). Similarly, the Arabidopsis histidine kinase AHK1 has been implicated as a positive regulator in the stress response (Tran et al., 2007). Future studies will verify whether such proteins are involved in sensing hydrostimulation. B. MECHANISM FOR HYDROSTIMULATION SIGNAL TRANSMISSION
The gravistimulation sensing apparatus resides in the columella cells of the root tip. Interactions between hydrotropism and gravitropism may occur in roots, as both stimuli are sensed by the same or nearby cells. The hydrotropism of maize is affected by different intensities of gravistimulation (Takahashi and Scott, 1991), and the starch granules that play a role in Arabidopsis graviperception are degraded upon hydrostimulation (Takahashi et al., 2003). Indeed, a starchless mutant of Arabidopsis shows not only decreased gravitropism but also enhanced hydrotropism (Takahashi et al., 2003). However, the commonality of this phenomenon among plant species awaits further study. Calcium ions may be involved in the interaction between hydrotropism and gravitropism. Several studies have identified calcium ions as important signal transducers for gravitropism (Plieth and Trewavas, 2002; Sedbrook et al., 1996; Toyota et al., 2008), and treatment with a calcium ion chelator leads to the inhibition of both hydrotropism and gravitropism in pea and Arabidopsis roots (Takahashi and Suge, 1991; Kaneyasu et al., unpublished
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results). However, the exact role that calcium ions play in such an interaction remains unclear. Phospholipase D proteins may also be involved in the interaction between these tropisms. Phospholipase Ds are known to be involved in cellular responses to biotic and abiotic stresses (Bargmann and Munnik, 2006; Li et al., 2009). A recent report showed that the mutation of a gene encoding phospholipase D2 produces defects in both gravitropism and hydrotropism in Arabidopsis roots (Taniguchi et al., 2010). As the gene encoding phospholipase D2 is expressed in root tip cells, it is proposed that this enzyme functions as a signal transducer for drought stress (Taniguchi et al., 2010). However, the phenotypes of these ahydrotropic mutants are quite subtle and, thus, comprehensive studies of multiple phospholipase D mutants will be needed to fully understand the role that these enzymes play in hydrotropism. Although these findings represent important clues towards a future understanding of the mechanisms underlying hydrotropic signalling, the contributions of calcium and/or phospholipase Ds to hydrotropic signalling remain no more than speculation at this time. C. MECHANISM FOR HYDROTROPIC ROOT BENDING
Auxin plays a central role in many aspects of plant morphogenesis, including gravitropism. In the gravitropic response of roots, auxin plays an important role by transmitting the gravity signal from sensing cells, that is, columella cells, to the root elongation zone. When gravity is sensed, auxin is transported preferentially to the lower side of the root, which leads to the accumulation of auxin in the elongation zone (Muday, 2001). Auxin influx and efflux carriers play critical roles in this transport system. When seedlings are treated with inhibitors of either auxin influx or efflux, gravitropism is severely inhibited. Recent molecular genetic analyses of Arabidopsis have identified the auxin-related molecules that function in the gravitropic response in roots (Abas et al., 2006; Friml et al., 2002; Swarup et al., 2005). Classical studies on root hydrotropism have shown that the auxin efflux inhibitor TIBA inhibits the hydrotropic response in pea (Takahashi and Suge, 1991). In cucumber roots, the auxin efflux inhibitor also caused a reduction in the hydrotropic response (Morohashi et al., unpublished). Moreover, hydrotropic bending of pea seedling roots occurs when auxin is redistributed and accumulated at the concave side of the elongation zone (Takano, 1999). In cucumber, hydrotropic bending is associated with differential accumulation of mRNA for the auxin-inducible gene CsIAA1, with the higher mRNA levels being detected on the concave side than convex side of the elongation zone (Mizuno et al., 2002). These results strongly suggest that differential auxin transport from the root tip to the elongation zone is crucial for the hydrotropic response, at
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least in the plant species examined thus far. However, the hydrotropic responses of some auxin influx- or efflux-associated gravitropic mutants of Arabidopsis are comparable to the wild type (Takahashi et al., 2002). Moreover, this hydrotropic response was not reduced by inhibitors of auxin influx or efflux (Kaneyasu et al., 2007). While polar auxin transport is unnecessary for the hydrotropic response in Arabidopsis, an inhibitor of the auxin response substantially reduces both gravitropism and hydrotropism (Kaneyasu et al., 2007). These findings imply that the auxin response is necessary for both gravitropism and hydrotropism, but that the regulatory mechanisms for auxin dynamics may differ between them. At present, we do not know why only some plant species require polar auxin transport for hydrotropism. There might be an as-yet unidentified mechanism that regulates auxin redistribution or activity in a species-specific manner. To understand root hydrotropism from both an ecological and evolutionary point of view, we should make further efforts to determine this species specificity. Thus, experimental systems for hydrotropism must be established in multiple species for the requisite physiological studies to be performed. D. MOLECULAR IDENTIFICATION OF GENES RESPONSIBLE FOR HYDROTROPISM IN ARABIDOPSIS ROOTS
As described above, Arabidopsis uses a mechanism for hydrotropism that differs from that for gravitropism. Two strategies were adopted to clarify this molecular mechanism, transcriptomic identification of genes responsible for hydrotropism and isolation and analyses of mutants showing abnormal hydrotropism. Recent developments in DNA microarray technology have enabled comprehensive profiling of genes that are up- or down-regulated in response to environmental stresses, gravity and light (Killan et al., 2007; Kimbrough et al., 2004; Ma et al., 2003). Public release of the Arabidopsis transcriptome database has allowed researchers to determine the uniqueness or commonalities of different signalling pathways. To investigate the transcriptional changes associated with root hydrotropism, a microarray analysis was performed on hydrostimulated roots of Arabidopsis seedlings (Moriwaki et al., 2010). Among the 22,810 genes identified, 322 were up-regulated and 468 were down-regulated under hydrostimulated condition. Despite the intimate relationship between hydrotropism and gravitropism, there was little overlap between the genes responsible for these two tropisms. Thus, the transcriptional regulation of hydrotropism differs from that of gravitropism. However, a significant overlap was observed between the genes responsible for hydrostimulation, ABA and water stress, including drought, salt and osmotic stresses (Moriwaki et al., 2010). Thus, it was concluded that ABA and water-stress
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responses contribute to the regulation of hydrotropism at the transcriptional level. In addition, Ponce et al. (2008) reported that the application of ABA disrupted the hydrotropic response in the roots of wild-type Arabidopsis seedlings. Although these results have furthered our knowledge of hydrotropism at the molecular level, they raise two questions: (1) How are ABA and water-stress signals integrated into the signalling pathway of root hydrotropism? (2) Is there a similar molecular system in other plant species? Comparative genetic analyses will need to be performed in other plant species to answer these questions. The establishment of an experimental system to examine hydrotropism in the roots of Arabidopsis seedling has led to genetic screening for defects in root hydrotropism (Eapen et al., 2003; Kobayashi et al., 2007). Cassab and her colleagues reported two mutants named no hydrotropic response1 and 2 (nhr1, 2; Eapen et al., 2003). Although the genes responsible for these mutants have not yet been identified, physiological studies of the semidominant mutant, nhr1, showed abnormal root tip cells and enhanced gravitropism (Eapen et al., 2003). Subsequent reports have suggested that nhr1 exhibits pleiotropic phenotypes, most of which can be explained by enhanced accumulation of the phytohormone ABA (Ponce et al., 2008; QuirozFigueroa et al., 2010). In addition, these authors showed that ABA treatment of wild-type roots not only reduces the starch granules in the root tip but also produces enhanced gravitropism. Although these findings have led to the hypothesis that ABA negatively regulates root hydrotropism (Ponce et al., 2008; Quiroz-Figueroa et al., 2010), the roots of some ABA-deficient and insensitive mutants have shown reductions in both hydrotropism and gravitropism (Takahashi et al., 2002). As hydrostimulation is related to mechanisms for combating water stress, it is possible that NHR1 might play a role in the drought response and/or drought-triggered ABA signalling. Clearly, ABA synthesis and signalling are important for root hydrotropism (Moriwaki et al., 2010; Takahashi et al., 2002); however, more detailed studies of the nhr1 mutant will be necessary to determine the molecular mechanism underlying root hydrotropism in its pleiotropic phenotypes, as well as the identification of the gene responsible for the phenotype. At the same time as work was being undertaken on nhr1, our group isolated two ahydrotropic mutants, mizu-kussei1 (miz1) and miz2 (Kobayashi et al., 2007; Miyazawa et al., 2009a). Although these mutants were completely ahydrotropic, they retained normal gravitropism, which enabled the dissection of molecular mechanisms unique to root hydrotropism. These are the only ahydrotropic mutants in which the responsible genes have been identified. MIZ1 encodes a protein of 297 amino acids that contains a domain of unknown function (DUF617), and MIZ2 encodes a guanine exchange factor for ADP-ribosylation factor (ARF-GEF), which is known as GNOM
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(Kobayashi et al., 2007; Miyazawa et al., 2009a). Thus far, our investigations have indicated that MIZ1 expression is restricted to the root tip, the mature region of primary roots and leaf hydathodes (Kobayashi et al., 2007). As hydrostimulation sensing is thought to occur at the root tip, it is likely that MIZ1 functions during an early stage of the root hydrotropic response. As stated above, MIZ1 contains a domain of unknown function, which we termed the MIZ domain. Genes encoding this domain are found in terrestrial plants but not in algae, fungi, bacteria or animals. Searches of the Arabidopsis genome revealed 12 genes encoding the MIZ domain; however, as yet, there are no reports describing its function. The missense mutation in miz1-1 is found in a conserved amino acid within the MIZ domain, which suggests that this domain plays an important role in root hydrotropism (Takahashi et al., 2009). Moreover, amino acid sequences outside the MIZ domain are not conserved among the gene family. With the exception of slightly reduced root phototropism, the miz1 mutant shows no obvious morphological defects other than the ahydrotropic root phenotype (Kobayashi et al., 2007). Phototropism of the hypocotyl appears normal in the miz1 mutant. Moreover, normal root hydrotropism is observed in nph1 mutants, which lack phototropin1. Although much of the relationship between root phototropism and hydrotropism remains to be elucidated, these findings suggest that MIZ1 may function in a common signalling pathway (Takahashi et al., 2002). Further investigations of MIZ1 will shed new light on the as-yet unidentified relationships between hydrotropism and phototropism in roots. Unlike MIZ1, MIZ2 encodes a well-described protein, GNOM (Miyazawa et al., 2009a). GNOM plays important roles in membrane trafficking and it is responsible for the dissociation of GDP from ARFs (Anders and Ju¨rgens, 2008). The guanine exchange activity of ARF-GEFs resides within the Sec7 domain, which exhibits strong homology to the yeast protein Sec7p. Embryonic lethality is observed in most severe gnom alleles that contain mutations inside the Sec7 domain. The conserved domains, DCB and HUS, are located upstream of the Sec7 domain, while the conserved domains, HDS 1–3, are located downstream. Interestingly, miz2 is a missense mutation that causes a single amino acid change in the less conserved region flanking the HDS1 domain. The phenotype of this mutant was surprising, as nearly all the defects associated with gnom alleles involve altered auxin transport. Proper localization of the auxin efflux facilitator requires GNOM function, and defects in localization cause agravitropic root growth, as well as abnormal organ patterning and vein formation (Geldner et al., 2003, 2004). The miz2 mutants were not defective in organ patterning or vein formation and showed normal localization of the mutant GNOM (gnommiz2), as well as the auxin efflux
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facilitator, PIN1 (Miyazawa et al., 2009b). Pharmacological and genetic studies support the suggestion that polar auxin transport is unnecessary for hydrotropism in Arabidopsis roots; however, these findings imply that GNOM performs an as-yet undiscovered role in root hydrotropism, as well as being instrumental for polar auxin transport. When a primary root of Arabidopsis is grown on an inclined agar plate, it exhibits an oscillatory pattern called waving (Okada and Shimura, 1990). Although we do not understand the exact reason why this root movement is induced, it is likely that the graviresponse and/or touch response is involved in the phenomenon (Oliva and Dunand, 2007). This root movement can be interpreted as reflecting the obstacle-avoidance response. It would be interesting to know whether or not there is any integration between the signalling pathways for root hydrotropism and wavy root growth. Some mutants with a short wavelength phenotype also demonstrate enhanced hydrotropism (Takahashi et al., 2002). Moreover, all reported ahydrotropic mutants also exhibit abnormal wavy growth. However, the waving phenotypes of the ahydrotropic mutants vary. For example, nhr1 shows enhanced wavy growth, while miz1 and miz2 exhibit decreased wavy growth (Eapen et al., 2003; Kobayashi et al., 2007; Miyazawa et al., unpublished result). Currently, we do not know why these opposite phenotypes are found in ahydrotropic mutants. However, it is clear that the signalling pathway for root hydrotropism interacts with many other root navigation signalling pathways.
IV. MECHANISMS FOR OTHER ROOT TROPISMS RELATED TO DROUGHT AVOIDANCE Plant roots show positive gravitropism and negative phototropism. Under normal conditions, soil water conditions reflect the effects of gravity on precipitation and the soil surface is assumed to be arid when exposed to sunshine. Thus, roots show positive gravitropism and negative phototropism to orient growth appropriately. In this section, we will give a brief summary of the molecular mechanisms underlying these two tropisms. As positive gravitropism and negative phototropism are particularly important for avoiding drought conditions, we will also discuss their regulation under water-stressed conditions. A. ROOT GRAVITROPISM
Physiological analyses of roots in which the root cap was removed surgically indicated that the gravity sensor for root gravitropism resides within the root cap. The root cap is organized by specialized cells, namely columella and lateral
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root cap cells. Laser ablation experiments have indicated that columella cells are essential for gravitropism (Blancaflor et al., 1998). Columella cells contain starch-filled plastids, termed amyloplasts. Due to the dense nature of starch, amyloplasts sediment under the force of gravity and accumulate at the lower side of the cell. As this sedimentation is observed immediately after gravistimulation, it is thought to be the trigger for gravi-sensing (Sack, 1991). This hypothesis is supported by genetic analyses of starchless Arabidopsis mutants and Kiss et al. (1989, 1996) showed that the roots of starchless mutants exhibit a weaker response to gravity than the wild type. Amyloplast sedimentation generates a physical signal that is converted into a biochemical signal, which initiates the graviresponse. However, the mechanisms underlying these processes are not fully understood. One possibility is that the sedimented amyloplasts activate mechano-sensitive ion channels in intercellular membranes or the plasma membrane. The activated channels lead to changes in Hþ and Ca2þ flow, and these ions have been postulated to play important roles in gravity signal transduction within root cap statocytes (Fasano et al., 2001; Perera et al., 2001). The starchless mutant pgm1 does not show pH changes after gravistimulation, which suggests that changes in root cap pH depend upon amyloplast sedimentation (Fasano et al., 2001). ALTERED RESPONSE TO GRAVITY1 (ARG1) and its homologue ARL2 play key roles in gravity signal transduction (Boonsirichai et al., 2003). These genes encode DnaJ-like proteins. Following gravistimulation, no changes in cytosolic pH were detected in the roots of an arg1 mutant; however, the mutant did exhibit reduced gravitropism. Moreover, the arg1 mutant phenotype was recovered by the expression of ARG1 in the root cap, which suggests that ARG1 functions during early gravisignal transduction (Boonsirichai et al., 2003). According to the Cholodny-Went hypothesis, gravistimulation induces an asymmetrical auxin gradient within gravistimulated organs, and auxin-dependent growth inhibition results in a downward curvature of roots. Indeed, an asymmetric pattern of auxin-responsive gene expression occurs rapidly in columella and lateral root cap cells following gravistimulation (Ottenschla¨ger et al., 2003). Pharmacological and genetic disruption of auxin transport inhibits the asymmetrical auxin redistribution and tropic responses in the root (Fujita and Syono, 1996; Li et al., 1991; Luschnig et al., 1998; Marchant et al., 1999). A combination of auxin influx and efflux transporters mediates auxin transport through the root cell files. In Arabidopsis roots, AUX1/LAX family proteins function as auxin influx carriers. As the mutation of AUX1 completely disrupts root gravitropism, it is likely that the AUX1-mediated influx of auxin is essential for gravitropism (Marchant et al., 1999). Localization analyses have shown that AUX1 is expressed in lateral root cap and stele cells, and that the expression of AUX1 in lateral root cap cells alone can rescue
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gravitropism in an aux1 mutant (Swarup et al., 2001, 2005). These results indicate that gravitropism requires auxin redistribution via the lateral root cap. The PINFORMED (PIN) auxin transporters have been shown to play critical roles in auxin efflux. PIN3 localizes symmetrically in the plasma membrane of columella cells but will relocalize to the lower side of the cell upon gravistimulation (Friml et al., 2002). This gravity-induced relocalization was not observed in mutants of ARG1 or ARL2, which suggests that ARG1/ARL2-dependent signalling is required for lateral relocalization of PIN3 (Harrison and Masson, 2007). Surprisingly, a pin3 mutant only exhibited a weak reduction in the root gravitropic response (Friml et al., 2002); however, this finding may be due to the redundancy of function among the PIN protein family (Vieten et al., 2005). In roots, PIN2 localizes to the apical membrane of epidermal cells and the basal membrane of cortical cells, and pin2 mutants completely lack root gravitropism (Luschnig et al., 1998; Muller et al., 1998). Following gravistimulation, epidermal PIN2 proteins present on the upper side of the cell were rapidly internalized and degraded (Abas et al., 2006). This degradation process results in an asymmetric distribution of PIN2, which is consistent with the lateral auxin distribution in the root. It is well known that the subcellular localization of PIN proteins is strictly determined by regulatory proteins. For instance, basal localization is regulated by GNOM, and apical localization is regulated by PINOID protein kinase (Friml et al., 2004; Geldner et al., 2003). Indeed, root gravitropic responses are disrupted by mutations in either GNOM or PINOID (Geldner et al., 2004; Sukumar et al., 2009). Later phases of gravitropism depend upon auxin-regulated transcription, which is controlled by a combination of two transcriptional regulators, AUXIN RESPONSE FACTOR (ARF) and AUX/IAA protein. The Arabidopsis genome contains 23 genes that encode ARF proteins, but two closely related genes, ARF7 and ARF19, are required for gravitropism (Okushima et al., 2005). Under low auxin concentrations, AUX/IAA binds ARF and represses its activity. In the presence of high auxin concentrations, auxin binds and activates the SCFTIR1 complex, which degrades AUX/IAA proteins. As expected, a mutation that stabilizes AUX/IAA proteins will also disrupt gravitropism (Fukaki et al., 2002; Weijers et al., 2005). B. REGULATION OF GRAVITROPISM BY WATER STRESS
Many researchers have demonstrated that the gravitropic response is modulated by water conditions. For instance, Sharp and Davies (1985) showed that water depletion accelerates root distribution in deeper soils. Osmotic stress also
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enhances the gravitropic response of maize roots (Leopold and LaFavre, 1989). These observations led to the hypothesis that water stress enhances the gravitropic response. Drought stress is known to induce ABA biosynthesis in roots (Zhang and Davies, 1987). ABA functions not only as a signal molecule for different types of drought response, but also as a regulator of root gravitropism. ABA treatment increases the gravitropic curvature of maize roots in a dose-dependent manner (Wilkins and Wain, 1976). In Arabidopsis, the ABA synthesis and signalling mutants, aba1 and abi2, show slightly reduced gravitropic responses (Takahashi et al., 2002). In contrast, Moore (1990) found a normal graviresponse in ABA-deficient mutants of maize, which suggests that while changes in ABA homeostasis may enhance the gravitropic response, ABA is not essential for gravitropism. Although numerous findings have described relationships between ABA and gravitropism, we have little understanding of how ABA functions in gravitropism under drought conditions. Ethylene is another important stress response hormone in plants. Usually, high concentrations of ethylene up-regulate auxin biosynthesis and, thus, inhibit the elongation of root cells (Swarup et al., 2007). To maintain root elongation under drought conditions, ABA represses ethylene synthesis (Sharp and LeNoble, 2002; Spollen et al., 2000). However, the mutation of EIN2, which is a positive regulator of ethylene signalling, causes a reduction of root growth under osmotic stress conditions (Wang et al., 2007). This finding suggests that ethylene signalling is an important factor in root growth under drought conditions. In addition, there is increasing evidence to indicate that ethylene also regulates the gravitropic response. Studies have shown decreased gravitropic responses following early phase application of ethylene gas or the ethylene precursor ACC in maize or Arabidopsis roots, respectively (Buer et al., 2006; Lee et al., 1990). Pharmacological inhibition of ethylene synthesis also reduced the gravitropism of maize roots (Chang et al., 2004; Lee et al., 1990). Inhibition of ethylene synthesis by drug treatments also reduced the gravitropism of maize roots (Chang et al., 2004; Lee et al., 1990). Thus, it appears that ethylene can both positively and negatively affect gravitropism. As the auxin transport inhibitor NPA abolishes the effect of ethylene on gravitropism (Lee et al., 1990), it is likely that ethylene affects the gravityinduced asymmetrical distribution of auxin. Recent work has demonstrated that flavonoids may function as ethylene-dependent regulators of gravitropism (Buer et al., 2006). In Arabidopsis roots, ethylene treatment inhibits the gravitropic response; however, this gravitropic inhibition is lost in chalcone synthase-deficient tt4 mutants (Buer and Muday, 2004). Flavonoid synthesis is modulated by environmental changes such as gravistimulation, and ACC treatment delays gravity-induced flavonoid synthesis (Buer and Muday, 2004; Buer et al., 2006). In addition, PIN mRNA expression patterns are
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altered in the tt4 mutant, which shows disruption to the polar localization of some PIN proteins (Peer et al., 2004). These results indicate that flavonoids are important for maintaining the auxin flow required for gravitropism, and that ethylene inhibits root gravitropism in Arabidopsis via modulation of flavonoid synthesis. Osmotic stress is also known to induce flavonoid accumulation (Chalker-Scott, 1999), which suggests that under water-stress conditions, flavonoid regulation of the gravitropic response occurs downstream of ethylene. Although many researchers have pointed out that drought signals modulate the gravitropic response, the obvious ecological benefits of root gravitropism under drought conditions are not fully understood. Mutants in signalling or synthesis of the stress signal hormones ABA and ethylene show poor tolerance to water deficits or salinity (Cao et al., 2007). However, ABA and ethylene play multiple roles in stress tolerance and, thus, a reduced gravitropic response would not be the sole factor contributing to the phenotypes observed with ABA and ethylene signalling or synthesis mutants under stress conditions. In addition, the roots of aux1 mutants exhibit a completely agravitropic phenotype, but no difference was detected in the survival ratio of aux1 and wild-type seedlings, even under drought conditions (Vartanian, 1996). As this field progresses, it is anticipated that novel approaches will assist in the elucidation of the ecological functions of the root gravitropic response. C. ROOT PHOTOTROPISM
Typically, roots show negative phototropism in response to blue or white light and this assists in the development of the root system in soil. In contrast, Arabidopsis roots also exhibit positive phototropism in response to red light; however, the ecological function of this response is not known (Ruppel et al., 2001). Arabidopsis has three major classes of photoreceptors, that is, the phytochromes, cryptochromes and phototropins. Genetic analyses of phototropic mutants have revealed that the phototropin family is essential for blue light perception and that these proteins are required for negative phototropism in roots (Christie et al., 1998; Sakai et al., 2001). It has been shown that two phytochromes, namely PHYA and PHYB, are required for positive phototropism in roots (Kiss et al., 2003). The mechanisms for signal transduction in root phototropism remain largely unknown. Isolation of the phototropism mutant nph3 led to the identification of NPH3, which encodes a plant-specific protein containing a BTB domain and a coiled-coil domain (Motchoulski and Liscum, 1999). NPH3 interacts with a phototropin, PHOT1, via the coiled-coil domain
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and with CULLIN3, which is a subunit of E3 ubiquitin ligase, via its BTB domain (Inada et al., 2004; Motchoulski and Liscum, 1999). In another phototropic mutant, rpt2, the NPH3-like gene RPT2 is disrupted. Inada et al. (2004) demonstrated that RPT2 interacts with both PHOT1 and NPH3. These results suggest that RPT2 functions as an adaptor between PHOT1 and NPH3. PHYTOCHROME KINASE SUBSTRATE1 (PKS1) was isolated originally from a screen using PHYA-interacting factor (Fankhauser et al., 1999). PKS1 also interacts with PHOT1 (Lariguet et al., 2006), and PKS1 mutants show reductions in both positive and negative root phototropism (Boccalandro et al., 2008; Molas and Kiss, 2008). Further, enhanced gravitropism was observed in a pks1 mutant, which indicates that PKS1 is involved in the transduction of both gravity and light signals (Boccalandro et al., 2008). In addition to gravitropism, pharmacological analysis has demonstrated that auxin-dependent asymmetric growth is required for root phototropism (Fujita and Syono, 1997). However, the molecular machinery required to establish an auxin gradient in response to light perception remains unclear. Recently, Ruzicka et al. (2010) demonstrated that PIS1 encodes an ABC transporter that can transport the IAA precursor, IBA. As the roots of PIS1 mutants exhibit slightly reduced phototropism and gravitropism (Fujita and Syono, 1997), it is possible that PIS1-mediated auxin transport may contribute to the establishment of a phototropic auxin gradient. D. ECOLOGICAL FUNCTION OF PHOTOTROPISM
In contrast to the research on gravitropism, there is little evidence from which to determine whether or not root phototropism is enhanced by drought or its related signals, such as salt, ABA and ethylene. Experiments on the phototropic mutant phot1 have indicated that root phototropism could contribute to the orientation of the root system, directing growth away from the soil surface to avoid drought conditions (Galen et al., 2007). Thus, root phototropism represents an important strategy for drought adaptation in plants.
V. CONCLUSIONS AND PERSPECTIVES Considering their sessile nature and the necessity for water acquisition, plants have evolved unique mechanisms to avoid or tolerate drought and to utilize the resources available at the site of germination. To improve plant survival under water-deficient conditions, it is necessary to understand and utilize drought tolerance and/or avoidance mechanisms. Intense study has led to many
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discoveries regarding the mechanisms underlying drought tolerance in plants, and these are described in the other chapters. However, it is important to develop plants with enhanced drought avoidance capabilities, as these seedlings would have the ability to acquire water for continued survival and to sustain growth. Roots exhibit tropisms in response to many environmental cues, including gravity, light and moisture gradients. Among these, the root hydrotropism response to moisture gradients is thought to function in water-stress avoidance as well as in the efficient uptake of water and nutrients from the soil (Takahashi et al., 2009). As described in this chapter, clarification of molecular mechanisms underlying root hydrotropism is just beginning, and our knowledge is too fragmented to determine the entire process. Nevertheless, we now have some clues to add to the puzzle, at least in Arabidopsis. Detailed studies that connect the physiological, genetic and molecular biological information of root hydrotropism will help develop a broader understanding of this phenomenon. In addition, it is important to clarify the mechanisms that integrate the different environmental cues that lead to the directional growth of roots. The ecological significance of root hydrotropism must also be elucidated. Thus far, contradictory results have been reported (e.g. Cole and Mahall, 2006; Tsuda et al., 2003) and these problems will need to be addressed using sophisticated experimental systems and a wide variety of plant species and/or ecotypes. Along with such studies, different types of hydrotropism mutants will be useful for developing a more detailed understanding of this phenomenon. We believe that future investigations will lead to the elucidation of all root tropism mechanisms, and especially hydrotropism. Once we understand root tropisms at the molecular level, these processes may be manipulated to develop plants with enhanced survival capabilities under water-stress conditions, improvements that will be beneficial for overcoming current climate changes.
ACKNOWLEDGEMENTS This work is supported by Grants-in-Aid for Scientific Research (B: 20370017) from JSPS, Grants-in-Aid for Scientific Research on Priority Areas (No. 22120004) from the MEXT, the Global COE Program J03 (Ecosystem Management Adapting to Global Change) of the MEXT to H. T., Special Research Grant of Global COE Program J03 for T. Y. (No. 27220004) and JSPS Research Fellowship for Young Scientists to T. M. (No. 09J06705). Y. M. is supported by the Funding Program for Next-Generation WorldLeading Researchers (GS002). This work was carried out as a part of the ‘‘Ground-based Research Announcement for Space Utilization’’, promoted by the Japan Space Forum.
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Roles of Circadian Clock in Developmental Controls and Stress Responses in Arabidopsis: Exploring a Link for Three Components of Clock Function in Arabidopsis
RIM NEFISSI,* YU NATSUI,* KANA MIYATA,* ABDELWAHED GHORBEL{ AND TSUYOSHI MIZOGUCHI*,1
*Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki, Japan { Biotechnology Center, Borj Cedria Science and Technology Park, Route Touristique Borj Ce´dria-Soliman, Hammam-Lif, Tunisia
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. ELF3, a Novel Protein without Significant Similarity to any Known Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. ELF3 as a Novel Nuclear Protein ........................................... B. ELF3-Like Genes in Non-Arabidopsis Plant Species ..................... C. Polyglutamine Tracts in ELF3 ............................................... III. Roles of ELF3 in Light Signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. ELF3 in the light input pathway to the clock .............................. B. ELF3 Interaction with PHYB ................................................ C. PHYB-Independent Roles of ELF3 ......................................... IV. Roles of ELF3 in the Control of Flowering Time . . . . . . . . . . . . . . . . . . . . . . . . . . A. Current View of Photoperiodic Control of Flowering Time Linked to the Circadian Clock in Arabidopsis....................................... B. Co-Independent Role of ELF3 in the Control of Flowering Time ..... C. Potential Role of ELF3 as a Scaffold for the Input, Oscillator and Output of the Arabidopsis Clock ............................................. D. Role of ELF3 as an Adaptor for COP1 and GI, Possibly in the CRY2-Dependent Flowering Pathway......................................
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Corresponding author: E-mail:
[email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00011-4
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E. Attempts to Identify Suppressors and Enhancers of ELF3 Phenotypes............................................................... V. Roles of ELF3 and Other Clock Proteins in Stress Tolerance. . . . . . . . . . . . . . A. Ambient Temperature Stress ................................................. B. Viability Under Very SD Regime ............................................ C. Microarray Analysis ........................................................... D. Gated induction of DREB/CBF by Low Temperature ................... E. Roles of PRR9, PRR7 and PRR5 in the Cold-Stress Response ........ F. PIF7 as a Possible Mediator between the Circadian Clock and DREB1/CBF in Stress Signalling ............................................ G. TOC1 as a Molecular Switch Connecting the Clock and Responses to ABA and Drought ............................................................. H. Release of Seed Dormancy.................................................... VI. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT Genetic analysis of the early flowering 3 (elf3) mutant of Arabidopsis thaliana indicates that ELF3 plays key roles in the regulation of plant morphology, flowering time and stress response, all of which are controlled by circadian clock. Although ELF3 appears to have multiple functions and has been shown to interact physically with the photoreceptor phyB, its ability to regulate several distinct signalling pathways has not been elucidated. This lack of information is attributable in part to the uniqueness of the ELF3 gene, which encodes a novel nuclear protein with no significant sequence similarity to any characterized protein in the existing public databases. Further, little is known about direct protein–protein interactions of ELF3, or about mutations that suppress elf3, phenotypes. Therefore, it is difficult to hypothesize about potential factors downstream of ELF3. In this chapter, we summarize recent progress on the characterization of ELF3 and discuss potential roles of ELF3 in plants. Several reports have demonstrated that a circadian clock affects stress responses in Arabidopsis and that DREB1A/CBF3 mediates between the clock and cold-inducible gene expression. Therefore, possible roles of clock genes such as ELF3, PRRs, LHY and CCA1 in the environmental stress responses of Arabidopsis are also discussed.
I. INTRODUCTION Developmental transitions in plants are strongly affected by light quality, intensity and duration. Arabidopsis is a facultative long-day (LD) plant, as it flowers earlier under LD; (e.g. 16 h light/8 h dark) than under short-day (SD; e.g. 8 h light/16 h dark) conditions (Simpson et al., 1999). In addition, exposure to blue or far-red light promotes flowering in Arabidopsis. Many genes are involved in the photoperiodic regulation of flowering in Arabidopsis. In many prokaryotic and eukaryotic organisms, including plants, biological clocks mediate the response of several physiological and molecular processes to diurnal changes in environmental conditions such as
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light quality and quantity, temperature and humidity. Circadian rhythms persist with a period close to 24 h in the absence of any environmental time cues and are generated by an endogenous timing mechanism. The basic principles of circadian clocks are known for many organisms such as cyanobacteria, Neurospora, Arabidopsis, mice and humans. The clock consists of oscillating molecules that form a negative, autoregulatory feedback loop (Bell-Pedersen et al., 2005; Young and Kay, 2001). Photoperiodic flowering is largely affected by the circadian clock. Genetic approaches have identified more than two dozen key genes for clock functions in Arabidopsis. Although phenotypic characterization of clock mutants and identification of the corresponding genes have been performed, the biochemical functions of most clock proteins are still largely unknown. For example, EARLY FLOWERING 3 (ELF3; Hicks et al., 2001; Zagotta et al., 1992, 1996), GIGANTEA (GI; Fowler et al., 1999) and EARLY FLOWERING 4 (ELF4; Doyle et al., 2002; Kikis et al., 2005) are proposed to perform key clock functions in Arabidopsis, and the genes for these clock proteins have been known for nearly a decade. However, the novel proteins encoded by the ELF3, GI and ELF4 genes have no significant sequence similarity to any characterized proteins in the existing public databases, providing few clues regarding their biochemical roles. It has recently been shown that GI interacts with FKF1 and ZEITLUPE (ZTL; Somers et al., 2000) and that these interactions are involved in the degradation of the proteins CDF1 and TIMING OF CAB EXPRESSION 1 (TOC1; Imaizumi et al., 2005; Kim et al, 2007; Ma´s et al., 2003; Sawa et al., 2007). The ELF3 gene of Arabidopsis is involved in the regulation of a wide variety of processes, including plant morphology, flowering time and circadian rhythm (Carre´, 2002). The elf3-1 mutation was the first loss-of-function allele of elf3 to be identified and was found in a screening for early flowering under an SD regime (Zagotta et al., 1996). Mutations in ELF3 result in the loss of both photoperiod sensitivity and circadian regulation, making ELF3 a candidate for linking circadian clock function and photoperiodic induction of flowering (Hicks et al., 2001). The elf3 mutant plants flower earlier than wild type under both SD and LD conditions (Zagotta et al., 1996). The mutant plants exhibit pale-green leaves and elongated hypocotyls and petioles, which are phenotypes associated with a defect in the perception or transduction of light signals (Coupland, 1997). Moreover, elf3-1 plants grow poorly compared with wild type under both LD and SD conditions, suggesting that they are more sensitive to other stresses in addition to light duration (Green et al., 2002). Taken together, these observations suggest that ELF3 is a multifunctional protein. Various approaches have been taken to uncover the mechanism by which the mysterious ELF3 protein regulates several important biological processes
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in Arabidopsis. The purpose of this chapter is to summarize recent advances in our understanding of the roles played by ELF3 and other clock proteins in the molecular mechanisms underlying the regulation of flowering time, organ elongation, and environmental stress responses in Arabidopsis. In the first section, we discuss the general characteristics of the amino acid sequence of ELF3 and ELF3-like proteins in plants. This is followed by an examination of the roles of ELF3 in light signalling (Section II) and in photoperiodic flowering (Section III). Finally, stress responses affected by clock genes, including ELF3, are summarized. For reference, all the genes discussed in this chapter are listed in Table I.
II. ELF3, A NOVEL PROTEIN WITHOUT SIGNIFICANT SIMILARITY TO ANY KNOWN PROTEINS Although the amino acid sequence of ELF3 shows no similarity to those of other proteins reported in the public databases, ELF3 and ELF3-like proteins in Arabidopsis and other plants share several conserved domains. Among these, the polyglutamine-repeat tracts (Q repeats) are thought to be responsible for several quantitative trait loci involved in responses to light, hypocotyl elongation and circadian rhythms. The possible functions of the Q repeats in ELF3 are discussed in more detail in this first section. A. ELF3 AS A NOVEL NUCLEAR PROTEIN
ELF3 is a novel protein of 695 amino acids, with little similarity to previously characterized proteins of Arabidopsis (Hicks et al., 2001). ELF3 was proposed to function as a transcription factor, as it is particularly rich in serine, proline and glutamine/threonine regions similar to those found in some transcriptional regulators (Carre´, 2002). As it contains a large number of potential phosphorylation sites, phosphorylation may be involved in ELF3 regulation (Hicks et al., 2001). B. ELF3-LIKE GENES IN NON-ARABIDOPSIS PLANT SPECIES
Genes similar to Arabidopsis ELF3 are found in other plant species such as rice, Lemna and Mesembryanthemum crystallinum (common ice plant). Rice has two genes similar to ELF3 (Izawa et al., 2003), and these two OsELF3like amino acid sequences show considerable similarities with the Arabidopsis ELF3 sequence. However, their expression profiles do not reflect the rhythmic expression pattern of Arabidopsis ELF3, whose expression is under
TABLE I Arabidopsis flowering time genes and their description Gene name
Accession no.
ELF3: EARLY FLOWERING 3
AT2G25930
GI: GIGANTEA
AT1G22770
ELF4: EARLY FLOWERING 4 FKF1: FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 ZTL: ZEITLUPE
AT2G40080 AT1G68050
CDF1: CYCLING DOF FACTOR 1
AT5G62430
TOC1/PRR1: TIMING OF CAB EXPRESSION 1/PSEUDORESPONSE REGULATOR 1 LHY: LATE ELONGATED HYPOCOTYL CCA1: CIRCADIAN CLOCK ASSOCIATED 1 SVP: SHORT VEGETATIVE PHASE PHYB: PHYTOCHROME B
AT5G61380
AT5G57360
AT1G01060 AT2G46830 AT2G22540 AT2G18790
TIC: TIME FOR COFFEE
AT3G22380
FHY3: FAR-RED ELONGATED HYPOCOTYLS 3
AT3G22170
Description A novel protein that is expressed rhythmically and interacts with phyB to control plant development, flowering and circadian rhythms A protein that regulates several developmental processes, including photoperiodic flowering, phyB signaling, circadian rhythms, carbohydrate metabolism and cold stress response A protein necessary for light-induced expression of both CCA1 and LHY A protein containing a PAS domain, kelch repeats and an F-box domain, involved in protein degradation A protein containing a PAS domain, kelch repeats and an F-box domain, involved in protein degradation A transcription factor with a Dof-type zinc-finger motif involved in repression of the expression of CO A pseudo-response regulator involved in the control of circadian rhythms A myb-related transcription factor involved in the control of circadian rhythms along with CCA1 A myb-related transcription factor involved in the control of circadian rhythms along with LHY A MADS-box protein that acts as a floral repressor A red/far-red photoreceptor involved in the regulation of de-etiolation, light promotion of seed germination and shade avoidance response A plant-specific clock regulator working close to the central oscillator and affecting the circadian gating of light responses A component of the PHYA signaling network that mediates the FR-HIR response to far-red light (continues)
TABLE I Gene name
Accession no.
PHYA: PHYTOCHROME A
AT1G09570
CO: CONSTANS
AT5G15840
FT: FLOWERING LOCUS T
AT1G65480
FLC: FLOWERING LOCUS C
AT5G10140
SOC1: SUPPRESSOR OF OVEREXPRESSION OF CO 1 CRY2: CRYPTOCHROME 2
AT2G45660
CIB1: CRYPTOCHROMEINTERACTING BASICHELIX-LOOP-HELIX 1 TFL1: TERMINAL FLOWER 1
AT4G34530
PIF7: PHYTOCHROMEINTERACTING FACTOR 7 DREB1A/CBF3: DEHYDRATION RESPONSE ELEMENT BINDING 1A/C-REPEAT BINDING FACTOR 3
AT5G61270
AT1G04400
AT5G03840
AT4G25480
(continued ) Description
A red/far-red light photoreceptor involved in the regulation of de-etiolation, gravitropism, flowering and phototropism A protein with zinc-finger motif involved in regulation of photoperiodic flowering under long days FT, together with LFY and SOC1, promotes flowering and is antagonistic with its homologous protein, TFL1 A MADS-box protein that functions as a repressor of floral transition and contributes to temperature compensation of the circadian clock A MADS-box protein that functions as activator of floral transition A blue-light receptor mediating blue-light-regulated cotyledon expansion, hypocotyl elongation and flowering time A transcription factor that interacts with CRY2 and acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation A protein required for the control of inflorescence meristem identity and involved in the floral initiation process A transcription factor with a basic helix–loop–helix (bHLH) motif that negatively regulates the phyB-mediated seedling de-etiolation A member of the DREB subfamily A-1 of the ERF/AP2 transcription factor family involved in response to low temperature, dehydration and ABA
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robust circadian control (Murakami et al., 2007). In contrast, the mRNA expression profile of LgELF3H1, an ELF3-like gene from Lemna species, is similar to that of Arabidopsis ELF3 under light/dark conditions (Miwa et al., 2006). Serikawa et al. (2008) showed that the role of LgELF3H1 in the circadian clock is comparable to that of ELF3 in Arabidopsis. In fact, the RNAi suppression of LgELF3H1 under constant light (LL) reduced the rhythmic expression amplitude of CCA1 and PRR1, and over-expression of LgELF3H1 lengthened the period of the circadian rhythm (Serikawa et al., 2008). These phenotypes appear similar to the phenotypes induced by elf3 mutation and ELF3 over-expression in Arabidopsis. McELF3, an ELF3-like gene in M. crystallinum (Boxall et al., 2005), displays an expression pattern similar to that of Arabidopsis ELF3, with transcription under circadian control and peaking in the evening. C. POLYGLUTAMINE TRACTS IN ELF3
Unlike other clock proteins in Arabidopsis, ELF3 possesses a motif known as Q repeats. Based on a comparison of ELF3 sequences reported from 60 different wild-type Arabidopsis plants, the number of Q-repeats in the C-terminal region is a polymorphic trait (Tajima et al., 2007). There was significant correlation between the number of Q repeats and two circadian markers, amplitude and period length, in Arabidopsis, suggesting that the length of the polyglutamine tracts may affect the function of ELF3 in the control of circadian rhythm (Tajima et al., 2007). The Q repeats are conserved in Arabidopsis lyrata subsp. lyrata (Q 19; NCBI Accession No. CAL85633) and subsp. petraea (Q 19, 20 and 21; CAL85631, CAL85630 and CAL85632), but not in M. crystallinum (AAQ73529), Vitis vinifera (CAO43769), Triticum aestivum (ABL11477), Oryza sativa (NP_001056770), Lemna gibba (BAD97872), Lemna paucicostata (BAD97868) or Pisum sativum (ABP81864). A far-red pulse at the end of day is a light signal that stimulates plants living in natural shade. Recent genetic mapping of natural variations in the shade-avoidance response (Coluccio et al., 2011) identified EODINDEX1 as the most significant quantitative trait locus involved in the response to the end-of-day signal. ELF3 was proposed as the most likely candidate gene underlying the natural variation in EODINDEX1.
III. ROLES OF ELF3 IN LIGHT SIGNALLING The circadian clock is composed of three components: the input pathway, a central oscillator and the output pathway (Fig. 1). Photoreceptors such as phytochromes and cryptochromes play major roles in the input pathway.
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Input pathways
Output pathways
Oscillator
LHY CCA1 Gl
PHYB ELF3
TOC1 ELF4
CO
FT
Flowering
Fig. 1. A model showing the molecular hierarchy that controls flowering time in Arabidopsis in response to photoperiod and the proposed interlocking feedback loops. ELF3 expression enables the oscillator to control the light sensitivity of both the resetting of the clock and the induction of circadian outputs. This suggests that ELF3 may mediate among the input, the oscillator and one of the outputs of the circadian clock in Arabidopsis.
Clock proteins are believed to function in the input pathway or as central oscillators, or both. In this section, we examine the possible roles of ELF3 in general light signalling and in the light input pathway. We also discuss the protein–protein interactions between ELF3 and phyB, and phyB-dependent and -independent roles of ELF3 in light signalling. A. ELF3 IN THE LIGHT INPUT PATHWAY TO THE CLOCK
An elf3 loss-of-function mutation (elf3-1) in Arabidopsis conferred arrhythmia for all rhythms tested under LL. However, when transferred to constant darkness (DD), elf3-1 plants retained the circadian rhythm. This indicates that ELF3 has little, if any, role in regulating circadian clock functions in the dark (Jeong and Clark, 2005; Liu et al., 2001; McWatters et al., 2000). Thus, instead of being an essential component of the clock itself, ELF3 may mediate an interaction between phototransduction and the circadian clock. This would be consistent with the light-dependent arrhythmia in plants with defective ELF3 (McWatters et al., 2000; Reed et al., 2000). The ELF3 gene product was proposed to function in a light input pathway to the circadian oscillator, and the absence of ELF3 was hypothesized to alter the coordination between the light and circadian regulatory pathways, resulting in the altered flowering time and photoperiodic intensity observed in elf3 mutants (Barak et al., 2000; Hicks et al., 2001; Searle and Coupland, 2004). In Arabidopsis, there are two major types of photoreceptors, the red-lightabsorbing phytochromes and blue-light-absorbing cryptochromes (Covington
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et al., 2001). The hypocotyl elongation response under blue light in plants overexpressing ELF3 (ELF3-OX) is similar to that in wild-type plants, suggesting that ELF3 is not involved in the blue-light inhibition mediated by cryptochromes. Under continuous red light, ELF3-OX plants do not exhibit the long hypocotyl phenotype that is seen in phyB mutants (Liu et al., 2001). This indicates that the ELF3 gene product is a negative regulator of phyB-mediated induction of circadian outputs (Covington et al., 2001; Kikis et al., 2005; Salome´ and McClung, 2005). Further, ELF3 must affect some downstream aspect of phyB signalling or a phyB-independent regulatory pathway (Reed et al., 2000). Other genes have been shown to be involved in the light input pathway to the clock. The mutation of TIME FOR COFFEE (TIC) also causes a defective gating of light, but at a different time of the cycle. TIC acts at middle to late night (Ding et al., 2007; Hall et al., 2003), whereas ELF3 acts with maximal effect during early night (Hicks et al., 1996). The double mutant tic;elf3 showed additive morphological, rhythmic and gene expression phenotypes, indicating that ELF3 and TIC have independent functions (Hall et al., 2003). Recently, another component of the light gating pathway, FAR-RED ELONGATED HYPOCOTYL 3 (FHY3), was identified in Arabidopsis (Allen et al., 2006). FHY3 regulates light signalling during the day; fhy3 mutants conferred arrhythmia of central oscillator genes and disrupted resetting of the clock under continuous red light (Allen et al., 2006). B. ELF3 INTERACTION WITH PHYB
The N-terminal region of ELF3 was shown to physically interact with phyB, suggesting that ELF3 may function as a direct modulator of signal information from phyB via this interaction (Fig. 2; Liu et al., 2001). Yeast two-hybrid system experiments demonstrated that ELF3 interacts with the C-terminal domain of phyB, but not that of phyA, suggesting that its role is specific to the regulation of phyB (Carre´, 2002; Liu et al., 2001). The ELF3–phyB complex may regulate multiple signalling pathways as well as gene expression. A phyB-mediated modification of ELF3 may allow interactions with other proteins to control gene transcription (Liu et al., 2001). C. PHYB-INDEPENDENT ROLES OF ELF3
In constant red light, elf3-1;phyB-9 double mutants had longer hypocotyls, more elongated petioles and earlier flowering compared with single mutants. These additive phenotypes suggest that ELF3 and phyB may act independently of each other, at least in part (Kim et al., 2005; Reed et al., 2000).
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Hypocotyl elongation 3 1
PhyB
1
3 4
PIF/PILs
DREB1/CBF
1
CK2A
2
Cold stress response
CK2B PRR9
2
2
ABA signaling
PRR7 1
5
2
ELF3
2
CCA1
8
2
PRR5
LHY
5
TOC1/PRR1
LHY/CCA1family
4
4
6 8
FLC
8
GI
SVP
CDFs 7
7
CO FT Flowering time
ZTL
6 6
4
SKPs 7
6
6
Drought stress response
6
FKF1
8
8
TOC1/PRR family
7
7
LKP2 ZTL/FKF family
26S Proteasome Protein degradation
Fig. 2. Functional interactions between various clock proteins and input/output pathways for the regulation of flowering time, organ elongation, protein degradation and stress responses. (1) Ni et al. (1998) and Liu et al. (2001). (2) Barak et al. (2000), Mizoguchi et al. (2002) and Mizoguchi et al. (2006). (3) Kurup et al. (2000). (4) Ma´s et al. (2003) and Yamashino et al. (2003). (5) Kidokoro et al. (2009). (6) Imaizumi et al. (2005), Nakasako et al. (2005), Kim et al. (2007), Rubio and Deng (2007) and Sawa et al. (2007). (7) Calderon-Villalobos et al. (2007). (8) Yoshida et al. (2009).
The elf3 mutations eliminated circadian rhythm outputs in LL, whereas the phyB mutations had quantitative effects on the amplitude and period without eliminating rhythmicity. This suggests that ELF3 cannot be considered as simply a unique phy-signalling component (Reed et al., 2000). In LL, elf3 mutations abolished the rhythmic expression of clock-controlled genes such as LHY, CCA1, TOC1, GI, CO, FT and CAB, which suggests that these genes may be downstream targets of transcriptional regulation by ELF3 (Barak et al., 2000; Hall et al., 2003; Hazen et al., 2005; Hicks et al., 2001). As physiological studies have previously related circadian dysfunction to chlorosis, the pale-green phenotype of elf3 may be due to the loss of circadian control over chloroplast functions, in addition to abnormal gene regulation (Dowson-Day and Millar, 1999).
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IV. ROLES OF ELF3 IN THE CONTROL OF FLOWERING TIME Photoperiodic flowering is one of the clock output pathways and is arguably the most studied developmental process under circadian control in plants. Lossand gain-of-function of ELF3 accelerates and delays flowering time in Arabidopsis, respectively, indicating the importance of ELF3 in this process. A short history and the current state of research on photoperiodic flowering are summarized in this section. We discuss the roles of ELF3 in both CO-dependent and -independent pathways and the potential role of ELF3 as a scaffold or adaptor protein. Finally, our recent attempts to identify new proteins that may function in tandem with ELF3 in the control of flowering are discussed. A. CURRENT VIEW OF PHOTOPERIODIC CONTROL OF FLOWERING TIME LINKED TO THE CIRCADIAN CLOCK IN ARABIDOPSIS
Garner and Allard classified plants into different day-length response types (Garner and Allard, 1920): LD plants (LDPs) require a shorter time to flower when light exposure exceeds a critical day length, and SD plants (SDPs) flower sooner when light exposure is shorter than a critical day length. Subsequent experiments demonstrated that SDPs actually measure the length of the night, which must exceed a critical length to induce flowering, and that these plants do not flower when grown under LL. Photoperiodic control of flowering time is tightly linked to the circadian clock, which acts as the time-keeping mechanism that measures the durations of day and night (Ma´s, 2005; Sua´rez-Lo´pez et al., 2001; Yanovsky and Kay, 2002). The circadian clock is an endogenous oscillator with an approximate period of 24 h and can be synchronized, or entrained, to the exact period of daily oscillations in light and temperature (Dunlap, 1999). This process phases the biological activities to the correct time of day. The LDPs are classified into two types, those that only flower (an absolute LDP) and those that flower most rapidly (a facultative LDP) when light exposure exceeds a certain number of hours during a 24-h period (Thomas and VincePrue, 1997). As a facultative LDP, Arabidopsis flowers much earlier with a daily regime of a long light period and short dark period (e.g. 16 h light/8 h dark) than with a short light period and long dark period (e.g. 8–10 h light/16–14 h dark). In Arabidopsis, the regulatory MYB proteins LHY and CCA1, close relatives of each other, are essential clock components with redundant functions in controlling the rhythmic expression of flowering-time genes for photoperiodic flowering (Carre´ and Kim, 2002; Mizoguchi et al., 2002, 2005). In particular, LHY and CCA1 regulate a flowering pathway, composed of the genes GI, CO
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and FT, in light/dark cycles (Ma´s, 2005; Mizoguchi et al., 2002, 2005). FT gene expression is activated under LDs primarily through a conserved pathway consisting of GI and CO (Mizoguchi et al., 2005). Several other Arabidopsis genes, whose mutation also delays or accelerates flowering, have been previously identified (Ma´s, 2005). The relationship between flowering and day length in Arabidopsis involves rhythmic, circadian clock-controlled expression of CO mRNA. In this model, CO mRNA levels rise and fall over the course of a day, producing unstable levels of the CO protein. If CO mRNA levels are high when the plant is exposed to light, CO protein is stabilized and activates the expression of FT (Ma´s, 2005; Sua´rez-Lo´pez et al., 2001; Valverde et al., 2004). Changing the timing of CO expression can alter the length of daylight that triggers flowering (Bo¨hlenius et al., 2006; Yanovsky and Kay, 2002), but does not alter the photoperiodic response type of Arabidopsis from facultative LDP to SDP. A comparative analysis of Arabidopsis and rice, an SDP plant, suggests that functional differences between Arabidopsis CO and its rice orthologue (Heading date1, Hd1) may be the basis for the reversal of response type (Hayama and Coupland, 2004). In rice, Hd1 represses flowering under LD by repressing the expression of the rice FT orthologue (Heading date3, Hd3a), whereas in Arabidopsis, CO activates flowering by activating FT expression (Hayama and Coupland, 2004). FT and Hd3a are candidates for the floral hormone florigen (Corbesier et al., 2007; Tamaki et al., 2007). B. CO-INDEPENDENT ROLE OF ELF3 IN THE CONTROL OF FLOWERING TIME
The expression of GI, CO and FT was increased in the elf3-1 mutant, indicating that ELF3 negatively regulates the transcript levels of all three genes (Kim et al., 2005). CO mediates between GI and FT to control flowering time (Mizoguchi et al., 2005). CO loss-of-function, as in co-1 and co-2 (Kardailsky et al., 1999; Kobayashi et al., 1999), decreased the FT expression level. Surprisingly, the elf3-1;co-1 double mutant flowered much earlier than co-1 in LD, although FT expression remained very low (Kim et al., 2005). These results indicate that elf3-1 partially suppresses late flowering in co-1 through a CO-independent mechanism. ELF3 may be involved in post-transcriptional regulation of FT protein or the transcriptional regulation of a gene downstream of FT, such as SOC1. C. POTENTIAL ROLE OF ELF3 AS A SCAFFOLD FOR THE INPUT, OSCILLATOR AND OUTPUT OF THE ARABIDOPSIS CLOCK
Recently, we reported that mutations in the circadian clock genes LHY and CCA1 caused Arabidopsis to show characteristics of an SDP (Fujiwara et al., 2008; Mizoguchi and Yoshida, 2009; Yoshida et al., 2009). The lhy;cca1
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mutant flowered later under LL than under SD, a behaviour pattern exhibited by SDPs. Characterization of suppressors of the late-flowering phenotype of lhy;cca1 under LL indicated that inversion of the response appeared to involve enhanced activities of ELF3 and two floral repressors, SHORT VEGETATIVE PHASE (SVP) and FLOWERING LOCUS C (FLC; Fujiwara et al., 2008; Mizoguchi and Yoshida, 2009; Yoshida et al., 2009). ELF3 directly interacted with both CCA1 and SVP, which are part of the central oscillator and output pathway of the Arabidopsis circadian clock, respectively (Fig. 2; Yoshida et al., 2009). As described in the previous section, ELF3 interacts with the photoreceptor phyB (Liu et al., 2001; Reed et al., 2000), which plays key roles in the input pathway. These findings suggest that ELF3 may function as a scaffold to form a clock protein complex in Arabidopsis. We have proposed that the circadian clock may control pathways that promote and repress flowering and that altering the balance among these pathways can switch the photoperiodic response type of a single species. D. ROLE OF ELF3 AS AN ADAPTOR FOR COP1 AND GI, POSSIBLY IN THE CRY2-DEPENDENT FLOWERING PATHWAY
Yeast two-hybrid analysis and co-immunoprecipitation assays have demonstrated molecular interactions between ELF3 and the E3 ubiquitin-ligase CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), and between ELF3 and GI (Yu et al., 2008). It was proposed that ELF3 acts as an adaptor protein between COP1 and GI. The blue-light receptor CRY2 may negatively regulate COP1 via a direct interaction between the two (Wang et al., 2001). Genetic analysis under LD and SD regimes support this idea. For example, the late-flowering phenotype of cry2 was suppressed by cop1 under LD, suggesting that COP1 may be a downstream factor of CRY2 under various light/dark cycles. Thus, blue-light-activated CRY may stabilize the CO protein in the GI-independent pathway by inhibiting COP1 (Valverde et al., 2004; Yu et al., 2008). Indeed, Jang et al. (2008) and Liu et al. (2008a,b) have shown that COP1 triggers degradation of the floral inducer CO. In this way, the cry2 loss-of-function mutation would cause increased accumulation of COP1, thereby inhibiting the stabilization of CO. E. ATTEMPTS TO IDENTIFY SUPPRESSORS AND ENHANCERS OF ELF3 PHENOTYPES
To understand the roles of ELF3 as a multi-functional protein, a reasonable approach is to isolate mutations that suppress or enhance all or some of the phenotypes seen in elf3 mutants under different growth conditions. Using
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this general approach, we looked for mutations that delayed flowering under LL, but not LD or SD, in genetic backgrounds lacking functional ELF3, in order to identify genes required for inversion of the photoperiodic flowering type in lhy;cca1 mutants of the ELF3–SVP/FLC-independent pathway (Fujiwara et al., 2008; Mizoguchi and Yoshida, 2009; Yoshida et al., 2009). We isolated seven elf3-suppressors (sel1, sel3, sel5, sel7, sel14, sel15 and sel20) and one enhancer (E#1) by mutagenesis of elf3-1 (Col accession) with a heavy-ion beam (Natsui et al., 2010; Neffisi et al., unpublished). Two additional suppressors were obtained with EMS mutagenesis of elf3101 (Ler accession) (Natsui et al., 2010). One mutant, the elf3-1;sel20 plants, exhibited accelerated floral transition under LD and SD regimes but repressed flowering under LL. This photoperiodic flowering response of elf31;sel20 was similar to that of lhy;cca1 and completely different from that of wild type. The sel20 mutation was determined to be a new deletion allele consisting of a mutation in the gene for the blue-light receptor CRY2 (Neffisi et al., unpublished). CRY2 has been shown to interact with and negatively regulate COP1, thereby accelerating flowering time via CO degradation (Liu et al., 2008a,b). Delay of flowering in cry1;cry2 was largely suppressed by cop1, and co mutation repressed the acceleration of flowering time caused by cop1. CRY2 was proposed to regulate the COP1–ELF3–GI pathway for the control of flowering time, as explained in Section IV.D. We believe this pathway is unlikely to play a major role in the delay of flowering in lhy; cca1 under LL; the downregulation of GI activity would decrease CO expression (Mizoguchi et al., 2005; Sua´rez-Lo´pez et al., 2001), but there was no significant difference in the CO expression level between lhy;cca1 and wildtype plants under LL (Fujiwara et al., 2008). The sel20/cry2 mutation also did not affect the CO expression level in elf3-1 under LL. These results suggest that late flowering of both lhy;cca1 and elf3;cry2 under LL was unlikely to depend on decreased GI activity. An additive effect of lhy;cca1 and gi on late flowering under LL supports this idea. We prefer a model based on the function of CIB1 (Liu et al., 2008a,b) to explain LL-specific delay of flowering in the elf3;cry2 double mutant. According to this model, CIB1 directly interacts with CRY2. CIB1 activity requires CRY2 and is increased by blue light. CIB1 binds to the FT gene and activates flowering via the control of FT expression. The increase of FT expression in CIB1 overexpressing plants was much more pronounced under the LL regime, but not under the LD regime. Without CRY2, the effect of the loss-of-function of CIB1 would be stronger under constant white light than under LD. It would be interesting to test whether downregulation of CRY2 and CIB1 can explain, at least in part, the delay of flowering in lhy;cca1 under LL.
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V. ROLES OF ELF3 AND OTHER CLOCK PROTEINS IN STRESS TOLERANCE Photoperiodic flowering and organ elongation are well-characterized developmental processes controlled by a circadian clock in plants. Signal transducers and transcription factors that mediate between the clock and these developmental processes have been identified, and molecular mechanisms underlying these processes are beginning to be understood (see Section II). After a microarray analysis demonstrated that the expression of a certain set of stress-inducible genes was under the control of a circadian clock (Harmer and Kay, 2000), our knowledge of the circadian clock control of plant stress responses rapidly advanced. In this section, we summarize the reported roles of clock genes, including ELF3, in responses to environmental stresses, including ambient temperature, very short days, low temperature and drought. A. AMBIENT TEMPERATURE STRESS
Sub-optimal, but not near-freezing, temperatures affect flowering through the thermosensory pathway, which overlaps with the autonomous pathway (Bla´zquez et al., 2003). Two distinct pathways regulate ambient temperaturedependent flowering (Strasser et al., 2009). One pathway requires ELF3, while the other requires TFL1. Delayed flowering at lower temperatures was partially suppressed in single elf3 and tfl1 mutant plants. Further, elf3; tfl1 double mutants were insensitive to temperature, suggesting that both ELF3 and TFL1 are important in the control of flowering under ambient temperature stress. B. VIABILITY UNDER VERY SD REGIME
The over-expression of either CCA1 or LHY in Arabidopsis (CCA1-ox and lhy-1) resulted in a loss of circadian rhythmicity under constant conditions such as LL or DD. A loss-of-function mutation in ELF3 (elf3) or in both CCA1 and LHY (lhy;cca1) also produced an arrhythmic phenotype under LL. However, all of the CCA1-ox, lhy-1, lhy;cca1 and elf3 plants retained the ability to respond to diurnal changes in light (Green et al., 2002; Hicks et al., 2001; Song and Carre´, 2005; Yoshida et al., 2009). The transcript levels of clock-controlled genes oscillated robustly in these plants, even under light/dark cycles. In wild-type plants, the expression of clock-controlled genes changes in anticipation of light/dark transitions. However, the CCA1-ox, lhy-1, lhy;cca1 and elf3 plants lost the ability to anticipate this daily change in their
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environment. Green and co-workers examined the effects of loss of circadian regulation on the fitness of Arabidopsis under different photoperiodic conditions: 16 h light/8 h dark (16L8D), 8L16D and 4L20D. The elf3, CCA1-ox and lhy-1 plants were less viable under very SD (4L20D) conditions. It would be interesting to test the viability of lhy;cca1 and other clock mutants under a very SD regime. Photoperiods with different T-cycles should also be used.
C. MICROARRAY ANALYSIS
The possible involvement of the circadian clock system in stress tolerance was suggested by microarray analysis (Covington et al., 2008; Harmer and Kay, 2000, Kant et al., 2008, Kreps et al., 2002). Many stress-inducible genes, as well as genes for key regulators of stress signalling pathways, were shown to be affected by a circadian clock. The cis-element required for induction by dehydration stress was identified and named the dehydration responsive element (DRE; YamaguchiShinozaki and Shinozaki, 1994). The core sequence of the DRE is AGCCGAC. The DRE was shown to be required for induction by low temperature. A transcription factor that specifically binds to the DRE was identified in a yeast one-hybrid system and named DRE BINDING 1 (DREB1; Liu et al., 1998). Sakuma et al. (2002) grouped 145 Arabidopsis DREB/ERF-related proteins into five subfamilies (AP-2, RAV, DREB, ERF and others) based on amino acid sequence similarities among the AP2/ERF domains. DREB1related genes were classified into three subgroups (DREB1, 2 and 3). DRE and DREB were also identified as C-box and CBF, respectively (Stockinger et al., 1997). The clock-controlled gene DREB1A/CBF3 was elucidated by Harmer and Kay (2000).
D. GATED INDUCTION OF DREB/CBF BY LOW TEMPERATURE
The expression levels of CBF3 and certain CBF-regulated genes exhibit circadian cycling under unstressed conditions (Harmer and Kay, 2000). Fowler et al. (2005) found that the cold induction of CBF/DREB was gated by a circadian clock, suggesting that the expression of genes for key regulators of cold-stress responses may be fine-tuned by several layers of regulation. The extent to which the CBF/DREB mRNA level increased in response to cold stress depended on the time of day that the plants were exposed to cold, with the highest and lowest levels at 4 and 16 h after subjective dawn,
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respectively. Constitutive over-expression of CCA1 abolished gating of CBF/ DREB induction by the circadian clock in response to cold stress.
E. ROLES OF PRR9, PRR7 AND PRR5 IN THE COLD-STRESS RESPONSE
In a comparative microarray analysis between the triple mutant prr9-11;prr710;prr5-10 (d975) and wild-type plants, Nakamichi and co-workers found significant overlap between the set of upregulated genes in d975 and the set of cold-responsive genes (Nakamichi et al., 2009). The expression level of DREB1/CBF was higher in d975 than in wild-type plants. Consistent with this, d975 plants were more tolerant to cold, high salinity, and drought compared with wild-type plants. Raffinose and L-proline, which are usually increased under stress conditions, accumulated at higher levels in d975, even under normal conditions. Based on these results, Nakamichi et al. have proposed that PRR9, PRR7 and PRR5 may be involved in a mechanism that anticipates diurnal cold stress and initiates a stress response by mediating cyclic expression of stress response genes, including DREB1/CBF.
F. PIF7 AS A POSSIBLE MEDIATOR BETWEEN THE CIRCADIAN CLOCK AND DREB1/CBF IN STRESS SIGNALLING
Although several research groups have independently shown clock-controlled expression of DREB1/CBF, the mechanism by which clock proteins affect DREB1/CBF expression has not been elucidated. There are six conserved motifs, boxes I–VI, in the promoter sequences of DREB1s. Kidokoro et al. (2009) demonstrated that box V with the G-box sequence negatively regulated the clock-controlled expression of DREB1C. Using yeast one-hybrid screens, they identified phytochrome-interacting factor 7 (PIF7) as a factor that specifically binds to the G-box of the DREB1C promoter (Fig. 2). Transactivation experiments with Arabidopsis protoplasts indicated that PIF7 functions as a repressor for DREB1C expression. PIF7 interacts with the clock protein TOC1/PRR1 and the red-light photoreceptor phyB (Kidokoro et al., 2009; Leivar et al., 2008; Yamashino et al., 2003), and the activity of PIF7 is enhanced by co-expression of TOC1 or phyB (Kidokoro et al., 2009). In a PIF7 loss-of-function mutant, the circadian rhythms of DREB1B and DREB1C expression were disrupted under LL; the transcript levels of both were elevated, and the amplitudes of the rhythmic expression were much lower in pif7, compared with wild-type plants. Kidokoro et al. (2009) proposed that the negative regulation of DREB1 expression may be important for
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avoiding growth retardation of plants owing to an accumulation of DREB1 protein under unstressed conditions. G. TOC1 AS A MOLECULAR SWITCH CONNECTING THE CLOCK AND RESPONSES TO ABA AND DROUGHT
The expression of genes for the biosynthesis and signalling of plant hormones such as ABA, auxin, ethylene, cytokinine, GA and brassinosteroid are controlled by a circadian clock (Hanano et al., 2006). TOC1/PRR1 is a critical component of the circadian clock system in Arabidopsis. Recently, Legnaioli et al. (2009) demonstrated that TOC1/PRR1 protein was bound to the promoter of the ABA-related gene (ABAR/CHLH/GUN5) and controlled its circadian expression (Fig. 2). TOC1/PRR1 expression was acutely induced by ABA, before TOC1/PRR1 binding to the ABAR promoter. The circadian control of ABAR expression was modulated by ABA. The gated induction of TOC1/PRR1 by ABA was suppressed in ABAR RNAi plants, suggesting that reciprocal regulation between TOC1 and ABAR was important for ABA signalling. The over-expression of TOC1/PRR1 significantly reduced the plant tolerance to drought, whereas plants with loss-of-function of TOC1 (toc1-2 and TOC1 RNAi) responded to water-deficient conditions better than wild-type plants. ABA INSENSITIVE 3 (ABI3), a transcription factor with a B3 domain, functions as a major regulator of ABA signalling (Giraudat et al., 1992). TOC1/PRR1 was identified as the ABI3-interacting protein 1 (AIP1; Kurup et al., 2000). These results suggest that clock-dependent gating of plant hormone functions is vital for cellular homeostasis under various environmental conditions. H. RELEASE OF SEED DORMANCY
The dormant stage of seeds is highly tolerant to stress conditions, and seed dormancy and germination are controlled by plant hormones such as ABA and GA. Recently, Penfield and Hall demonstrated that clock genes such as LHY, CCA1, GI and TOC1 play key roles in responses to the signals that break seed dormancy in Arabidopsis (Penfield and Hall, 2009).
VI. PERSPECTIVES Loss-of-function of the ELF3 gene produced pleiotropic phenotypes, suggesting that ELF3 may have several functions in more than two signalling pathways. A protein–protein interaction between ELF3 and phyB was
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LHY/ CCA1
ELF3
PhyB
SVP/ FLC
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MAPK cascades
PhyA/ PhyB
FKF
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Ste11
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Fig. 3. Analogy between clock components and MAPK cascades, showing a possible role of ELF3 as a scaffold protein. For details, see the text.
reported. Recently, two groups demonstrated several additional protein– protein interactions of ELF3 (Fig. 3; Yoshida et al., 2009; Yu et al., 2008). Mitogen-activated protein kinases (MAPKs) play central roles in many distinct signal transduction pathways (Mizoguchi et al., 1997) and are activated by environmental stimuli (e.g. high or low osmotic stress, dehydration, high or low temperature), growth substances (e.g. growth hormones, pheromones) and biotic stresses (e.g. infection by bacteria or viruses, attack by insects or nematodes). MAPKs are good research models for studying both specificity and diversity of signalling pathways. For activation, MAPKs require phosphorylation of both the threonine and tyrosine residues in the conserved TXY motif, by their upstream factors called MAPK kinases (MAPKKs). MAPKKs themselves are also phosphorylated and activated by their upstream factors, MAPKK kinases (MAPKKKs). In Saccharomyces cerevisiae, three types of MAPKs participate in three different signalling pathways (Schwartz and Madhani, 2004). Fus3/Kss1, Hog1 and Mpk1 function in the responses to mating pheromone, osmotic stress and cell wall biosynthesis, respectively. These three MAPKs are activated by three different MAPKKs and MAPKKKs. To achieve this kind of specificity, a scaffold protein interacts with all three kinases. For example, Ste5 is a scaffold protein for the MAPK cascade of the mating-pheromone pathway (Seeliger and Kuriyan, 2009) and Ste11 (MAPK), Ste7 (MAPKK) and Fus3/Kss1
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(MAPKs) all interact with Ste5 (Fig. 3). There is no epistatic relationship between Ste5 and any other cascade of protein kinases. In the Ssk2/Ssk22– Pbs2–Hog1 pathway of S. cerevisiae and in the AtMEKK1–AtMEK/ AtMKK2–AtMPK4 pathway, Pbs2 and AtMEKK1, respectively, appear to function as scaffold proteins (Fig. 3; Ichimura et al., 1998; Maeda et al., 1995; Mizoguchi et al., 1998; Posas and Saito, 1997). In Arabidopsis, there are 20 MAPKs, 10 MAPKKs and more than 50 MAPKKKs (MAPK Group, 2002). Specificity for the interaction and activation in each MAPK cascade may be controlled by unidentified scaffold proteins. Many of the clock proteins in Arabidopsis interact with other proteins and are proposed to function in signalling complexes (Fig. 2). These putative complexes include photoreceptors (phyA, phyB, CRY2, FKF1 and ZTL), F-box proteins involved in protein degradation (FKF1 and ZTL) and transcription factors (SVP, FLC, CDFs and PIFs/PILs). We have recently identified phyB mutations as suppressors of the late-flowering phenotype of lhy; cca1 under LL (Miyata and Mizoguchi, unpublished). In a manner similar to Ste5, ELF3 may function as a scaffold protein for phyB, LHY/CCA1 and SVP/FLC in the control of flowering time (Figs. 2 and 3; Mizoguchi and Yoshida, 2009). ELF3 was proposed to function as an adaptor protein for COP1 and GI (see Section IV.D), and CRY2 directly interacts with ELF3 and COP1. Thus, ELF3 appears to function as a scaffold protein for different sets of partners in distinct signalling pathways, which may explain the pleiotropic phenotype of elf3 mutants. FKF1 and ZTL, closely related F-box proteins (Baudry et al., 2010), interact with and are regulated by GI. Molecular targets for protein degradation of FKF1 and ZTL are CDFs, which control CO expression, and TOC1, which is a central oscillator, respectively (Kim et al, 2007; Sawa et al., 2007). CDFs and TOC1 bind to ZTL and FKF1, and therefore, these two F-box proteins appear to function as scaffold proteins. In the phyA/phyB–PRRs–PIFs/PILs pathway, the PIFs/PILs probably function as scaffold proteins. The expression of many genes, including stress-inducible genes, is under the control of a circadian clock (Covington et al., 2008; Harmer and Kay, 2000, Kant et al., 2008, Kreps et al., 2002). The molecular mechanism underlying the clock-controlled response to low temperature is starting to be understood; however, the machinery behind the responses to other stressors such as desiccation, high temperature, UV and high/low osmolarity remains unknown. Transcriptional as well as post-transcriptional and posttranslational regulation may play key roles in clock-controlled responses to environmental stress. The identification of enzymatic activities that are positively or negatively regulated by a circadian clock is the next important challenge.
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ACKNOWLEDGEMENTS This work was supported in part by the Bilateral Joint-Lab Project between Japan and France of the Ministry of Education, Culture, Sports, Science and Technology (MEXT; to T. M.).
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Engineering Salinity and Water-Stress Tolerance in Crop Plants: Getting Closer to the Field
ZVI PELEG,* MARIS P. APSE{ AND EDUARDO BLUMWALD*,1
*Department of Plant Sciences, University of California, Davis, California, USA { Arcadia Biosciences, Davis, California, USA
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Plant Responses to Drought and Salinity Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plants Response to Water Deficit ............................................ B. Plant Response to Salinity Stress ............................................ C. Plant Adaptations to Abiotic Stress ......................................... D. New Technologies to Study Plant Response to Abiotic Stress .......... III. Engineering of Drought and Salinity-Tolerant Crop Plants . . . . . . . . . . . . . . . . A. Genes Involved in Osmoregulation .......................................... B. Genes for Mitigating Oxidative Damage ................................... C. Genes for Ionic Balance ....................................................... D. Regulatory and Signalling Genes ............................................ IV. Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT Abiotic stress is the primary cause of crop plant yield losses worldwide. Drought and salinity stress are the major environmental challenges faced by agriculture. Improving yield production and stability under stressful environments is needed to fulfil the food 1
Corresponding author: E-mail:
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Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00012-6
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demand of the ever-growing world population. Numerous genes associated to plant response(s) to drought and salinity stress have been identified and characterized, in most cases, in the model plant Arabidopsis. However, while many of these genes are potential candidates for improving tolerance to abiotic stress, only a small proportion were transferred into crop plants. Further, transgenic crop plants overexpressing the genes of interest were, in most cases, tested under artificial conditions in the laboratory or controlled greenhouse. Thus, while many reports on drought and salinity tolerance in transgenic plants have been published, there is urgent need to test these traits under field conditions. In this chapter, we discuss recent advances in engineering drought and salinity tolerance in crop plants with emphasis on yield and the needs to close the gaps between the laboratory and the field conditions.
ABBREVIATIONS ABA CAT CDPK CIPK CK DREB ERF GB GST IPT JA LEA MAPK MtlD NAM P5CS PEG PIP RLK ROS RWC SOD SOS TE TIP TF TPS OA WUE
abscisic acid catalase calcium-dependent protein kinase calcineurin B-like protein-interacting protein kinase cytokinin dehydration-responsive element binding protein ethylene responsive factor glycine betaine glutathione S-transferase isopentenyltransferase jasmonic acid late embryogenesis abundant mitogen-activated protein kinase mannitol-1-phosphate dehydrogenase no apical meristem D1-pyrroline-5-carboxylate synthetase polyethylene glycol plasma membrane intrinsic protein receptor-like kinase reactive oxygen species relative water content superoxide dismutase salt overly sensitive transpiration efficiency tonoplast intrinsic protein transcription factor trehalose-6-phosphate synthase osmotic adjustment water-use efficiency
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I. INTRODUCTION Crop plants are often grown under unfavourable environmental conditions that prevent the full expression of their genetic yield potential. The most frequently occurring abiotic stress conditions with adverse effects on crop yield are water, deficit or excess; ions, deficit or excess; temperature, low or high; and light, deficit or excess. The ever-increasing human population, concomitant with loss of agricultural land (due to urbanization processes) and diminishing water availability (associated with climate change) pose serious challenges to world agriculture (reviewed by Mittler and Blumwald, 2010). A significant increase (an estimated 50%) in grain yield of major crop plants such as rice (Oryza sativa L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) is required to fulfil the food supply requirements for the projected population by 2050 (Godfray et al., 2010). The average production of major U.S. crops (corn, wheat, soybean, sorghum, oat, barley, potato and sugar beet) is only 21.6% of the highest yields attained under optimal conditions (Boyer, 1982). Diseases, pests and weed competition losses account for 4.1% and 2.6% yield reductions, respectively, with the remainder of the yield reduction (69.1%) attributed to unfavourable physicochemical (abiotic) environments induced by problematic soils and erratic climate patterns. Certainly, some of these losses are caused by inherently unfavourable environments and some by suboptimal management practices by farmers, often due to economic constraints or lack of training. Nevertheless, there is no doubt that a large fraction of potential crop productivity is lost to abiotic stress factors. Plants respond to abiotic stresses at multiple levels such as molecular, cellular, tissue, anatomical, morphological and whole-plant physiological levels (Bartels and Sunkar, 2005; Bray, 1993, 1997; Chaves et al., 2003; Munns, 2002; Munns and Tester, 2008; Witcombe et al., 2008). The response to stress depends on the duration and severity of the event, as well as the age and developmental stage of the plant, which varies with the species and genotype level (Bray, 1997). For crop plants, tolerance to abiotic stresses is measured by yield loss rather than survival. Typically, early plant establishment (germination and seedling) and the reproductive stage are the most sensitive in determining yield under stress (Barnabas et al., 2008). However, a large segment of the research on abiotic stress in model systems (particularly Arabidopsis) in the past has focused primarily on the vegetative phase and strived to identify survival phenotypes. This has hindered our ability to readily translate the discoveries into improved yield in crop plants.
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II. PLANT RESPONSES TO DROUGHT AND SALINITY STRESS A. PLANTS RESPONSE TO WATER DEFICIT
Among the various abiotic stress conditions, water deficit is the most devastating factor (Araus et al., 2008; Boyer, 1982). About one-third of the world’s arable land suffers from chronically inadequate water availability for agriculture, and in virtually all agricultural regions, crop yields are periodically reduced by drought (Bruce et al., 2002). While currently 80% of the world’s useable water resources are consumed by irrigated agriculture (Condon et al., 2004), within a few decades, the expanding world population will require more water for domestic, municipal, industrial and environmental needs (Hamdy et al., 2003). This trend is expected to accentuate due to global climatic change and increased aridity (Vorosmarty et al., 2000). Thus, to meet the projected food demands, more crop per drop is required (Condon et al., 2004). B. PLANT RESPONSE TO SALINITY STRESS
Salinity (see definition of saline and sodic soils; Richards, 1954) is a major constraint on crop-plant productivity (reviewed by Apse and Blumwald, 2002; Flowers, 2004; Munns and Tester, 2008; Witcombe et al., 2008). More than 800 million hectares of land throughout the world are salt affected, which accounts for 6% of the world total land area (Munns and Tester, 2008). In most cases, salinity results from natural causes (salt accumulation over long periods of time). In addition, a significant portion of the cultivated agricultural land is becoming saline due to deforestation or excess irrigation and fertilization (Shannon, 1997). Current estimates indicate that 20% of the roughly 230 million hectares of irrigated land is affected by salinity. Given that a third of the food production comes from irrigated agriculture, salinity is becoming a serious problem for crop-plant productivity. C. PLANT ADAPTATIONS TO ABIOTIC STRESS
Plant resistance to stress conditions may arise from escape, avoidance or tolerance strategies (Levitt, 1972). Escape relies on successful completion of reproduction before the onset of severe stress (i.e. developmental plasticity), achieved by early flowering and/or short growth duration (Mooney et al., 1987). Avoidance involves the prevention or decreasing the impact of the stress on the plant, such as minimizing water loss and maximizing water
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uptake (Chaves et al., 2003) or exclusion of salt ions, a feature observed in halophytes (Munns and Tester, 2008). Tolerance relies on the inherent ability of the plant to sustain growth (likely at a reduced rate) even when the conditions are unfavourable for the maintenance of basic plant processes. This strategy involves coordination of physiological and biochemical alterations at the cellular and molecular levels, such as osmotic adjustment (Morgan, 1984) and the sequestration of ion in the plants, in the vacuole or leaf sheath and/or older leaves (Mimura et al., 2003). In most cases, plants subjected to stress conditions combine a suite of responses, exhibiting a number of physiological and biochemical responses at the molecular, cellular and whole-plant level (Bohnert et al., 1995; Bray, 1993, 1997; Chaves et al., 2003). D. NEW TECHNOLOGIES TO STUDY PLANT RESPONSE TO ABIOTIC STRESS
New technologies are providing opportunities to address the challenging problem of maintaining high-yield crop production under stressful and changing climates. The information provided by high-resolution transcript profiling, the identification of large-scale specific protein networks and their association with the plant responses to environmental perturbations are allowing the application of a systems-level approach to uncover the bases of plant responses to environmental changes. Model plants, such as Arabidopsis thaliana, Brachypodium distachyon and Medicago truncatula, have been and will continue to offer insights into the genetic and biochemical basis of abiotic stress adaptations (Bohnert et al., 2006; Hirayama and Shinozaki, 2010). Further, the identification of stress-related genes and pathways has been facilitated by introducing new tools and resources developed in these model plants. Numerous genes related to plant response to drought and salinity stress have been identified and characterized (Ashraf, 2010; Pardo, 2010; Shinozaki and Yamaguchi-Shinozaki, 2007; Umezawa et al., 2006). Many of the genes so identified are considered as potential candidates for improving tolerance to abiotic stress. In the majority of cases, these genes are overexpressed in the target plant(s), whether with a strong constitutive promoter or a stress-responsive promoter. Early generations (T1–T3) are screened for responses to stresses to assess the efficacy of the construct. However, the vast majority of these studies were conducted under laboratory conditions (i.e. dehydration) in the vegetative phase (i.e. seedling, or plate assays) using artificial stress (e.g. PEG, mannitol), with very high concentration (i.e. osmotic shock) and for short periods (i.e. hours). Moreover, most of these studies showed stress tolerance and/or survival, but not the effects of the different stress conditions on plant productivity (Parry et al., 2005).
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Under rain-fed drought prone agriculture, water stress at the reproductive stage is the most prevalent problem as in most rain-fed ecosystems, the crop season’s rains diminish towards flowering and harvest time (Blum, 2009). Thus, more emphasis should be given to the study of the response of crop plants to abiotic stress at the reproductive stage and under field conditions.
III. ENGINEERING OF DROUGHT AND SALINITYTOLERANT CROP PLANTS Plant responses to abiotic stress affect all aspects of plant physiology and metabolism, leading to severe yield losses. Thus, tolerance mechanisms depend on the prevention or alleviation of cellular damage, the re-establishment of homeostatic conditions and the resumption of growth. Discovering and understanding the molecular/genetic basis of these tolerance components have been the focus of crop biotechnology in the past 2 decades. Despite these enormous research efforts, the role of very few genes in enhancing abiotic stress tolerance has been demonstrated under field conditions. However, this is expected to change primarily because research is increasingly focused on high yields under stress rather than plant survival. Other factors include better facilities for testing the transgenic materials and the increasing acceptance of genetically engineered plants. Genetic engineering of candidate genes for abiotic stress was found to be successful in model plants growing under controlled conditions and provided insights on the role of these genes in key physiological and biochemical processes (reviewed by Pardo, 2010; Umezawa et al., 2006; Vinocur and Altman, 2005). In this chapter, we have focused on efforts towards the improvement of drought and salinity stresses tolerance in crop plants with emphasis on field trials. A. GENES INVOLVED IN OSMOREGULATION
The biosynthesis and accumulation of compatible solutes in is an adaptive response of plants to both drought and salinity stress (Munns, 2002). Compatible solutes are non-toxic small molecules which do not interfere with normal cellular metabolism. A variety of substances have been identified in plants as compatible solutes, including sugars (trehalose, fructan), sugar alcohols (galactinol, trehalose and mannitol), amino acids (proline) and amines (glycine betaine, GB). There are many examples in the literature of increasing compatible solute synthesis as a strategy to improve tolerance to abiotic stress. In most cases, tolerance to either water or salinity stress has been reported as comparisons of plant recovery from treatments of rapid
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drying or high salinity. Survival, protection of photosynthetic activity, degree of lipid peroxidation and membrane leakage are common parameters for assessing the effects of increased synthesis of compatible solutes. In rare cases, evaluations are made over longer term growth, but even so, effects on yield are rarely reported, and we are aware of no reports of field performance under both normal and stress conditions of transgenic plants engineered to produce increased amounts of compatible solutes. In this section, we highlight some of the promising candidate technological approaches that remain to be substantiated with field testing for yield performance.
1. Proline The accumulation of proline in response to osmotic stress has been reported in many plant species (Delauney and Verma, 1993). Proline is believed to act as a store of carbon and nitrogen, as a scavenger of reactive oxygen species (ROS), a molecular chaperone and even as a signal for other adaptive responses to abiotic and biotic stresses (Verbruggen and Hermans, 2008). Transformation of chickpea (Cicer arietinum) with the osmoregulatory gene P5CSF129A (under 35S promoter) encoding the mutagenized 1-pyrroline5-carboxylate synthetase (P5CS) for the overproduction of proline showed significantly higher proline accumulation. However, the transgenic plants resulted only in a modest increase in transpiration efficiency (TE), suggesting that enhanced proline had little bearing on the components of yield in chickpea (Bhatnagar-Mathur et al., 2009). Wheat plants overexpressing P5CS (under the control of a stress-induced promoter complex-AIPC) showed accumulation of proline, which resulted in improved tolerance to water deficit (Vendruscolo et al., 2007). Likewise, transgenic rice overexpressing P5CS showed significantly higher tolerance to salinity and water stress produced in terms of faster growth of shoots and roots (Su and Wu, 2004). Rice plants overexpressing the ZFP252 gene, resulted in increased amount of free proline and soluble sugars, elevated the expression of stress defence genes and enhanced tolerance to salt and drought stresses (Xu et al., 2008). Soybean plants expressing 1-pyrroline-5-carboxylate reductase (P5CR) under control of an inducible heat shock promoter were found in greenhouse trials to accumulate proline without deleterious effects and to retain higher relative water content (RWC), and higher glucose and fructose levels than the antisense and control plants (de Ronde et al., 2004). Field trials have been conducted in South Africa with apparent yield advantages for the proline accumulating soybean transgenic plants under reduced watering conditions and heat stress (ARC Research Highlights, 2006). However, these results have yet to appear in a scientific peer-reviewed publication.
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2. Mannitol Mannitol is accumulated as a compatible solute in many plants and organisms of other kingdoms, although its accumulation in celery is often cited, perhaps because in celery up to half of fixed CO2 is converted to mannitol (Stoop et al., 1996). The overexpression of mannitol-1-phosphate dehydrogenase (the Escherichia coli locus mtlD) resulted in the accumulation of a small amount of mannitol and also in the improved tolerance to salinity and drought in Arabidopsis (Thomas et al., 1995) and tobacco (Karakas et al., 1997). In wheat, where mannitol is normally not synthesized, constitutive expression of the mtlD (under the control of the ZmUbi-1 promoter) improved growth and tolerance to water stress and salinity, although growth in the absence of stress was accompanied with sterility, stunted growth and leaf curling at levels of mannitol higher than 0.7 mmol/gFW (Abebe et al., 2003). As with other compatible solutes discussed above, the concentration of mannitol in the transgenic plants that showed better response to water and salinity stress at the whole-plant level was too small to be osmotically relevant. Rather, the ameliorative effect of mannitol was likely to be exerted through the scavenging of hydroxyl radicals and stabilization of macromolecular structures (see Abebe et al., 2003, and references therein). 3. Glycine betaine GB, a fully N-methyl-substituted derivative of glycine, accumulates in the chloroplasts and plastids of many species such as Poaceae, Amaranthaceae, Asteraceae, Malvaceae and Chenopodiaceae, in response to drought and salinity. In some species, GB accumulates to concentrations that would contribute to cellular osmotic pressure (Munns and Tester, 2008), but in most cases, plants accumulate less than this amount. At lower concentrations, GB stabilizes the quaternary structures of enzymes and complex proteins and protects the photosynthetic machinery via ROS scavenging (Chen and Murata, 2008). Transgenic maize expressing the betA locus of E. coli, encoding choline dehydrogenase, showed more GB accumulation under drought and salinity in the field (Quan et al., 2004). Under drought stress, imposed at the reproductive stage, transgenic maize lines that showed the highest amounts of GB accumulation (between 5.4 and 5.7 mmol/gFW) also had a 10–23% higher yield than wild-type plants under the same treatment (Quan et al., 2004). Quantitative data describing yields in the field in the absence of stress were not reported. Cotton plants (Gossypium hirsutum L.) expressing betA were also described as more drought tolerant (Lv et al., 2007). Under water-stress conditions, the transgenic cotton lines had higher RWC, OA, increased photosynthesis, reduced ion leakage and lower lipid membrane peroxidation than wild-type plants. As with the transgenic maize
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(Quan et al., 2004), GB levels in the transgenic cotton were up to threefold greater than that measured in the wild-type controls. Yield was tested in pots in the greenhouse and one line showed a reduced loss of yield on water-stress treatment at anthesis. Recently, betA was transformed (under control of a maize ubiquitin promoter) into bread wheat and resulted in improved salt tolerance (He et al., 2010). Under 200 mM NaCl treatment, the transgenic wheat seedling (five-leaf stage) had higher levels of GB and chlorophyll, lower Naþ/Kþ ratios and solute potential, and less cell membrane damage. Further, in a field experiment under saline conditions (0.42–0.47% NaCl w/w), the transgenic plants dramatically outyielded the wild-type control plants (He et al., 2010). A CMO gene (AhCMO), cloned from Atriplex hortensis, was introduced into cotton, showing enhance resistance to salinity stress (Zhang et al., 2009). GB levels in the leaves of the transgenic cotton plants were on the high end of the range of GB reported in transgenic plants (43 mmol/gFW). While yield in the absence of stress was approximately 10% lower in the transgenic lines, these were T3 generation materials that were being compared to untransformed controls. At least one backcross to the wild type would be useful to make comparisons with wild type and to minimize tissue culture effects in the transgenic lines. Seed cotton yields of the transgenic lines were 20–30% higher than wild type in three seasons of field trials on what was reported as saline soil (Zhang et al., 2009); however, no description of the salinity level was provided in the publication. Transgenic potato (Solanum tuberosum L.) plants, developed via the introduction of the bacterial choline oxidase (codA) gene, expressed under the control of an oxidative stress-inducible SWPA2 promoter and directed to the chloroplast with the addition of a transit peptide at the N-terminus, showed enhanced tolerance to NaCl and drought stress at the whole-plant level (Ahmad et al., 2008). While not yet tested under field conditions, greenhouse testing with transgenic potato plants having relatively low levels of GB (0.9–1.4 mmol/gFW) showed greater dry weight accumulation after recovery from 150 mM NaCl treatment and water withholding stress treatments. Recently, wheat plants overexpressing a BADH gene, encoding betaine aldehyde dehydrogenase (BADH), were shown to be more tolerant to drought and heat, by improving the photosynthesis capacity of flag leaves (Wang et al., 2010). 4. Trehalose Trehalose ( -D-glucopyranosyl-(1!1)- -D-glucopyranoside) is a nonreducing disaccharide composed of two molecules of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi and invertebrates (Goddijn and van Dun, 1999).
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Trehalose is not thought to accumulate to detectable levels in most plants, with exception of the desiccation-tolerant ‘‘resurrection plants’’. However, there is thought to be a signalling role for trehalose at least in part through its inhibition of SNF-1-related kinase (SnRK1), which results in an up-regulation of biosynthetic reactions supporting photosynthesis and starch synthesis, among others (reviewed by Iturriaga et al., 2009). Transgenic tomatoes (Solanum lycopersicum) overexpressing the yeast trehalose-6-phosphate synthase (TPS1) gene (under control of 35S promoter) showed higher tolerance to salt, drought and oxidative stresses (Cortina and Culia´n˜ez-Macia`, 2005). The transgenic plants exhibited pleiotropic changes such as thick shoots, rigid dark-green leaves, erected branches and an aberrant root development and higher chlorophyll and starch content compared to wild-type plants. The alteration of soluble carbohydrate content suggests that the stress tolerance phenotype in trehalose genetically engineered plants could be partly due to modulation of sugar sensing and carbohydrate metabolism (Fernandez et al., 2010). In rice, the overexpression of a synthetic fusion of E. coli trehalose biosynthetic genes (otsA and otsB), under the control of tissue-specific (rbcS) and rice stress-dependent promoter ABA-inducible), resulted in sustained plant growth, less photo-oxidative damage and more favourable mineral balance under salt and drought stress conditions. The transgenic rice plants accumulate up to 3–10 times more trehalose than the wild-type plants (Garg et al., 2002). A similar fusion construct was made with the constitutive promoter maize ubiquitin, and used to transform rice (Jang et al., 2003). Incredibly, the transgenic rice accumulated up to 1000 mg/g FW trehalose, which was attributed to the increased efficiency of the fusion protein over two separate enzymes (Jang et al., 2003). Even more surprising was the absence of abnormal developmental and morphological phenotypes, given the high level of trehalose and the occurrence of such deleterious phenotypes in Arabidopsis, potato and tobacco (Goddijn and van Dun, 1999). Jang et al. (2003) suggested that the fusion protein would reduce the amount of the trehalose-6-phosphate intermediate, which is the metabolite responsible for signalling cytosolic carbon status and regulation of chloroplastic starch synthesis (reviewed by Paul et al., 2008). However, constitutive expression of such fusion proteins in potato (Jang et al., 2003) and alfalfa (Suarez et al., 2009) results in a range of stunted plant growth phenotypes. It may be the case that sensitivity to trehalose and the synthetic pathway intermediates are different for monocots and dicots. The use of inducible promoters has been an approach that appears to circumvent the deleterious effects of trehalose synthesis and accumulation in alfalfa (Suarez et al., 2009). A fusion of yeast trehalose biosynthetic genes, TPS1 and TPS2, was driven either by the constitutive strong promoter 35S or by the drought-inducible
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promoter rd29A. Stunting of growth in the absence of stress was apparent for the alfalfa plants harbouring the constitutive expression of the fusion gene, but was not apparent for plants with the inducible construct. Both rice and alfalfa were tested in controlled growth conditions for tolerance to water and salinity stresses and were found to outperform the wild-type controls (Jang et al., 2003; Suarez et al., 2009). Though promising as tools for the application to abiotic stress tolerance in agriculture, we are not aware of field trials or testing of this technology as yet. 5. Osmotin genes Osmotin is a stress-responsive multifunctional 24-kDa protein with roles in plant response to fungal pathogens and osmotic tolerance. Overexpression of a heterologous osmotin-like protein (under control of 35S) in potato (S. tuberosum) improved tolerance to salinity stress (Evers et al., 1999). The tobacco osmotin gene (driven by the 35S promoter) was transformed into tomato and was reported to enhance tolerance to salt and drought stresses (Goel et al., 2010). Estimation of several physiological traits such as RWC, chlorophyll, leaf proline, leaf expansion and plant height was observed in transgenic lines as compared to the wild-type plants. Yield of potted plants grown in the greenhouse showed a dramatic advantage for the transgenic osmotin tomatoes after recovery from 150 mM NaCl treatment for 3 weeks. Strawberry (Fragaria ananassa Duch) plants overexpressing osmotin gene of Nicotiana tabacum (driven by the 35S promoter) showed increased accumulation of proline and higher chlorophyll content compared with wild-type plants (Husaini and Abdin, 2008). Under salinity stress conditions, transgenic plants perform better than the wild-type control plants; however, under normal conditions, growth rate was slower. B. GENES FOR MITIGATING OXIDATIVE DAMAGE
Another physiological and biochemical cellular component common to a suite of abiotic stresses including drought and salt stress is oxidative stress. Oxidative stress involves the generation of ROS during stress. The most common ROS are hydrogen peroxide (H2O2), superoxide, the hydroxyl radical and singlet oxygen. Under normal conditions, ROS are continuously produced through cellular metabolism and plant cells are well equipped with antioxidants and scavenging enzymes to keep their levels low (Jaspers and Kangasja¨rvi, 2010). Under stress conditions, increased ROS production results from an increased production of superoxide due to reduced CO2 availability and the over reduction of the photosynthetic electron transport chain. Increased photorespiration also generates more H2O2, which, if not
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adequately balanced by scavenging molecules and enzymes, can lead to further generation of ROS via lipid peroxidation. Oxidative damage is believed to be a consequence of inadequate ROS scavenging, which might be mitigated by the inducible or constitutive overexpression of enzymes that can reduce ROS under stress. McKersie et al. (1996) reported that alfalfa constitutively expressing a tobacco MnSOD directed at either chloroplasts or mitochondria had improved survival and yield over 3 years of field trials, relative to the untransformed control plants. Increased SOD activity in the transgenic plants was accompanied by increased photosynthetic efficiency (Fv/Fm) and shoot regrowth during water-deficit stress treatments in controlled growth conditions. A wheat mitochondrial MnSOD, regulated by either constitutive (35S) or the stress-inducible (COR78) promoter, was used to transform canola (Gusta et al., 2009). In both constitutive and stress-inducible MnSOD transgenic canola plants, SOD activity was increased by 25–45% over that in control plants, and survival and recovery from water withholding was greater. Field experiments showed that the MnSOD transgenic canola had superior germination and emergence, as well as earlier time to flowering; yield testing is to occur in future trials using these transgenic plants (Gusta et al., 2009). Improving the antioxidant capacity in plants has also been accomplished indirectly, with the overexpression of proteins involved in signalling upstream of ROS scavenging. Recently, a rice gene coding for a receptor-like kinase (RLK) was reported to improve the drought and salt tolerance (DST) of transgenic plants overexpressing the RLK (OsSIK1) (Ouyang et al., 2010). The transgenic plants had higher activity of peroxidases, SOD and catalase (CAT) during stress, as well as reduced stomatal density. The improved tolerance to osmotic stress treatments (using very high concentrations of NaCl or water withholding) of the transgenic plants may be attributed to reduced stomatal density as much as to the increased antioxidant activity (Ouyang et al., 2010). What cannot be determined from the data provided by Ouyang et al. (2010) is whether the changes in antioxidant activity are dependent on the changes in stomatal density, or vice versa, or if the two are independent. Overexpression of the Arabidopsis gene GF14l, encoding a 14-33 protein that interacts with proteins involved in numerous metabolic processes, including antioxidant activity, demonstrated a ‘‘stay-green’’ phenotype and improved tolerance to moderate water stress in cotton (Yan et al., 2004). CAT is one of the major endogenous enzyme antioxidants. It catalyses H2O2 decomposition and is up-regulated at the transcriptional level upon exposure to high salinity stress. In cyanobacteria, introduction of a CAT gene of E. coli, katE, was found to reduce ROS production under salt stresses
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and confer salt tolerance (Kaku et al., 2000). Transgenic rice plants’ constitutive overexpression of the katE gene showed improved growth under salinity stress (Nagamiya et al., 2007). Plants were evaluated at the vegetative and reproductive stages for salt tolerance. T1 seedlings were soaked in 0, 50, 100, 150, 200, 250, 300, 400, 500 or 600 mM NaCl and surviving rate (green tissue) was recorded. In addition, flowering T1 transgenic lines grown under normal conditions were soaked in 250 mM NaCl solution for 14 days. The transgenic rice seedlings showed improved growth under high salinity (250 mM), and were able to form flower and produce seeds in the presence of 100 mM NaCl. CAT activity in the transgenic rice plants was 1.5- to 2.5fold higher than in nontransgenic rice plants. Pyramiding of ROS-scavenging genes may provide more effective tolerance of oxidative stress resulting from drought or salinity. Two genes (from Suaeda salsa) coding GST (glutathione S-transferase, EC 2.5.1.18) and CAT (EC 1.11.1.6) were transformed under the control of a constitutive promoter into rice plants. Transgenic rice seedlings showed a marked enhanced tolerance to salinity and oxidative stresses (Zhao and Zhang, 2006). Expression of three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD), ascorbate peroxidase (APX) and dehydroascorbate (DHA) reductase (DHAR), in tobacco chloroplasts resulted in a higher tolerance to oxidative stress induced by salinity stress (Lee et al., 2007). These studies suggested that the simultaneous expression of multiple antioxidant enzymes could be more effective than the expression of single genes for developing transgenic plants with enhanced tolerance to abiotic stresses. ROS, and H2O2 in particular, also play a role in the signalling pathways involved in the adaptation to the stress response (Miller and Mittler, 2006). Samis et al. (2002) combined the mitochondrial and chloroplastic SOD expression by crossing the transgenic alfalfa plants that had shown superior field performance in earlier trials (McKersie et al., 1996). The plants carrying both constructs had higher SOD activity than either of the sibling controls that carried only one of the MnSOD transgenes, but biomass production in the field of the plants carrying both genes was reduced, relative to the single gene siblings (Samis et al., 2002). The authors suggested that there might be an optimum level of SOD activity, above which processes such a H2O2 signalling might be impaired. The use of inducible promoters for driving the expression of antioxidant enzymes is also being tested as an alternative to constitutive expression. In rice, transformation of chloroplast-targeted manganese superoxide dismutase isolated from pea (MnSOD) under the control of an oxidative stress-inducible SWPA2 promoter resulted the improvement of indicators of oxidative stress tolerance in T1 plants tested in the greenhouse (Wang et al., 2005a).
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In most saline soils, Naþ and Cl are the predominant ions in the soil solution. At sufficiently high concentrations, both ions contribute to an unfavourable osmotic gradient between the soil solution and the plant roots. Both ions also cause ion-specific toxicity when accumulated in saltsensitive plants. And while it is clear that the exclusion of Naþ or Cl, or both, is correlated with improved salinity tolerance in some species (and the accumulation of both with others), the state knowledge of Naþ transport mechanisms is more advanced than that for Cl transport (Teakle and Tyerman, 2010). 1. Decreasing Naþ uptake In both glycophytes and halophytes, the net uptake of sodium into the roots is the sum of sodium influx and efflux. The negative electrical membrane potential difference at the plasma membrane of root cells ( 140 mV) favours the passive transport of sodium into root cells, and especially so when sodium concentrations increase in the soil solution. The entry of sodium into root cells is mediated by uniporter or ion channel-type transporters, like HKT, LCT1 and NSCC (reviewed in Plett and Moller, 2010). The reduction of Naþ uptake might be accomplished by decreasing the number or activity of these transporters in the roots. Reduction of TaHKT2;1 expression in wheat by antisense suppression resulted in lower net sodium uptake of transgenic roots and higher fresh weight of plants grown under salinity stress in controlled growth conditions (Laurie et al., 2002). Similarly, Arabidopsis T-DNA knockout mutants of AtCNGC3, a cyclic nucleotide gated channel which catalyses Naþ uptake, had lower net influx of Naþ and were more tolerant to salinity at germination (Gobert et al., 2006). The efflux of sodium from the roots is an active process, which is presumed to be mediated by plasma membrane Naþ/Hþ antiporters. These secondary transporters use the energy of the proton gradient across the plasma membrane to drive the active efflux of sodium from the cytosol to the apoplast. The Naþ/Hþ antiporter, SOS1 (identified in a mutant screen as salt overly sensitive 1), is the only Naþ efflux protein at the plasma membrane of plants characterized so far. The overexpression of AtSOS1, a plasma membranebound Naþ/Hþ antiporter, improved the ability of the Arabidopsis transgenic plants to grow in the presence of high NaCl concentrations (Shi et al., 2003). And the rice orthologue, OsSOS1, is able to complement the Arabidopsis sos1 mutant (Martinez-Atienza et al., 2007). The SOD2 (Sodium2) gene was identified in yeast, Schizosaccharomyces pombe, as a Naþ/Hþ antiporter on the plasma membrane involved in salt tolerance. Transformation of rice with
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the SOD2 gene (under 35S promoter) resulted in accumulation of more Kþ, Ca2þ, Mg2þ and less Naþ in the shoots compared with wild type (Zhao et al., 2006b). The transgenic rice plants were able to maintain higher photosynthesis level and root proton exportation capacity, whereas reduced ROS generation. Although yield data were not reported, the trials were conducted outdoors, which is the closest to field level study of a crop plant for this approach in the literature. 2. Decreasing root to shoot translocation of Naþ The accumulation of sodium in shoots occurs via the translocation of sodium from the roots along the transpirational stream. The removal of sodium from the xylem, which reduces the rate of sodium transfer to the shoot tissue, has been shown to be mediated by members of the HKT gene family (reviewed in Plett and Moller, 2010). AtHKT1;1 in Arabidopsis, OsHKT1;5 in rice, and HKT1;4 in wheat are all critical in reducing Naþ shoot concentrations by transporting Naþ from the xylem into the root stele (reviewed in Hauser and Horie, 2010). One strategy for improving salinity tolerance is to increase the expression of such genes to further reduce sodium concentrations in the xylem (Plett et al., 2010). The overexpression of AtHKT1;1 under the control of the constitutive promoter CaMV35S leads to increased salt sensitivity, presumably because Naþ fluxes are increased in inappropriate cells and tissues (Moller et al., 2009). However, when expressed under the control of a promoter directing expression in root epidermal and cortical cells, both in rice and in Arabidopsis, HKT1;1 overexpression causes an increase in root cortical sodium, a decrease in shoot sodium and a higher accumulation of fresh weight during the course of the experiment (Plett et al., 2010). 3. Sequestering Naþ The accumulation of Naþ ions into vacuoles through the operation of a vacuolar Naþ/Hþ antiporter provided an efficient strategy to avert the deleterious effect of Naþ in the cytosol and maintain osmotic balance by using Naþ (and Cl) accumulated in the vacuole to drive water into the cells (Apse et al., 1999; Apse and Blumwald, 2002). Transgenic plants overexpressing an Arabidopsis vacuolar Naþ/Hþ antiporter, AtNHX1, exhibited improved salt tolerance in Brassica napus (Zhang et al., 2001), tomato (Zhang and Blumwald, 2001), cotton (He et al., 2005), wheat (Xue et al., 2004), beet (Yang et al., 2005) and tall fescue (Zhao et al., 2007). The transformation of an orthologue gene (AgNHX1) from halophytic plant Atriplex gmelini into rice improved salt tolerance of the transgenic rice (Ohta et al., 2002). Maize plants overexpressing rice OsNHX1 gene accumulated more biomass, under 200 mM NaCl in greenhouse (Chen et al., 2007). Moreover, under field trail
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conditions, the transgenic maize plants produced higher grain yields than the wild-type plants. Transformation of another Naþ/Hþ antiporter family member, AtNHX3 (from Arabidopsis), in sugar beet (Beta vulgaris L.) resulted in increased salt accumulation in leaves, but not in the storage roots, with enhanced constituent soluble sugar contents under salt stress condition (Liu et al., 2008). The introduction of genes associated with the maintenance of ion homeostasis in halotolerant plant into crop plants confirmed salinity tolerance. The yeast gene HAL1 was introduced into tomato (Gisbert et al., 2000), watermelon (Citrullus lanatus (Thunb.); Ellul et al., 2003) and melon (Cucumis melo L.; Bordas et al., 1997), which confirmed higher level of salt tolerance, with higher cellular Kþ to Naþ ratio under salt stress. Likewise, the introduction of the yeast HAL2 gene into tomato resulted in improved root growth under NaCl conditions, contributing to improved salt tolerance (Arrillaga et al., 1998). Overexpression of HAL3 (from S. cerevisiae) homologue NtHAL3 in tobacco increased proline biosynthesis and the enhancement of salt and osmotic tolerance in cultured tobacco cells (Yonamine et al., 2004). The electrochemical gradient of protons across the vacuolar membrane is generated by the activity of the vacuolar Hþ-translocating enzymes, HþATPase and Hþ-pyrophosphatase. Increasing vacuolar Hþ pumping might be required to provide the additional driving force for vacuolar accumulation via sodium/proton antiporters. A gene coding for a vacuolar Hþ-pyrophosphatase proton pump (AVP1) from Arabidopsis was overexpressed in tomato (Park et al., 2005), cotton (Pasapula et al., 2011) and rice (Zhao et al., 2006a) and induced improved growth during drought and salt stress. Interestingly, the overexpressed AVP1 resulted in a more robust root system which could possibly improve the plants ability to absorb more water from the soil (Pasapula et al., 2011). D. REGULATORY AND SIGNALLING GENES
1. DREB/CBF Dehydration-responsive element (DRE)/C-repeat (CRT) was identified in Arabidopsis, a cis-acting element regulating gene expression in response to dehydration (drought, salinity and cold stress; Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994). Several DRE-binding proteins (DREB)/CRT-binding factor (CBF) were isolated and identified as key players in dehydration (drought, salinity and cold stress) responsive gene expression (Yamaguchi-Shinozaki and Shinozaki, 1994). Using transgenic approaches, the DREB/CRF signalling pathway is one of the most studied in
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numerous plant species. The overexpression of these genes activated the expression of many downstream genes with the DRE elements in their promoters, and the resulting transgenic plants showed improved stress tolerance (Agarwal et al., 2006). In Arabidopsis, two classes of DREBs were isolated: DREB1 expression was found to be highly up-regulated during cold stress, and DREB2 expression was responsive to drought and salinity. Transgenic rice lines overexpressing OsDREB1A and OsDREB1B under the control of a constitutive ubiquitin promoter showed more tolerance to drought and salinity conditions (in term of survival rate); however, under normal conditions, the transgenic lines showed reduced growth (Ito et al., 2006). In this experiment, rice seedlings (17–19 days) that were grown in very small pots under continuous light were exposed to high salinity (250 mM NaCl, 3 days) or drought (withholding water for 9 days), followed by rewatering. While drought associated traits (as proline) were measured, no data on yield were reported. Further, the transgenic rice plants overexpressing OsDREB1 or DREB1 showed growth retardation under normal growth conditions (Ito et al., 2006). Constitutive (35S promoter) overexpression of AtDREB1A in transgenic rice resulted in increased tolerance to drought (Oh et al., 2005). Transgenic plants were grown in small pots for 4 weeks and exposed to 4 days of drought followed by re-watering. Survival rate was measured. In contrast to previously reported reduction in growth, in this experiment, neither growth inhibition nor visible phenotypic alterations were noted, despite constitutive expression of DREB gene. Overexpression of two other OsDREB genes, OsDREB1G and OsDREB2B, also showed significantly improved survival rate under water-deficit stress in rice seedling (Chen et al., 2008). Overexpression of DREB1A/CBF3, driven by the stress-inducible RD29A promoter in bread wheat, improved drought tolerance in greenhouse (Pellegrineschi et al., 2004). Small seedlings (six leaf stage) grown in pots (5 5 cm) of T 2 plants were exposed to 10–12 days of withholding water and re-watering. Survival rate was used to measure tolerance, but no yield was reported. Transformation of AtDREB1A into peanut (Arachis hypogaea L.) was reported to improve TE under water-limited conditions (BhatnagarMathur et al., 2007). T3 plants were grown in pots and water stress was applied after 28 days. Interestingly, most transgenic events had higher TE than the wild type under well-watered conditions, and one event showed 40% improvement than wild-type plants under water stress. While P35S::DREB1A plants exhibited stunted growth even under control conditions, the transgenic Prd29A::DREB1A peanut plants did not show any growth retardation (Bhatnagar-Mathur et al., 2007). In contrast, transgenic potato expressing the same Prd29A::DREB1A gene showed growth retardation (Behnam et al.,
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2006). Overexpression of a soybean DREB orthologue, GmDREB1, in alfalfa (Medicago sativa L.) plants under the control of Arabidopsis Rd29A promoter was tested in greenhouse pot experiment (Jin et al., 2010). Four-week-old plants were watered with NaCl solution (0, 100, 200, 300 and 400 mM) for 60 days at 5-day intervals. The transgenic lines showed improved tolerance to salinity in terms of survival as compared with wild-type plants; however, no biomass production data were reported. Tomato plants overexpressing the AtDREB1B/CBF1 under constitutive 35S promoter showed a higher level of proline, as compared with the wildtype plants grown under normal or water-deficit conditions (Hsieh et al., 2002). T1 plants, grown in controlled greenhouse conditions, were exposed to water deficit (after 3 months) for 3 weeks and survival rate was calculated. However, severely reduced growth was found in the transgenic tomato plants. Further, the transgenic tomato plants showed a decrease in fruit, seed number, and fresh weight as compared with wild-type plants under normal conditions (Hsieh et al., 2002). HARDY (HRD), a gene encoding AP2/ethylene response factor (ERF)like transcription factor (TF) that belongs to the BREB/CRB family, was identified as a gain-of-function mutation in Arabidopsis (Karaba et al., 2007). The hrd mutant showed abnormally dense root system, increased mesophyll cell layer and enhanced tolerance to drought and salinity (Karaba et al., 2007). Overexpressing of the HRD gene in rice resulted in increased water-use efficiency (WUE) in controlled greenhouse conditions. Rice plants of T3 generation lines were grown in pots under 100% and 70% field capacity. Under control conditions, the transgenic lines showed no growth reduction, an increase in leaf biomass and an increase in bundle sheath cells. The HRD expression in rice caused significant increases of instantaneous and wholeplant WUE in well-watered and drought conditions, with a very remarkable increase of 100% in absence of drought and a consistent 50% increase under drought stress (Karaba et al., 2007). The efficiency of this approach still needs to be tested for yield under greenhouse and field conditions. 2. Protein kinase Several studies have suggested that many protein kinases are involved in drought resistance, among them, members of the calcium-dependent protein kinase (CDPK), calcineurin B-like protein-interacting protein kinase (CIPK) and mitogen-activated protein kinase (MAPK) families. Ca2þ cytosolic levels increase rapidly in plant cells in response to environmental stress, including drought and salinity (Sanders et al., 1999). This Ca2þ influx is likely to be mediated by a combination of protein phosphorylation/dephosphorylation cascades involving members of the CDPK family. In rice, overexpression of
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OsCDPK7 (under the control of the 35S promoter) resulted in increased seedling recovery rate after a salt treatment (Saijo et al., 2000). T1 seedlings (10 days) old treated with 150/200 mM NaCl and transferred again to a normal nutrient solution. The transgenic plants showed normal development and yield. It was suggested that OsCDPK7 underwent post-translational regulation, since the presence of OsCDPK7 was not sufficient to induce expression of stress-associated target genes. Overexpression of three CIPK genes (OsCIPK03, OsCIPK12 and OsCIPK15) enhanced tolerance to cold, drought and salt stress, respectively, in transgenic rice (Xiang et al., 2007). Overexpression of a MAPK family gene OsMAPK5a in rice leads to increased OsMAPK5a kinase activity and enhanced tolerance to drought and salt stresses (Xiong and Yang, 2003). Overexpression of another rice MAPK family, OsMAPK44, resulted in increased tolerance to salt stress (Jeong et al., 2006). Recently, overexpression in rice of DSM1 (drought-hypersensitive mutant1), a putative MAPK kinase kinase (MAPKKK) gene, increased the tolerance of the seedlings to dehydration stress (Ning et al., 2010). It was suggested that DSM1 might be a novel MAPKKK functioning as an early signalling component in regulating mechanisms of ROS scavenging in rice Expression of a MAPKKK gene was shown to activate an oxidative signal cascade and led to the tolerance to environmental stress in transgenic tobacco. The catalytic domain of Nicotiana protein kinase 1 (NPK1) activated a bypass of BCK1-mediated signal transduction pathway in yeast, which was found to be conserved among different organisms (Banno et al., 1993). NPK1 was reported to be upstream of oxidative pathways inducing expression of heat shock proteins and GST (Kovtun et al., 2000). Constitutive overexpression of the tobacco MAPKKK in maize enhanced the drought tolerance of the transgenic plants (Shou et al., 2004). Under drought conditions, the transgenic plants maintained significantly higher photosynthesis rates and kernel weight as compared with wild-type plants. However, the effect of NPK1 on yield components was less apparent. 3. Nuclear factor Y-B subunit In Arabidopsis, AtNF-YB1, a nuclear factor Y (NF-Y complex), was found to mediate transcriptional control through CCAAT DNA elements and confer tolerance to abiotic stress when constitutively expressed in Arabidopsis (Nelson et al., 2007). NF-Y is a conserved heterotrimeric complex consisting of NF-YA (HAP2), NF-YB (HAP3) and NF-YC (HAP5) subunits (Mantovani, 1999). An orthologous NF-YB gene was found in maize with similar response to drought. Transgenic maize lines constitutively overexpressing ZmNF-YB2 showed improved drought tolerance under field conditions (Nelson et al., 2007). Under water-limited conditions, transgenic plants
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show tolerance to drought based on grain yield and on the responses of a number of stress-related parameters, including chlorophyll content, stomatal conductance, leaf temperature, reduced wilting and maintenance of photosynthesis. 4. NAC proteins Several NAC [NAM (No Apical Meristem), ATAF1-2 and CUC2 (cupshaped cotyledon)] domain proteins, which are one of the largest plant TF families (Riechmann et al., 2000), have been reported to be associated with abiotic stresses. Of the 140 putative rice NAC genes, the expression of 40 NAC genes increased with drought or salinity stress (Fang et al., 2008). Twenty of these genes were induced at least twofold with stress treatment and a majority of these form the group III clade of NAC genes, called SNAC or the stress-responsive NACs (Fang et al., 2008). The overexpression of a stress-responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) in rice significantly enhanced the drought tolerance (22–34% increase in seed setting) of the transgenic plants under severe water-stress conditions at the reproductive stage in the field (Hu et al., 2006). Biomass accumulation at the vegetative stage was improved in rice plants overexpressing SNAC1 under both salinity and drought stress (Hu et al., 2006). The phenotype was partially attributed to increased stomatal closure and ABA sensitivity in the transgenic plants (Hu et al., 2006). Overexpression of OsNAC45 in rice improved tolerance to drought and salt treatments as discussed in more detail in Section 5 (LEA gene expression). Recently, the overexpression of OsNAC10 in rice, under the control of the constitutive promoter GOS2 and the root-specific promoter RCc3, improved tolerance to drought and salinity of the transgenic plants at the vegetative stage. However, only the rootspecific overexpression of OsNAC10 (PRCc3::OsNAC10) significantly enhanced drought tolerance at the reproductive stage, increasing grain yield (25–42%) in the field under drought conditions (Jeong et al., 2010). The yield advantage in the PRCc3::OsNAC10 plants was attributed to the larger root diameter in these plants, which were approximately 20% larger than both the wild type and PGOS2::OsNAC10 plants (Jeong et al., 2010). 5. Increasing LEA gene expression Late embryogenesis abundant (LEA) proteins are low-molecular weight proteins that, in molar excess, and synergistically with trehalose, prevent protein aggregation during desiccation or water stress (Goyal et al., 2005). The overexpression of OsLEA3-1 under the control of a strong constitutive
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promoters (35S and Actin1) and a stress-inducible promoter (HVA1-like promoter isolated from the upland rice IRAT109) in a drought-sensitive Japonica (lowland) rice resulted in improved drought tolerance (Xiao et al., 2007). Transgenic rice plants with 35S and HVA1-like promoters displayed improved yields when grown in PVC pipes and under field conditions without yield penalties. The improved yield under drought conditions was primarily due to improved spikelet fertility under stress (Xiao et al., 2007). Spring wheat lines expressing the barley HVA1 gene (under the control of the ubiquitin promoter) tested across multiple years and locations in dry land cultivation yielded better than the untransformed controls (Bahieldin et al., 2005). In an earlier study, wheat lines were taken to the T4 generation and compared to newly developed lines using the same construct (Sivamani et al., 2000). Yields of the transgenic HVA1 lines were not significantly different than the wild-type and non-transformed control lines under irrigated conditions; however, under dry land conditions, the HVA1 lines produced 7–35% more yield. The yield under water stress was correlated with the amount of HVA1 protein detected in leaf extracts of the transgenic lines (Bahieldin et al., 2005). Increasing LEA gene expression under stress, and presumably LEA protein abundance, has also been accomplished indirectly, with the overexpression of NAC genes. LEA gene expression under stress may account for improved tolerance to drought and/or salinity stress in plants overexpressing OsNAC5 and OsNAC6 (Takasaki et al., 2010), and OsNAC45 (Zheng et al., 2009). The overexpression of the stress-responsive proteins OsNAC5 and OsNAC6 resulted in enhanced stress tolerance by up-regulating the expression of stress-inducible rice genes such as OsLEA3, although the effects of these proteins on plant growth were different. However, the tolerance of the UBIpro::OsNAC5 transgenic rice plant to salinity was measured in 2-weekold transgenic plants that were grown in 250 mM NaCl for 3 days and then grown for 30 days under normal conditions (i.e. survival rate), and no yield data were presented. The overexpression of OsNAC45 leads to increased LEA3 and PM1 gene expression Zheng et al. (2009). Preliminary assays of the response to drought stress showed that young seedlings overexpressing OsNAC45 had improved survival rates, relative to wild-type controls, 10 days after recovery from a 9.5-h period of root drying (Zheng et al., 2009). Although these hydroponic assays on T2 generation transgenics are not sufficient to assess the response of the transgenic plants to drought under field conditions, the increased expression of LEA3, taken together with the results of Xiao et al. (2007), provides an incentive to take later generations of these transgenic rice plants to field testing.
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6. Aquaporins Aquaporins are intrinsic membrane proteins that mediate the transport of water, small neutral solutes and CO2 (Tyerman et al., 2002). The regulatory role of aquaporins in cellular water transport had been demonstrated (Knepper, 1994). The stress-induced expression of the aquaporin, RWC3, a member of the plasma membrane intrinsic protein 1 (PIP1) subfamily, resulted in improved water status of lowland rice (Lian et al., 2004). Fourweek-old plants grown hydroponically in nutrient solution were exposed to a osmotic shock treatment of 20% polyethylene glycol (PEG) 6000 for 10 h (Lian et al., 2004). However, transgenic tobacco plants constitutively expressing the Arabidopsis plasma membrane aquaporin PIP1b displayed enhanced growth vigour under well-watered conditions, but the transgenic plants wilted rapidly during water stress (Aharon et al., 2003). A comparison between the results obtained by overexpressing PIP-type aquaporins in tobacco and rice is difficult. In addition to the difference between the constitutive (tobacco) and stress-inducible (rice) expression, two different treatments (osmotic shock vs. gradual dehydration) were applied. Further, transgenic rice plants constitutively overexpressing a barley HvPIP2;1 (a plasma membrane aquaporin) showed more sensitivity (reduction in growth rate) to salinity stress (Katsuhara et al., 2003). T2 rice plants were grown hydroponically and exposed to 100 mM NaCl after 4 weeks. Although the growth of transgenic rice plants was similar to that of control plants under normal conditions, the growth of the transgenic plants was greatly inhibited and eventually withered and died under a salinity treatment (Katsuhara et al., 2003). Recently, tomato plants’ constitutive overexpressing of atonoplast SlTIP2;2 showed increased cell water permeability and whole-plant transpiration (Sade et al., 2009). The expression of SlTIP2;2 resulted in increased transpiration under normal growth conditions, limited transpiration reduction under drought and salt stresses and also accelerate transpiration recovery after stress Two field experiments of F1 hybrids of transgenic MicroTom and M82 plants were conducted in commercial net-house. Salinity was applied by irrigation with saline water (80–200 mM NaCl) and in parallel, the same F1 hybrids were grown under well-watered and water-limited conditions. Transgenic plants showed significant increases in fruit yield, harvest index and plant mass relative to the control under both normal and waterstress conditions (Sade et al., 2009). It was postulated that overexpression of the SlTIP2;2 could bypass the stress-induced down-regulation of the endogenous aquaporins genes of the tonoplast and thus prevent the slowdown of tonoplast osmotic water permeability (Sade et al., 2009).
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7. Hormonal homeostasis and abiotic stress Hormones play a major role in stress signalling. One of the fast responses of plants to soil water stress is the accumulation of ABA in the roots (Thompson et al., 2007), which is transported through the xylem to the shoot (Wilkinson and Davies, 2010) causing stomatal closure reducing water loss via transpiration (Schroeder et al., 2001) and eventually restricting cellular growth. ABA can also be synthesized in leaf cells and transported through the plant (Wilkinson and Davies, 2010). In Arabidopsis, a large number of genes associated with ABA metabolic pathway have been characterized, and genes coding ABA receptors and downstream signal relays have been recently reviewed (Cutler et al., 2010; Huang et al., 2008). However, in crop plants, only one gene involved in ABA metabolism (LOS5/ABA3, a key enzyme in the last step of ABA biosynthesis) has been manipulated in rice with enhanced drought tolerance (Xiao et al., 2009). LOS5 gene was overexpressed under the control of constitutive or drought-inducible promoters and tested in the field. Plants were grown under normal conditions for 1 month and then water was stopped during the initiation of panicle development. The improved yield of the transgenic lines under field conditions was a result of a significant increase in the spikelet fertility (Xiao et al., 2009). While many reports on the development of transgenic plants with improved tolerance to drought or salinity by manipulating the expression of stress-related genes in laboratory or greenhouse conditions are available, only few studies were tested under natural field condition. In tomato, the constitutive overexpression of LeNCED1 (drought-inducible and a rate-limiting enzyme for ABA biosynthesis) resulted in increased ABA accumulation (Thompson et al., 2007). Plants were grown to a four- to five-leaf stage in a controlled environment cabinet in 500-mL free-draining pots and exposed to drought treatment. The constant elevation in ABA level resulted in physiological and morphological changes in the transgenic plants. Under well-watered conditions, plants showed reduction in assimilation rates, leaf flooding and chlorosis, but under water-deficit conditions, these effects were insufficient to reduce biomass production, presumably because of counteracting positive effects on leaf expansion through improvements in water status, turgor and antagonism of epinastic growth (Thompson et al., 2007). Cytokinins (CKs) have been found linked to a variety of abiotic stresses (Hare et al., 1997). In Arabidopsis, examination of public microarray expression data revealed many genes encoding proteins associated with CK signalling pathways that were differentially affected by various abiotic stresses (reviewed by Argueso et al., 2009). CK is an antagonist to ABA, and the exposure of plants to drought results in decreased levels of CK. Elevated CK levels could promote survival under water-stress conditions, inhibit leaf
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senescence and increased levels of proline (Alvarez et al., 2008). The manipulation of endogenous CK levels was effective in delaying senescence (Gan and Amasino, 1997). Isopentenyltransferase (IPT, mediating the rate-limiting step in CK biosynthesis) has been overexpressed in several plant species. However, drought tolerance varied with the type of promoter used to drive IPT expression (Ma, 2008). Transgenic tobacco (N. tabacum) expressing the IPT gene under control of a drought-induced promoter (SARK, senescenceassociated receptor kinase) resulted in increased drought tolerance, photosynthetic capacity and yield (Rivero et al., 2007, 2009, 2010). Recently, we have showed that transgenic rice plants expressing PSARK::IPT resulted in enhanced drought tolerance and superior yields (Peleg et al., 2011). Transgenic Cassava (Manihot esculenta Crantz), expressing IPT under control of a senescence-induced promoter, SAG12, were tested for drought tolerance under field conditions (Zhang et al., 2011). The transgenic cassava plants displayed higher tolerance to drought due to the inhibition of stress-induced leaf abscission and fast recovery from stress. Creeping bentgrass (Agrostis stolonifera) expressing PSAG12::IPT was tested hydroponically using osmotic stress induced by different PEG concentrations (Merewitz et al., 2011). The transgenic plants were able to maintain higher osmotic adjustment, chlorophyll content, WUE and greater root viability under osmotic stress compared with the wild-type plants (Merewitz et al., 2011). However, these results should be taken with caution since the use of PEG to stimulate osmotic stress is artificial, and did not represent the multidimensional response of plants to water deficit under natural conditions. Jasmonic acid (JA) is involved in plant development and the defence response. Transgenic rice plants overexpressing the Arabidopsis JA carboxyl methyltransferase gene (AtJMT) under the control of the Ubi1 promoter showed increased JA levels in panicles (Kim et al., 2009). Plants were grown in the greenhouse and were subjected to 2 weeks of drought before panicle initiation. The PUbi1::AtJMT plants resulted in significantly grain yield reduction, due to a lower numbers of spikelets and lower filling rates than wild-type plants (Kim et al., 2009). Rice plants overexpressing the ERF, AP37, under the control of the constitutive promoter OsCc1, displayed increased tolerance to drought and high salinity at the vegetative stage (Oh et al., 2009). More importantly, when these transgenic lines were tested in the field, the POsCc1::AP37 plants showed increased grain yield over controls under severe drought conditions, while no significant differences were noted under well-watered conditions (Oh et al., 2009). Overexpression in rice of another ERF gene, a protein TSRF1 that binds to the GCC box, showed enhanced osmotic and drought tolerance in seedlings (Quan et al., 2010). T2 rice seedlings (10 days old) were exposed
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osmotic shock (20% PEG for 3 days) or withholding water for 6 days followed by recovery under control conditions. Under normal conditions the transgenic TSRF1 plants did not show any differences in growth or development. In another experiment, 2-week-old seedlings overexpressing TERF1 (a tomato ERF protein) were exposed to drought by withholding water for 9 days, or salinity by immersing in 200 mM NaCl. The transgenic plants showed improved survival rate after exposure to drought or salinity (Gao et al., 2008). Further study is needed to test the efficiency of this strategy under field experiment and more critical growth phases (i.e. reproductive stage). Plant hormone crosstalk and the regulation of various hormone-regulated biosynthetic pathways (see Nemhauser et al., 2006) during water stress play important roles in abiotic stress adaptation. The homeostatic regulation of phytohormones could play significant roles in the regulation of source/sink relationships and its manipulation could provide a significant avenue for the development of abiotic stress tolerance in plants. 8. The regulation of the stomatal response to stress Reducing transpiration rates without affecting CO2 assimilation would result in increase WUE and may contribute to improve yields. It was postulated recently that reductions in stomata density and stomatal aperture can reduce transpirational water loss while maintaining sufficient CO2 uptake to sustain biomass and yield under water-deficit conditions (Yoo et al., 2009). There are a handful of examples where the modification of a single gene resulted in reduced stomatal aperture and stomatal density, and consequently increasing WUE (reviewed in Yoo et al., 2009). These modifications also resulted in improved plant resistance to water-deficit stresses like salinity and drought. Some of these modifications have been tested in crop plants and in some cases, under field conditions. ERA1 is a negative regulator of the ABA response in Arabidopsis, and was found in a screen for hypersensitivity of seed germination to ABA (Cutler et al., 1996). era1 rosettes were slower to wilt under severe water deficit, owing to the smaller stomatal aperture in the mutant plants (Pei et al., 1998). The ERA1 locus is the beta subunit of farnesyltransferase, which adds a farnesyl group to proteins containing a CaaX motif (Andrews et al., 2010). In era1 plants, and to a lesser degree in plants expressing a constitutive AtFTB (farnesyltransferase B) hairpin construct, growth and development are impaired, owing to the loss (or reduction) of function of FTB in other aspects of plant development, including meristem organization (Bonetta et al., 2000), among others. An agriculturally relevant application FTB down-regulation was accomplished by the use of a stress-inducible promoter, rd29. While early seedling development was impaired in canola plants expressing Prd29::antiFTB, yields of the field
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grown transgenic plants were no different that wild-type controls under sufficient water conditions (Wang et al., 2005b). Down-regulation of FTB in canola provided improved yield relative to wild-type controls under mild and moderate water-deficit stress conditions in the field (Wang et al., 2005b). The concept of reducing stomatal aperture and transpiration during drought stress was further refined and confirmed by Wang et al. (2009) by using the hydroxypyruvate reductase (HPR1) promoter to drive the expression of an RNAi construct directed against the farnesyltransferase A (FTA) subunit. The HPR1 promoter is up-regulated by drought stress and is preferentially expressed in the shoot tissues. PHPR1::antiFTA transgenic canola seedlings were not impaired in early shoot and root growth, as was the case with Prd29:: antiFTB seedlings, and PHPR1::antiFTA plants had no yield drag relative to wild-type controls under water-sufficient conditions in the field (Wang et al., 2009). Under water-deficit conditions, experienced primarily during flowering and pod filling, PHPR1::antiFTA plants yielded 14–16% greater seed than wild-type controls, which experienced yield losses of 20% (Wang et al., 2009). Whether this technology can be applied to crops other than canola is yet to be reported. However, the successful application of SNAC1 overexpression to improving rice yields under drought and salinity stress, by increasing stomatal closure without decreasing CO2 assimilation, shows the concept viability. Loss of function of the zinc finger protein DST resulted in reduced stomatal aperture and stomatal density, and increased drought and salt tolerance in rice (Huang et al., 2009). While field testing has not been reported for the dst plants, under controlled growth conditions, they retained a higher RWC under soil drying conditions and recovered more rapidly on re-watering than the wild-type control plants (Huang et al., 2009). DST negatively regulates the expression of hydrogen peroxide scavenging enzymes in guard cells, which balances the ROS signalling for stomatal closure that is propagated through the ABA signal. Therefore, in the dst mutant, the ROS signal was less attenuated and stomatal apertures remained smaller than in the wild type. While CO2 assimilation was not measured, Huang et al. (2009) reported that seed yields were not reduced in the dst mutant. Genetic modifications, where stomatal aperture and stomatal density reduce water loss under stress, but do not reduce CO2 assimilation in the absence of stress, are attractive targets for engineering abiotic stress tolerance. 9. Other transcription factors Although multiple TFs have been well characterized in various plant species, transcriptional reprogramming under drought and stress is not fully understood. Overexpression of the AtMYB2 gene (from Arabidopsis) in rice under the control of an ABA-inducible promoter conferred salt stress tolerance to
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the transgenic plants, with higher biomass and decreased ions leakage (Malik and Wu, 2005). Overexpression of OsWRKY11 (encoding a TF comprising a WRKY domain), under the control of a HSP101 promoter, conferred heat and drought tolerance at the seedling stage (slower leaf wilting and higher survival rate of green parts of plants; Wu et al., 2009). Recently, it was shown that the constitutive overexpression of two members of a family of bacterial RNA chaperones, CspA (from E. coli) and CspB (from Bacillus subtilus), conferred abiotic stress tolerance to transgenic Arabidopsis, rice and maize (Castiglioni et al., 2008). The transgenic maize plants showed yield benefits of up to 15% (0.75 t/ha) as compared to the nontransgenic controls, under water-stressed environment. Importantly, the observed yield improvements in water-limited field trials were not associated with a yield penalty in non-stressed (high-yielding) environments (Castiglioni et al., 2008). These results suggested that chaperones molecules may be good candidates for abiotic stress enhancement in crop plants.
IV. CONCLUSIONS AND PERSPECTIVES Developing drought and salinity tolerance crop plants using conventional plant breeding methods had limited success during the past century. New technologies are providing opportunities to address the challenging problem of maintaining high-yield crop production under stressful environmental conditions and changing climates. The information provided by high-resolution transcript profiling, the identification of large-scale specific protein networks and their association with the plant responses to environmental perturbations are allowing the application of a systems-level approach to uncover the bases of plant responses to environmental changes. The application of an integrated approach is of paramount importance because the crops of the future are likely to be stacked with multiple traits (water deficit, nitrogen use efficiency, pathogen challenges, etc.). However, a review of the different transgenic crops produced so far revealed very limited success in producing drought- and salinity-tolerant cultivars through genetic transformation. Most transgenic plants developed with improved tolerance based on the performance of transgenic lines under controlled conditions in growth room or greenhouse, while only few lines were tested under field conditions (Flowers, 2004). Numerous genes related to plant response to abiotic stress have been identified and characterized (Araus et al., 2008; Wang et al., 2005b). However, the vast majority of these studies were conducted on the model species such as Arabidopsis and tobacco under laboratory conditions (reviewed by Ashraf and Akram, 2009; Pardo, 2010; Umezawa et al., 2006). While for
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crops, the reproductive stage in the most critical stage for productivity, in the majority of studies cited here, stress tolerance has been assessed at the initial growth stages, that is, germination and seedling stage, using survival rate as the main trait to represent tolerance to stress. In many of these experiments, artificial extreme conditions were applied (i.e. high salinity, osmotic shock, etc.). Under field conditions, plants have to cope with multiple stresses (as water deficit and heat) for longer periods. Hence, more emphasis should be given to the study of the responses of crop plants to a combination of environmental stresses at the reproductive stage and under field conditions.
ACKNOWLEDGEMENTS This study was supported by Grant from NSF-IOS-0802112, CGIAR GCP#3008.03, UC Discovery #bio06-10627 and the Will W. Lester Endowment of University of California. Z. P. was supported by Vaadia-BARD postdoctoral Fellowship Award No. FI-419-08 from the United States— Israel Binational Agricultural Research and Development Fund (BARD).
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Drought Stress: Molecular Genetics and Genomics Approaches
MELDA KANTAR, STUART J. LUCAS AND HIKMET BUDAK1
Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The Molecular Biology of Drought . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Growth Responses ............................................................. B. Signalling Pathways ............................................................ C. Compatible Solutes............................................................. D. Protective Proteins ............................................................. E. Antioxidants..................................................................... F. Other related Molecules ....................................................... III. Transcriptional Regulation of Drought. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Post-Transcriptional Regulation of Drought . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MicroRNAs ..................................................................... B. Other Post-Transcriptional Mechanisms ................................... C. Post-Translational Modifications ............................................ V. Molecular Methods of Drought Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Model Organisms............................................................... B. Conventional Breeding ........................................................ C. Identification of QTLs and Marker-Assisted Breeding ................... D. ‘Omics’ Studies.................................................................. E. Transgenic Approaches and Functional Studies........................... F. Bioinformatics and Databases................................................ VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding author: E-mail:
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Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00013-8
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ABSTRACT Agriculture faces a constant challenge to increase crop production annually in response to human population growth. As land and water resources become limiting, high-yielding crops even in environmentally stressful conditions will be essential. Drought is the single largest abiotic stress factor leading to reduced crop yields, and as such, has been a target of research for some decades. Recently, however, the rapid advance of molecular biological, transgenic and functional genomics technologies has facilitated significant progress in identifying some aspects of the drought response in plants. This chapter summarizes the current state of knowledge of the molecular events that take place when a plant is under drought stress, starting with the mechanisms by which the plant perceives drought and the intracellular signalling pathways that are engaged in initiating the drought response. Next, the functional importance of various biomolecules that are synthesized or activated to protect the plant from cellular damage during drought are considered. The differing capacity of varieties of the same species to respond to drought stress is associated with differing gene expression patterns, so the mechanisms by which drought-responsive gene expression is regulated are discussed at the transcriptional and post-transcriptional levels. A large number of genes and gene products have been implicated in the drought response, but identifying which are most useful for breeding drought-resistant crop varieties remains a significant technical challenge. The second half of the chapter, therefore, surveys the molecular methods that are currently in use for drought research, and ways in which they can be applied to accelerate breeding for drought resistance. Particular focus is given to post-genomic techniques—transcriptomics, proteomics and metabolomics—assessing the relative strengths and weaknesses of each approach and how to make use of the large datasets they produce.
I. INTRODUCTION Increasing demands on land, water and petroleum mean that simply ploughing in more resources is not feasible—we need to produce more from the existing resources. This is not a new situation; 50 years ago, population growth threatened to overtake food production. At that point, it was discovered that semi-dwarf mutants of wheat produced much more grain than their taller relatives. Selective breeding for this and other important traits over the past half century has led to steady annual increases in grain production, the so-called Green Revolution. Unfortunately, this growth may no longer be sufficient to meet future demand (Tester and Langridge, 2010). Climate change, with the prospect of increasing environmental stresses, makes stabilizing yields as much of a challenge as increasing them. This is an important aspect especially for crops which are of great economic importance (Habash et al., 2009). Drought, arguably the biggest single abiotic stress factor impacting agricultural production (Ergen and Budak, 2009), is increasing globally, affects more than 10% of arable land and reduces average crop yield by more than 50%
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(Bray et al., 2000). Ironically, the semi-dwarfism trait that boosted grain yields in the past makes wheat more vulnerable to drought in many cases. Therefore, the next Green Revolution must develop plant varieties that produce high yields even under environmental stresses such as drought. In this chapter, we present an overview of recent gains in understanding of how plants acclimatize to drought, before outlining new molecular approaches that promise to facilitate rapid development of drought-tolerant varieties.
II. THE MOLECULAR BIOLOGY OF DROUGHT Plants have frequently evolved in habitats where drought occurs, and so have developed multiple strategies to cope with drought stress. Drought tolerance is defined as the ability of a plant to live, grow and reproduce satisfactorily with limited water supply. Tolerance strategies can be divided into resistance mechanisms, which enable plants to survive dehydration, and avoidance mechanisms, which are growth habits that prevent the exposure of plant to osmotic stress, such as deeper rooting or a shorter growth season. The capacity of a plant to tolerate drought depends largely on the droughtadaptation mechanisms present within its genome, and how efficiently it can activate them. Unfortunately, the domestication of modern crops has greatly reduced the genetic diversity of elite cultivars, and may even have promoted accumulation of deleterious mutations in their stress response mechanisms (Tang et al., 2010). Exposure to drought stress in plants leads to cellular dehydration, causing decreased cytosolic and vacuolar volumes and osmotic stress. Drought responses of plants includes attenuated growth, altered gene expression, changes in hormone levels, accumulation of osmoprotective solutes and proteins, increased levels of antioxidants and suppression of core metabolism. Drought tolerance is a quantitative trait, with a complex phenotype including all of these responses and involving a number of genes. A. GROWTH RESPONSES
Plant response in relation to growth varies according to the tissue, mode/ severity/time scale of the stress and species of concern. Mild osmotic stress can cause growth arrest in leaves and stems, but no inhibition in root growth. The growth arrest can be a mechanism for either energy conservation with reduced metabolism for better subsequent recovery or a support for osmotic adjustment (Osorio et al., 1998; Sharp et al., 1988; Westgate and Boyer, 1985). Several lines of evidence have supported the role of cyclin-dependent
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kinases (CDKs) and cyclin-dependent kinase inhibitors (ICKs) in the regulation of cell division under drought conditions (Schuppler et al., 1998). There is also evidence linking ICKs with abscisic acid (ABA)-dependent mechanism of drought (Kang et al., 2002; Wang et al., 1998). In a recent study, it has been shown that Arabidopsis thaliana MYB, discussed further in this chapter, limits cell expansion since its constitutive expression results in a dwarf phenotype and small cells (Cominelli et al., 2008). However, droughttriggered growth sustenance of roots can be an adaptive mechanism for water uptake. Expansin genes, involved in cell wall loosening, a parameter involved in cell expansion, were shown to alter their expression patterns in response to water deficit (Jones and McQueen-Mason, 2004; Wu et al., 2001). B. SIGNALLING PATHWAYS
1. Signal perception Plants perceive drought prior to initiating a signalling cascade for appropriate response. A number of plant osmosensor candidates were proposed. A receptor-like protein, NTC7, was suggested in a study in which its transcripts were induced in response to osmotic stress and its overexpression induced osmotic stress tolerance (Tamura et al., 2003). Additionally, Arabidopsis cytokinin receptor, Cre1 (cytokinin response 1), which has a similar structural organization to yeast osmosensor SLN1 was proposed as an osmosensor (Reiser et al., 2003). Further, Arabidopsis homologue of SLN1, a plasma membrane nonethylene receptor histidine kinase, ATHK1, was shown to complement yeast SLN1 mutant (Urao et al., 1999). Recently another study showed that AHK1/ATHK1 positively regulates ABA-related drought response while other nonethylene receptor kinases called cytokine receptors (CK) including AHK2, AHK3 and CRE1 are involved in drought-related negative regulation (Tran et al., 2007b). Further analysis of ATHK1 revealed that it is involved in drought response not only during early vegetative stages of growth but also during seed formation. This research showed that it is co-regulated with several Arabidopsis response regulators and its overexpression induced water deficit tolerance (Wohlbach et al., 2008). Recently, Oryza sativa receptor-like kinase (RLK), OsSIK1, was cloned, characterized in relation to kinase activity, and transgenic work has shown that it is involved in drought tolerance modulating stomata and activating antioxidative system (Ouyang et al., 2010). 2. Signal transduction Signalling pathways consist of signalling molecules and a network of protein interactions which are mediated by reversible phosphorylation in response to environmental factors including drought. Several components of the signal
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TR-4 h 1,4,5-trifosfat signal transduction mechanism
Secondary signal mechanisms based on the changes on Ca concentration
Increase in TF depending on ABA (bZIP, HD-ZIP)
TTD-4 h Increase in TF (EREBP) Ethylene synthesis 1,4,5-trifosfat signal transduction mechanism
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Fig. 1. A proposed model for the ABA and ethylene syntheses of wild emmer wheat, TRt (a tolerant genotype) and TTD (a sensitive genotype) under shock drought stress (4 and 8 h stress).
transduction have been identified although their interactions and positions along the pathway remain unknown. Differences in signalling between related genotypes can effect their drought response (Fig. 1).
3. MAPKinases Mitogen-activated protein kinase (MAPK) cascade includes three protein kinases (MAPK, MAPKK and MAPKKK) which are activated by serial phosphorylation, resulting in specific localization of the module in cell compartments, phosphorylating and regulating transcription factors and other proteins. In Arabidopsis, using sequence information, a number of MAPKinases were identified (Ichimura et al., 2002). Some MAPKinases were transcriptionally up-regulated and others were shown to be post-translationally activated by drought stress (Ichimura et al., 2000; Jonak et al., 1996; Mizoguchi et al., 1996). Additionally, ADR1, a CC–NBS–LRR gene which is a homologue of serine/threonine protein kinases was shown to confer dehydration tolerance consistent with dehydration responsive gene expression (Chini et al., 2004). Recently, a rice drought-hypersensitive mutant (dsm1) of a putative MAP kinase kinase kinase (MAPKKK) was identified. DSM1 protein was shown to belong to Raf-like MAPKKK, localize in the nucleus, induced in response to water deficit/ABA and confer seedling drought resistance. It was also proposed as an early signalling component, a regulator of scavenging of reactive oxygen species (ROS; Ning et al., 2010).
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Taking into consideration the above evidence that one MAPKinase can respond to different stress conditions and there are different numbers of proposed and identified MAPKinases from each of the three categories up to date, there should be a convergence in the signalling of MAPK cascade, possibly different stress factors activating MAPKinases to different levels (Bartels and Sunkar, 2005). 4. SNF1-like kinases SNF-1-like kinases, classified into three families, SnRK1, SnRK2 and SnRK3 are another family of protein kinases which are activated by the phosphorylation of their serine or threonines (Halford and Hardie, 1998). In various plant species, several SNF-1 like kinases were predicted and shown to be expressed in response to dehydration or ABA, including Arabidopsis OPEN STOMATA1 (OST1) protein kinase (Bartels and Sunkar, 2005). Arabidopsis OST1 protein kinase was shown to be involved as a positive regulator of ABA-induced stomatal closure and regulated negatively as a substrate of protein phosphatases 2C (PP2C) HAB1. There are lines of evidence on ABA-bound receptor inhibiting protein phosphatases resulting in activation of OST1 (Mustilli et al., 2002; Vlad et al., 2009). A recent study brings evidence for the involvement of distinct phosphorylation mechanisms in the activation of the two subgroups of SnRK2s. This can be related to their ABA responsiveness because members of SnRK2 are responsive to osmotic stress, but only some to ABA (Vlad et al., 2010). In line with these evidences, analysis of the phosphoproteome in response to ABA treatment leads to the identification of increases in the phosphorylation states of SNF1-related kinases after ABA treatment (Kline et al., 2010). 5. Phosphatases Phosphatases are classified based on their substrates into two major groups phosphoprotein (serine/threonine) phosphatases (PPases) including PP1, PP2A, PP2B and PP2C; tyrosine phosphatases (PTPases), receptor-like, intracellular or dual specific. Phosphatases aid in counteracting the action of kinases as noted above (Bartels and Sunkar, 2005). As noted above, there is intense research on the negative role of serine threonine PP2Cs including ABI1, ABI2 and HAB1 in ABA signalling. Studies with ABI1 and ABI2 mutants have shown in guard cells that ABA activation of Caþ-permeable channels requires intermediate steps of first ABI1 action, then ROS, finally ABI2 action (Murata et al., 2001). Two recent independent studies have revealed substrates of HAB1 as OST1 (as noted above) and PYL5 from the Bet v1-like superfamily, which was shown to be a cytosolic and nuclear ABA receptor that activates ABA signalling through direct
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inhibition of HAB1 and ABI enhancing drought tolerance (Santiago et al., 2009; Vlad et al., 2009). In another study, PTPases were shown to be involved in stomatal closure, downstream of Ca2þ signalling, most probably aiding in dephosphorylation of an unidentified protein, resulting in subsequent ion flux from guard cells and stomatal aperture (MacRobbie, 2002). Moreover, there are several independent studies on various pyrophosphatases. One of them is plastidial soluble pyrophosphatases (psPPs), which is involved in plastidial pyrophosphate (PPi) degradation for continuation of several metabolic pathways. This work involves transient down-regulation of psPPs in the leaves of tabacco (Nicotiana benthamiana) resulting in drought sensitivity, probably resulting from inability of plants in ABA synthesis (George et al., 2010). In another study, overexpression of vacuole membrane-bound proton-translocating inorganic pyrophosphatase (H(þ)PPase), AVP1, was shown to induce drought tolerance in Arabidopsis and tomato (Lycopersicon esculentum). In tomato, increased pyrophosphatedriven cation transport into root vacuoles was also observed (Park et al., 2005). Increased (H(þ)-PPase) activity is thought to acidify the vacuoles triggering secondary active transport of ions into vacuoles resulting in a decrease in vacuolar osmotic potential powering water uptake. 6. Phospholipid signalling One recognized class of osmotic stress-signalling secondary messengers are phospholipid-derived signalling molecules of the phosphoinositide pathway which are cleaved from membrane phospholipids by phospholipases. Several phospholipid-derived secondary messengers, especially inositol 1,4,5triphosphate (IP3), diacylglycerol (DAG) and phosphatidic acid (PA), were shown to be drought related. Phospholipases in the context of drought are phospholipase C (PLC) and phospholipase D (PLD). PLC cleaves phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), which is synthesized by a phosphatidylinositol-kinase, PIP5K, into IP3 and the membrane protein DAG. PIP5K, PIP, PLC and IP3 levels were shown to be induced in response to water deficit or ABA in several plant species (El-Maarouf et al., 2001; Hirayama et al., 1995; Kopka et al., 1998; Mikami et al., 1998; Pical et al., 1999; Takahashi et al., 2001). Two independent studies together support that phospholipid signalling involving this pathway is activated through both ABA-dependent and -independent mechanisms. The current hypothesis is that drought activation of PLC leads to higher IP3 levels, a subsequent release of Ca2þ from intracellular stores to cytoplasm, and activation of Kþ ion channels resulting in stomatal closure (Staxen et al., 1999; Takahashi et al., 2001). In addition, its initial synthesis, an additional regulatory mechanism for inositol phosphate levels, involves the action of
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5-phosphatases (5Ptases) or inositol polyphosphate 1-phosphatases. Arabidopsis SAL1 belongs to the latter group mentioned and recently droughttolerant Sal1 mutants along with omics studies have supported it as a negative regulator of both ABA-independent and also -dependent drought-response pathways (Wilson et al., 2009). In another recent study, an inositol phosphatelacking transgenic plant was generated by the expression of inositol polyphosphate 5-phosphatase (InsP 5-ptase; Perera et al., 2008). These plants exhibited higher drought tolerance and ABA-induced stomatal closure. Moreover, SAL1 was suggested as a regulator of an ABA-independent pathway since expression of dehydration-responsive element binding protein (DREB), which will be discussed below, is induced in transgenic plants (Wilson et al., 2009). Another important secondary messenger is PLD which cleaves phospholipases producing PA which contains a Ca2þ-binding domain and can activate PLC (Katagiri et al., 2001). PLDs were shown to be drought or ABA induced in several plant species (El-Maarouf et al., 2001; Frank et al., 2000; Jacob et al., 1999; Katagiri et al., 2001; Sang et al., 2001). Interaction of PLD with ABA effectors supports that PLD is involved in ABA-dependent pathway. There is supportive evidence of the role of PLD in stomatal closure such as its interaction with ABA effectors including ABI1 (Gampala et al., 2001; Jacob et al., 1999; Sang et al., 2001). One study has also shown the simultaneous accumulation of PLD and PA in water deficit (Katagiri et al., 2001). Non-specific phospholipase C (NPC4) hydrolyses phospholipids in a calcium-dependent manner, producing DAG. In another recent study, transgenic studies of this messenger reveal that it is converted to PA and involved in ABA sensitivity, drought tolerance and stomatal closure (Peters et al., 2010). 7. Secondary messengers and calcium Calcium functions (Ca2þ) as a secondary messenger since different extracellular stimuli eliciting specific and Ca2þ signatures results in a variety of intracellular response. Several Ca2þ sensors were implicated to be involved in drought signalling. Spatial and temporal dynamics of Ca2þ transients in response to drought were studied revealing increase in cytosolic Ca2þ due to release of Ca2þ from the vacuole and cell-type specificity of Ca2þ transients (Kiegle et al., 2000; Knight et al., 1997). One Ca2þ sensor is calcium-dependent protein kinases (CDPKs) which was reported to be drought induced and the importance of specific CDPK isoforms in mediating the effects of stress was demonstrated (Ozturk et al., 2002; Seki et al., 2002). Arabidopsis genome encodes 34 CDPKs and recently one of them, CPK10, was shown to confer drought tolerance via its interaction with a heat shock protein (HSP) aiding in ABA/Ca(2þ) inhibition of the inward K(þ) currents resulting in guard cell stomatal closure (Zou et al.,
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2010). Another Ca2þ sensor is calmodulin which is Ca2þ-binding protein activated by increased calcium concentrations and then modulates Ca2þ concentrations further by activating specific kinases. A family of calmodulinbinding transcription activators were first discovered in drought-stressed Brassica napus (Bouche et al., 2002). Further research revealed osmotic stress or ABA-activated calmodulins in Arabidopsis and rice (O. sativa) and suggested it as a negative regulator of osmotic stress (Frandsen et al., 1996; Perruc et al., 2004). Another Ca2þ-binding protein is calcineurin B-like protein (CBLs), and CBL1 was shown to be the only drought induced CBL among 10 identified in Arabidopsis CBLs (Kudla et al., 1999). Further transgenic studies supported the role of CBL1 in conferring drought tolerance (Albrecht et al., 2003). There is also lines of research on annexins which are a family of Ca2þdependent membrane-binding proteins. There is supportive evidence that certain annexins may be targets of abiotic stress-induced cytoplasmic Ca2þ. Their up-regulation in response to ABA or osmotic stress is well documented. In a recent transgenic study, annexin AnnAt1 was shown to reduce accumulation of H2O2, and confer increased drought tolerance. AnnAt1 was also shown to be highly susceptible to oxidation-driven S-glutathionylation, which decreases its Ca2þ affinity and occurs after ABA treatment. This mechanism probably aids in post-translational regulation of annexins in their modulation of ROS (Konopka-Postupolska et al., 2009). Another area of research comprises Arabidopsis AKT1 and Grapevine (Vitis vinifera) VvK1.1 which are homologous K(þ) channels and were shown to be regulated by CBL-interacting protein kinase (CIPK) and Ca(2þ)-sensing CBL partners. Kþ channel expression was shown to be sensitive to drought and ABA. Loss of function of Arabidopsis CIPK23 and overexpression of rice CIPK12 increased drought tolerance (Cue`llar et al., 2010). 8. Salicylic acid and nitric oxide The role of salicylic acid (SA) in drought signalling is based on the observation of reduced necrosis in the seedlings of SA-deficient transgenic lines in comparison to wild types in the presence of mannitol. The general view is that SA amplifies the effects of water deficit by enhancing the generation of ROS in photosynthesis (Borsani et al., 2001). Supportive data was established with studies on Arabidopsis lesion-mimic mutants which misregulate programmed cell death and used for studying hypersensitive response. A line with constitutive expression of pathogenesis-related (PR) genes 22 (cpr22) was shown to have elevated levels of SA which was further induced with water deficit. In the mutant, elevated SA levels were shown to change ABA levels and ABA-related gene expression. Mutant exhibited reduced responsiveness to ABA and suppressed responses to drought (Mosher et al., 2010). In a recent
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study, functional characterization of phenylalanine ammonia-lyase (PAL) was undertaken. PAL catalyses the first step of the phenylpropanoid pathway producing flavonoids, precursors of several secondary metabolites. The study revealed Arabidopsis pal1 pal2 mutants as drought tolerant and pal1, pal2, pal3, pal4 mutants as SA deficient, probably resulting from alterations in their signalling pathways (Huang et al., 2010). Nitric oxide (NO) is another signalling molecule which was suggested to have a role in drought tolerance although the nature of its role is unclear. Exogenous application of NO was shown to confer dehydration tolerance; conversely, mutants of NO biosynthetic enzymes exhibited reduced NO content and resistance to dehydration (Lozano-Juste and Leon, 2010; Mata and Lamattina, 2001). In the initial study, NO application triggered higher accumulation of late embryogenesis-abundant (LEA) transcripts, which will be discussed later in detail, and increased stomatal closure. In the latter study, ABA hypersensitivity of the mutants deficient in NO synthesis supported the role of NO in ABA-dependent stomatal closure. C. COMPATIBLE SOLUTES
Compatible solutes are nontoxic molecules that accumulate in the cytoplasm in response to drought stress and do not interfere with metabolism. Major compatible solutes are sugars (sucrose, hexose, raffinose-type oligosaccarides, trehalose); sugar alcohols including cyclic polyols (pinitol, D-ononitol); glycine betaine; and amino acids, most importantly proline. Current hypothesis on their mode of action ranges from conferring osmotic adjustment, scavenging ROS, stabilizing proteins and cell structures and adaptive value of metabolic pathways. The accumulation of several compatible solutes was observed to be drought induced and engineering the synthesis of compatible solutes has been relatively successful (Bartels and Sunkar, 2005). A recent striking evidence is several truncated/recombinant transcripts of betaine aldehyde dehydrogenase (BADH), the enzyme involved in glycine betaine synthesis, were observed in monocots. Surrounding the deletion/ insertion sites of these transcripts, sequence similarities, named short, direct repeats (SDR), were detected (Niu et al., 2007). These can possibly be recognition sites for post-transcriptional silencing. D. PROTECTIVE PROTEINS
1. Late embryogenesis-abundant proteins LEA proteins are a diverse group of proteins expressed normally during embryogenesis, or in vegetative tissues, in response to ABA or drought stress. Accumulation of LEA proteins correlates with ABA levels and desiccation
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tolerance (Dure et al., 1981; Ergen et al., 2009; Galau et al., 1986). Evidence from expression profiles and overexpression studies supports a role for LEA proteins as protective molecules in water deficiency. LEA proteins were grouped based on conserved structural features (Dure, 1993; Dure et al., 1989). Group 1 LEA proteins harbour high hydrophilicity and are thought to be soluble aiding in water binding or replacement. The group 2 (dehydrins) and group 4 LEA proteins may contribute to the maintenance of protein and membrane structures. Early response to dehydration proteins of the dehydrin family was recently shown to have disordered 3D structure enabling them to maintain in low water concentrations, probably enabling them to act as chaperones in high ionic strength (Kovacs et al., 2008; Mouillon et al., 2008). Additionally, a research on spatial and temporal accumulation patterns of group 4 LEA proteins was conducted. This was further followed by a bioinformatics analysis revealing origination of subgroups of group 4 LEA with gene duplication events. Generation of transgenic plants confirmed that the role of group 4 LEA proteins is indispensable in dehydration tolerance and recovery (Olvera-Carrillo et al., 2010). The group 3 and group 5 LEA proteins which harbour the most hydrophobicity are thought to sequester ions, which accumulate due to water deficit. A supporting evidence came from a recent study in which Arabidopsis LEA5 was shown to confer ABAdependent protection against oxidative stress by decreasing photosynthesis (Mowla et al., 2006). Moreover, lately Medicago truncatula seed desiccation tolerance (DT) was linked to 11 mostly seed-specific LEA proteins from different groups (Boudet et al., 2006). Homeostasis of indole-3-acetic acid (IAA), the main form of active auxin in plants, is maintained through the conjugation of free IAA to sugars, amino acids or methyl groups. GH3 family are responsible for converting active IAA to its inactive form via the conjugation of IAA with amino acids. Recently, OsGH3.13, which encodes IAA-amido synthetase, exclusively induced by drought stress, was cloned from a gain-of-function mutant tld1-D. It was shown that activation of TLD1/OsGH3.13 in tld1-D mutant rice results in down-regulation of IAA leading to the accumulation of LEA proteins (Zhang et al., 2009). 2. Aquaporins Changes in water flow is crucial to drought and the rate of water flux into or out of cells can be determined either by diffusion resulting from water potential gradient or by aquaporin proteins facilitating osmosis by forming water-specific pores which increase water permeability of the membrane shown by aquaporin antisense experiments (Kaldenhoff et al., 1998; Siefritz et al., 2002). Aquaporins are members of a large superfamily of membrane
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spanning proteins called the major intrinsic proteins (MIPs) including tonoplast intrinsic proteins (TIPs) and plasma membrane intrinsic proteins (PIPs; Weig et al., 1997). There are several reports that aquaporin genes are induced by dehydration enhancing water uptake (Fray et al., 1994; Guerrero et al., 1990; Sarda et al., 1999; Yamada et al., 1997; Yamaguchi-Shinozaki et al., 1992) and some showing aquaporins are reduced by dehydration which can allow water conservation (Johansson et al., 1998; Smart et al., 2001; Yamada et al., 1997). Likewise, most classes of Arabidopsis MIPs are up-regulated in response to drought, but only some are down-regulated. A recent expression profiling study of Arabidopsis PIPs revealed that this regulation is consistent through accessions excluding the special case for three PIP genes. The relation of PIP genes to drought was further supported by linking variation of drought-related PIP expression to leaf water content and demonstrating the presence of drought stress response elements in the promoters of two PIPs (Alexandersson et al., 2010). Additional supportive evidence on the interspecies conservation of the aquaporin gating mechanism came from a recent work on structural dynamic simulations of spinach aquaporin SoPIP2;1 (To¨rnroth-Horsefield et al., 2006). Recently, 10 PIPs from rice were cloned, classified into OsPIP1 and OsPIP1 based on amino acid sequence similarity and shown to be drought responsive having distinct roles in response to stress with transgenic studies (Guo et al., 2006). In V. vinifera roots, there are also two classes of PIPs with different functions: VvPIP2;2 functions as a water channel and VvPIP1;1 interacts with it to induce water permeability. In a recent work, it was shown that waterinduced diurnal changes in VvPIP1;1 expression follow the same trend with the amplitude of changes in hydraulic conductance-related variables. This supports that VvPIP1;1 may dynamically account for water transport capacity across roots to meet transpirational demand occurring on a diurnal basis in response to water stress (Vandeleur et al., 2009). Another study supported the role of PIPs in root hydraulic conductivity which was dropped in the roots of transgenic plants with low level of PIPs. In this study, antisense RNA was used to knock down ABA biosynthesis, resulting in transcript and protein level down-regulation of four PIPs in root tissue, suggesting ABA promotes root water uptake through aquaporins (Parent et al., 2009). 3. Ion channels Aside from water uptake and efflux, transporters that regulate ion flow are also implicated in the drought response, especially in the regulation of stomatal opening and closure. One player in this sense is proton (Hþ)ATPases in guard cells, which are known to drive hyperpolarization of the plasma membrane to initiate stomatal opening. It was previously observed
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that Hþ-ATPase activity is diminished by ABA. Recently, mutations in the OST2 locus which encode Arabidopsis H(þ)-ATPase AHA1 causing constitutive activity were reported (Merlot et al., 2007). Another protein implicated in this response is Arabidopsis glycine-rich protein 7 (GRP7), which is abundantly expressed in guard cells and involved in regulation of stomatal closure. When overexpressed, it conferred freezing tolerance on plants but retarded their germination and growth under drought or high salinity conditions (Kim et al., 2008). Anion efflux from guard cells precedes stomatal closure and involves slow, weak voltage-dependent, deactivating (S-type) and rapid (R-type) anion channels. Recently, SLAC1 gene has been shown to encode a slow, voltageindependent anion channel component. In a later study, AtALMT12, a member of the aluminium-activated malate transporter, was found to represent a guard cell voltage-dependent R-type anion channel. Plants lacking the transporter were shown to be impaired in stomatal closure in response to ABA (Meyer et al., 2010). Ca(2þ)-independent OST1 was identified as an interaction partner of SLAC1 leading to its activation via phosphorylation. In one study, OST1 was shown to interact with ABI1 which deactivates the SLAC1/OST1 complex via dephosphorylation (Geiger et al., 2009). Similarly, PP2CA was shown to inhibit the activity of SLAC1 by either direct interaction with SLAC1 itself or interaction with OSTI leading to inhibition of the kinase independently of phosphatase activity (Lee et al., 2009b). In another study, mutants lacking calcium-dependent protein kinases (CDPKs) were observed to be impaired in ABA stimulation of ion channels. So a Ca(2þ)-sensitive CDPK was found to interact with SLAC1 and activate it in an ABI1dependent manner. Overall, the CDPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex (Geiger et al., 2010). Recently, a K(þ) channel from the Shaker family, named VvK1.1, was identified from grape (V. vinifera). It was found to be the counterpart of the Arabidopsis AKT1 channel with similar functional properties such as Kþ uptake from soil, regulation by CIPK and Ca(2þ)-sensing CBL partners. Unlike AKT1, its expression was found to be sensitive to drought and ABA supporting its role in K(þ) loading upon drought stress (Cue`llar et al., 2010). 4. Heat Shock Proteins HSP are important for efficient cellular functions since they are chaperones that aid in folding and assembly of correctly structured proteins during synthesis, their maintenance by preventing aggregation by binding and stabilizing denatured proteins and in the removal and disposal of non-functional and
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degraded proteins (Lee et al., 1995). Low water content impairs protein structure and HSPs are usually only present in vegetative tissues under stress conditions. HSPs were shown to be dehydration induced in several plants (Alamillo et al., 1995; Campalans et al., 2001; Coca et al., 1996; Wehmeyer and Vierling, 2000). Their role in conferring tolerance was shown with transgenic studies and (Sun et al., 2001) chaperone binding protein from HSP70 protein family is involved in targeting and was shown to be water deficit induced and shown to confer tolerance to drought (Alvim et al., 2001; Cascardo et al., 2000). Since HSPs are dehydration-induced genes, there has been research on the transcriptional mechanisms of drought regulating its expression. One report revealed DREB 2A, discussed in chapter 7, activates HS-related gene expression (Sakuma et al., 2006). This was supported by a study in sunflower (Helianthus annuus) in which small stress protein (sHSP) is activated by the binding of DREB2 to sHSP promoter and interacting with its known regulator heat stress factor A9 (HaHSFA9; Diaz-Martin et al., 2005). Very recently, HSP was also identified as an interacting partner of CDPKs and involved in ABA and Ca(2þ)-mediated stomatal closure leading to drought tolerance (Zou et al., 2010). E. ANTIOXIDANTS
Drought stress leads to increased accumulation of ROS, generated mostly in chloroplast and to some extend in mitochondria, causing oxidative stress. Major ROS molecules are singlet oxygen, superoxide anion radicals, hydroxyl radicals and hydrogen peroxide (H2O2). To detoxify ROS, plants can intrinsically develop different types of antioxidants reducing oxidative damage and conferring drought tolerance. ROS scavengers are either nonenzymatic (ascorbate (vitamin C), glutathione, tocopherol (vitamin E), flavonoids, alkaloids, carotenoids) or enzymatic containing superoxide dismutase, peroxidases and catalase. Free radical-mediated lipid peroxidation results in complex, highly reactive and toxic aldehydes, which are scavenged by either aldehyde dehydrogenases or aldose/aldehyde reductases. There are lines of evidence revealing the involvement of these enzymes in drought response (Kirch et al., 2001; Mundree et al., 2000; Oberschall et al., 2000; Ozturk et al., 2002; Seki et al., 2002; Sunkar et al., 2003). The osmotic stress involvement of peroxiredoxins which detoxify toxic peroxides was also shown (Seki et al., 2001; Mowla et al., 2002). Peroxiredoxin is also a potential target of DREB1A (Kasuga et al., 1999). Thioredoxins function as hydrogen donors, and their role in water deficit was studied (Broin et al., 2000; Pruvot et al., 1996; Rey et al., 1998). Peptide-methionine sulphoxide reductases (MsrA) can
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counteract the damage caused by the modification of methionine-containing proteins to methionine sulphoxide [Met(O)] making them vulnerable to protease degradation and causing loss of function. Their involvement in drought stress was confirmed with expression and transgenic studies (Moskovitz et al., 1999; Rodrigo et al., 2002). Recently, it was demonstrated that changes in the auxin substrate of a H2O2-responsive enzyme involved in anthocyanin production conferred drought tolerance (Tognetti et al., 2010). In another study, mutants with reduced anthocyanin levels were more dehydration tolerant (Huang et al., 2010). These data support a role of anthocyanins, which are antioxidants, in drought tolerance related to its interplay with ROS and phytohormones. Further independent studies have raised new insights into the regulation of ROS homeostasis in conferring drought tolerance. The implicated molecules include glutathione peroxidase and cellulase-synthase-like protein (Zhu et al., 2010). Squalene epoxidase implicated in sterol biosynthesis was also shown to have a role the localization of NADPH oxidases required for regulation of ROS (Pose´ et al., 2009). Isoprene, a product of photosynthesis, which was also proposed to have antioxidant activity, was suggested to have a role in drought response (Fortunati et al., 2008).
F. OTHER RELATED MOLECULES
A number of other small molecules have putative roles in the drought response, although most of these await further confirmation. Recently, an Arabidopsis mutant was identified with lower content of dolichols, a type of polyisoprenoid, and increased drought tolerance (Zhang et al., 2008). Subtle differences were observed between tocopherol (vitamin E) mutant and controls in tolerance to drought stress, but the main focus of the study was on low-temperature stress (Maeda et al., 2006). Lipocalins are small ligandbinding proteins and recently an A. thaliana lipocalin AtCHL was functionally characterized. Its transcript and protein were found to be induced upon drought and ABA. With transgenic studies, it was shown that AtCHL aids in coping with stress conditions, especially damage upon photo-oxidative stress induced by drought (Levesque-Tremblay et al., 2009). Recently, an Arabidopsis mutant defective in fatty acid elongase condensing enzyme was isolated. This defective protein was shown to be involved in suberin biosynthesis which is a polyester found in cell walls. It was transcriptionally activated upon polyethylene glycol-induced drought (Franke et al., 2009). Also, two transgenic Arabidopsis plants were engineered with altered enzyme levels resulting in high/low glucosamine (GlcN) levels. Decreased
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GlcN resulted in enhanced sensitivity to drought stress, consistent with previous findings of GlcN-induced ROS generation (Chu et al., 2010). In transgenic Arabidopsis with higher levels of methyl jasmonate (MeJA) and wild-type plants with drought-induced MeJA levels, ABA was also increased. This suggests that MeJA induces ABA biosynthesis under drought conditions. With further expression analysis, genes that were regulated by both were identified, including genes involved in ABA or MeJA biosynthesis in rice (Kim et al., 2009a).
III. TRANSCRIPTIONAL REGULATION OF DROUGHT Many plants can gradually acclimatize to drought conditions, indicating that the genetic basis of tolerance is also present in non-tolerant plants to some extent, and changes in expression of drought-related genes leads to tolerance acquisition (Zhu, 2001). The expression of many drought-induced gene products is regulated at the transcriptional level. Two transcriptional regulation circuits induced by drought have been studied in detail, and are labelled the ABA-dependent and -independent pathways reviewed in detail previously in this book (in Chapters 6 and 7). However, transcriptional regulation in response to drought is complex; the two pathways can overlap since cis elements for both are known to reside in some genes. It is also highly possible that there are other cis elements yet to be discovered (Seki et al., 2002). Further, recent studies demonstrated that transcriptional regulation of abiotic stress converges with biotic stress regulation, as well as that several additional transcription factors are involved in drought transcriptional regulation. In particular, overexpression of a NAC family transcription factor, SNAC1, was shown to significantly improve drought resistance in rice (Hu et al., 2006). Another rice NAC, OsNAC6, similarly conferred resistance to drought as well as to rice blast infection (Nakashima et al., 2007) while OsNAC10 gave enlarged roots and improved yield during drought under field conditions (Jeong et al., 2010). Examination of NAC genes in Arabidopsis showed that ATAF1 could be induced both by ABA and by drought in ABA-deficient mutants, and its overexpression conferred greatly enhanced drought tolerance (WU et al., 2009). One gene that contains the NAC recognition sequence, ERD1, also contains a zinc-finger homeodomain (ZFHD) recognition sequence, and yeast-1-hybrid screening identified transcription factor ZFHD1, which was drought inducible, interacted with NAC proteins and gave a drought-tolerant phenotype on overexpression (Tran et al., 2007a).
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In maize, the B3 domain-containing transcription factor Viviparous1 (Vp1) was found to be inducible by drought stress and ABA, an effect mediated by an ABRE and coupling element-binding motifs in its promoter (Cao et al., 2007). Arabidopsis NFYA5 was also ABA-induced and conferred drought resistance, but in this case did not contain an ABRE; instead, the induction was achieved by down-regulation of miR169 (Li et al., 2008b). Another member of the NFY family, AtNFYB1, was found to confer improved performance under drought conditions, as did an orthologous maize transcription factor (Nelson et al., 2007). Additionally, during a screen for gain-of-function mutants leading to increased drought tolerance, an HDSTART transcription factor was identified (Yu et al., 2008). All of these factors are likely to act downstream of AREB or DREBmediated induction, but there are also proteins that function to modulate either or both pathways. Transcriptional co-activators are proteins which enhance the binding of transcription factors to the basal transcription machinery, and overexpression of one such, MBF1c, enhanced osmotic and heat stress tolerance (Suzuki et al., 2005). The plant-specific histone deacetylase HD2, whose function is not well understood, was found to be repressed by ABA and when overexpressed confer an ABA-insensitive phenotype (Sridha and Wu, 2006). Among nonethylene receptor histidine kinases, AHK1/ATHK1 has been shown to up-regulate AREB1, ANAC and DREB2A, while AHK2, AHK3 and CRE1 seem to be negative regulators of the same pathways (Tran et al., 2007a,b). Loss-of-function mutants for the disease-response regulator OCP3 were also found to gain improved drought resistance in an ABA-dependent manner (Ramirez et al., 2009). WRKY transcription factor ABO3 was isolated from a screen for mutations giving altered ABA sensitivity, and shown to modulate the transcription of AREB1/ ABF2 (Ren et al., 2010). Transgenic plants with disrupted IP3 signalling showed basal up-regulation of DREB2A and a subset of its targets, suggesting that IP3 is a negative regulator of this pathway; surprisingly, the transgenic guard cells also had altered responsiveness to ABA, suggesting that both pathways contribute to the regulation of stomatal closure (Perera et al., 2008). Again in guard cells, overexpression of a novel nuclear protein NPX1 was shown to decrease stomatal response to ABA, and it was proposed that this was due to transcriptional repression of an ABA-inducible NAC transcription factor, TIP1 (Kim et al., 2009b). Another important signal for stomatal closure is the H2O2 level. Recently, a zinc-finger transcription factor DST (drought and salt tolerance) was cloned and was shown to negatively regulate stomatal closure by modulating expression of genes related to H2O2 homeostasis (Huang et al., 2009). Conversely, ABA was shown to increase H2O2 production by signalling through
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AtMKK1 and AtMPK6 to enhance catalase expression (Xing et al., 2008). Overexpressing AtMKK1 conferred an increase in drought tolerance. In summary, the plant response to drought stress involves changes in the transcriptional levels of literally hundreds of genes, most of which are regulated by multiple transcription factors. Therefore it is perhaps unsurprising that for many different transcription factors, constitutive overexpression or loss of function result in a changed drought phenotype. Productive future research directions should include temporal studies to determine the sequence in which different transcriptional units are activated during drought, as well as dissecting the downstream pathways to determine which transcriptional programmes improve drought resistance without negatively impacting yield.
IV. POST-TRANSCRIPTIONAL REGULATION OF DROUGHT A. MICRORNAS
Plant genes involved in responses to stresses such as drought may also be regulated at the post-transcriptional level, and microRNAs (miRNAs) are small regulatory non-coding RNA molecules that have a major role in posttranscriptional regulation, usually by repressing target transcripts. The involvement of miRNAs in drought is supported by several lines of evidence: (1) Several elements related to miRNA metabolism have been shown to be involved in ABA signalling (Nishimura et al., 2005; Xiong et al., 2001). (2) In plants, miRNAs are involved in several cellular processes including response to environmental stress (Chen et al., 2005; Dugas and Bartel, 2004; JonesRhoades et al., 2006; Zhang et al., 2006). (3) Several studies show changes in the expression level of multiple miRNAs in response to abiotic or biotic stress (Sunkar and Zhu, 2004). Further, analysis of the Sorghum bicolor genome has indicated that recent duplication of miRNA genes may contribute to this species’ drought tolerance (Paterson et al., 2009). Recently, miRNA expression profiling in response to drought was performed for O. sativa, Populus trichocarpa, Arabidopsis and Triticum dicoccoides (Kantar et al., 2010a,b; Liu et al., 2008; Lu et al., 2008; Zhao et al., 2007). Arenas-Huertero and his team constructed a small RNA library of drought and ABA-treated Phaseolus vulgaris (Arenas-Huertero et al., 2009). Independent studies showed drought stress responsiveness of miRNAs in Triticum aestivum, Hordeum vulgare, Brachypodium distachyon and Euphorbiaceae (Jia et al., 2009; Kantar et al., 2010a,b; Wei et al., 2009; Yao
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et al., 2010; Zeng et al., 2010). Analysis of Arabidopsis transcriptome under drought stress with the whole-genome tiling techniques has also revealed several unannotated non-coding stress-induced RNA molecules including miRNAs, splicing and processing variants (Matsui et al., 2008; Zeller et al., 2009). The overall results indicate that miRNA expression profiles are conserved to some extent in different species, but further studies are required because the details of their response often differ (Sunkar, 2010). B. OTHER POST-TRANSCRIPTIONAL MECHANISMS
There are other possible mechanisms of post-transcriptional regulation, although none have yet been demonstrated to have a role in drought responses. In several cereal species, numerous transcripts with truncations and/or recombination around SDRs in the 50 exonic region of the two BADH genes were detected. These unusually processed transcripts were not found in dicotyledonous plants, so this may represent a cereal-specific regulation mechanism for biosynthesis of the important osmoprotectant glycine betaine (Niu et al., 2007). C. POST-TRANSLATIONAL MODIFICATIONS
Ubiquitination is a eukaryotic post-translational protein modification that is mediated by the cascade of E1, E2 and E3 ubiquitin (Ub) ligases, most often targeting ubiquitinated proteins for degradation by the 26S proteasome. It is involved in regulating numerous cellular functions and has been implicated in various aspects of the drought response. For example, an E3 Ub ligase from hot pepper, Rma1H1 and its Arabidopsis orthologue were both shown to confer strongly increased drought tolerance when overexpressed in Arabidopsis. This phenotype was mediated by targeting the aquaporin isoform PIP2:1 for proteasomal degradation, thus reducing water loss (Lee et al., 2009a). In contrast, E3 Ub ligases PUB22 and PUB23 had a negative effect on drought responses, apparently by interacting with the proteasome regulator RPN12a (Cho et al., 2008). Two further E3 Ub ligases, SDIR1 and AtAIRP1, have been shown to be ABA and drought inducible and act as positive regulators of the ABA signalling pathway, acting upstream of ABFs (Ryu et al., 2010; Zhang et al., 2007). SUMOylation enzymatically resembles ubiquitination, with the difference that the peptide added to the modified protein is SUMO rather than ubiquitin. In the Arabidopsis genome, SIZ1 is the only E3 SUMO ligase. During drought stress, plants accumulated higher levels of SUMOylated proteins, while null mutant siz1-3 plants were highly drought sensitive. Subsequent
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analysis of the entire transcriptome under drought stress in wild-type and siz1-3 mutants indicated identified 262 drought-inducible genes that required SIZ1 for their induction, including components of the ABA response, and some that were induced independently of ABA or DREB2A (Catala et al., 2007). Finally, C-terminal modification of proteins by isoprenylcysteine methyltransferase (ICMT) seems to inhibit ABA signalling, although which proteins are isoprenylated remains unclear. Overexpression of isoprenylcysteine methylesterase (ICME), which removes isoprenylation, increased ABA sensitivity, with ABA inducing ICME expression in a positive feedback mechanism (Huizinga et al., 2008).
V. MOLECULAR METHODS OF DROUGHT RESEARCH Drought-sensitive and -resistance model plants, conventional breeding, marker-assisted breeding, omics studies, transgenics and functional methods are all major research areas with the aim of developing drought-tolerant crops to increase crop productivity. In two recent independent reviews, conventional and molecular tools of drought research are discussed (Fleury et al., 2010; Reynolds and Tuberosa, 2008). A. MODEL ORGANISMS
Studies on water deficit have advanced by focusing on drought-tolerant plants, the most remarkable of which are ‘resurrection plants’ such as Craterostigma plantagineum (Bartels and Salamini, 2001). These can tolerate desiccation in environments with long arid periods, appearing dried out and dead but recovering rapidly during seasonal rainfall. Interestingly, a relative of C. plantagineum that is not found in arid environments, Lindernia brevidens, exhibits the same desiccation tolerance, showing that this adaptation does not severely impact growth in non-arid habitats (Phillips et al., 2008). The fern Mohria caffrorum has been proposed as a valuable model organism for verifying protection mechanisms because it cycles seasonally between desiccation-tolerant and -sensitive phenotypes (Farrant et al., 2009). Equally valuable is the wild germplasm of wild relatives of crop species which have adapted to a broad range of environments and contain rich genetic diversity (Nevo and Chen, 2010). For example, the progenitors of cultivated wheat and barley, T. dicoccoides and Hordeum spontaneum, have drought-response traits which have been identified and transferred to
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cultivated species leading to improved drought tolerance. To collect and preserve the genetic diversity of wild relatives, seed germplasm banks have been established as a source of genes for improving agricultural crops (Tanksley and McCouch, 1997). Other crops including soybean are also major lines of research (Manavalan et al., 2009). B. CONVENTIONAL BREEDING
Utilization of this wild germplasm requires first screening for a wild donor line with the desired trait (such as drought resistance) and then crossing this line with an elite cultivar with good agronomic characteristics. Traditionally, screening was carried out by observation of phenotypes (conventional breeding). By crossing two lines with different advantages and shortcomings, selecting for physiological traits and interspecies/intergeneric-wide crossing/ backcrossing techniques, conventional breeding has played an important role in the past century for improving drought stress tolerance (reviewed by Ashraf, 2010). Drought-tolerant lines of crops such as peanut, common bean, safflower, chickpea, wheat, tall fescue, soybean, wheatgrass, barley and maize have been developed using conventional breeding techniques. One challenge to applying this approach is identifying phenotypes that correlate well with drought tolerance. Recently through an Arabidopsis genetic screen, Xiong et al. (2006) proposed that drought/ABA inhibition of lateral root growth is a good indicator of drought tolerance. Meanwhile in Brassica rapa, early flowering was shown to be rapidly evolved adaptation to escape drought (Franks et al., 2007). C. IDENTIFICATION OF QTLS AND MARKER-ASSISTED BREEDING
Conventional breeding has major limitations, including the need for multiple backcrosses to eliminate undesirable traits, restriction to loci that give a clearly observable phenotype and inadequacy if the gene pool lacks sufficient variation in the trait of interest. Therefore, the focus is currently on markerassisted breeding, which allows ‘pyramiding’ of desirable traits for more rapid crop improvement with less input of resources. Marker-assisted breeding is of great value for drought-related studies due to the complex and additive polygenic nature of the trait (Mohammadi et al., 2005; Thi Lang and Chi Buu, 2008; Zhao, 2002). This approach is made possible by the development of DNA markers that can be used to construct detailed genetic linkage maps. A plethora of different types of DNA markers that have been described for stress tolerance such as RFLPs, RAPDs, CAPS, PCRindels, AFLPs, microsatellites (SSRs) and SNPs (reviewed by Ashraf
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et al., 2008). A fundamental application of linkage maps is to localize the sequences encoding drought-related, quantitatively inherited traits (quantitative trait loci, QTLs). QTL mapping and identification of drought-associated QTLs has been performed for several crops including maize, wheat, barley, cotton, sorghum and rice (Bernier et al., 2008; Quarrie et al., 1994; Sanchez et al., 2002; Saranga et al., 2001; Sari-Gorla et al., 1999; Teulat et al., 1997). However, the complexity of the intergenic and gene–environment interactions in drought makes robust identification of QTLs difficult, as shown by the contrasting results of similar QTL mapping studies in rice (Babu et al., 2003; Bernier et al., 2008; Kamoshita et al., 2008; Kumar et al., 2007). Of several crops, rice has been the major focus of marker-assisted breeding for drought tolerance, which led to the release of the first highly droughttolerant rice variety, Birsa Vikas Dhan 111 (PY 84; Bernier et al., 2009; Steele, 2009). Selection of QTLs to breed drought-tolerant crop cultivars has also been performed in pearl millet, cotton, maize, barley and sorghum (Baum et al., 2003; Bidinger et al., 2007; Harris et al., 2007; Levi et al., 2009a, b; Ribaut and Ragot, 2006; Serraj et al., 2005). QTL analysis is now being used alongside genomic approaches. For example, Street et al. (2006) combined phenotyping and QTL analysis of a poplar mapping population with transcript microarrays to identify drought-inducible genes that co-localized with QTLs known to be associated with drought resistance. D. ‘OMICS’ STUDIES
The complex nature of the drought response means that, to understand it, the plant must be viewed as a complete system, rather than just looking at individual components. This is made possible by the use of ‘omics’ techniques, that examine all or representative subset of a plant’s genes, transcripts, proteins or metabolites (Urano et al., 2010). The availability of genome sequence data for crop species is undoubtedly important for drought research, but as the drought response is largely mediated by changes in protein expression, the current focus is on other ‘omic’ methods such as transcriptomics, proteomics and metabolomics to elucidate plant responses to drought. In general, the most technically straightforward and by far the most widely used approaches are those which survey RNA transcripts. 1. Transcriptomics Transcriptomics techniques include microarrays (e.g. AtGenExpress; Kilian et al., 2007) and next-generation sequencing-based profiling methods (e.g. RNA-Seq), which measure the abundance of thousands of transcripts in parallel. To date, over 100 publications have used microarrays to describe
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the transcriptomes of a total of 28 species exposed to osmotic stress (reviewed by Deyholos, 2010). With their high potential, microarrays have several limitations including working with a predefined probe set and a high rate of false positives necessitating confirmation of results with RT-PCR or other techniques. Some of the limitations such as sensitivity, resolution and narrow probe sets have been overcome with high-throughput sequencing. To date, a few such analyses of plants have been reported, in addition to previous reports of serial analysis of gene expression (SAGE) studies of several abiotic stresses (Barakat et al., 2009; Byun et al., 2009; Eveland et al., 2008; Molina et al., 2008; Moon et al., 2007). High-throughput sequencing is also the most powerful method available for detecting novel small, non-coding RNAs such as miRNAs (reviewed by Unver et al., 2009) and natural antisense transcript RNAs (Zhou et al., 2009). A major weakness of transcriptomics techniques is that steady-state transcript levels often do not accurately represent gene or protein expression because of post-transcriptional regulation (Branco-Price et al., 2008; Kawaguchi et al., 2004). One approach to offset this is by the selection of polysome (i.e. multiple ribosomes) associated transcripts during RNA extraction, by immunopurification of epitope-tagged ribosomes (Arava et al., 2003; Ederth et al., 2009; Mustroph et al., 2009; Zanetti et al., 2005). In transcriptomics studies, the exact stress treatment applied can have a large effect, as shown by comparative analyses of different drought treatments (Bray, 2004; Talame et al., 2007). Moreover, the importance of consistency in tissue sampling was demonstrated by a microarray analysis of the effects of water stress in four serial, transverse segments cut from the maize root apex (Spollen et al., 2008). Methods of selecting individual cells or tissues for transcript analysis, such as fluorescence-activated cell sorting (FACS) and microdissection, are also being developed (Deyholos, 2010). Comparative transcriptome analysis of related species, genotypes or ideally near isogenic lines differing in their drought tolerance is a powerful strategy and has been reported in Arabidopsis, rice, wheat, sugarcane and Andean potato (Aprile et al., 2009; Ergen et al., 2009; Mane et al., 2008; Mohammadi et al., 2007, 2008; Rabello et al., 2008; Rodrigues et al., 2009; Wong et al., 2006). For example, von Korff et al. (2009) undertook profiling of allele-specific expression variations in different barley hybrids, revealing that expression levels of the same stress-related genes frequently varied between hybrids because of variation between different alleles in their cis-regulatory sequences. In another transcriptomics study, Ergen et al. generated a subtractive cDNA library from drought applied and control wheat tissues sequencing 13,000 expressed sequence taqs (ESTs; Ergen and Budak, 2009).
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As we have noted above, drought stress responses primarily involve transcriptional regulation of gene expression. This can depend on the interaction of transcription factors with cis-regulatory sequences, or post-transcriptional regulatory mechanisms such as miRNA function. One use of microarrays is to identify cis-regulatory elements adjacent to transcripts co-regulated by drought (Ma and Bohnert, 2008). Transcriptomic approaches are also useful in describing the phenotype associated with a mutation in a transcription factor (Fowler and Thomashow, 2002; Hu et al., 2006; Jiang and Deyholos, 2009; Jiang et al., 2009; Li et al., 2008a,b; Maruyama et al., 2004; Sakuma et al., 2006; Yokotani et al., 2009), alongside validation techniques such as chemically inducible transcription factors and chromatin immunoprecipitation (Waters et al., 2009). Further, transcript profiling can confirm whether other endogenous or exogenous signalling molecules affect the drought response by detecting changes in expression of known drought-induced genes (de Torres-Zabala et al., 2007; Kim et al., 2009a; Perera et al., 2008; Suzuki et al., 2005; Tran et al., 2007a,b; Wilson et al., 2009). The relative ease with which a microarray, once set up, can be probed with multiple samples allows time-course analysis of the drought response, which provides greater resolution by identifying time-specific expression patterns as well as core genes up-regulated throughout the test period (Harb et al., 2010; Wilkins et al., 2009, 2010). In theory, it should be straightforward to characterize novel, proteincoding, stress-related genes with transcriptomic approaches. An overview of drought-induced transcriptomics results indicates a decrease in the abundance of transcripts related to primary energy metabolism, photosynthesis and protein synthesis, and an increase in stress-signalling, transport protein, hydrophilic, osmoprotective and antioxidative-related transcripts (Bray, 2002; Catala et al., 2007; Gong et al., 2005; Jia et al., 2006; Jiang and Deyholos, 2006; Sahi et al., 2006). Transcriptome analysis of a triple knockout mutant for AREB/ABFs identified drought-regulated genes that had not been detected in single or double knockouts (Yoshida et al., 2010). However, despite its theoretical utility, no pathway or confirmed major component of the stress response was first identified through transcriptomics (Munns and Tester, 2008; Takeda and Matsuoka, 2008; Yamaguchi-Shinozaki and Shinozaki, 2006). This could be due to the limitations of transcriptomics highlighted above, especially the lack of correlation between transcript levels and function. So transcriptomics is an indispensable tool especially when studying gene networks, but it should be used as only an initial screening tool of validated with subsequent functional assays. Transcriptome data should be integrated with other types of linkage or expression data to identify
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candidate genes. For example, drought stress-induced transcript expression profiles have been used to identify candidate genes associated with QTLs (Diab et al., 2008; Golkari et al., 2009; Gorantla et al., 2005; Street et al., 2006). 2. Proteomics and metabolomics Protein and metabolite profiling, typically carried out using separation techniques such as 2D gel electrophoresis or chromatography followed by analysis by mass spectrometry (MS), allow direct assessment of changing levels of proteins or metabolites. Protein abundance is not perfectly correlated with functional activity because of post-translational modifications, localization and association with other molecules, but is generally more representative of function than transcript levels. For example, a protein phosphatase profiling strategy was important in establishing the role of SNF1-related kinases in the ABA signalling pathway, by showing that one such kinase, OST1, is a target for the PP2C HAB1 (Vlad et al., 2009). Further, use of 14N/15N labelling and MS to profile the phosphorylation state of Arabidopsis proteins following ABA treatment not only identified the proteins known to be involved in the ABA signalling pathway but also 20 proteins not previously known to be regulated by ABA (Kline et al., 2010). Meanwhile, metabolite profiling and the measurement of 30 different enzyme activities found that Arabidopsis drought adaptation did not involve major changes to carbon metabolism, with Kþ and organic acids being the main contributors to osmotic adjustment (Hummel et al., 2010). In M. truncatula seed radicles, proteomic analysis highlighted 15 polypeptides linked to DT (Boudet et al., 2006). A further proteomic analysis of Medicago root nodules was able to identify subsets of both plant and bacterial proteins involved in the drought response (Larrainzar et al., 2007). Assessment of mild drought stress effect on key metabolites showed rapid accumulation of respiratory substrated succinate and sucrose as well as antioxidant enzymes (Naya et al., 2007). This was taken to indicate a loss of bacterial respiration caused by oxidative damage, leading to the loss in nitrogen fixation in root nodules under even mild drought stress. Recently, proteomic analysis has also been carried out in bread wheat (Peng et al., 2009) identifying 93 root and 65 leaf proteins that show differential expression under stress, and demonstrating the substantial overlap between responses to drought and salt stress. Despite their limitations, microarrays tend to identify an order of magnitude more gene products than reported in proteomic studies (Baginsky, 2009). Therefore, integration of transcriptomic data with proteomic and metabolomic studies can be an effective way of confirming the inferences derived from
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both methods. For example, metabolomic profiling of an Arabidopsis NCED3 knockout mutant indicated that during drought, accumulation of osmoprotective amino acids was dependent on ABA synthesis, whereas that of raffinose was not. Additional transcriptomics studies revealed that biosynthesis of branched-chain amino acids during drought was controlled by ABA at the transcriptional level (Urano et al., 2009). A combination of transcriptomic and metabolomic approaches were used to investigate the ability of A. thaliana leaves to grow and develop at a similar rate to controls in spite of mild osmotic stress (Skirycz et al., 2010). Under these conditions, ethylene and gibberellin but not ABA-based regulatory circuits were implicated, and changes in mitochondrial function were shown to be important. In particular, overexpression of a mitochondrial alternative oxidase led to increased drought resistance. Accordingly, metabolite and transcript profiling in the absence of alternative oxidase 1a (Giraud et al., 2008) showed that these plants suffered acute sensitivity to drought stress and widespread perturbations in the expression of stress response genes. However, datasets produced by ‘omic’ methods are not always easy to interpret. In a comprehensive study of Populus euphratica drought acclimation combining transcript, protein and metabolite profiling under arid field conditions, no correlation was found between levels of drought-responsive proteins and their corresponding transcripts (Bogeat-Triboulot et al., 2007). E. TRANSGENIC APPROACHES AND FUNCTIONAL STUDIES
Transgenic methodology is an alternative to ongoing breeding programmes, and has the advantage of transferring only the desired genes from one species to another. Potentially, this allows only the drought tolerance genes from stress adapted wild relatives to be incorporated into cultivated lines, without adjacent loci that might reduce yield. Additionally, transgenic plants provide the clearest way to study the function of a candidate drought-resistance gene, allowing modification of a single gene on an otherwise identical genetic background. Functional relevance of the candidate gene can be demonstrated by confirming that loss-of-function and overexpressing mutants have opposite phenotypes. These functional studies are most frequently carried out in A. thaliana, due to the availability of a large collection of sequence-tagged insertional knockout mutants (e.g. Salk Lines), its rapid generation time and ease of genetic transformation (Alonso et al., 2003). However, there are certainly functional differences in the drought response between Arabidopsis and some crop species, particularly monocots; for this reason, efforts are underway to develop other transgenic systems, such as the model grass B. distachyon (Vogel et al., 2006). Of course, even validation in a
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model system does not guarantee that a transgene will perform identically in a crop plant. For example, transgenic tomato plants overexpressing NCED3 to increase ABA biosynthesis surprisingly did not appear to be more resistant to soil drying than controls, but rather showed better water-use efficiency under well-watered conditions (Thompson et al., 2007). In other cases, the performance of a gene in Arabidopsis does correlate well with its action in a crop plant, such as transgenic expression of the AP2-type transcription factor HARDY in rice (Karaba et al., 2007). Many of the studies described in the above text used transgenic techniques to elucidate the function of all kinds of drought-related genes, and are detailed under the relevant sections. Many other functional approaches have also proved valuable in dissecting drought tolerance: .
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X-ray crystallography was important in demonstrating the function of the ABA receptor PYL1 (Miyazono et al., 2009; Nishimura et al., 2009) and the conformational changes undergone by spinach aquaporin (To¨rnroth-Horsefield et al., 2006). Protein–protein interactions have been investigated using yeast two-hybrid, in vitro pull-down, in vivo co-immunoprecipitation experiments (Cho et al., 2008) and isothermal titration calorimetry (Santiago et al., 2009). Use of reporter gene constructs to detect changes in expression (Li et al., 2008b; Zhang et al., 2007). Transactivation assays using yeast assay and yeast one hybrid systems (Guan et al., 2009; Liao et al., 2008). Colocalization with GFP-tagged proteins (Wormit et al., 2006). Physiological assays such as stomatal bioassay (Perera et al., 2008) and patch clamp (Meyer et al., 2010). Protein-promoter element binding by Northern blot (Ren et al., 2010) and electrophoretic mobility shift assays (Cao et al., 2007). Quantitative PCR analysis (Kim et al., 2009a). Fourier transform infrared spectroscopy to detect changes in LEA proteins (Boudet et al., 2006).
F. BIOINFORMATICS AND DATABASES
Some of the online resources and/or tools that have proved useful or applicable in drought-related research are The Generation Challenge Programme (GCP, a comparative plant stress-responsive gene catalogue), AthaMap, MultiGO and DRASTIC-INSIGHTS. Their applications include elucidating orthologous/paralogous relationships; identifying co-regulated genes,
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deducing functional gene sets from clustered expression data; and constructing signal transduction pathways (Button et al., 2006; Galuschka et al., 2007; Kankainen et al., 2006; Wanchana et al., 2008). Online databases contain valuable material for drought analysis. For O. sativa, Lee and colleagues have assembled available transcriptomic data into a database called RiceArrayNet, and using it demonstrated a correlation between expression patterns of different abiotic stress-inducible genes (Lee et al., 2009c). Tools are now being developed to aid in mining these databases; for instance, Geisler et al. (2006) developed an algorithm to verify putative cis-regulatory elements by correlating their occurrence in genomic DNA sequences with results of transcriptome profiling studies, and confirmed that it worked for the ABRE and DRE/C-repeat in Arabidopsis. Similarly with bioinformatic tools, Arabidopsis genes that are differentially regulated by drought were shown to contain unusually high levels of Gquadruplexes, which are tandem stretches of guanines that can associate in hydrogen-bonded arrays stabilized by K(þ) ions (Mullen et al., 2010). The complexity and multivariate nature of drought stress also makes computer simulation of drought responses an attractive approach, and there is now enough data to construct useful models (reviewed by Tardieu and Tuberosa, 2010). These frequently focus on one aspect of the drought response. For example, a dynamic model of guard cell signal transduction network for ABA-induced stomatal closure, including 40 previously identified network components, has been assessed. This can be used as a tool for the identification of candidate manipulations for conferring drought tolerance (Li et al., 2006). More recently, photosynthesis in the C3 plants, which can thrive in optimal environments, was modelled using method called Minimization of Metabolic Adjustment Dynamic Flux Balance Analysis (M_DFBA). Then its performance was assessed under drought conditions showing highly cooperative regulation resulting in robust photosynthesis even under stress (Luo et al., 2009).
VI. CONCLUSION Drought research has been underway for many years, but the past decade has seen an explosion of the molecular tools and techniques available to tackle this and other intractable biological questions. The best prospect for facing future demands on agriculture will be to combine the best of traditional breeding techniques with the latest innovations and complementary research in model organisms.
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AUTHOR INDEX
A Aalen, R.B., 327 Abad, M., 431 Abas, L., 357, 363 Abdalla, K.A., 335–336 Abdelly, C., 109, 175, 177, 183–185 Abdin, M.Z., 415 Abdul Jaleel, C., 300 Abebe, T., 118, 412 Abe, H., 68, 225, 299, 392, 458 Ablett, S., 322 Abo-Ogiala, A., 23 ´ braha´m, E., 114, 121–123 A Abrams, G.D., 205 Abrams, S.R., 205–206, 209, 252, 427 Abreu, E.F., 112, 118 Abreu, I.A., 464, 468 Acar, O., 296 Achard, P., 276 Ache, P., 72–73, 222, 231–232, 268, 457 Acosta, M., 7 Adam-Blondon, A.F., 453 Adam, E., 78 Adam, L., 77, 424 Adams, K.L., 229 Adams, M.W.W., 301 Adams, P., 158, 160, 163, 181 Adams, T.R., 423, 461 Addicott, F.T., 202 Adler, L.N., 327 Aebersold, R., 330 Aeschbacher, R.A., 111, 116–117 Afif, D., 299 Agarie, S., 81, 159–160, 163, 165, 181, 184 Agarwal, M., 76, 271, 296, 303 Agarwal, P., 421 Agius, F., 296, 303 Agnes, C., 273 Agorio, A., 461 Aharon, G.S., 18, 51, 174, 419 Aharoni, A., 422, 471 Aharon, R., 60, 352, 426 Ahmadpour, D., 298 Ahmad, R., 413 Ahmed, I.A., 425 Ahn, J.H., 371, 388 Ahn, S.J., 60 Ahuja, I., 106–107 Aı¨nouche, A., 109, 113, 126, 128 Ainsworth, E.A., 65, 80 Akhtar, J., 3, 11
Akhtar, N., 3, 11 Akiyama, K., 68, 77, 115, 229, 299, 352, 452, 458, 460 Akpo, H., 35, 40 Akram, N.A., 431 Alamillo, J., 458 Alatalo, E.R., 299 Albacete, A., 7–8, 20 Alberston, T.M., 379–380, 384, 386, 391 Albert, R., 163, 472 Albrecht, V., 223, 453 Alcamo, J., 294 Alcon, C., 265, 456 Aldaya, M.M., 50 Alegre, L., 326 Aleman, F., 167 Alexandersson, E., 456 Alexieva, V., 114 Alfred, S.E., 68, 72, 211, 213–214, 218, 229–230, 256–260 Alia, A., 114–116, 122 Allard, F., 115 Allard, G., 78 Allard, H.A., 387 Allen, C.D., 35 Allen, G.J., 57, 73, 221, 268, 276, 301, 304–305, 427 Allen, R.D., 305, 416 Allen, T., 385 Almeida, A.D., 203 Almeida, R.S., 458 Almeraya, R., 421 Almoguera, C., 328, 458 Almogura, C., 458 Al-Niemi, T., 425 Aloni, R., 253 Alonso, J.M., 221–222, 263, 294, 363, 366, 393, 470 Alonso, R., 115, 124 Alpert, P., 108, 320 Alquezar, D., 327, 329, 337 Al-Rasheid, K.A.S., 72–73, 222, 231–232, 268, 457, 471 Al-Sady, B., 393 Alsina, M.M., 53 Altman, A., 23, 53–54, 299, 410, 470 Altmann, T., 108 Alvarez, M.J., 391 Alvarez, R., 177 Alvarez, S., 42, 428 Alves, G., 60
496
AUTHOR INDEX
Alvim, F.C., 458 Alvino, A., 57, 65 Amasino, R.M., 227, 379, 385–386, 428 Ambavaram, M.M., 468 Ammerer, G., 273 Ampatzidou, H., 271 Amrhein, N., 259 Amtmann, A., 55, 110, 164, 166–169, 171, 418 Amuti, K.S., 328 Anacleto, M., 472 Anderberg, R.J., 220 Anders, N., 360, 363 Anderson, K.M., 324, 326, 334 Anderson, T.A., 462 Anderson, W.P., 164, 170, 172 Andersson, C.R., 78 Andralojc, P.J., 64–65, 80 Andreas, G., 115, 129 Andreev, I.M., 172 Andreoli, S., 221–222 Andrews, J., 427, 471 Andrews, M., 429 Andrews, T.J., 67 Andriankaja, A., 55 Andriankaja, M., 470 Andronis, C., 384, 386 Angelopoulos, K., 64 Angus, J.F., 2, 12 An, G.Y., 70, 472 An, L.-Z., 305 Anne, E., 107–108 Ansari, S.A., 59 Anstrom, D.C., 423, 431, 461 Antonioli, B., 277 Antoni, R., 214, 230, 259, 263, 269, 451, 471 Aono, M., 70, 266 Aoyama, T., 357 Apel, K., 324 Apostolova, N., 203, 217 Appel, L., 328 Aprile, A., 467 Apse, M.P., 18, 51, 174, 405, 408, 419 Aragao, F. J., 112, 118 Arakawa, H., 121 Araki, E., 417 Araki, T., 388 Arau´jo, S.A.M., 65 Araujo, S.D., 55 Araus, J.L., 408, 431 Arava, Y., 467 Arbona, V., 127–128, 130 Arcy-Lameta, A., 451 Arenas-Huertero, C., 462 Arendall, B., 412 Argamasilla, R., 127–128, 130 Argueso, C.T., 427 Argyrokastritis, A., 116 Arias, C., 116, 182
Ariel, F.D., 79 Arima, K., 363 Armstrong, F., 264 Arnott, H.J., 322 Aroca, R., 60 Aronova, E.E., 185 Aronso, J., 220 Arrese-Igor, C., 469 Arrillaga, I., 420 Arroyo, A., 203, 209, 253, 274 Artsaenko, O., 62, 253 Arvai, A.S., 214, 259–260, 471 Asada, K., 296 Asami, T., 203, 205–206, 209, 217–218, 227, 253, 274, 359, 462 Asano, A., 72, 214, 259–260, 471 Asayama, M., 70, 266 Asch, F., 11 Ashraf, M., 39, 107, 409, 465–466 Aslam, Z., 6 Asli, S., 44 Aspinal, D., 114 Assaad, F.F., 258–259, 280 Assareh, M.H., 178 Assmann, S.M., 57, 66, 68–69, 73, 209–211, 213, 215, 220, 228, 254, 262–263, 266, 452, 472 Atamov, V., 294 Atanassov, A., 114 Atare´s, A., 420 Athar, H.R., 39, 465–466 Atherton, N.M., 328 Atkins, C.A., 11 Atlin, G.N., 466 Auld, D., 419–420 Ausubel, F.M., 261 Auton, M., 113 Ayala, F., 164, 173–175, 177–178 Ayaydin, F., 114, 121–123 AydoJdu, M., 294 Azaizeh, H., 38 Azevedo, L., 69 Azmi, A., 35, 40 B Babbar, S., 415 Babiychuk, E., 226 Babu, C.R., 466 Babu, P.R., 299, 469 Babu, R.C., 466 Baburina, O.K., 176 Bacic, A., 109 Backes, G., 466 Back, K.-H., 413 Back, S., 431 Baczek-Kwinta, R., 184 Badawi, G.H., 305 Badeck, F.W., 3
AUTHOR INDEX Badger, M.R., 25, 55–56 Bae, G., 79 Baek, K.H., 424, 460 Bae, M.S., 335 Baena-Gonzalez, E., 107–108 Baginsky, S., 469 Bahieldin, A., 425 Bahk, J., 303 Baho, M.N., 11, 18 Bahrami, A.R., 76 Baier, K., 467 Bai, J., 53 Bailey-Serres, J., 300, 352, 447, 458, 467 Bailly, A., 366 Bailly, C., 324 Bai, S., 389, 395 Bajji, M., 127, 129, 178 Baker, B., 335–336 Baker, E.H., 329 Baker, N.R., 50, 67, 324 Baker, S.S., 420 Bak, G., 304 Bakker, E.P., 166 Bakulina, E.A., 185 Balderas, E., 167, 169, 174, 182 Balling, A., 181 Ball, J.T., 66 Ball, L.D., 472 Ball, M.C., 63, 177 Balnokin, Y.V., 171–172, 176–177 Balog, J., 273 Ban, A., 225 Bandyopadhyay, A., 365 Bang, J.-W., 417 Banks, S.W., 301 Banno, H., 423 Bansal, K., 415 Ban, T., 469 Banuelos, M.A., 166 Banu, N.A., 115 Ba´nyai, E., 110, 127, 129 Bao, Y.-M., 411 Barajas, P., 294 Barakat, A., 467 Barak, S., 184, 227, 384, 386, 392, 396 Barak, V., 466 Barbero, G.F., 225–226 Barbier-Brygoo, H., 220, 230 Barbier, G.G., 109 Barbour, M.M., 57, 59 Bardor, M., 322 Bargmann, B.O., 357 Barigah, T.S., 60 Barker, P.J., 449 Barker, T.C., 408 Barkla, B.J., 163–164, 167–168, 170, 173–176, 181–182, 186 Barlow, E.W.R., 8 Barnabas, B., 407
Barnavon, L., 297 Baron, M., 53, 58, 464 Barrero, J.M., 203, 253 Barrett-Lennard, E.G., 157 Barrett-Wilt, G.A., 450, 469 Barrieu, F., 60 Barr, J.E., 305 Barron, C., 275 Barroso, M.L., 359, 361 Barta, C., 459 Bartel, B., 273, 462 Bartel, D.P., 462 Bartels, D., 4, 51, 107, 110, 295, 298, 320, 323, 327–329, 333, 337, 407, 450, 452, 454, 458–459, 464 Bartholomew, D.M., 78 Bartlett, S.G., 59 Bartley, G.E., 78 Bartsch, S., 325–326, 333 Basile, G., 278 Baskakov, I. V., 113 Bassaganya-Riera, J., 277–278, 281 Bassani, M., 42 Basset, G., 125, 132 Bassuk, N.L., 352, 354 Bastow, R.M., 379, 384–386, 389 Batelli, G., 213, 296, 303 Bates, B.C., 294 Bathula, S., 70, 266 Batistic, O., 223, 358, 453, 466 Bauder, J.W., 156–157 Baudouin, E., 273 Baudry, A., 396 Bauer, H., 72, 231, 268, 457 Bauer, R., 451 Baumann, K., 159–160, 163, 165, 181 Baum, M., 115, 129, 466–467 Bavestrello, G., 277 Bayliss, P., 429 Beaith, M., 430 Beaudoin, N., 217, 262 Becana, M., 469 Becher, M., 108 Beck, E., 66 Becker, A., 175 Becker, D., 73, 179–180 Beckett, A., 352 Beckett, R.P., 109 Becraft, P.W., 461 Becuwe-Linka, N., 59 Becuwe, N., 458 Beddington, J.R., 50, 81, 407 Beeckman, T., 363 Beemster, G.T.S., 357, 363–364 Beerling, D.J., 57 Begre, L., 468 Behnam, B., 421 Beis, A., 8
497
498
AUTHOR INDEX
Belin, C., 218, 221, 229–230, 262–263, 450–451, 469 Bellaire, B.A., 301 Belle´s, J.M., 117, 203 Bell-Pedersen, D., 379 Belton, P.S., 322 Ben Amor, N., 183–184 Bendov, R., 60, 352, 426 Be´ne´detti, H., 298 Benedito, V., 55 Benes, S.E., 156–157, 165 Benfey, P.N., 42, 275 Bengough, A.G., 25 Ben Hamed, K., 183–184 Ben Hassine, A., 119, 164–166 Benjamins, R., 209, 357, 363 Benkova´, E., 357, 363 Benlloch, M., 163–164 Bennet, M.J., 357, 363 Bennett, A., 456 Bennett, M.H., 363, 468 Bennett, M.J., 362, 364 Bennetzen, J.L., 299 Benning, G., 73, 260, 268, 301, 304–305 Benning, N., 416 Benscher, D., 469 Bensen, R.J., 423, 431, 461 Berger, B., 24 Bergmann, D.C., 75, 77 Bergmann-Honsberger, A., 366 Berjak, P., 320–322, 324, 328–329 Berkowitz, G.A., 420, 451 Berlett, B.S., 459 Bernal-Lugo, I., 328 Bernard, C., 458 Bernasconi, P., 365 Bernhardt, C., 125 Bernier, J., 466 Bernstein, N., 274, 364 Berry, J.A., 62, 66 Berthomieu, C., 184 Berthomieu, P., 166 Bertrand, C., 273 Be´thencourt, L., 116–117, 414 Bethke, G., 224, 273 Betts, S.A., 11, 18 Betz, C., 55, 70 Bevilacqua, P.C., 472 Bevington, K.B., 11 Beyschlag, W., 61–62, 64 Bezerra, I.C., 227 Bhalerao, R.P., 357, 362–364 Bhalu, B., 114, 122 Bhatnagar-Mathur, P., 411, 421 Bhuiyan, M.N.H., 39 Bhushan, D., 335 Bianchi, G., 328 Bidard, J.N., 277 Biderre-Petit, C., 461
Bidinger, F.R., 466 Biel, C., 54 Biesiada, H., 109 Bijl, F., 172 Bingman, C.A., 214 Bird, D., 279 Biricolti, S., 14 Birnbaum, K., 42 Biro, R.L., 354 Birtic, S., 324, 326, 334 Bittner, F., 205 Bjorkman, O., 55 Black, C.R., 427, 471 Blackman, S.A., 328–329 Black, R.F., 158 Bladenopoulos, K., 271 Blakeslee, J.J., 365–366 Blamey, F.P.C., 22, 24 Blancaflor, E.B., 362 Blanco-Melo, D., 462 Blatt, M.R., 179, 264–265 Blazevic, D., 453 Bla´zquez, M.A., 111, 371 Blomstedt, C.K., 320, 323, 327, 329–330 Bloom, R.E., 70, 268, 296, 304, 306 Blum, A., 113, 352, 354, 410 Blumwald, E., 18, 36, 51, 109–110, 152, 155–157, 162, 165, 174–175, 177–178, 405, 407–408, 419–420, 428, 468 Boccalandro, H.E., 366 Bochicchio, A., 109, 328 Bock, R., 223, 453 Bodde, S., 70, 268, 296, 304, 306 Bodrato, N., 277 Boerjan, W., 459 Bogeat-Triboulot, M.B., 54, 299, 470 Bogenschutz, N.L., 75, 218 Bogorad, L., 67 Bogoslavsky, L., 41 Bo¨gre, L., 273 Bo¨hlenius, H., 388 Bohlman, M. C., 109 Bo¨hmer, M., 53, 68, 73, 82, 208–209, 212, 264 Bohnert, H.J., 55, 107, 110, 118–119, 126–127, 130, 153, 157–158, 160, 163, 166–167, 169, 174, 181, 253, 272, 295, 299, 383, 409, 412, 452, 455, 458, 467–468 Bohnert, J.H., 167, 169, 174, 182 Bohrer, A.-S., 264 Boisson-Dernier, A., 53, 70, 82, 217 Boivin, K., 55, 267, 457 Bolarin, M.C., 420 Bolen, D.W., 113 Boller, T., 111, 116–117, 258 Boman, J., 298 Bonales-Alatorre, E., 185
AUTHOR INDEX Bones, A.M., 106–107, 115 Bonetta, D., 68, 72, 211, 213–214, 218, 229–230, 256–260, 429 Bongi, G., 58 Bonierbale, M., 110, 127, 130 Boonsirichai, K., 362 Boopathi, N.M., 466 Borchert, C., 299 Bordallo, P., 423 Bordas, M., 420 Borevitz, J.O., 386 Borisy, G.G., 356 Bor, M., 293, 296 Bornberg-Bauer, E., 358, 466 Borner, T., 78 Borrell, A., 466 Borrie`s, C., 466 Borsani, O., 453, 459 Bortner, C.D., 185 Boschetti, C., 329 Bossche, R.V., 225–226 Boss, W.F., 358, 362, 452, 461, 468, 471 Bota, J., 51, 53, 57–59, 64 Botella, M.A., 453, 459 Botia, P., 54 Botran, L., 203 Bots, M., 35, 40 Botto, J.F., 383 Bouchabke, O., 109 Bouche, N., 453 Bouchereau, A., 106, 108–110, 113, 119, 126–128, 130 Boucherez, J., 69 Bouchez, D., 69, 453 Boudet, J., 455, 469, 471 Boudsocq, M., 220, 230 Bourbousse, C., 230 Boursiac, Y., 36 Bouteau, F., 264 Bouteille´, M., 8, 352, 469 Boutin, J., 203 Boutry, M., 42, 267 Bouvier-Durand, M., 216, 260 Bouvier, F., 203 Bouzid, S., 164–166 Bo, W., 463 Bowers, J.E., 462 Bowley, S.R., 416–417 Bowlus, R.D., 107, 110, 113 Boxall, S.F., 383 Boyer, J.S., 19, 106, 113, 407–408, 447 Brackenbury, W.J., 166 Brader, G., 55, 70, 109, 472 Bradford, K.J., 329 Bradshaw, H.D., 55 Brady, S.M., 275 Bragato, C., 178 Brambilla, V., 228 Bramley, H., 60, 426
499
Bramley, P.M., 337 Branco-Price, C., 467 Brandsto¨tter, M., 273 Brandt, A., 298 Brandt, W.F., 109, 116, 320, 322–329, 331, 334, 337 Braud, J., 301 Brault, M., 230, 450 Bra¨utigam, A., 327 ˜ ¤utigam, K., 468 BrA Bray, E.A., 271, 329, 352, 407, 409, 447, 458, 467–468 Brazille, S., 299 Brearley, C., 73, 256 Bre´da, N., 65 Brenner, C., 327 Bresler, A.P., 326–327 Bressan, R.A., 55, 107, 110, 119, 130, 167, 170, 173, 272, 295, 299–301, 303 Breton, G., 221–222 Brewer, P.B., 209 Briand, J., 264 Briantais, J.M., 64 Briens, M., 128 Briesen, I., 459 Briggs, W.R., 365 Brilli, F., 459 Brinker, M., 23 Brito, R.M., 421 Brix, H., 178 Brklacich, M., 106 Broadhurst, D., 109 Brock, A.K., 224, 273 Brodmann, A., 116–117 Bro, E., 62 Broin, M., 458 Bromham, L., 9, 153–155 Bronson, P., 323 Brosche´, M., 23, 54, 109, 231, 266, 299, 470 Brossier, F., 277 Brown, A.J., 157 Brown, C.S., 358 Brownfield, D. L., 121 Brown, H., 57 Browning, L.S., 156–157 Brown, J.J., 155–157, 162, 165, 174, 177–178 Brown, L., 50–51 Brownlee, C., 57, 300, 422 Brown, P.O., 156, 467 Brown, R.H., 59 Brown, S.J., 418 Browse, J., 108 Bruce, W.B., 408 Bruggemann, W., 173 Bruggmann, R., 462 Brugnoli, E., 57 Brunel, N., 60 Brunner, A.M., 388 Bruskiewich, R.M., 472
500
AUTHOR INDEX
Brusslan, J.A., 215 Bruzzone, S., 277–278, 291 Bryant, J.A., 329 Buchala, A., 273 Buchanan, B.B., 72, 223, 231, 268, 457 Buchanan, C.D., 299 Bu¨chel, C., 325 Buch-Pedersen, M.J., 267 Buckley, T.N., 61, 64 Budak, H., 445–446, 455, 462, 467 Buell, C.R., 299 Bueno, P., 301 Buer, C.S., 364 Bu, H.H., 184 Buhler, J., 330 Buitink, J., 320–321, 324, 328–329, 353, 455, 469, 471 Bulard, C., 277 Bu¨low, L., 472 Bumgarner, R., 330 Bunce, J.A., 65 Bundy, J. G., 108 Bu, Q., 226 Burgie, E.S., 214 Burg, M. B., 113 Burla, B., 69, 255, 279 Burnet, M., 111, 115, 126–128 Busch, H., 179, 264 Busch, W., 228 Butowt, R., 325, 330, 333 Button, D.K., 472 Buzeli, R.A.A., 458 Bych, K., 223, 267 Byrd, G.T., 59 Byrt, C., 17 Byu, M.O., 117 Byun, M.-O., 299, 423 Byun, Y.J., 467 C Caboche, M., 305 Caddell, D.F., 70, 72, 214, 230 Caetano, T., 411 Cagnac, O., 304 Cai, X.L., 17 Cai, Y.F., 53 Cakmak, I., 186 Caldas, D.G.G., 467 Calderon, C., 414–415 Calderon-Villalobos, L., 386 Calestani, C., 466 Califano, A., 391 Callis, J., 226, 229 Calvi, D., 448 Camacho-Emiterio, J., 163–164, 170, 173, 175, 181–182 Camara, B., 203 Cambrolle, J., 177
Cameron, K.D., 456 Cameron Schiller, K., 116 Campalans, A., 458 Campbell, M.M., 69, 74, 468 Campos, F., 455 Campos, M.E., 359, 361 Campos, P.S., 65 Cannesan, M.A., 322 Cantero, A., 453 Canvin, D.T., 11 Cao, G.H., 327 Cao, J., 455 Cao, S., 115–116 Cao, W.H., 365 Cao, X., 461, 471 Cao, Z.Y., 55, 68, 158, 169, 177–178, 213, 215–216, 218, 260–261, 280 Capper, R.G., 78, 468 Caprioli, M., 320 Caragea, D., 53 Cardinale, F., 273 Cardon, Z.G., 62 Carle, C., 393 Carlson, J.E., 467 Carmi, A., 355 Carmody, J., 301 Carneiro, R.T., 467 Carninic, P., 458 Carolino, S.M.B., 458 Caro, M., 420 Carpaneto, A., 277 Carpenter, J.F., 328 Carpin, S., 298 Carpita, N.C., 299, 303 Carptenter, J.F., 328 Carra, A., 8 Carre´, I.A., 379–380, 385–388, 391 Carroll, A.J., 306, 452, 468, 470 Carter, A.B., 277 Carter, C., 162–164, 169 Carvalho, I., 52, 64, 294 Carvalho, P.C., 70, 72, 214, 230 Carvalho, S.M.P., 51 Casagran, O., 281 Casal, J.J., 366 Cascardo, J.C.M., 458 Caspar, T., 362 Cassab, G.I., 359, 361 Casse, F., 456 Cassone, V.M., 379 Casson, S.A., 76 Castagna, A., 183–184 Castanedo, I., 459 Castellanos, E.M., 163–164, 177–178 Castelli, S., 14, 52 Castiglioni, P.P., 423, 431, 461 Castillo, J.M., 153 Castonguay, Y., 125 Catala, R., 464, 468
AUTHOR INDEX Cattivelli, L., 3, 467 Cavalieri, A. J., 119 Ceccarelli, S., 465–467 Celebi-Toprak, F., 421 Cellier, F., 166, 456 Centritto, M., 459 Cerda´n, P.D., 391 Cerrano, C., 277 Cescatti, A., 58 C ¸ etin, E., 294 Ceulemans, R., 55 Chaerle, L., 68 Chaignepain, S., 230 Chai, J., 295, 298 Chakrabortee, S., 329 Chakraborty, N., 335 Chakraborty, S., 335 Chalifa-Caspi, V., 392, 396 Chalifoux, M., 276, 430–431 Chalker-Scott, L., 365 Chalmandrier, R., 11, 18 Chalmers, K.J., 11, 18 Chamarerk, V., 466 Chamberlin, B., 115, 124, 127–128 Chan, A., 363 Chan, C.W., 222 Chang, C.A., 214, 363 Chang, F., 109 Chang, H.S., 109, 294, 299, 352, 392, 396 Chang, I.F., 467 Chang, Q., 422 Chang, S.C., 362, 364 Chang, W.-K., 364 Chang, X., 39 Chan, M.T., 422 Chan, R.L., 79 Chan, W.-Y., 231, 266 Chao, D.Y., 17, 70, 74, 430, 461 Chapman, D., 117 Charbonnel-Campaa, L., 388 Charng, Y.Y., 422 Charpenteau, M., 453 Charrier, A., 466 Chattopadhyay, A., 335 Chaumont, F., 456 Chavarria-Krauser, A., 63–64 Chaves, M.M., 2, 7, 49–54, 56–57, 64–67, 77, 80, 107, 294, 407, 409, 447 Chazen, O., 38 Chee, P.W., 466 Chefdor, F., 216, 260, 298 Chen, B., 425 Chen, F., 209, 299, 471–472 Chen, G., 464 Cheng, H., 276 Cheng, J., 215, 278 Cheng, W.H., 203, 209, 253, 274 Chen, H.C., 203, 209, 226, 253, 274, 294, 463, 471
501
Chen, J.-G., 215, 219, 268, 278, 419–421, 456 Chen, J.-Q., 419–421 Chen, L., 223 Chen, M., 123, 129, 419 Chen, N., 295, 298 Chen, Q.-J., 419, 462 Chen, R.J., 362, 365 Chen, S.H., 174 Chen, S.X., 42 Chen, S.Y., 365, 416, 448, 471 Chen, S.-Y., 115–116, 267, 305 Chen, T.H., 110, 114–116, 305 Chen, T.H.H., 412 Chen, W., 109 Chen, X., 223, 327, 427, 429, 461, 463, 468 Chen, Z.G., 123, 126, 129, 169, 454, 459, 461, 471 Chen, Z.H., 155, 169 Chen, Z.L., 456 Chen, Z.Z., 70, 77 Cheong, J.J., 74 Cheong, Y.H., 223, 294 Chevalier, L.M., 322 Chevone, B., 299 Chezhian, P., 466 Chiang, V.L., 462 Chiatante, D., 271 Chi Buu, B., 465 Chico, J.M., 225–226 Chien, C.T., 116 Chien, S., 299 Chikayama, E., 52 Chinchilla, D., 258 Chini, A., 449 Chini, E.N., 277 Chinnusamy, V., 72, 76, 214, 230, 263, 269, 271, 294–295, 303 Chiu, W.-L., 423 Chiwocha, S., 459 Chmara, W., 158, 160, 163, 181 Cho, D., 70, 224, 273, 304, 306 Cho, E.J., 335 Cho, H.-T., 304 Choi, E.Y., 335 Choi, G., 79, 303 Choi, H., 222, 225, 448 Cho, I.J., 42 Choi, J.H., 222 Choi, S.M., 212, 253 Choi, W.-B., 117, 414–415 Choi, Y.B., 69, 255, 304 Choi, Y.D., 74, 117, 414–415, 428, 460, 468, 471 Cho, M.H., 256, 364 Chory, J., 70, 72, 78, 214–215, 229–230, 250, 261, 280, 366, 384–386, 388–389, 429 Cho, S.K., 352, 463, 471 Choudhary, M.K., 335
502
AUTHOR INDEX
Choudhary, N.L., 119, 126, 129 Choumane, W., 467 Cho, W.-S., 423 Chow, T.F., 68, 72, 211, 213–214, 218, 229–230, 256–260 Chrispeels, M.J., 60, 299, 456 Christensen, K.C., 327 Christensen, S.K., 388 Christie, J.M., 365 Christmann, A., 38, 68, 72, 207–209, 211, 213–214, 218, 229, 252–253, 256–260, 274, 280, 305–306 Christmas, R., 330 Christophe, A., 8, 352, 469 Chuang, W.-I., 304 Chua, N.H., 225–226, 228, 396, 464, 468 Chueng, F., 55 Chu, L.Y., 55 Chung, G.C., 60 Chung, J.S., 301 Chung, W.-I., 413 Chung, Y.Y., 428, 460, 468, 471 Chun, S.C., 39 Churin, Y., 78 Chu, S.H., 460 Chu, S.P., 221 Cidlowski, J.A., 185 Cifre, J., 80 Ciftci-Yilmaz, S., 38, 295–296, 304–306 Cimato, A., 52 Claeys, H., 470 Clarke, S.G., 327 Clark, G., 453 Clark, M.E., 107, 110, 113 Clark, S.E., 384 Cleland, R.E., 38, 42 Cle´ment, C., 116–117, 414 Clerkx, E.J., 228 Clifton, R., 306, 470 Cline, K., 206–207, 253, 274 Clipson, N.J.W., 162, 171–172, 177–178 Cloos, K., 63–64 Close, T.J., 329 Cnop, M., 281 Coates, J.C., 17, 419 Cobb, G.P., 462 Coca, M., 458 Cochard, H., 60 Coego, A., 461 Cohen, G., 304 Cole, E.S., 367 Collett, H., 320, 324–331, 333 Collins, M.J., 8 Collins, N.C., 11, 18 Collins, R., 50–51 Colmer, T.D., 11, 14, 16, 26, 153–155, 157, 162–163, 165, 169, 173–174, 177, 181 Coluccio, M.P., 383
Colville, L., 183 Colvis, C., 335 Cominelli, E., 69, 74, 448 Comstock, J.P., 35 Condon, A.G., 3, 12–14, 19, 23, 25, 53, 408 Conejero, G., 166 Connolly, E.L., 115, 306 Conn, S., 155 Conoly, E. L., 115 Conrad, U., 62, 253 Conroy, J.P., 67 Conte, M., 472 Conti, L., 69, 74 Cook, D., 109, 392 Cooney, S., 429 Cooper, K., 322–323, 325, 331, 337, 464 Copley, M.J., 78 Corbesier, L., 388 Cordonnier-Pratt, M.M., 299 Corkidi, G., 359, 361 Cornic, G., 51, 57–61, 63–64 Correll, M.J., 365 Cortes, D., 337 Cortina, C., 414 Cosgrove, D.J., 36, 42–43, 448 Costa, A., 59 Costa, J.M., 49–50, 56 Costa, J.M.R.C., 63 Costa, L.M., 461 Costa, M., 55, 267, 457 Costantini, L., 56 Coupland, G., 379, 384, 386–391, 395 Coutts, K.B., 185 Coutts, M.P., 355 Coutu, A., 306 Coutu, J., 306 Covarrubias, A.A., 455, 462 Covington, M.F., 384–386, 389, 392, 396 Cowan, I.R., 66 Cowan, L.R., 66 Cowan, R., 66 Cowley, K., 157 Crabbe, M.J., 472 Crafts-Brandner, S. J., 115 Craig, A.D., 157 Cramer, G.R., 7, 9 Cramer, G. R., 109 Crane, P.R., 154 Cranlucas, M., 301 Creelman, R.A., 68, 77, 213, 276, 303, 423–424, 461 Cremer, F., 387–388 Cress, W.A., 65, 107, 110, 113–114, 122, 125, 427 Crick, F.H.C., 34 Cronk, Q., 55 Crosby, W.L., 448 Cross, J. M., 109 Crouch, M., 455
AUTHOR INDEX Crowe, J.H., 110, 117, 328 Crowell, D.N., 429, 464 Crowe, L.M., 110, 117, 328 Crute, I.R., 407 Cse´plo, A., 114, 121–123 Csintalan, Zs, 325 Csisza´r, J., 114, 121–123 Csonka, L.N., 120, 124 Cubero, B., 18 Cue`llar, T., 453, 457 Cuevas, J., 216, 394 Cuine, S., 458 Cuin, T.A., 11, 18, 53, 114, 153, 155, 164, 166, 169, 176, 185, 223, 267 Cui, W.E., 456 Cui, X., 303, 459, 461, 471 Culia´n˜ez-Macia`, F.A., 117, 327, 414 Cullmann, A.D., 23 Cumbes, Q.J., 114 Cunningham, G.L., 178 Curtis, M.D., 69 Cushman, J.C., 78, 159–160, 163, 165, 181, 184, 306, 383, 412 Cutler, A.J., 205, 427, 448 Cutler, S.R., 70, 72, 203, 208–209, 213–214, 230, 252–253, 259–260, 263, 269, 427, 429, 451, 471 Cuypers, A., 455 Czempinski, K., 70, 73 D Dabauza, M., 420 Dace, H., 325, 330 Dae-Jin, Y., 110, 130 Dagenais, N., 388 Dahkeel, A.J., 11 Dai, J., 335 Dai, M., 424, 460, 468 Dallaire, P., 472 Dalmadi, A., 227 Dalmay, T., 295 Dal Santo, S., 109 Damm, B., 117 Damonte, G., 277–278, 291 Damour, G., 65 Damsz, B., 55, 158, 169, 177–178 Dana, W., 108–109 D’Angelo, C., 223, 358, 453, 466 Dangl, J.L., 70, 250, 268, 296, 304, 306 Danielson, J.A., 456 Danyluk, J., 299 Das, A.B., 110, 113, 178 Dasgupta, S., 467 da Silva, A.B., 55 da Silva, J.M., 55 Dassa, E., 279 Datta, A., 335 Dauzat, M., 8, 352, 469
503
Davanture, M., 55, 73, 231, 268, 305 Davenport, R.J., 10–11, 17–18, 153, 159, 161, 164, 166–167, 171, 186 Davey, M.P., 108 David, K., 379, 386, 396 David, M.M., 67 Davidson, A., 9, 35, 39 Davies, B., 2, 50 Davies, J.M., 169, 185 Davies, W.J., 50, 55, 68, 207–209, 263, 304, 363–364, 427 Davis, A.M., 385 Davis, J., 55 Davis, S.J., 379, 385–386, 394 Davletova, S., 52, 306 Davy, A.J., 153, 163–164, 177–178 Davydov, O., 392, 396 Day, C.L., 262, 279 Deak, K.I., 275 Deak, M., 458 Dean, C., 378 De Bellis, L., 467 Debez, A., 173, 175, 177, 184 De Block, M., 35, 40 De Bodt, S., 470 De Boer, A.H., 172 Debski, J., 453 de Carbonnel, M., 366 de Carvalho, M., 467 Decat, J., 276 de Cires, A., 153 De Clercq, I., 459, 470 Decourteix, M., 60 Deepa, J., 17 Deepak, S., 125 De Flora, A., 277–278, 291 de Franco, P., 230 Degl’lnnocenti, E., 52 de Graaf, P. T., 117 De Grauwe, L., 276 de Kerchove dÆExaerde, A., 267 de Laia, M.L., 467 De la Rosa, C., 462 Delauney, A. J., 110–111, 120, 122, 124 Delauney, A.J., 411 Delbarre, A., 360, 362–363 De Leonardis, A.M., 467 Delfine, S., 57, 65 Delgado-Enciso, I., 185 DellaPenna, D., 459 Dellaporta, S.L., 69, 74 Dellinger, M., 303, 459 Delmotte, F., 298 DeLong, A., 219 Delong, A., 363 del Rio, L.A., 185 Deltour, R., 328 De Meester, S., 267 Demidchik, V., 166, 169, 185
504
AUTHOR INDEX
Demiral, T., 114, 293, 296, 301, 303 Demmel, S., 258–259, 280 Denby, K.J., 320, 324–331, 333 Deng, W.T., 206–207, 253, 274 Deng, X.W., 78, 267, 323, 327, 330–331, 333–334, 336, 358, 386, 389, 459 Deng, Z., 460 Denne, F., 64 Dennis, D.T., 276, 430–431 Dennis, E.S., 17 DeNoma, J., 116 Denter, D., 42 Depierreux, C., 298 Dequin, S., 297 DeRidder, B. P., 115 De Ronde, J. A., 123 de Ronde, J.A., 411 De Rycke, R., 470 Desikan, R., 231, 266, 301 De Silva, D.L.R., 179 DeSimone, N.A., 329 De Simone, S.N., 366 de Souza, C.R., 65 Deswarte, C., 456 de Torres-Zabala, M., 468 Deuschle, K., 111, 121 Deutch, C.E., 125 Devi, M., 421 Devlin, P.F., 385 de Vos, Ric C. H., 106–107 Deyholos, M., 299, 452, 458 Deyholos, M.K., 121, 467–468 Deyholosn, M.K., 468 Dey, N., 461 Dezar, C.A., 79 d’Harlingue, A., 203 Dhonukshe, P., 209 Diab, A.A., 469 Diaz, C., 77, 299, 467 Diaz-Espejo, A., 57–58 Diaz, F., 157, 165 Diaz-Martin, J., 458 Diber, A., 426 Dichio, B., 54, 64 Diego, C., 106, 108–110 Dietrich, K., 115, 124 Dietrich, M.A., 175–176, 223 Dietrich, P., 179–180 Dietz, K.J., 170, 182, 212, 253 Di Fonzo, N., 466 Dillon, M.O., 111, 126–128 DiLoreto, D.S., 467 DiMichele, M., 271 Dingkuhn, M., 11 Ding, X.-S., 426 Ding, Z., 385 Dinkins, R., 462 Dinneny, J.R., 275 Dionisio-Sese, M.L., 66
Dispa, L., 229 Ditzer, A., 329 Dixit, S., 422, 471 Dixon, R.A., 108 Di, Y., 298 Dizon, M.B., 70, 217–218 Djaoui, M., 55, 73, 231, 268, 305 Djilianov, D., 114, 299 Dobrev, P., 428 Dobrovinskaya, O.R., 185 Do Choi, Y., 424, 460 Doczi, R., 273 Dodd, I.C., 7–8, 69, 74, 208 Doi, M., 266 Dolan, L., 459 Dolferus, R., 3, 12–14, 25, 53 Do¨ll, P., 294 Domagalska, M.A., 394 Domingo, R., 54 Dominguez-Ferreras, A., 116 Dominy, P.J., 164, 167–169 Domrachev, M., 294 Dong, C., 227 Dong, F.C., 268, 301, 305 Dong, H., 413 Dong-Ha, O., 110, 130 Dong, J., 75 Dong, P., 13, 25 Donnarummo, M.G., 423, 461 D’Ordine, R., 431 Do¨rffling, K., 11 Do¨rmann, P., 108 dos Santos, T.P., 65 Douglas, C.J., 55 Dove, S.K., 451 Dowd, P.E., 362 Downton, W.J.S., 62 Dowson-Day, M.J., 384–386, 389 Doyle, M.R., 379, 385–386 Dreger, M., 335 Dreher, K., 229 Drennan, P.M., 116 Dreyer, E., 65, 299 Dreyer, I., 69, 232, 265 Drincovich, M.F., 79 Driouich, A., 109, 116, 320–322, 326, 337, 353 Driscoll, S.D., 64 Driscoll, S.P., 455 Dro¨ge-Laser, W., 115, 124, 225 Droillard, M.J., 230, 450 Drouin, S., 299 Duan, X.G., 419 Duan, Y., 364 Dubchak, I., 462 Dubey, R.S., 122 Dubos, C., 69, 74 Dubouzet, J.G., 392 Dubrovsky, J.G., 359, 361
AUTHOR INDEX Duby, G., 42, 267 Ducruet, J.M., 53, 58 Dudits, D., 458 Dugas, D.V., 462 Duhaze´, C., 109–110, 113, 119, 126, 128 Dulai, S., 227 Dunand, C., 361 Dunkel, M., 180 Dunlap, J.C., 387 Dupasquier, M., 228 Dupeux, F., 214, 259, 451, 471 Dupont, F.M., 174 Dupuis, J.M., 456 Durand-Tardif, M., 109 Dure, L.I.I.I., 329, 455 Du, S.-Y., 68, 213, 215–216, 218, 222, 260–261, 280 Dvora´k, J., 17 Dyer, W.E., 425 E Eapen, D., 359, 361 Earl, H.J., 54 Earnest, D.J., 379 Ebercon, A., 352, 354 Eckardt, N.A., 55 Ecker, J.R., 220–222, 263, 363, 366, 386, 393 Eckstein, J., 61–62, 64 Ederth, J., 467 Edgar, R., 294 Edmeades, G.O., 408 Edsga, D., 327, 329, 337 Edwards, G.E., 112, 118 Edwards, K.S., 363 Efremova, N., 459 Egea, G., 208 Egertsdotter, U., 299 Ehleringer, J., 408 Ehlert, A., 115, 124 Ehlert, B., 223 Ehlting, J., 216 Eing, C., 155 Einset, J., 115 Eissa, H.F., 425 Eizirik, D.L., 281 Ekberg, K., 267 Elich, T.D., 366 Eliopoulos, E., 116 El-Itriby, H.A., 425 Ellenberg, J., 179 Ellis, B.E., 70, 215, 224, 273, 278 Ellis, M., 299 Ellul, P., 420 El-Maarouf, H., 451–452 Else, M.A., 252, 274 Elstner, E.F., 324 Eltayeb, A.E., 305 Elumalai, R.P., 384–386, 389
505
Elzenga, J.T.M., 170, 174, 186 Emami, M., 215 Emborg, T.J., 226 Emsermann, M., 459 Endler, A., 457, 471 Endo, A., 203, 207, 209, 253, 274 Endo, T.A., 55, 299, 463 Eng, J.K., 330 Engstro¨m, P., 327 Enju, A., 68, 77, 299, 352, 452, 458, 460 Epron, D., 65 Erban, A., 108 Ergen, N.Z., 446, 455, 467 Ergu¨l, A., 109 Eriksson, S.K., 455 Erismann, N.D., 57 Esaka, M., 39 Escalona, J.M., 64 Eshel, A., 179 Espinosa, J.M., 458 Essah, P.A., 17 Estavillo, G., 306, 470 Estavillo, G.M., 452, 468 Estelle, M., 213, 226, 250 Estrada-Navarrete, G., 462 Etterson, J. R., 106 Evans, J., 109, 323 Evans, J.R., 57, 59 Evans, M.L., 362, 364 Evans, P.M., 157 Eveland, A.L., 467 Evers, D., 110, 127, 130, 415 Evlagon, D., 38 Eymery, F., 183, 458 Ezura, H., 386, 388–391, 395 F Fabre, N., 59, 82 Fabro, G., 119, 123 Facchinelli, F., 464 Fahy, G.H., 323 Fairbairn, D.J., 298 Fallahi, H., 25, 55 Fan, B., 454, 459 Fang, Y., 424 Fan, H., 72, 214, 259–260 Fankhauser, C., 366, 384–386, 389 Fan, L., 42–43 Fan, Q., 388 Fan, R.-C., 68, 213, 215, 218, 222, 260–261 Farı´as-Rodrı´guez, R., 116 Faria, T., 52, 64, 294 Farmer, L.M., 226 Farquhar, G.D., 57, 61–63, 66–67, 75–76, 408, 427, 471 Farrant, J.M., 109, 116, 319–331, 333–334, 336–337, 353–354, 458, 464 Farrona, S., 388
506
AUTHOR INDEX
Fasano, J.M., 362 Fay, P., 53 Fayyaz, P., 23, 54, 470 Federica, C., 110, 130 Fedoroff, N.V., 224, 227 Feeney, K.A., 418 Feher, A., 407, 458 Feifer, I., 471 Fekih, R., 386, 388–391, 395 Felix, G., 258 Feller, U., 53 Feltus, F.A., 469 Feng, C.P., 55 Feng, J.C., 184 Fenske, R., 459 Fenzi, F., 55, 72, 220, 263, 267–268, 305, 450, 457 Ferjani, A., 115 Fernandez-Munoz, F., 163–164, 177–178 Fernandez, O., 116–117, 414 Fernie, A.R., 108–109, 451, 470 Ferrandino, A., 8 Ferrante, A., 60 Ferrare, K., 456 Ferrari, F., 467 Ferreira, C., 298 Ferreira, F.J., 427 Ferrer, A., 459 Ferrer, G., 281 Ferry, J.G., 59 Feury, D., 464 Fevereiro, M.P.S., 55 Fiehn, O., 108, 111, 117, 337 Field, K.J., 452, 468 Fieldler, U., 253 Figueras, M., 330 Figueroa, M.E., 153, 163–164, 177–178 Figueroa, P., 78 Fijolek, B., 453 Filipowicz, W., 228 Finkelstein, R.R., 203, 208–209, 213, 225, 252, 295, 328, 427 Fink, G.R., 362–363 Fink, J.L., 336 Firnhaber, C., 336 Fischer, E., 464 Fischer, K.L., 81 Fisher, D.B., 10 Fitter, D.W., 78 Fitzgerald, T. L., 115 Fleurat-Lessard, P., 60 Flexas, J., 7, 51, 53–54, 57–61, 64–65, 80, 218, 409 Flexus, J., 8 Floras, K., 453 Flo´rez-Sarasa, I.D., 53, 58 Flors, V., 461 Floss, D.S., 212 Flowers, S.A., 182
Flowers, T.J., 9, 11, 14, 16, 18–19, 26, 152–155, 158, 162–165, 169, 171–174, 176–179, 181–182, 408, 431 Fluhr, R., 205, 303, 305 Fontana, P., 56 Fontes, E.P.B., 458 Fontes, M., 456 Fooland, M.R., 152 Forestier, C., 75, 253, 256, 279 Forlani, G., 121 Fornara, F., 388 Forrester, G., 57 Forstheoefel, N.R., 78 Fortunati, A., 459 Foster, J.M., 383 Foster, T.J., 322 Fougere, F., 119 Fowke, L.C., 448 Fowler, S.G., 379, 387–388, 392, 468 Fowler, T.E., 301 Fowler, T. J., 125 Foyer, C.H., 65, 183, 268, 300–301, 305, 326, 455 Franceschini, L.M., 467 Francia, E., 3, 271 Francisco, R., 56 Franco, L., 277 Frandsen, G., 453 Franke, R., 459 Franklin, K.A., 76 Franks, P.J., 64, 76 Franks, S.J., 465 Frank, W., 452 Franza, B.R., 330 Fraser, P.D., 337 Fray, R.G., 456 Frebort, I., 7 Fredericks, D.P., 320 Frei dit Frey, N., 277 Frelet, A., 69, 256 Frelet-Barrand, A., 73 Fresia, C., 278 Frey, A., 203, 206 Frey, N.F.D., 55 Fricke, W., 6, 164, 167–169 Fridovich, I., 296 Friml, J., 209, 357, 363 Frison, M., 111 Fromm, H., 453 Frova, C., 466 Fruscione, F., 277–278 Fry, S.C., 42 Fuentes, S., 8 Fuglsang, A.T., 169, 223, 267 Fu, J., 468 Fujii, H., 68, 72, 75, 211, 213–214, 218, 220–221, 229–230, 256–260, 263–264, 269
AUTHOR INDEX Fujii, N., 355, 357–361, 367 Fujimori, T., 386, 393 Fujita, H., 362, 366 Fujita, M., 68, 77, 109, 115, 216, 221, 225, 263, 299–300, 352, 409–410, 431, 452, 458, 460 Fujita, T., 124 Fujita, Y., 72, 115, 124, 214, 216, 221, 225, 230, 263, 270, 300, 386, 393, 409–410, 425, 431, 460–461, 468, 471 Fujiwara, M., 72, 231, 268 Fujiwara, S., 379, 386–391, 395–396 Fujiwara, T., 112, 115, 118 Fukai, S., 466 Fukaki, H., 363 Fukao, Y., 72, 231, 268 Fukayama, H., 81 Fukui, K., 115–116 Fukushima, A., 393 Fu, L., 419 Funayama, T., 355 Funck, D., 121 Fung, P., 68, 72, 211, 213–214, 218, 229–230, 256–260 Furbank, R.T., 6, 20–21, 23–25, 67 Furihata, T., 225, 230, 270 Furuichi, T., 256, 356 Futsaether, C.M., 63 Fu, X., 301 Fux, B., 277 Fu, Y., 228 G Gadjdanowicz, P., 265 Gad, M., 106, 108–110 Gadrinab, C., 294 Gaedeke, N., 69, 256 Gaff, D.F., 320, 323, 325, 327–330 Gage, D.A., 111, 115–116, 124, 126–128, 203 Gagneul, D., 109, 113, 126, 128 Gaillard, I., 453 Galal, H.K., 9, 153–155 Galau, G.A., 329, 455 Galaug, J.P., 453 Galaz-Avalos, R.M., 359 Galbiati, M., 69, 74 Galbraith, D.W., 42, 227, 268, 299, 301, 305, 452, 458, 462, 467, 472 Galen, C., 366 Galiba, G., 110, 127, 129, 467 Galili, G., 60, 352, 426 Gallardo, K., 336 Galle´, A., 53–54, 58, 218 Galme´s, J., 53–54, 57–58, 60, 80 Galuschka, C., 472 Galweiler, L., 363 Gamba, A., 328
507
Gambale, F., 232 Gampala, S.S.L., 295, 452 Gandullo, J., 177 Gan, S., 428 Gao, F., 419 Gao, J.F., 268, 301, 305 Gao, J.-P., 17, 70, 74, 430, 461 Gao, Q., 413 Gao, S., 429 Gao, T., 226, 463, 471 Gao, Y., 78, 215, 278 Garbarino, J., 174 Garcia, A. B., 129 Garcı´a-Casado, G., 219, 225–226 Garciadeblas, B., 166, 418 Garcia-Ramirez, L., 163–164, 167–168, 170, 173, 175–176, 186 Garciarrubio, A., 455 Garcia-Sanchez, F., 54 Gardeur, T.L., 334 Gardiner, D., 336 Gardner, M., 327, 329–331, 333 Gareil, M., 451 Garey, W., 171–172 Garg, A.K., 117, 414 Garland, D., 335 Garner, W.W., 387 Garreton, V., 226 Garrett, K.A., 53 Garthwaite, A.J., 11 Gartland, J.S., 472 Gartland, K.M., 472 Ga¨rtner, T., 108 Garvin, D.F., 470 Gaspar, L., 325 Gassmann, W., 166 Gaur, J.P., 114, 119 Gaxiola, R.A., 362–363, 420, 451 Gaymard, F., 69, 265 Geerinck, J., 225–226 Geiger, D., 72–73, 222, 231–232, 268, 457, 471 Geisler, M., 69, 279, 472 Geissler, N., 177 Ge, L., 462 Geldner, N., 360, 363 Gendall, A.R., 378 Genty, B., 55, 57, 59, 61–62, 64, 69, 252, 263, 267, 457, 459 Gentzbittel, L., 299 George, G.M., 451 Georgieva, K., 325 Gepstein, A., 428 Gepstein, S., 42–43, 428 Gerhart, V.J., 156 Getzoff, E.D., 213–214, 221, 259–260, 471 Gevaudant, F., 42 Ge´vaudant, F., 267 Ghanem, M.E., 7–8, 20, 164–166
508
AUTHOR INDEX
Ghannoum, O., 57, 67, 80 Ghasempour, H.R., 328 Ghashghaie, J., 64 Ghassemian, M., 70, 109, 253, 429 Gherardi, F., 252, 274 Ghorbanli, M., 178 Ghorbel, A., 378–396 Ghosh Dastidar, K., 129 Giakountis, A., 388 Gianello, R.D., 327–328, 330 Gianfagna, T., 428 Gibon, Y., 8, 352, 469 Gibouin, D., 321–322 Gidley, M.J., 322 Gierth, M., 166 Gigon, A., 354 Gilbert, J., 469 Gilbert, T., 327, 329, 337 Gil, E., 225–226 Gilliham, M., 17, 56, 419, 456 Gillon, J.S., 59 Gil-Mascarell, R., 420 Gilmore, S.R., 75 Gilmour, S.J., 392 Gilroy, S., 362, 452 Gimeno, J., 428 Ginzberg, I., 123 Giordo, R., 70, 224, 273 Giovine, M., 277 Giraudat, J., 55, 69, 72, 209, 216–217, 220, 230, 252, 256, 260, 262–263, 267–268, 274, 295, 305, 394, 396, 450, 457 Giraudat, M., 55 Giraud, E., 306, 470 Giri, J., 116 Girke, T., 352, 467 Gisbert, C., 420 Gissot, L., 388 Giuliano, G., 467 Glaser, W., 273 Glenn, E.P., 155–157, 162, 165, 174, 177–178 Glisic, O., 65, 354 Gobert, A., 70, 73, 418 Goch, G., 453 Goday, A., 330 Goddijn, O.J.M., 117, 413–414 Godfray, H.C.J., 407 Godoski, J., 53, 82 Goel, D., 415 Goffeau, A., 267 Goh, C.H., 457 Goldberg, R., 322 Golden, S.S., 379 Golding, G.B., 77, 299, 467 Goldsworth, D., 116 Golkari, S., 469 Golldack, D., 167, 169–170, 174, 182 Gollery, M., 182, 296, 301, 303
Golovian, E.A., 321, 324, 328, 353 Go´mez-Cadenas, A., 127–128, 130, 220 Gomez, T.A., 327 Gomis-Ruth, F.X., 125 Gong, D., 222–223, 227 Gong, F.C., 467 Gong, H., 116 Gong, M., 119 Gong, Q.Q., 110, 127, 153, 167, 409, 468 Gong, Z.H., 70, 77, 209, 223, 227, 295, 299, 461–462, 471 Gong, Z.Z., 183–184 Gonneau, M., 203 Gonugunta, V.K., 72, 213, 252, 305–306 Gonzalez, E.M., 469 Gonzalez-Garcia, M., 217 Gonzalez-Guzman, M., 203, 217, 253 Gonza´lez, J.A., 108 Goodacre, R., 109 Goodger, J.Q.D., 207–208 Goodlett, D.R., 330 Goodman, H.M., 394 Goodwin, I., 81 Goodwin, S.M., 55, 158, 169, 177–178 Goodwin, W., 125 Goo, J. H., 125 Gooley, A.A., 335 Gorantla, M., 299, 469 Gordon, M., 227, 392, 396 Gorham, J., 3, 11, 17, 158 Gortan, E., 64 Gossett, D.R., 301 Gosti, F., 72, 166, 217, 262 Goto, D.B., 72, 231, 268 Goto, K., 388 Goto, N., 355, 358–361, 364 Gotz, S., 253 Goud, S., 227 Gowdaa, R.P.V., 466 Goyal, K., 329, 424 Goyal, S., 17 Gozukirmizi, N., 299, 452, 458 Grams, T.E.E., 184 Grando Baum, M., 465 Grandol, S., 466 Grando, M.S., 56 Grando, S., 115, 129, 467 Grant, J.J., 223, 449 Grant, M., 468 Grant, W.J.R., 62 Grassi, G., 57, 61, 65 Grattan, S.R., 157, 165 Grava, A., 355 Gray, G.R., 77, 299, 467 Gray, J.E., 57, 75–76, 451 Gray, W.M., 366 Greco, R., 422, 471 Green, P., 73, 408 Green, P.J., 256
AUTHOR INDEX Green, R.M., 379, 384, 386, 391 Greenway, H., 6, 114, 153 Greenway, S., 455 Grefen, L., 224, 273 Gregory, P.J., 25, 106 Greppin, H., 415 Greyling, M.M., 411 Gribskov, M., 222 Grierson, C.S., 76 Grierson, D., 456 Grieve, A.M., 11 Griffith, M., 77, 299, 467 Griffiths, C.A., 320 Griffiths, H., 158, 160, 163, 181 Griffiths, R., 430 Grignon, C., 183–184 Grill, E., 38, 68, 72–73, 207–209, 211, 213–214, 216, 218–219, 221–222, 229, 231–232, 252–253, 256–260, 268–269, 272, 274, 280, 301, 304–306, 457 Grillo, S., 296, 303 Grimplet, J., 109 Grimwood, J., 462 Grinev, D., 25 Grisafi, P., 362–363 Grob, H., 73, 256 Grondin, A., 36 Grossman, A.R., 55 Grote, K., 455 Grover, A., 468 Grozio, A., 278 Gruissem, W., 428, 453 Grunewald, W., 225–226 Grzesiak, M.T., 54, 57 Grzesiak, S., 54, 57 Guan, C., 363 Guan, L.M., 301, 305–306 Guan, Q., 59 Guan, Y., 471 Gu, D., 55, 70, 73, 115–116, 224, 231, 268, 273, 305 Gude, H., 172 Guegan, J.P., 106, 108–110, 127–128, 130 Guenzi, A.C., 412 Guerrero, F.D., 456 Guerrier, D., 72, 216, 260, 262 Guevara, D., 77, 299, 467 Guevara-Garcia, A., 224 Gu, H., 456 Guida, L., 277–278, 291 Guidi, L., 14, 52 Guihua, B., 115, 129 Guilfoyle, T.J., 362 Guilliot, A., 60 Guimaraes, C.M., 467 Guisez, Y., 35, 40 Gulias, J., 64 Gulick, P.J., 299
509
Gulli, M., 466 Gulzar, S., 178 Gu, M., 454, 459 Gunderson, C.A., 80 Gundlach, H., 462 Gunsu, I., 110, 130 Guo, G., 295, 298 Guo, H., 226, 463, 471 Guo, J., 39, 215, 278 Guo, L., 456 Guo, P., 115, 129 Guo, S.L., 174 Guo, S.-Q., 411, 419 Guo, S.Z., 58 Guo, Y., 115–116, 218, 222–223, 227, 267, 271, 462 Gupta, D.K., 185 Gupta, N.K., 11, 18 Gupta, R., 294 Guri, A.J., 277–278, 281 Gusmaroli, G., 448 Gussin, A.E., 111 Gust, A.A., 224, 273 Gusta, L., 416 Gusta, M., 416 Guthridge, K., 323 Gutierrez, M., 110, 127, 130 Gutierrez-Marcos, J.F., 461 Gutteridge, J.M.C., 38, 324 Guttikonda, S.K., 465 Gu, X.F., 419 Guy, C.L., 107–108, 116 Guy, M., 39 Guyot, S., 109 Gygi, S., 228, 330 H Habash, D.Z., 446 Haberer, G., 72, 216, 272, 462 Habricot, Y., 273 Hachez, C., 456 Hack, E., 120 Hacke, U.G., 35 Haddad, L., 407 Haesendonckx, B., 35, 40 Hafsi, C., 185 Hagenbeek, D., 364, 452 Hagen, G., 362 Hager, A., 42 Hagiwara, S., 264 Hahn, G.T., 127, 129 Ha, J., 225 Hajibagheri, M.A., 158, 162, 164, 172, 177–179, 182 Hajji, M., 183–184 Hakoshima, T., 214 Haldimann, P., 53 Halford, N.G., 222, 450
510
AUTHOR INDEX
Halfter, U., 218, 223, 271 Halket, J.M., 337 Hall, A.E., 66, 384–386, 394 Hallak-Herr, E., 305 Halliday, K.J., 385–386 Halliwell, B., 38, 324 Hall, J.L., 158, 164 Hall, R.D., 106–107 Hamada, A., 170, 175, 419 Hamada, N., 355 Hamann, T., 363 Hamant, O., 35 Hambler, D.J., 325 Hamdy, A., 408 Hamelin, J., 114–115 Hamill, J.D., 320, 327, 329–330 Hamilton, N., 336 Hammel, H.T., 171–172 Hammill, J.D., 323 Hanano, S., 385–386, 394 Han, B., 299, 425 Hanba, Y.T., 60 Han, C., 364 Hancock, J., 301 Handa, S., 119 Hand, S.C., 107, 110, 113 Hangarter, R.P., 365, 379 hang, X.-F., 174, 216, 222, 226, 261, 268, 280, 301, 305, 420, 464, 468 Han, L., 379, 386, 396 Han, M., 224, 227 Hannah, M. A., 108–109 Hanna, W-W., 107–108 Han, S.E., 117 Han, S.J., 228 Hanson, A.D., 111, 115–116, 124–128, 132 Hanson, J., 124 Han, S.W., 74 Hans-Werner, K., 109 Han, Y.S., 457 Hanzawa, Y., 386–388 Hao, Q., 72, 214, 259–260 Harada, J., 455 Hara, K., 75 Hara-Nishimura, I., 76 Harb, A., 468 Harberd, N.P., 276 Harbinson, J., 63 Hardie, D.G., 219, 450 Hardin, P.E., 379 Hare, P.D., 65, 107, 110, 113–114, 122, 125, 427 Hare, R.A., 171 Harjanto, E., 416–417 Harmer, S.L., 391–392, 396 Harmon, A., 221–222 Harmon, H.G., 379, 386 Harper, J.F., 221–222, 299–300, 352, 392, 396, 422
Harrington, C.L., 221 Harris, K., 466 Harrison, B.R., 363 Harrison, J., 431 Harrison, M.J., 388 Harris, P.J.C., 107, 465–466 Harryson, P., 455 Hart, A., 299 Harter, K., 115, 124, 223, 358, 453, 466 Hartung, W., 38, 73, 208–209, 212, 253 Hartwell, J., 383 Harvey, D.M.R., 172 Harvey Millar, A., 452, 468 Hasegawa, P.M., 55, 107, 110, 167, 170, 173, 225, 272, 295, 299–301, 303, 429 Haseloff, J., 17, 357, 363, 419, 452 Hase, Y., 390 Ha, S.H., 424, 460 Hash, C.T., 11, 466 Hashimoto, M., 55, 69–70, 231, 255, 266 Hashimoto, T., 55 Haskell, D.W., 116 Haslam, R.P., 80 Haswell, E.S., 356 Hata, S., 423 Hattori, E., 417 Hattori, T., 115, 220, 225, 230, 269 Hauben, M., 35, 40 Haudecoeur, E., 119 Hauge, B.M., 394 Hauser, F., 14, 17–18, 419 Hau¨sler, R.E., 81 Hausman, J.-F., 54, 110, 127, 130, 415, 470 Havaux, M., 52, 184, 459 Hayakawa, T., 60, 170, 175, 419, 426 Hayama, R., 388 Hayashida, N., 449 Hayashi, K., 386, 388–391, 395 Hayashi, N., 460 Hayashi, S., 216, 221, 229–230, 262–264 Hayashi, Y., 60, 170, 175, 419, 426 Hayashizaki, Y., 336, 458 Hayden, D.M., 470 Hazen, S.P., 386 Heard, J., 77, 424 Heard, P.J., 418 Hearshaw, M., 109, 323, 325, 327 Heath, L.S., 299 He, C.X., 115, 413, 416, 419 Hedfalk, K., 456, 471 Hedrich, R., 72–73, 179–180, 222, 231–232, 264, 268, 457, 471 Hegie, A., 306 Hehl, R., 472 Heidari, H., 178 Heide, T., 299 Heilmann, I., 362 Heilmeier, H., 208 Heimer, Y.M., 392, 396
AUTHOR INDEX Heinonen, T.S., 327 Heino, P., 218 Heja´tko, J., 366 Hejlek, L.G., 253 He, K., 78 He, L., 223 Heller, C., 294 Heller, M., 335 Hellsten, U., 462 Hemavathi, U.C.P., 39 Hemmingsen, E., 171–172 Hendry, G.A.F., 324, 328 Henkle, M., 53 Hennig, J., 453 Henrichs, H., 294 Henriques, R., 396 Henry, R.J., 115 Henry, S.A., 112, 118 Henry, Y., 449 Henz, S.R., 463 He, P.-H., 448 Herbert, B.R., 335 Herbette, S., 60 Hermans, C., 122, 124, 132, 411 Herna´ndez-Domı´nguez, E., 359 Hernandez, L.D., 328 Herrera-Cervera, J.A., 111, 116 Herschlag, D., 467 Herskowitz, I., 297 Hertel, R., 362 Herve´ du Penhoat, C., 322 He, S.J., 416, 448 Hetherington, A.M., 55, 57, 68–69, 74, 76, 263, 451 Heuvelink, E., 50 He, W., 39 Hewezi, T., 299 He, X.J., 365, 461, 471 Heyer, A. G., 108–109 Hibberd, J.M., 81 Hibberd, V., 385–386 Hibino, T., 417 Hibi, T., 72, 231, 268 Hicks, K.A., 379–380, 384–386, 388, 391 Hicks, L.M., 277, 423, 449 Hideyuki Takahashi, 349 Higashitani, A., 356 Higashitani, H., 355, 359–361 Higgins, C.F., 210 Hilal, M., 108 Hill, L.M., 320 Hilton, H.W., 427, 471 Himmelbach, A., 72, 216, 218, 272 Hincha, D.K., 108–109 Hinchey, B.S., 423, 461 Hirai, N., 205–206 Hirano, K., 423 Hirano, Y., 214 Hiraoka, G., 232
511
Hirasawa, T., 356, 367 Hiratsu, K., 225 Hirayama, T., 52, 201, 203, 213, 216–219, 221, 226–227, 229–230, 232, 262–264, 298, 409, 448–449, 451, 462 Hirji, R., 115 Hirochika, H., 468 Hirose, S., 81, 417 Hirsch, H.J., 81 Hirschi, K.D., 420, 451 Hirt, H., 216, 273, 324, 449 Hishinuma, H., 232 Hitomi, C., 214, 259–260, 471 Hitomi, K., 213–214, 221, 259–260, 471 Hobo, T., 220, 225, 230, 263, 269 Hochstrasser, D.F., 335 Hodgson, D., 366 Hodson, J.N., 419 Hoekstra, F.A., 110, 321, 324, 328–329, 353, 455, 469, 471 Hofer, J.M.I., 55 Ho¨fer, R., 459 Hoffmann, L., 110, 127, 130 Hoffmann, T., 218, 221, 252–253, 274 Hohener, B., 218 Hohmann, S., 297–298 Hoisington, D., 421 Ho, L.A., 379, 386 Holaday, A.S., 419 Holaday, S.A., 305, 416 Holappa, L.D., 220 Holbrook, N.M., 208 Holdsworth, M.J., 386, 394 Ho, L.H., 470 Ho, L.H.M., 306 Hollington, P.A., 407–408 Holm, L., 472 Holmstrom, K. O., 115 Holroyd, G.H., 55, 69, 74, 76, 263 Holtan, H.E., 467 Holthauzen, L. M., 113 Hong-bo, S., 300 Hong, F., 229, 280, 429 Hong, J.C., 225, 303 Hong-mei, M., 300 Hong, S.W., 460 Hong, X.H., 70, 76–77, 461, 471 Hong, Y.Y., 357, 461 Hong, Z., 113–114, 122 Hontecillas, R., 277–278, 281 Hood, L., 330 Hooper, P.L., 276 Hooykaas, P.J., 363 Hopia, A.I., 327 Hoque, M.A., 122 Horak, J., 358, 466 Horbowicz, M., 328 Horie, T., 14, 17–18, 419
512
AUTHOR INDEX
Hori, K., 115 Hornberg, C., 256 Horres, R., 467 Horridge, J.S., 427, 471 Horst, R.J., 109 Horvath, G.V., 458 Hose, E., 209, 212 Hosy, E., 69 Ho, T.D., 220 Ho, T.H.D., 455 Ho, T.-H.D., 425 Hoth, S., 73, 179 Hotta, Y., 323 Houde, M., 115 Hou, P., 420 Howarth, C.J., 407–408, 466 Howell, K.A., 306, 452, 468, 470 Hrabak, E.M., 222 Hra, K., 298, 448 Hrmova, M., 18 Hsiao, T.C., 113, 447 Hsieh, T.H., 422 Huai, J., 468 Huang, A.H., 119 Huang, B.R., 59, 82, 428 Huang, C.X., 10, 39, 186 Huang, D., 427 Huang, J., 223, 411, 454, 459 Huang, L.F., 64 Huang, P., 174 Huang, R.F., 79, 428–429 Huang, S.Z., 17, 229 Huang, T., 388 Huang, W.X., 17, 452, 454, 463 Huang, X.-Y., 70, 74, 430, 461 Huang, Y., 36, 39, 276, 423, 425, 430–431 Hua, X., 121 Hubbard, K.E., 213, 221 Hu, C.A., 110, 120, 124 Hu, C.A.A., 113, 122 Huchzermeyer, B., 173, 175, 177 Hughes, B., 363 Hughes, D.W., 329, 455 Hughes, S.J., 157 Hugouvieux, V., 57, 70, 227 Hugueney, P., 203 Hu, H.-C., 53, 55, 73, 208–209, 212, 231, 264, 268, 305, 364, 424, 460, 468 Hu, H.H., 53, 68, 73, 82 Huijser, P., 363 Hui, Z., 413 Huizinga, D.H., 429, 464 Hulbert, S.H., 53 Hu, L.X., 59, 118 Hummel, I., 8, 352, 469 Huner, N.P., 78 Huner, N.P.A., 115 Hung, C.Y., 452, 461, 468, 471 Hunt, L., 75
Hura, K., 57 Hura, T., 57, 74 Hurry, V., 108 Hurst, A.C., 184 Husaini, A.M., 415 Hu, S.J., 79, 428 Huskisson, N.S., 449 Hussain, S., 327, 329, 337 Hussin, S., 177 Hu, X.W., 76, 301, 304–305 Hu, Y., 6, 464, 468 Hu, Z., 295, 298, 327, 331, 333–334, 336 Hwa, C., 224 Hwang, E.W., 299 Hwang, I., 212, 222, 225, 253, 352, 463 Hwang, J.-U., 68–69, 209–211, 254 I Iannacone, R., 327 Iba, K., 55, 69–70, 231, 255, 266 Ichikawa, T., 468 Ichimura, K., 220, 224, 263, 395–396, 449 Ichiyanagi, T., 115 Ideker, T., 330 Ide, T., 262 Iida, K., 115, 229, 461, 471 Ikeda, A., 109 Ikegami, K., 207, 209 Iljin, W.S., 321 Illing, N., 320, 324–331, 333 Illingworth, C.J., 278 Imai, R., 111, 116 Imai, Y., 76 Imaizumi, T., 379, 386, 396 Imaseki, H., 456 Imes, D., 457, 471 Im, M., 222, 225, 448 Impa, S., 466 Imura, Y., 386, 393 Inada, S., 366 Inagaki, N., 163, 178 Inanaga, S., 305 Inan, G., 55, 127–128, 130, 158, 169, 177–178 Indu Rupassara, S., 110, 127 Infante, J.M., 58 Ingle, R.A., 325, 327, 329, 331, 333–334, 336 Ingram, J.S., 106, 110 Interdonato, R., 108 Inze´, D., 35–36, 218, 301, 305, 470 Iordachescu, M., 116 Iribarne, C., 111, 116 Irie, K., 396, 423, 449 Irigoyen, M.L., 389, 395 Isayenkov, S., 70, 73 Ishida, J., 68, 77, 115, 299, 352, 452, 458, 460, 463 Ishihama, Y., 216, 221, 229–230, 262–264
AUTHOR INDEX Ishihara, K., 367 Ishikawa, H., 362, 417 Ishikawa, T., 174 Ishitani, M., 69, 118, 295, 300 Ishiyama, K., 169, 183, 216, 221, 263, 299 Islam, M.M., 122 Ismail, A.M., 66, 114, 300 Israelsson, M., 55, 70 Israelsson-Nordstrom, M., 53, 82 Itoh, H., 366 Ito, S., 393, 396 Ito, T., 217 Ito, Y., 68, 299, 336, 361, 421, 425, 431, 460 Iturriaga, G., 414–415 Iuchi, S., 69, 109, 203, 205 Ivanov, A., 115 Ivanova, A., 306, 470 Ivanov, S., 114 Ivanov, Y.V., 185 Iwabuchi, M., 79, 388, 468 Izawa, T., 380 Izui, K., 423 J Jablonowski, D., 70, 77 Jacchetti, E, 278 Jacobs, A., 419 Jacobs, C.I., 379 Jacobsen, S.E., 250 Jacob, T., 452 Jae Cheol, J., 110, 130 Jaffe, M.J., 354 Jaffer, M., 322 Jagendorf, A.T., 39, 223, 303 Ja¨ger, K.E., 363, 407 Jahnke, S., 63–64 Jahoor, A., 466 Jaindl, M., 113 Jakab, S., 216 Jakoby, M., 124, 225 James, R.A., 3, 6, 8, 10–11, 17–23, 25, 65, 171, 186, 208 Jamin, M., 214 Jammes, F., 55, 70, 224, 273, 277 Jang, C.J.H., 467 Jang, F., 253 Jang, H.-J., 421 Jang, I.-C., 117, 301, 414–415 Jang, J.Y., 60 Jang, S., 388–389 Janiesch, P., 173 Jan, L., 107–108 Jansson, S., 55, 388, 466, 469 Janz, D., 23 Jarvis, M.C., 322 Jasik, J., 114, 121–123 Ja´sik, J., 272 Jaspers, P., 300, 303, 415
513
Jaspert, N., 267 Jauneau, A., 322, 453 Jawali, N., 122 Jeannette, E., 273 Jean-Richard-dit-Bressel, L., 111, 117 Je, B.I., 223 Jefferies, R.L., 164 Jefferies, S., 464 Jeknic, Z., 114–116 Jelinek, H., 273 Jenkins, G. I., 125 Jennings, D.H., 158 Jenny, F.E., 301 Jensen, D.R., 68, 72, 211, 213–214, 218, 229–230, 256–260 Jensen, R.G., 118–119, 157–158, 160, 163, 181, 409 Jeon, B.W., 69, 255, 304, 306 Jeong, J.C., 272 Jeong, J.S., 424, 428, 460 Jeong, M.-J., 299, 423 Jeong, S., 384 Jerzmanowski, A., 272 Jeschke, W.D., 6, 160, 181 Jeyaprakash, P., 466 Jha, B., 183–184 Jha, D., 17, 169, 419 Jhurreea, D., 414 Jia, J., 468 Jian, F., 253 Jiang, C.X., 77, 424, 466 Jiang, F., 208, 212 Jiang, G.Q., 174, 327, 331, 333–334, 336 Jiang, H., 226 Jiang, M.Y., 301, 303–306 Jiang, T., 216, 261, 280 Jiang, X.S., 335, 418 Jiang, Y.Q., 468 Jian-Hua, Z., 301 Jia, W., 224, 273, 300, 304, 462 Jia, X., 462 Jimenez, A., 183–184 Jimenez, M., 53, 58 Jin, H., 461, 467, 471 Jin, J.B., 225 Jin, L., 472 Jin, T., 422 Jin, Y., 462 Jithesh, M.N., 183 Joachim, K., 116 Joachim, S., 107–108 Johannesson, H., 327 Johanson, U., 352, 456, 471 Johansson, E., 298 Johansson, I.I., 265, 352, 456 John, P.C.L., 448 Johnson, A., 419 Johnson, H.E., 109 Johnson, M.D., 112, 118
514 Johnson, R.R., 230, 269 Johnsson, A., 63 Jolivet, Y., 114–115 Joly, R.J., 127, 129, 178 Jonak, C., 109, 273, 449 Jones, A.M., 73, 215, 250, 262 Jones, G.P., 122 Jones, H.D., 386, 394 Jones, H.G., 6, 20–21, 23, 25, 62 Jones, J.D., 70, 268, 296, 304, 306 Jones, J.D.G., 258 Jones, J.T., 456 Jones, L., 323, 448 Jones, R., 81 Jones, R.G.W., 158 Jones-Rhoades, M.W., 462 Jones, R.L., 177 Jordan, D., 466 Jordan, F.L., 156 Jordano, J., 328, 458 Jose-Estanyol, M., 125 Joseph, L.M., 206–207, 253, 274 Joseph, M.P., 20, 55 Joseph, R.A., 354 Jouve, L., 54, 470 Jou, Y., 160, 181 Julien, J.L., 60 Jungas, C., 184 Jung, C., 74, 228 Jung, E., 335 Jung, H.J., 424, 457, 460 Jung, J.W., 42, 275 Jung, K.H., 472 Ju¨rgens, G., 360, 363 Jyostna Devi, M., 411 K Kacperska, A., 300 Kaczanowski, S., 272 Kadota, Y., 232 Kagawa, T., 365 Kagaya, Y., 220, 225, 230, 269 Kagiampakis, I., 299 Kagiyama, M., 214 Kahkonen, M.P., 327 Kai, C., 336 Kaines, S., 25, 55 Kaiser, B.N., 56, 456 Kaiser, K.A., 467 Kaiser, W.M., 53, 64 Kajita, R., 75 Kakar, K., 55 Kakimoto, T., 75, 356, 448, 461, 468 Kakimoto, Y., 355, 359–361 Kakizaki, T., 355 Kakubari, Y., 69, 205 Kaku, N., 417 Kaldenhoff, R., 60, 455
AUTHOR INDEX Kalinkina, L.G., 176 Kalmar, E., 455 Kaltsikes, P.J., 116 Kamada, H., 383, 387–388 Kami, C., 366 Kamies, R., 327 Kaminaka, H., 305 Kaminek, M., 428 Kaminuma, E., 299, 463 Kamiya, A., 68, 77, 210–211, 254, 299, 352, 452, 458, 460 Kamiya, Y., 205–207, 209, 212, 253 Kamoshita, A., 466 Kanamori, N., 216, 221, 263 Kanaoka, M.M., 75, 218 Kanczewska, J., 267 Kanechi, M., 163, 178 Kane, R., 156 Kaneyasu, T., 355, 358–360 Kangasja¨rvi, J., 23, 55, 70, 109, 231, 266, 300, 303, 415 Kang, H., 60, 79, 214, 227–228, 457 Kang, H.-J., 72, 259–260, 471 Kang, J., 68–69, 209–211, 225, 254, 448 Kang, K.Y., 472 Kang, M.M., 184 Kang, S.M., 223 Kang, Y.L., 70 Kankainen, M., 472 Kanna, M., 70, 266 Kanrar, S., 76 Kantar, M., 445, 462 Kantety, R.V., 469 Kant, M., 273 Kant, P., 184, 227, 392, 396 Kant, S., 130, 184, 227, 392, 396 Kao, T.H., 362 Kapazoglou, A., 271 Kaplan, F., 108, 116 Kappen, L., 320 Kapulnik, Y., 60, 352, 426 Karaba, A., 422, 471 Karakas, B., 412 Karanov, E., 114 Karchi, H., 426 Kardailsky, I., 388 Kargul, J., 362 Karimi, G., 178 Karim, S., 117 Karlsson, M., 352, 456, 471 Karolewski, P., 183 Karpinska, B., 184 Karpinski, S., 184, 472 Karssen, C.M., 69, 260 Kartashov, A.V., 185 Karunaratne, S., 336 Kasahara, M., 365 Kasamo, K., 60, 426 Kaspar, F., 294
AUTHOR INDEX Kasuga, M., 392, 421, 458, 468 Kasulin, L., 383 Katagiri, T., 216, 221, 263, 356, 451–452 Kathuria, H., 115–116 Katiyar-Agarwal, S., 296, 303 Katori, T., 109 Kato, T., 69, 205, 356, 386, 393 Katsuhara, M., 60, 426 Katsura, K., 68, 299, 421 Kaufman, P.B., 362, 364 Kav, H.N.V., 467 Kavi Kishor, P.B., 122 Kav, N.N., 467 Kawaguchi, R., 352, 467 Kawaide, H., 253 Kawai, K., 299 Kawai-Yamada, M., 69, 231, 255, 266 Kawano, N., 305 Kawasaki, S., 299 Kawashima, M., 299, 463 Kaya, H., 232, 388 ¨ .F., 294 Kaya, O Kay, S.A., 78, 379, 384–388, 391–392, 396 Kazanaviciute, V., 273 Kbhaya, B., 461 Keddie, J., 77, 424 Kehel, Z., 446 Keicher, J., 360, 363 Keller, F., 328 Kemmerling, B., 258 Kempa, S., 109 Kemp, P.R., 178 Kenrick, P., 154 Kepka, M., 258–259, 280 Kerepesi, I., 110, 127, 129, 411 Kerkmann, K., 452 Kervazo, L., 106, 108–109 Ke, S.D., 461 KetenoJlu, O., 294 Keurentjes, J., 107 Kevresan, S., 65 Keys, A.J., 64–65, 80 Kfzma, M., 276 Khafif, M., 230, 450 Khailova, G.F., 172 Khalilova, L.A., 176 Khan, A., 39 Khan, M.A., 68, 156, 178, 299 Khanna, R., 379, 385 Khan, S., 64–65 Khedr, A.H., 122 Kiba, T., 396 Kiddle, G., 455 Kido, K., 420 Kidokoro, S., 109, 216, 221, 263, 270, 386, 393, 425, 468 Kieber, J.J., 427 Kiegerl, S., 449 Kiegle, E., 452
515
Kielland-Brandt, M.C., 298 Kientz, M., 363 Kikis, E.A., 379, 385 Kikuchi, A., 421 Kikuchi, J., 52 Kilian, J., 466 Killan, J., 358 Kim, B.-G., 223, 423 Kimbrough, J.M., 358 Kim, C.H., 74, 117, 414–415 Kim, C.J., 294 Kim, C.M., 223 Kim, D.H., 39, 303 Kim, E.H., 428, 460, 468, 471 Kim, E.J., 166 Kim, H.J., 467 Kim, H.-S., 413 Kim, H.Y., 212, 253 Kim, J.G., 301 Kim, J.K., 117, 460, 468, 471–472 Kim, J.-K., 117, 379, 386, 396, 414–415, 421, 424, 428 Kim, J.M., 463 Kim, J.-M., 294–295, 299 Kim, J.S., 227–228, 457 Kim, J.Y., 379–380, 385, 387 Kim, K.A., 79, 223, 227, 299, 457 Kim, K.H., 460 Kim, M.J., 223, 413, 421, 424, 460–461, 472 Kim, S.-H., 417, 460 Kim, S.-K., 364 Kim, S.S., 389, 395 Kim, S.Y., 222, 225, 364, 421, 448 Kim, T.H., 68, 73, 208–209, 212, 218, 264 Kim, T.-W., 364 Kimura, K., 53 Kimura, S., 232 Kim, W.T., 352, 463, 471 Kim, W.Y., 379, 385–386, 388, 396 Kim, Y.K., 472 Kim, Y.-K., 379, 386, 396, 421 Kim, Y.O., 222, 225, 228 Kim, Y.S., 117, 123, 296, 303, 414–415, 421, 424, 428, 460, 468, 471 Kim, Y.-S., 364 Kim, Y.Y., 68–69, 209–211 Kim, Y.-Y., 254–255 Kinet, J.-M., 20, 127, 129, 178 Kingston, D.G., 277 Kini, R. K., 125 Kinoshita, N., 225–226 Kinoshita, T., 228, 262, 264, 266 Kinraide, T.B., 22, 24 Kintisch, E., 106 Kirakosyan, A., 364 Kirch, H.-H., 327, 329, 458 Kirk, D.A.W., 228 Kirkegaard, J.A., 25 Kirschbaum, M.U.F., 54
516
AUTHOR INDEX
Kishigami, A., 356 Kishitani, S., 115–116, 431 Kishor, P., 113, 122 Kishor, P.B.K., 122 Kiss, J.Z., 362, 365–366 Kitagawa, Y., 426 Kitahata, N., 217–218, 227, 359, 462 Kita, M., 393 Kitamura, S., 205–206 Kiyosue, T., 111, 121, 123–124 Kizis, D., 300 Kjellbom, P., 352, 456, 471 Kleczkowski, L.A., 472 Klein, M., 69, 73, 256, 279 Klein, P.E., 299, 466 Klein, R.R., 299, 466 Klejnot, J., 389–390 Kline, K.G., 450, 469 Klingler, J.P., 213 Klusener, B., 73, 268, 301, 304–305 Knapp, A.K., 53 Knepper, M.A., 426 Knight, H., 299, 303, 452 Knight, M.R., 55, 263, 299, 303, 452 Knight, T.A., 354 Knox, K., 357, 363 Kobayashi, A., 355–361 Kobayashi, M., 69, 109, 169, 183, 203, 205–207, 216, 221, 263, 299, 421, 470 Kobayashi, Y., 220, 225, 230, 269, 355, 388 Koca, H., 293, 296 Kochian, L.V., 117, 414 Koch, K.E., 253, 467 Koch, W., 121 Koczan, J.M., 275, 465 Kodaira, K., 214, 259–260 Kodaira, K.S., 72, 471 Koebner, R.M.D., 55 Koehl, K., 109, 126 Ko, E.Y., 39 Ko¨hler, B., 172 Kohl, K.I., 165 Koike, M., 262 Koiwa, H., 55, 295, 300 Koiwai, H., 205, 207–209, 253, 274 Koizumi, M., 456 Koizumi, N., 298, 448 Kojima, I., 356 Kojima, M., 428 Kolb, D., 224, 273 Kollist, H., 55, 70, 109, 231, 266 Kolukisaoglu, H.U., 69 Kolukisaoglu, U., 279, 453 Komamine, A., 417 Komatsu, S., 336 Komori, T., 456 Konagaya, A., 229 Koncz, C., 114, 121–123, 227, 272
Kondo, A., 159–160, 163, 165, 181 Kondo, T., 383 Kondou, Y., 468 Kong, Z., 299 Konopka-Postupolska, D., 453 Konstantinova, T., 114 Koops, A., 253 Koornneef, M., 69, 205, 228, 260, 305 Koo, Y.D., 301 Koo, Y.J., 74, 428, 460, 468, 471 Kopittke, P.M., 22, 24 Kopka, J., 65, 106–110, 126–128, 130, 451 Korkaric, M., 78, 468 Kornberger, W., 360, 363 Korn, M., 108 Korte, A., 68, 72, 211, 213–214, 216, 218, 229, 256–260, 272, 280 Koshiba, T., 203, 205–209, 253, 274 Koshio, K., 426 Kosmas, S.A., 116 Kossmann, J., 451 Koster, K.L., 328 Koukoumanos, M., 118, 126 Koussevitzky, S., 280, 306 Koustenis, A., 385 Kovach, A., 72, 214 Kovacs, D., 455 Kovacs, I., 458 Kovtun, Y., 423 Koyro, H.W., 175, 177 Kozbial, M., 272 Kozbial, P., 272 Kozma-Bogna´r, L., 78, 379 Kraepiel, Y., 305 Krajewski, P., 466 Kramer, E.M., 357, 363 Kranner, I., 109, 324, 326, 334 Krantz, M., 297–298 Krapp, A., 67, 79 Krasensky, J., 109 Krauss, A., 42 Kreps, J.A., 299, 352, 392, 396 Kretsch, T., 386 Kreuzaler, F., 81 Krishman, A., 277 Krishnamurthy, L., 11 Krishnamurthy, N., 472 Krishnan, A., 422, 468, 471 Kristensen, P., 50 Kristiansdottir, I., 115 Kristiansen, K.A., 223, 267 Krizek, B.A., 306 Krochko, J.E., 205 Kroj, T., 225 Kro¨l, M., 115 Kronebusch, P.J., 356 Krutwagen, R. W., 117 Ksouri, R., 109 Kuang, J., 327, 329–330
AUTHOR INDEX Kubo, A., 70, 266 Kubo, T., 456 Kubota, K., 72, 214, 259–260, 471 Kuchel, H., 464 Kuchitsu, K., 219 Kudla, J., 222–223, 358, 453, 466 Kuehl, R.O., 157 Kuhn, J.M., 53, 70, 82, 217 Kukavica, B., 328, 334 Kumar, A., 466 Kumar, D., 299 Kumar, G., 431 Kumar, P., 59 Kumar, R., 110, 113, 466 Kumar, V., 122 Kumimoto, R.W., 423, 461 Ku, M.S.B., 81 Kundzewicz, Z.W., 294 Kung, C., 356 Kuppu, S., 420 Kura-Hotta, M., 409 Kurata, N., 336 Kurepa, J., 226, 301 Kurihara, Y., 36, 109, 466 Kuriyan, J., 395 Kurkova, E.B., 172, 176–177 Kuromori, T., 68, 201, 210–211, 217, 227, 232, 254, 462 Kurup, S., 386, 394 Kusano, M., 393 Kushiro, T., 205–207 Kushnir, S., 226 Ku¨ster, H., 336 Kutzer, M., 464 Kuwata, S., 456 Kuzma, M., 430–431 Kuznetsov, V.V., 185 Kwak, C.M., 70, 429 Kwak, J.M., 55, 57, 59, 70, 73, 212, 219, 221–222, 227, 231, 253, 268, 276, 296, 304–306, 427, 463, 471 Kwak, K.J., 228 Kwak, S.S., 301 Kwak, S.-S., 303, 413, 417, 426 Kwiatkowska, A., 272 Kwon, C.-W., 428 Kwon, H.B., 117, 299 Kwon, H.-B., 423 Kwon, S.-Y., 301, 413, 417 Kwon, T.-R., 423, 465–466 Kyozuka, J., 423 L Labate, C.A., 467 Labbe, A., 77, 299, 467 Lachaal, M., 183–184 Lachagari, V.B.R., 469
517
Lacombe, B., 265 Ladrera, R., 469 LaFavre, A.K., 364 Lafforgue, G., 468 Laffray, D., 354 Lafontaine, P.J., 111, 115 Laga, B., 35, 40 Lagarias, J.C., 366 Lagudah, E.S., 17 Lai, J., 460 Lai, Y., 299 Lai, Z., 454, 459 Lajunen, H.M., 224, 273 Lake, J.A., 57, 452, 468 Lakkineni, K., 114, 122 Lamant, A., 119 Lamark, T., 115 Lamattina, L., 454 Lamaze, T., 456 Lambermon, M.H.L., 228 Lambert, B., 35, 40 Lambert, C., 166 Lambert, G.M., 42 Lammers, R.B., 408 Lamminmaki, A., 231, 266, 299 Lamond, A., 228–229 Lamoureux, D., 110, 127, 130 Lampard, G.R., 77 Langdale, J.A., 78, 468 Langebartels, C., 55, 70 Lange, B. M., 108–109 Lange, I., 109 Langhans, M., 253 Langowski, L., 366 Langridge, P., 11, 18, 446, 464 Lan, L., 299 Lanni, L.M., 329 Lan, S.S., 119 Lan, W.-Z., 72, 223, 231, 268, 457 Larher, F., 106, 108–110, 114–115, 127–128, 130 Larher, F.R., 106, 108–109, 113, 126, 128 Lariguet, P., 366 Larive, C.K., 467 Larkin, R., 215 Larrainzar, E., 469 Larre´, C., 336, 455, 469, 471 Larsson, C., 266, 352, 451, 456 Lash, A.E., 294 Laubinger, S., 228, 463 La¨uchli, A., 10–11, 65, 186 Laukens, K., 54, 470 Lauriano, J.A., 65 Laurie`re, C., 218, 220–221, 229–230, 262–263, 450–451, 469 Laurie, R.N., 411 Laurie, S., 418 Lavanya, M., 411, 421 Lawlor, D.W., 51, 58, 60–61, 64
518
AUTHOR INDEX
Lawrence, D., 407 Lawson, T., 61, 63–64, 67 Lax, A.R., 334 Layzell, D.B., 11 Lazdunski, M., 277 Leach, J.E., 53 Leach, R.P., 164, 177, 179 Leake, J.E., 181 Lea, P.J., 64–65 Lebaudy, A., 69, 265 Lee, B.-H., 18, 51, 76, 303, 418, 459 Lee, C.H., 79 Lee, D.H., 79, 467 Lee, G., 458 Leegood, R.C., 81 Lee, H.J., 457 Lee, H.K., 352, 463 Lee, H.-S., 69, 78, 227, 295, 300, 413, 417, 460 Lee, I.-J., 78, 212, 223, 253, 389, 395, 428, 460, 468, 471 Lee, J.A., 324 Lee, J.H., 300 Lee, J.-O., 78, 180, 224–225, 254, 273, 423 Lee, J.S., 364 Lee, J.T., 117, 422 Lee, J.Y., 364 Lee, K.H., 212, 253–254, 379, 387–388 Lee, M., 68–69, 209–211, 254–255 Lee, N.Y., 389, 395 Lee, O., 303 Lee, S.C., 55, 70, 72–73, 214, 223–224, 230–231, 268, 273, 305, 457 Lee, S.-K., 423 Lee, S.Y., 60, 303, 389, 395 Lee, T.H., 472 Lee, W.S., 228 Lee, Y.-P., 68–69, 209–211, 254–255, 279, 304, 417 Lefebvre, V., 55, 69, 206, 252, 263 Lefe´vre, I., 110, 127, 130, 181 Legay, S., 110, 127, 130 Le´ger, M., 299 Legnaioli, T., 216, 394 Lehmann, S., 122–123 Lehner, A., 264, 323, 325, 337, 464 Lehner, B., 294 Leidi, E.O., 18 Lei, G., 416, 448 Leigh, R.A., 418 Leisse, T.J., 294, 470 Leister, D., 55 Leivar, P., 393 Lenoble, M.E., 42–43, 252, 274, 364 Le, N.T., 109, 116, 320, 322, 326–327, 329–330, 337 Leon, D., 38 Leong, O.M., 470 Leonhardt, G., 59
Leonhardt, N., 55, 59, 69–70, 74–75, 219, 224, 253, 256, 267–268, 273, 296, 304, 306, 457 Leon, J., 454 Leon-Kloosterziel, K.M., 305 Leon, P., 203, 209, 253, 274 Leopold, A.C., 328–329, 364 Leopold, C., 324 Le Page-Degivry, M.T., 277 Leponce, I., 229 Leport, L., 106, 108–110, 127–128, 130 Leprince, O., 320–321, 328–329, 416–417, 455, 469, 471 Lerouge, P., 322 Lerouxel, O., 322 Le Rudulier, D., 119 Leshem, Y., 304 Lespinase, Y., 109 Le Thiec, D., 54, 470 Le, T.-J., 323 Leube, M.P., 218, 259–260 Leung, J., 55, 73, 209, 216, 218, 221, 228–229, 231, 253, 258, 260, 262–263, 267–268, 270, 277, 295, 305, 450–451, 457, 469 Leuning, R., 66–67 Levesque-Tremblay, G., 459 Levi, A., 466 Levine, A., 304 Levitt, J., 321, 408 Lewis, M.W., 306 Lew, R.R., 155 Leydecker, M.T., 305 Leyser, H.M.O., 55, 263, 357, 363 Liang, H.J., 52, 306, 461 Liang, J.S., 209, 299 Liang, R., 462 Liang, Y.K., 69, 74 Liang, Z., 116 Lian, H.-L., 389–390, 426 Liao, Y., 471 Li, B., 68, 213, 215 Libik, M., 183 Li, C.H., 455, 469 Lichtenthaler, H.K., 325 Liese, A., 72–73, 222, 231–232, 268, 457 Li, F., 413, 429, 468–469 Ligterink, W., 449 Li, H., 223, 226 Li, J., 72, 81, 214, 220, 228, 263, 420, 451 Li, J.M., 389 Li, J.Y., 174 Li, K., 364, 389–390 Li, L.G., 17, 223, 265 Li, M., 109, 268, 299, 357, 452 Lima, J.P.M.S., 65 Lim, J.J., 280 Lim, S., 299 Lin, C., 389–390
AUTHOR INDEX Linder, P., 227 Lindsey, G.G., 109, 320, 322–329, 331, 334 Lingle, W.L., 329 Lin, H.-Q., 419, 456, 462 Lin, H.-X., 17, 70, 74, 430, 461 Linker, R., 42–43 Lin, P., 58 Linster, C.L., 327 Lin, T.P., 116 Li, P.-H., 55, 110, 127, 158, 169, 174, 177–178, 409, 420, 468 Lipka, V., 81 Lips, S.H., 305 Li, Q.B., 206–207, 253, 274 Li, Q.H., 389–390 Li, R, 115, 129 Liscum, E., 365–366 Lisec, J., 108 Lisiero, D., 389–390 Li, S.Y., 53, 472 Liu, A., 121 Liu, B., 422 Liu, C.L., 364, 467 Liu, C.W., 305 Liu, D., 267, 278, 281, 305 Liu, H.H., 70, 226, 228, 295, 298, 389–390, 419–420, 462 Liu, H.Y., 461 Liu, J., 299, 365, 454, 463 Liu, K., 265 Liu, L.J., 206–207, 214, 223, 389–390, 395, 422, 472 Liu, M.S., 116, 364, 456 Liu, P., 416, 448 Liu, Q., 392, 458, 461, 471 Liu, S., 59, 169, 305 Liu, T., 75 Liu, W.X., 121, 298, 452–453, 458 Liu, X.L., 68, 213, 215, 295, 298, 330, 384– 386, 389, 416 Liu, Y.D., 76, 113, 295, 298, 323, 454, 461, 463, 471 Liu, Y.F., 416, 448 Liu, Z.H., 55 Liu, Z.-Q., 216, 261, 280 Livingston, N.J., 64 Liwosz, A., 273 Li, W.X., 68, 72, 109, 213–215, 259–260, 413, 422, 452, 461–462, 471 Li, X.-Y., 215, 260, 364, 423–424, 449, 460, 468 Li,Y., 272 Li-ye, C., 300 Li, Y.J., 77, 222, 226, 228, 299, 362, 460, 462–463, 467, 471–472 Li, Y.Y., 183–184 Li, Y.Z., 167 Li, Z., 422, 459 Ljung, K., 363–364
519
Llamas, M. R., 50 Lloyd, J.R., 451 Lluch, C., 111, 116 Loake, G.J., 449 Lobst, S., 299 Lockhart, J.A., 35 Loescher, W.H., 112, 118 Loewen, M.K., 205 Loffell, D., 325–326, 333 Lohmann, J.U., 228 Lohr, C., 471 Long, M.J., 10 Long, S.P., 55, 65, 80–81 Longstreth, D.J., 158 Long, T.A., 275 Lopes, C.M., 56, 65 Lopes, M.S., 13, 25 Lopez, A., 461 Lopez-Coronado, J.M., 420 Lopez, M.J., 111, 116, 163–164 Lopez-Molina, L., 225–226 Lorenzo, O., 217 Loreto, F., 51, 54, 57–59, 62, 64–65, 459 Lorkovic, Z.J., 228 Lothar, W., 107–108 Loukas, M.G., 116 Lourenc¸o, T., 77, 295 Lovelock, C.E., 177 Loveys, B.R., 25, 62 Lovisolo, C., 8, 60, 455 Lozano-Juste, J., 454 Luan, S., 17, 72, 222–223, 231, 265, 268, 294, 300–301, 453, 457 Lu¨, B.R., 209, 299, 454, 463 Lucas, C., 298 Lucas, S.J., 445, 462 Luchi, S., 451 Lu, C.M., 116, 163, 183–184, 224, 227 Ludwig-Mu¨ller, J., 116–117 Lu, G., 223, 425 Lugan, R., 106, 108–110, 113, 126–128, 130 Lu, H., 469 Lui, A., 363 Lui,L., 253, 274 Lu, J., 301, 304–305 Lumba, S., 68, 72, 211, 213–214, 218, 229–230, 256–260 Lumbreras, V., 300 Luna, C., 182 Lundqvist, M., 183–184 Lunkov, R.V., 172 Luo, D., 454, 463 Luo, L., 472 Luo, R., 472 Luo, X., 416 Lu, Q., 110, 116, 121, 123–124 Luque, C.J., 153 Luque, T., 153, 163–164, 177–178 Lu, S., 396, 462
520
AUTHOR INDEX
Luschnig, C., 357, 362–363 Lutes, J., 109 Luttge, U., 160, 163, 174–175, 183–184 Lutts, S., 7, 20, 127, 129, 164–166, 178 Lu, X.Y., 429, 461, 471 Lu, Z., 35–36, 38, 305 Lva, D., 219 Lv, D., 268 Lv, S., 412 Lycett, G.W., 456 Lynch, T.J., 225 Lyon, G.D., 472 Lyon, J.L., 202 M Maathuis, F.J.M., 70, 73, 165, 167, 170–171, 174, 176–177, 186, 418 Ma, B., 416, 448, 471 MacAlister, C.A., 75, 77 Mace, D., 275 Machado, E.C., 57 Machida, Y., 423 Mackay, A., 151 Mackay, I., 467 Macknight, R.C., 262, 279 MacRobbie, E.A.C., 451 Madgwick, P.J., 80 Madhani, H.D., 396 Madkour, M.A., 425 Maeda, H., 459 Maeda, S., 468 Maeda, T., 396 Maeda, Y., 262 Maeder, D., 329 Maehashi, K., 109 Maeshima, M., 69, 255, 409 Magee, L.J., 56 Maggio, A., 178 Magnani, F., 57 Magnone, M., 277–278, 291 Mahajan, S., 295, 300, 303 Mahall, B.E., 172, 367 Mahfouz, H.T., 425 Mahony, D., 336 Maitani, T., 53 Ma, J., 174 Majee, M., 119 Major, F., 472 Majumder, A.L., 112, 118, 129 Makam, S.N., 365 Maktabi, M.H., 70, 72, 217 Malacarne, G., 56 Malagoli, M., 178 Malamy, J., 275 Malcolm, C.V., 157 Maldiney, R., 273 Malenica, N., 357, 363 Ma, L.G., 68, 213, 215, 358, 389
Malik, V., 431 Malloch, A.J.C., 180 Maloof, J.N., 392, 396 Manavalan, L.P., 465 Manavella, P.A., 79 Mancuso, S., 44, 185 Mandal, A., 115 Mandava, C.S., 467 Mane, S.P., 467 Manfre, A.J., 329 Mansfield, J.W., 468 Mansfield, T.A., 179–180, 186 Mantovani, R., 423 Mao, G., 275, 465 Mao, J., 389–390 Mao, Z.H., 416 Ma, Q.-H., 428 Marchal, V., 389 Marchant, A., 362–363 Marcon, C., 267 Marco, S., 267 Marcotte, L., 274 Marcotte, W.R. Jr., 329 Marcum, K.B., 164–165, 181 Mare`, C., 3 Marenco, R.A., 63 Maria, V. K., 115, 129 Marin, E., 203, 253, 365 Marion-Poll, A., 202–203, 205–206, 212, 253 Marmiroli, N., 466 Maroco, J.P., 7, 51–52, 57, 64–65, 80, 107, 294, 407, 409 Maroti, I., 325 Marquardt, S., 360, 363 Ma´rquez, J.A., 214, 259, 451, 471 Marrocco, K., 386 Marsch-Martinez, N., 422, 471 Marsh, E.L., 42, 428 Marten, I., 72–73, 222, 231–232, 268, 457, 471 Martina, B., 107–108 Martin, B., 412 Martin, D.J., 78 Martinelli, T., 328 Martı´nez-Andu´jar, C., 7, 20 Martinez-Atienza, J., 418 Martinez, C.A., 458 Martinez-Canellas, S., 53 Martinez, J.P., 126, 128, 178 Martinez-Santos, P., 50 Martinez, V., 167 Martin, G., 77, 424 Martinoia, E, 254 Martinoia, E., 68–69, 73, 209–211, 255–256, 279, 457, 471 Martins, A., 298 Martre, P., 60 Marur, C.J., 411
AUTHOR INDEX Maruyama, K., 68, 79, 109, 115, 205, 216, 220–221, 224–225, 230, 263, 270, 299, 356, 386, 393, 421, 425, 448, 458, 460–461, 468, 470 Maser, P., 166 Masia, A., 54 Masle, J., 75 Maslenkova, L., 325 Mason Pharr, D., 412 Ma´s, P., 216, 379, 386–388, 394 Ma, S.S., 110, 127, 153, 409, 468 Massai, R., 14, 52 Masson, P.H., 356, 362–363, 365 Masters, D.G., 156 Mastrangelo, A.M., 3, 467 Masuda, H., 363 Mata, C.G., 454 Mateos-Naranjo, E., 153, 163–164, 177–178 Matilde Paino, D.U., 110, 130 Matoh, T., 174 Matos, A.R., 354 Matos, M.C., 65 Matschi, S., 72–73, 222, 231–232, 268, 457 Matsuda, F., 110 Matsuda, O., 69, 231, 255, 266 Matsui, A., 294–295, 299, 463 Matsui, M., 468 Matsukura, S., 109 Matsumoto, C., 205–206 Matsumoto, K., 396, 423, 449 Matsuoka, D., 386 Matsuoka, K., 256 Matsuoka, M., 468 Matsuo, S., 388 Matsushika, A., 386, 393 Matthias, S., 107–108 Matysik, J., 114, 122 Mauch, F., 273 Maudoux, O., 267 Mauleon, R., 472 Maurel, C., 36, 60 Maurel, K., 60 Ma, W.Y., 167, 227 Maxwell, B.B., 78 Maxwell, D.P., 306 Ma, Y., 68, 71–72, 211, 213–214, 218, 229, 256–260, 280 Mayama, T., 366 Mayer, C.S., 115, 124 Mayer, F., 109 Mayer, U., 360, 363 Mayo, G.M., 17, 56, 419, 456 May, S.T., 362 Ma, Z., 471 Mazzucotelli, E., 3 McAbee, J.M., 75 McAinsh, M.R., 451 McArthur, C., 276, 430–431 McCann, M.C., 322
521
McCarty, D.R., 203, 206–207, 253, 274, 461, 467 McClung, C.R., 385 McCord, J.M., 296 McCourt, P., 68, 70, 213, 253, 276, 429–431 McCully, M., 10, 186 McCully, M.E., 56 McDonald, G.K., 22–23, 53 McDonnell, E., 158 McDowell, N., 35 McHughen, A., 416 McIntosh, L., 306 McKee, J.M., 471 McKee, J.M.T., 427 McKersie, B., 417 McKersie, B.D., 416–417 McNeil, S.D., 116 McQueen-Mason, S.J., 323, 448 McWatters, H.G., 379, 384–386 Medina, E., 178 Medrano, H., 8, 53–54, 57–58, 61, 64, 80, 409 Meeks-Wagner, D.R., 379, 385 Megdiche, W., 183–184 Mehta, S.K., 114, 119 Mei, C., 216, 261, 280 Meijer, A.H., 327 Meimoun, P., 264 Meinhard, M., 219, 269, 305 Melamed-Book, N., 304 Melcher, K., 72, 214 Melgar, J.C., 52 Meller, A., 457 Mellor, R.B., 116 Mendel, R.R., 205 Mendes, P., 108 Mendgen, K., 357, 363 Mendham, N.J., 169 Mendoza, I., 418 Mendoza, M., 420 Mendu, V., 462 Meng, X.-P., 421 Menzel, H., 42 Menzies, N.W., 22, 24 Menz, M., 466 Merewitz, E.B., 428 Merilo, E., 109 Merlot, S., 55–56, 69, 72–73, 218, 220–221, 229–231, 252, 262–263, 267–268, 270, 305, 450–451, 457, 469 Meshi, T., 79 Meskiene, I., 216, 273 Messdaghi, D., 212, 253 Messeguer, R., 458 Meurer, J., 55 Meyerhoff, O., 73 Meyer, K., 260 Meyerowitz, E.M., 250, 356 Meyer, P., 295
522
AUTHOR INDEX
Meyer, R.C., 107–108 Meyer, S., 61–62, 457, 471 Miao, C., 70, 219, 268 Miao, G.H., 113, 122 Miao, Y., 219, 268 Michaels, S.D., 227 Michalowski, C.B., 299, 452, 458 Michard, E., 265 Michen, V., 322 Michniewicz, M., 363 Mickelbart, M.V., 110, 130, 429 Micol, J.L., 203, 253 Miezan, K., 11 Miginiac, E., 273 Mikami, K., 451 Milborrow, B.V., 202 Millar, A.H., 306, 470 Millar, A.J., 379, 384–386, 389 Miller, A.J., 169, 185 Miller, G., 38, 121, 123–124, 182, 295–296, 300–301, 304–306, 337, 417 Miller, N.J., 334 Millgate, A., 67 Millo, E., 277–278, 291 Milnamow, M., 379 Mimura, M., 409 Mimura, T., 409 Minami, H., 220, 225, 230, 269 Minchin, F.R., 469 Ming-an, S., 300 Ming-Yi, J., 301 Minorsky, P., 277 Mishler, B.D., 320 Mishra, S., 122 Mistrik, I., 253 Misyak, S.A., 277 Miszalski, Z., 183–184 Mita, S., 227 Mitchell, R.A.C., 80 Mitchell, V.J., 64 Mithfer, A., 125 Mitros, T., 462 Mitsuhashi, W., 207–209, 253, 274 Mitsuya, S., 115 Mittal, A., 336 Mittler, R., 36, 38–39, 52–53, 108–110, 182–183, 280, 295–296, 300–301, 303–306, 337, 407, 417, 428, 461 Mittova, V., 39 Mittra, B., 178 Miura, K., 225 Miura, M., 409 Miura, S., 392, 458 Miura, T., 225 Miwa, K., 383 Miyaji, T., 68, 210–211, 254 Miyakawa, T., 72, 214, 232, 259–260, 471 Miyake, H., 72, 231, 268 Miyamoto, N., 367
Miyao, M., 81 Miyashita, K., 53–54 Miyata, K., 378–396 Miyata, S., 298 Miyauchi, Y., 72, 214, 259–260, 471 Miyazaki, J., 11, 18 Miyazaki, S., 272, 299 Miyazawa, Y., 349, 355, 358–361, 367 Miyazono, K.I., 72, 214, 259–260, 471 Mizoguchi, M., 216, 221, 229–230, 262–264, 386–388 Mizoguchi, T., 72, 220, 224, 263, 378–396, 449, 451 Mizoi, J., 270, 386, 393, 468 Mizuguchi, T., 263 Mizuno, H., 355, 357 Mizuno, T., 383, 386, 393 Mizutani, M., 205–206 Mochizuki, N., 215 Mochizuki, Y., 299, 463 Mockler, T.C., 280 Moeder, W., 453 Moes, D., 68, 72, 211, 213–214, 216, 218, 229, 256–260, 272 Moffatt, B.A., 77, 299, 467 Mohammadi, M., 465, 467 Mohanty, A., 115 Mohanty, P., 114–115, 122 Molas, M.L., 366 Moldau, H., 231, 266 Molina, C., 467 Molinari, H.B.C., 411 Møller, I.S., 17, 55–56, 418–419 Molloy, M.P., 335 Mongrand, S., 225–226 Monneveux, P., 466 Monnier, C., 109, 128 Monroe-Augustus, M., 273 Monroy, A.F., 299 Montagu, M., 458 Montanari, O., 55 Monte, E., 393 Montesinos, C., 420 Montgomery, L.T., 451 Moon, D.H., 467 Mooney, H.A., 408 Moon, H., 303 Moon, J., 219 Moore, C.A., 452 Moore, C.D., 452, 461, 468, 471 Moore, J.P., 109, 116, 320, 322–323, 325–327, 334, 337, 353 Moore, R., 364 Moparthi, V.K., 456 Morabito, D., 298 Moradi, F., 66, 114 Moreira, F.M., 56 Moreno, V., 420 Moreschi, I., 277
AUTHOR INDEX Morgan, J.M., 409 Morgante, M., 467 Moriguchi, K., 336 Mori, I.C., 70, 217, 221–222, 256, 268, 296, 304, 306, 450 Morillas, M., 298 Morillon, R.L., 36, 60, 299 Mori, M., 76, 468 Morino, K., 156 Morishige, D.T., 299 Morishita, Y., 109, 205, 470 Morison, J.I.L., 50, 61, 63–64 Moritz, T., 276 Moriwaki, T., 349, 358–359, 361 Moriyama, Y., 68, 210–211, 254 Morohashi, K., 355 Moroney, J.V., 59 Morosawa, T., 299, 463 Morreel, K., 459 Morris, B., 379 Morris, J., 420, 451 Morris, P.-C., 216, 260, 396 Morse, M.V., 319 Morsomme, P., 42, 267 Morsy, M.R., 306 Morvan, C., 322 Moshelion, M., 426 Mosher, S., 453 Moskal, W.A., 456 Moskovitz, J., 459 Motchoulski, A., 365–366 Motohashi, T., 417 Motte, P., 229 Mott, K.A., 60–64 Mouillon, J.M., 455 Mouline, K., 69 Moulinier-Anzola, J.C., 357, 363 Mouradov, A., 387–389 Moureaux, T., 305 Mousavi, A., 323 Mowla, S.B., 326–327, 455, 458 M’Rah, S., 183–184 Mtwisha, L., 328 Muck, A., 125 Muday, G.K., 357, 363–364 Mueller-Cajar, O., 80 Mueller-Roeber, B., 69, 256, 265 Mu¨hling, K.H., 42 Muir, J.F., 407 Mulako, I., 326–327 Mulholland, B.J., 427, 471 Mullen, J.L., 365 Mullen, M.A., 472 Muller, A., 55, 252–253, 267, 274, 363 Muller, B., 8, 352, 469 Muller, J., 111, 117 Muller, P., 360, 362–363 Muller-Rober, B., 179, 451 Mu¨ller-Uri, F., 453
523
Mullet, J.E., 299, 351, 354, 456, 466 Mullineaux, P.M., 50, 55, 70, 278 Mumm, P., 72–73, 222, 231–232, 268, 457, 471 Mundree, S.G., 323, 325–329, 331, 333–334, 336, 458 Mundy, J., 55, 327, 329, 337, 453, 455 Munekage, Y., 55 Munemasa, S., 70, 221–222, 224, 256, 273 Munne-Bosch, S., 326 Munnik, T., 357, 452 Munns, R., 1–11, 14, 16–23, 25–26, 36, 38–39, 51, 65, 106, 113–114, 208, 301, 407–410, 412, 448, 468 Munns, R.E., 153, 157, 171–172, 177, 186 Munoz-Mayor, A., 17 Murakami, M., 383 Murakeozy, E.P., 110, 119, 126, 128 Muramoto, Y., 115–116 Murata, M., 225, 230, 269 Murata, N., 110, 114–116, 412–413 Murata, Y., 73, 217, 219, 221–222, 256, 264, 268, 301, 304–305, 450 Murchie, E., 55, 263 Murdoch, C.L., 164–165, 181 Murelli, C., 328 Mur, L., 227 Mu, R.L., 365 Murphy, A.S., 279, 365 Murray, J., 55 Mustilli, A.-C., 55, 69, 72, 220, 252, 263, 268, 305, 450 Mustroph, A., 467 Muto, S., 166 Myasoedov, N.A., 171–172, 176 Myers, P.N., 456 Myouga, F., 216, 221, 229–230, 262–264 N Nachit, M.M., 446, 469 Nadeau, J.A., 75 Nadolska-Orczyk, A., 19 Nagamiya, K., 417 Nagamune, K., 277 Nagasawa, T., 69, 231, 255, 266 Nagata, K., 232 Nagatani, A., 215 Naghavi, M.R., 465 Nagpal, P., 384–386, 389 Nagy, F., 78, 379, 394 Nagy, R., 73, 256 Nagy, Z., 110, 119, 126, 128 Nahal, H., 468 Nahm, B.H., 74, 117, 414–415, 421, 472 Naidoo, G., 164 Naidu, S.L., 80 Nair, A., 327, 329, 458 Naito, S., 112, 118
524
AUTHOR INDEX
Nakabayashi, K., 205–206 Nakagawa, A., 420 Nakagawa, M., 214, 383, 386, 388–391, 395 Nakagawa, Y., 356 Nakahara, T., 159–160, 163, 165, 181 Nakajima, M., 68, 77, 299, 352, 452, 458, 460, 463 Nakajima, N., 70, 266 Nakaji, T., 70, 266 Nakamichi, N., 393 Nakaminami, K., 207–209, 253, 274 Nakamura, K., 227 Nakamura, T., 115–116, 166, 419, 423 Nakamura, Y., 121 Nakano, A., 360, 363 Nakao, K., 417 Nakasako, M., 386 Nakashima, A., 419 Nakashima, K., 115, 124, 216, 221, 232, 263, 386, 393, 425, 460–461, 468 Nakasone, S., 216, 221, 263 Nakayama, H., 420 Nakayama, M., 358 Nakazono, M., 207 Namara, E., 253 Nambara, E., 202–203, 205–207, 209, 212, 253, 274, 299, 463 Nam, H.G., 379, 386, 396 Nam, J., 303 Namuth-Covert, D.M., 467 Nanjo, T., 68, 77, 115, 299, 352, 452, 458, 460 Nano, R., 277 Nara, M., 232 Naramoto, M., 69, 205 Narasimhan, M.L., 167, 170, 173 Narasimhan, R., 452 Nardini, A., 64 Narusaka, M., 68, 77, 115, 169, 183, 227, 299, 352, 452, 458, 460, 462 Narusaka, Y., 169, 183, 227, 299–300, 386, 393, 462 Nassery, H., 177 Natanson, L., 472 Nataraja, K.N., 116, 422, 471 Natsui, Y., 378–396 Naumova, T.G., 176 Navari-Izzo, F., 65, 328, 334, 354 Navarro, S., 431 Naya, L., 469 Neale, A.D., 320, 323, 327, 329–330 Neales, T.F., 153, 164 Nefissi, R., 378–396 Negi, J., 55, 69–70, 231, 255, 266 Negishi, H., 355 Neill, S., 301 Nejad, A.R., 63 Nejad, R.A.K., 178 Nejedla´, E., 366
Nelsen, C.E., 115 Nelson, D.C., 68–69, 213, 215, 262 Nelson, D.E., 118–119, 126, 157–158, 160, 163, 181, 409, 423, 461 Nemchinov, L.G., 185 Nemhauser, J.L., 229, 429 Nepolean, T., 466 Netondo, G.W., 66 Neubert, A., 39 Neuhaus, H.E., 471 Neumann, P.M., 33, 35–36, 38–39, 41–44 Neutze, R., 456, 471 Neves, L., 298 Nevo, E., 464 Newman, I.A., 169 Newton, R.J., 118 Ng, C., 228 Ng, L.M., 72, 214 N’Guyen, A., 119 Nguyen, B.D., 466 Nguyen, D., 363 Nguyen, H.T., 42–43, 113, 253, 465–466 Nguyen, J.T., 388 Ngwenyama, N., 76 Nichols, P.G.H., 157 Nicolas, C., 217 Nicole, G., 116 Nicoll, B.C., 355 Nicotra, A.B., 9, 35, 39 Nielsen, B.H., 327, 329, 337 Nielsen, E., 115, 121 Nielsen, M., 453 Niemietz, C.M., 60, 426 Nieto, B., 459 Nieves-Cordones, M., 167 Niewiadomska, E., 183–184 Nie, Y., 68, 213, 215 Ni, F.T., 55, 216 Niinemets, U., 57–58 Niinuma, K., 386, 388–391, 395 Nill, C., 386 Nilson, S.E., 66, 69, 73, 75 Nilsson, O., 388 Nilsson, P., 466, 469 Ni, M., 386 Nimmo, H.G., 222, 383 Ning, J., 423, 449 Niogret, M.F., 106, 108–110, 127–128, 130 Nippert, J., 53 Nishimura, M., 264 Nishimura, N., 68, 70, 72–73, 208–209, 211–214, 217–218, 221, 227, 229–231, 256–260, 264, 266, 305, 453, 462, 471 Nishri, Y., 304 Nissan, H., 426 Nito, K., 70, 72, 214, 230 Niu, X.-G., 419, 454, 463 Niu, X.M., 167, 170, 173
AUTHOR INDEX Niwa, Y., 336 Niyogi, K.K., 55 Ni, Z., 462–463 Noaman, M.M., 17 Nobel, P.S., 60, 158 Noble, A.M., 281 Noctor, G., 65, 107, 183, 268, 300–301, 305, 326 Nodine, M.D., 224 Noh, E.W., 254 Noh, H.N., 460 Nolte, K.D., 116 Nomoto, S., 423 Nomura, M., 81 Nookaraju, A., 39 Norby, R.J., 80 Nordborg, M., 250 Nordeng, T.W., 327 Norman, H.C., 156 North, G.B., 60 North, H.M., 55, 69, 203, 206, 252, 263 Norwood, M., 328 Nose, A., 159–160, 163, 165, 181 Nothnagel, E.A., 303, 459 Nott, A., 280 Noutoshi, Y., 300 Nova-Franco, B., 414 Nublat, A., 166 Nuccio, M.L., 116 Nuin, P., 77, 299, 467 Nunes, C.C., 55, 458 Nunes-Nesi, A., 451 Nurnberger, T., 224, 258, 273 Nusinow, D.A., 379, 386, 396 Nussaume, L., 203 O Oba, K., 39 Obata, T., 470 Oba, Y., 69, 231, 255, 266 Obendorf, R.L., 328–329 Ober, E.S., 23 Oberschall, A., 458 Oda, A., 383, 386, 388–391, 395 Oda, K., 468 Oda, S., 467 Oecking, C., 267 Oelmuller, R., 55 Oesterhelt, C., 109 Offermanns, S., 261 Ogasawara, Y., 232 Ogata, Y., 205, 470 Oguri, Y., 115–116 Ohashi-Ito, K., 75 Oh, D.H., 167 Ohgishi, M., 366 Ohigashi, H., 205–206 Ohkawa, Y., 417
525
Ohkoshi, Y., 386 Ohme-Takagi, M., 225 Ohnishi, M., 409 Ohnishi, N., 114 Oh, S.H., 457 Oh, S.J., 117 Oh, S.-J., 414–415, 421, 428 Ohta, D., 205–206 Ohta, M., 76, 170, 175, 218, 271, 419 Ohto, C., 451 Ohto, M., 227 Oh, T.R., 227 Oiki, S., 166 Okada, K., 355, 358–361, 364–366 Okamoto, M., 52, 205–207, 209, 212, 299, 463 Oka, T., 357 Okawa, K., 76 Okazaki, Y., 409 Okushima, Y., 363 O’leary, J.W., 157, 164, 173–175, 177–178 Oliva, M., 361 Olivares, J., 116 Oliveira, M.M., 53, 77, 295 Oliveira, R., 298 Oliver, D.J., 306, 461 Oliver, F., 107–109 Oliver, M.J., 320, 333 Olson, K.J., 472 Olvera-Carrillo, Y., 455 O’Mahony, P., 320 Omery, B., 464 Omosegbon, O., 464 Onai, K., 227 Onelli, E., 271 Onodera, C., 363 Ono, K., 60, 81 Onouchi, H., 379, 387–388, 390 Onyango, J.C., 66 Ooba, A., 355 Oono, Y., 77, 115, 206–207, 299, 352, 452, 458, 460–461, 471 Oreb, M., 205 Oren, R., 35 Oritani, T., 205 Oron, E., 305 Oross, J.W., 159 O’Rourke, S.M., 297 Orsini, F., 110, 130 Ort, D.R., 62, 65, 80 Ortis, F., 281 Ortizlopez, A., 62 Osakabe, Y., 224, 386, 393, 458, 460–461, 468 Osborne, C., 109 Osmond, C.B., 62 Oso´rio, J., 67, 447 Oso´rio, M.L., 52, 64, 294, 447 Osswald, W., 324
526 Otoni, W.C., 458 Ottenschla¨ger, I, 362 Ottmann, C., 267 Otto´ T., 107–108 Ottow, E.A., 23, 299 Ouellet, F., 459 Ouerghi, Z., 175, 177, 183–184 Ouwerkerk, P.B.F., 327, 363 Ouyang, J., 464, 468 Ouyang, S.Q., 416, 448 Overbeek, J.H.M., 170, 174, 186 Overmyer, K., 55, 70 Overney, S., 415 Overvoorde, P.J., 363 Ovnat, L., 466 Owens, T.G., 117, 414 Ownbey, R.S., 172 Owttrim, G.W., 227 Oxborough, K., 56, 67 Oyama, T., 383 Oyanagi, A., 355 Ozaki, K., 115 Ozawa, K., 417 Ozdemir, F., 293, 296 Ozfidan, C., 296 Ozias-Akins, P., 412 ¨ zkum, D., 109, 128 O Ozkur, O., 296 Ozturk, N.Z., 467, 469 Ozturk, Z.N., 452, 458 Oztur, Z.N., 299 P Pacheco, M., 421 Paciorek, T., 357, 363 Paez-Valencia, J., 420 Paganga, G., 334 Page´s, M., 300, 330, 458 Pagter, M., 178 Paleg, L.G., 114 Pallas, J. A., 125 Pallotta, M., 18 Palme, K., 209, 357, 362–363 Palmgren, M.G., 169, 223, 266–267 Palombo, D., 277–278, 291 Palta, J., 13 Palutikof, J. P., 294 Palva, E.T., 115, 218, 472 Pammenter, N.W., 320–325, 328–329 Panda, S., 384–385 Pandey, A., 335 Pandey, G.K., 223, 303 Pandey, S., 68–70, 213, 215, 228, 262 Pandolfi, C., 185 Pang, C.H., 183–184 Pang, J.Y., 155 Pang, Y., 72, 214, 259–260 Panigrahi, K.C., 389
AUTHOR INDEX Pankovic, D., 65 Pan, S., 228 Pantin, F., 8, 352, 469 Pantoja, O., 163–164, 167–170, 173–176, 181–182, 186 Pantschitz, E., 80 Pan, X.P., 462 Papaefthimiou, D., 271 Papageorgiou, G.C., 114 Papdi, C., 20, 55 Papenbrock, J., 173 Papp, I., 227 Parcy, F., 225, 394 Pardo, J.M., 4, 14, 17–18, 172, 300, 409–410, 418, 431 Parent, B., 24, 456 Parida, A.K., 110, 113, 178, 183–184 Park, C.M., 227 Park, E.J., 114–116 Parkes, K.E., 278 Park, G., 418 Park, H.K., 222, 225, 428 Park, J.H., 222, 225 Park, O.K., 335 Park, S.-C., 299, 423 Parks, G.E., 175–176 Park, S.-H., 214, 223, 230, 420, 428, 451, 460, 468, 471 Park, S.J., 223 Park, S.W., 39 Park, S.Y., 68, 70, 72, 211, 213–214, 218, 229–230, 263, 269, 301, 451, 471 Park, S.-Y., 256–260 Park, Y.-T., 423 Parry, G., 363 Parry, M.A.J., 64–65, 80, 409 Parsons, D., 11, 18, 53 Parsons, H.L., 306 Parvanova, D., 114 Parvez, M.M., 225 Pasapula, V., 420 Pascaud, F., 265, 453 Pashkovskii, P.P., 185 Passarinho, J.A., 109 Passioura, J.B., 2, 12, 19, 21, 113, 294 Patakas, A., 8 Pate, J.S., 11 Patel, R.K., 337 Paterson, A.H., 447, 462, 465–466, 469 Patra, B., 119 Patrignani, G., 271 Paul, M.J., 65, 117, 414 Pauwels, L., 225–226 Pavlovkin, J., 253 Pawlikowska, K., 272 Peak, D., 60, 63 Pearce, R.S., 352–354 Pearcy, R.W., 52, 54, 408 Pechter, P., 109
AUTHOR INDEX Pedmale, U.V., 366 Pedras, M.S., 110 Peer, W.A., 365 Peeters, A.J.M., 205 Peeva, V., 58, 325 Peisker, M., 62, 253 Pei, Z.M., 70, 73, 217, 268, 296, 301, 304–306, 429, 450 Peleg, Z., 405, 428 Peli, E., 325 Pellegrineschi, A., 421 Pelletier, G., 109 Peltier, G.A., 458 Pen˜a-Cabriales, J. J., 116 Pence, H.E., 429 Penfield, S., 394 Peng, C.-C., 68, 213, 215, 218, 260–261 Peng, H., 462–463 Peng, J., 276 Peng, M., 463 Peng, X., 118 Peng, Z.Y., 110, 121, 123–124, 469 Pen, J., 117 Penney, J., 203, 209, 253, 274 Percey, W., 155 Pereira, A., 277, 422, 468 Pereira, A.C., 471 Pereira, J.S., 7, 51–52, 57, 64–65, 67, 80, 107, 294, 407, 409, 447 Perera, I.Y., 362, 452, 461, 468, 471 Perera, L.K.R.R., 179–180 Perez, A.C., 225–226 Pe´rez-Alfocea, F., 7–8, 20 Perez-Arellano, I., 120 Pe´rez-Arnedo, R., 116 Pe´rez, B., 462 Perez-Martin, A., 58 Perez, P., 461 Pe´rez-Pe´rez, J.G., 54 Perfus-Barbeoch, L., 69, 75, 215, 256 Pernas, M., 219 Perrier, A., 67 Perrone, I., 8 Perrot-Rechenmann, C., 362 Perruc, E., 453 Perry, P., 357, 363–364 Pervent, M., 8, 352, 469 Pesaresi, P., 55 Peters, C., 452 Peters, D.W., 157, 165 Peterson, F.C., 214 Peterson, J.Q., 63 Peters, S., 328 Petra´sek, J., 209 Peynot, P., 270 Pfanz, H., 62 Pfeifhofer, H.W., 109 Pham-Thi, A.-T., 354, 451 Pham, T.T., 472
527
Phang, J.M., 121 Phelps, S.D., 156–157 Phillips, G.N., 214 Phillips, J.R., 253, 295, 323, 327, 330–331, 333–334, 336, 464 Piao, H.L., 212, 223, 253 Pical, C., 451 Pierik, R., 52 Pieruschka, R., 63–64 Piette, L., 55, 267, 457 Pihlaja, K.K., 327 Pileggi, M., 411 Pilgrim, M., 77, 424 Pillitteri, L.J., 75, 218 Pilot, G., 265 Pin, A., 229 Pindo, M., 56 Pineda, O., 77, 424 Pinheiro, C., 7, 51–53, 64, 109, 294 Piqueras, A., 301 Piqueras, P., 203, 253 Piques, M., 8, 352, 469 Pitann, B., 42 Pitman, M.G., 162–164, 169 Pittman, J.K., 420, 451 Plancot, B., 322 Platten, J.D., 17 Plaut, Z., 355 Plaza, S., 69 Plesnicar, M., 65 Plett, D.C., 418–419 Plieth, C., 356 Pockman, W.T., 35 Pocs, T., 325 Podazza, G., 108 Poels, J., 117 Pogson, B.J., 306, 452, 468, 470 Pointer, S., 275 Poisson, G., 299 Pokala, N., 458 Poliakov, A., 462 Pollard, C.J., 328 Polle, A., 23, 299 Ponce, G., 359, 361 Ponce, M.R., 203, 253 Ponstein, A. S., 117 Pons, T.L., 57 Poole, D.S., 78 Poole, R.J., 176 Poolman, B., 299 Popp, M., 108, 113 Porat, R., 108 Poree, F., 69, 265 Pornsiriwong, W., 452, 468 Poroyko, V., 253 Portis, A.R., 55 Posas, F., 297–298, 396 Pose´, D., 459 Pospisilova, H., 7
528 Pospı´silova´, J., 60 Postaire, O., 36 Poston, J.M., 459 Potrykus, I., 39 Pottosin, I.I., 169, 185 Pou, A., 53, 58 Poustini, K., 9, 19–20 Powell, G.K., 365 Powell, J., 64 Powell, W.A., 467 Prado, C., 108 Prado, F.E., 108 Pramanik, M.H., 111, 116 Prasad, D.T., 303 Prashanth, S.R., 183–184 Pratt, L.H., 299 Premachandra, G.S., 127, 129 Pretty, J., 407 Price, A.H., 25 Prieto-Dapena, P., 458 Primavesi, L.F., 414 Prinsen, E., 459 Prins, H.B.A., 170–171, 174, 186 Prior, L.D., 11 Prior, R.L., 327 Prismall, L., 185 Pritchard, H.W., 324, 326, 334 Proctor, M.C.F., 325 Procunier, J.D., 469 Prodhan, S., 417 Provart, N., 179 Provart, N.J., 468 Pruneda-Paz, J.L., 386, 396 Pruvot, G., 458 Prytz, G., 63 Przyborowska, A.M., 337 Puigdomenech, P., 125 Pujal, J., 330 Pukacka, S., 326 Putterill, J., 379, 386–388, 396 Puzo˜ rjova, I., 109 Q Qin, C., 218 Qin, F., 356, 448, 458, 460–461, 468 Qin, X.Q., 203 Qin, Y., 267 Qi, Q., 448 Qiu, D.I., 58 Qiu, N., 163 Qiu, Q., 223, 267 Qiu, X., 420 Qi, Z., 356 Quach, H., 363 Quail, P.H., 379, 385–386, 393 Quan, R., 412–413, 428 Quan, R.D., 79 Quarrie, S.A., 466
AUTHOR INDEX Quartacci, M.F., 65, 354 Quatrano, R.S., 274, 455 Quero, A., 116–117, 414 Quesada, A., 203 Quettier, A.-L., 273 Quick, W.P., 57, 67, 81 Quigley, F., 167, 169, 174, 182 Quilici, D., 109 Quinet, M., 20 Quint, A., 363 Quintero, F.J., 18, 172, 418 Quirino, B.F., 448 Quiroz-Figueroa, F., 359 Quist, T.M., 55, 158, 169, 177–178 Qu, L.J., 456 Qu, R., 425 R Rabanal, F., 462 Rabbani, M.A., 68, 299 Rabello, A.R., 467 Rabenold, J.J., 366 Rachmilevitch, S., 305 Rademacher, T., 81 Radovic, S., 467 Radyukina, N.L., 185 Rafudeen, M.S., 319, 327, 335–336 Ragab, R., 408 Raghavendra, A.S., 72, 213, 252, 305–306 Ragot, M., 466 Rahman, A., 363 Rahmanzadeh, R., 464 Rahnama, A., 9, 19–20 Raibekas, A.A., 365 Rai, K.N., 11 Raines, C.A., 67, 80–81 Raitt, D.C., 297–298, 356, 448 Rajashekar, C.B., 109 Rakitin, V.Y., 185 Ramadan, A.M., 425 Ramakrishnan, N., 299 Ramalho, J.C., 65 Ramani, B., 173, 175, 177 Ramani, M., 64 Ramanjulu, S., 51 Ramati, A., 179 Rambo, R.P., 214, 259–260, 471 Ramirez, B.C., 453 Ramirez, V., 461 Rammesmayer, G., 112, 119 Ramos, J., 163–164, 469 Ranf, S., 180 Rangel, P.H.N., 467 Ranieri, A., 183–184 Ranish, J.A., 330 Ranjeva, R., 453 Ranney, T.G., 352, 354 Ranty, B., 453
AUTHOR INDEX Ranwala, A.P., 117, 414 Rascher, U., 253 Raschke, K., 66, 172, 180, 264 Rasgado, F.A., 359 Rasmussen, M., 327, 329, 337 Ratajczak, E., 326 Ratajczak, R., 163, 174–175, 183–184 Ratcliffe, O.J., 77, 424 Ratcliffe, R.G., 108 Rathinasabapathi, B., 111, 115–116, 124, 126–128 Ratnasekera, D., 452–453, 458 Ra¨tsch, G., 228, 463 Rausin, G., 229 Raveh, E., 184 Raven, J.A., 9 Ravenscroft, D., 388–389 Ravenscroft, N., 109, 323, 325–327, 334 Ravina, I., 38 Rawson, H.M., 10 Rayapati, P.J., 120–121 Rayle, D.L., 42 Ray, W., 115 Reader, S., 407–408 Rea, P.A., 176, 279 Rebetzke, G.J., 3, 12–14, 25, 53, 171, 408 Reddy, A.C., 39 Reddy, A.M.M., 299 Reddy, A.R., 299, 469 Reddy, D., 421 Reddy Lachagari, V.B., 299 Reddy, M.P., 178, 421 Redestig, H., 108 Redko, Y., 55, 228, 277 Redondo, E., 456 Redondo-Go´mez, S., 153, 163–164, 177–178 Reeck, T., 173 Reed, J.W., 363, 384–386, 389 Regalado, A.P., 56 Reguera, M., 405, 428 Reichert, D.L., 352, 354 Reichman, S.M., 22, 24 Reid, D., 328 Reid, R.J., 298 Reinhardt, D.H., 69, 158, 256 Reiser, V., 297–298, 356, 448 Reiter, I.M., 59 Remize, F., 297 Remorini, D., 52 Remus, D., 464 Renard, G.M.G.C., 322 Renaut, J., 54, 470 Ren, D.T., 184 Ren, G., 454, 463 Rengasamy, P., 3, 6, 22–24, 26, 53 Ren, H., 471 Ren, L., 462 Rensink, W.A., 299 Ren, X.Z., 70, 77, 461, 471
529
Ren, Z.H., 17 Repetti, P.P., 423, 461, 467 Repetto, O., 336 Rep, M., 297–298 Reuber, L., 77, 424 Reuling, G., 69, 260 Reuzeau, C., 424, 460 Reyes, J.L., 228, 455, 462 Reymond, P., 365 Reynolds, C.A., 278 Reynolds, M.P., 12–13, 25, 421, 464 Rey, P., 183, 458 Rhee, J.Y., 60 Rhodes, D., 19, 115, 119, 127, 129 Ribarits, A., 123 Ribas-Carbo, M., 53, 57–58 Ribaut, J.M., 466 Ricardo, C.P.P., 52, 64, 109, 294 Ricci, C., 320 Rice-Evans, C.A., 334 Richards, L.A., 408 Richardson, K., 379 Richards, R.A., 3–4, 12–14, 25, 53, 408 Rich, P.J., 19 Richter, A., 69, 328 Richter, J., 175 Richter, K., 258–259, 280 Richter, S., 360, 363 Riechmann, J.L., 77, 424 Rieger, M., 412 Riera, M., 228 Ries, A., 53, 82 Rigaud, J.-L., 267 Rigo´, G., 114, 121–123 Rios, G., 272 Rı´os, G., 420 Risk, J.M., 262, 279 Ritchie, S., 452 Rivandi, J., 18 Rivelli, A.R., 8 Rivero, R.M., 428 Rivoal, J., 111, 126–128 Rizhsky, L., 52, 305–306, 461 Rizvi, M.H.S., 466 Rizza, F., 3 Robatzek, S., 258 Robert, L.S., 78 Robert, N., 59, 72, 218, 221, 229, 262–263, 450–451, 469 Roberts, J.K., 329 Robinet, C.V., 467 Robinson, M.F., 179–180, 186 Robinson, S., 407 Robson, F., 387–388, 390 Rochon, Y., 330 Rock, C.D., 227, 295, 452, 462 Rockel, B., 175 Rodeghiero, M., 58 Røde, J., 456
530
AUTHOR INDEX
Rodermel, S.R., 67, 305 Ro¨ding, A., 325 Rodrigo, M.-J., 459 Rodrigues, A., 68, 72, 211, 213–214, 218, 221, 229–230, 256–260, 263, 269, 272, 450–451, 469, 471 Rodrigues, F.A., 467 Rodrigues, M.L., 52, 56, 64–65, 67, 294 Rodriguez, A., 262–263 Rodrı´guez-Acosta, A., 359 Rodriguez-Acosta, M., 466, 469 Rodriguez Egea, P., 468 Rodriguez-Navarro, A., 154, 166 Rodriguez, P.L., 72, 203, 208–209, 213–214, 217–219, 221, 229–230, 252–253, 259–260, 262–263, 269, 272–273, 427, 450–451, 469, 471 Roelfsema, M.R., 73 Roessner, U., 65, 107, 109–110 Rogers, A., 65, 80 Rogers, E.E., 43 Rogers, M.E., 157 Rogers, W.J., 164, 179 Rogniaux, H., 336, 455, 469, 471 Roig, L.A., 420 Rojo, E., 219 Rolland, F., 108 Rolland, G., 8, 352, 469 Romeis, T., 72–73, 222, 231–232, 268, 457 Romero, C., 117 Romero-Puertas, M.C., 185 Rona, J.-P., 264 Roncaglia, E., 467 Ronen, G., 304, 426 Ron, M., 106, 108–110 Rontein, D., 125, 132 Roosens, N.H., 121, 123 Rosado, A., 459 Rosales, R. O., 110, 127, 130 Rosa, M., 108 Rose, G.D., 113 Rosenow, D., 466 Rosenow, D.T., 466 Rosgen, J., 113 Rost, T.L., 158, 329 Roth, M., 452 Rotter, B., 467 Rouard, M., 472 Round, A., 214 Rouvier, E., 277 Roux, S., 453 Roy, C., 330 Roychoudhury, A., 126, 129, 330 Royo, C., 408, 431 Roy, S.J., 17, 419 Rozema, J., 172 Ruaha, J.P., 327 Ruan, K., 462 Ruberti, I., 327
Rubinsztein, D.C., 329 Rubio, F., 154, 166–167 Rubio, S., 218, 221, 229–230, 259, 262–263, 269, 272, 450–451, 469, 471 Rubio, V., 386, 389, 395 Rudolph, A., 328 Rughunanan, R., 164 Ruiz, M., 472 Ruiz-Sanchez, M.C., 54 Rumeau, D., 59, 458 Rumpho, M.E., 112, 118 Rupassara, S.I., 468 Ruppel, N.J., 365 Rus, A.M., 17, 420 Russell, B.L., 116 Russouw, P.S., 329 Ruzicka, K., 366 Ryel, R.J., 62 Rygol, J., 181 Ryu, J.C., 117 Ryu, M.Y., 463, 471 Ryu, S.H., 301 Rzepka, A., 57 S Saadaoui, D., 175, 177 Saad, A.S.I., 12 Saa, L., 458 Sabbagh, A., 466 Saborowski, J., 23 Sachetto-Martins, G., 280 Sachsenberg, T., 228, 463 Sack, F.D., 75, 362 Sade, N., 426 Sadhasivam, V., 184 Saez, A., 72, 217–218, 259, 272, 451, 471 Safwat, G., 419 Sage, R.F., 80 Sage, T.L., 459 Sagi, M., 205, 303, 305 Sahi, C., 468 Saibo, N.J.M., 49, 68, 77, 295 Saijo, Y., 423 Saint Pierre, C., 12 Sairam, R. K., 119, 126, 129 Saito, H., 297–298, 356, 396, 448 Saito, K., 110, 205, 393, 470 Saito, S., 205–206, 297 Saji, H., 70, 266 Saji, S., 70, 266 Sajnani, C., 53, 58 Sakac, Z., 65 Sakai, T., 365–366 Sakakibara, H., 393, 428 Sakamoto, A., 115–116 Sakamoto, H., 79 Sakashita, T., 355 Sakata, K., 205–206
AUTHOR INDEX Sakata, Y., 109 Sakr, S., 60 Sakuma, Y., 79, 109, 392, 458, 460–461, 468 Sakurai, N., 205, 470 Sakurai, T., 68, 77, 115, 169, 183, 229, 299, 352, 452, 458, 460 Salamini, F., 55, 327–328, 452, 459, 464 Salamo´, I.P., 20, 55 Sala, T., 69, 74, 448 Salazar-Blas, A., 359 Saleh, A., 53, 330 Saleh, O.M., 425 Salem, E., 183–184 Sales, E., 420 Salinas, J., 327 Salinas-Mondragon, R., 358 Salis, A., 277 Salisbury, J., 408 Salleo, S., 64 Salome´, P.A., 385 Salvador, A., 420 Salvador, N., 64 Salvador, S., 431 Salvatierra, G.R., 467 Salvatore, C., 115, 129 Salvucci, M.E., 80 Salzman, R.A., 167, 170, 173, 299 Samach, A., 379, 388–389 Samaha, R.R., 77, 424 Samis, K., 417 Sample, A., 276, 430–431 Samuelsson, G., 59 Samuels, T.D., 274, 364 Sanchez, A.C., 466 Sanchez-Bravo, J., 7 Sanchez, D.H., 65, 107, 109–110 Sanchez, F., 462 Sanchez, J.C., 335 Sanchez, S.E., 383 Sa´nchez-Serrano, J.J., 219 Sandalio, L.M., 185 Sandberg, G., 362–364 Sanders, D., 166–167, 179–180, 186, 300, 418, 422 Sanekata, T., 305 Sangwan, R.S., 116–117, 414 Sang, Y., 109, 389–390, 452 Sanjua´n, J., 116 Sano, H., 298, 448 Santiago, J., 68, 72, 211, 213–214, 218, 229– 230, 256–260, 272, 451, 471 Santner, A., 213, 226 Santos, T., 56 Santrucek, J., 60 Sanyal, S., 467 Saradhi, P.P., 119 Sarah, F., 109 Saranga, Y., 466 Sara, S., 110, 130
531
Sarda, X., 456 Sarhan, F., 115, 299 Sari-Gorla, M., 466 Sarkar, S., 329 Sarkeshik, A., 70, 72, 214, 230 Sarnowski, T.J., 272 Sarvas, C., 276, 430–431 Sasaki, R., 109 Sasaki, T., 256 Sato, A., 72, 231–232, 268 Satoh, R., 115, 124, 225, 298, 448 Sato, S., 356, 386, 393 Satou, M., 68, 77, 115, 169, 183, 224, 229, 299, 352, 452, 458, 460, 463 Satour, P., 455, 469, 471 Sato, Y., 72, 231, 268 Saunders, N.J., 78, 468 Sauter, A., 209, 212, 253 Savarti, E., 325 Save, R., 54, 330 Savio, C., 166 Savitch, L.V., 78 Savoure, A., 106, 108–110, 122–123, 127– 128, 130, 132, 301 Sawada, Y., 207 Sawa, M., 379, 386, 396 Sawano, Y., 72, 214, 259–260, 471 Sayama, H., 270, 468 Sayre, R.T., 114, 122 Scainelli, D., 121 Scandalios, J.G., 182, 301, 305–306 Scapim, C.A., 411 Scarascia-Mugnozza, E., 408 Scarfi, S., 277–278, 291 Scazzocchio, C., 205 Schachtman, D.P., 11, 23, 42, 186, 207–208, 298, 428, 461 Schaeffer, H.J., 78 Scha¨ffner, A.R., 36 Schaffrath, R., 70, 77 Schafleitner, R., 110, 127, 130 Scharlat, A., 453 Scharr, H., 63–64 Schat, H., 119, 122 Schauer, N., 108–109, 267 Scheel, D., 180 Scheibe, R., 67 Scheikl, E., 273 Schepens, I., 366 Scheres, B., 209 Scherzer, S., 72–73, 222, 231–232, 268, 457 Schiefelbein, J., 275 Schierholt, A., 39 Schindler, M., 472 Schjoerring, J.K., 73, 256 Schlauch, K.A., 109, 306 Schlereth, A., 363 Schlichting, C.D., 9 Schluepmann, H., 117
532
AUTHOR INDEX
Schmelzer, E., 114, 121–123 Schmidhalter, U., 6 Schmidt, A., 173 Schmidt, J.E., 53 Schmidt, S., 25 Schmidt, U.G., 327, 329, 331, 333–334, 336, 471 Schmitter, J., 230 Schmutz, J., 462 Schnable, P.S., 53 Schnall, J.A., 274 Schneebeli, K., 13, 25 Schnitzler, J.P., 459 Schnorr, K., 305 Scholander, P.F., 171–172 Schomburg, F.M., 227 Schorr, P., 448 Schoutteten, H., 276 Schreiber, L., 459 Schroeder, J.I., 14, 53, 55, 57, 59, 68, 70, 72–73, 82, 166, 208–209, 212–214, 217–219, 221–222, 227, 230–231, 256, 259–260, 264, 266, 268, 276, 296, 301, 304–306, 427, 429, 450, 471 Schroeder, S.G., 428 Schubert, A., 8, 60, 455 Schubert, S., 39, 42, 81 Schuch, W., 76 Schuller, A., 116–117 Schu¨ltke, S., 223 Schultz, T.F., 379, 386 Schulz, A., 223, 267 Schulz, B., 69, 223, 453 Schulze, E.D., 66–67, 80, 208 Schumacher, K., 221 Schumaker, K.S., 173–176, 222–223, 267, 294–295 Schuppler, U., 448 Schurr, U., 36, 63–64, 67 Schuster, I., 411 Schu¨tze, K., 115, 124 Schuurink, R., 273 Schwalm, K., 253 Schwanninger, M., 273 Schwartz, M.A., 396 Schwartz, S.H., 203, 305 Schwechheimer, C., 386 Schweighofer, A., 216, 273 Scippa, G. S., 271 Scolnik, P.A., 78 Scotland, R.W., 78 Scott, P., 328 Scott, T.K., 355–356 Seaman, R., 156 Searle, I., 388 Searle, L., 384 Seckin, B., 296 Sedbrook, J.C., 356, 362
Sederoff, H.W., 358 Sederoff, R., 467 Seeliger, M.A., 395 Seel, W., 324 Seffino, L.G., 182 Seki, B., 448 Seki, M., 36, 68–69, 77–78, 109, 115, 169, 183, 205–207, 209, 224–225, 227, 229, 294–295, 298–299, 352, 421, 449, 452, 458, 460, 462, 466 Sekmen, A.H., 293, 296 Selbig, J., 108 Selvaraj, G., 115 Semel, Y., 109 Semple, W.S., 157 Sengupta, D.N., 330 Sengupta, S., 112, 118–119, 127, 129 Senn, M.E., 166 Sentenac, H., 265, 453 Senzaki, E., 232 Seo, H.H., 222, 225, 228 Seo, H.-S., 117, 414–415, 464, 468 Seo, J.-S., 74, 117, 414–415 Seo, M., 203, 205–209, 212, 253, 274 Sepahi, M., 412 Serikawa, M., 383 Seri, L., 304 Serizet, C., 217, 262 Serraj, R., 11, 25, 421, 466 Serrano, R., 72, 117, 203, 253, 266, 420 Serret, M.D., 408, 431 Setter, T.L., 299 Sevilla, F., 183–184 Sewell, J., 225–226 Sezen, U., 447 Sgherri, C., 328, 334 Shabala, L., 169, 185 Shabala, S.N., 11, 18, 53, 114, 151, 153, 155, 164, 166, 169, 176, 185, 223, 267 Shachar-Hill, Y., 108 Shahak, Y., 60, 352, 426 Shailasree, S., 125 Shaked, R., 227, 392, 396 Shalata, A., 39 Shamsutdinov, N.Z., 171–172 Shamsutdinov, Z.S., 171–172 Shang, H., 323, 327, 331, 333–334, 336 Shang, M., 412–413 Shang, Y., 68, 213, 215–216, 218, 222, 260–261, 280 Shanmugasundaram, P., 466 Shannon, M.C., 408 Shannon, S., 379 Shao, H.B., 55 Sharkey, P.J., 153, 164 Sharkey, T.D., 51, 57–59, 62, 64, 66 Sharma, A., 466 Sharma, K., 411, 421 Sharma, P., 122
AUTHOR INDEX Sharma, S.S., 119, 122–123 Sharp, R.E., 4, 23, 42–43, 113, 252–253, 274, 363–364, 447–448 Shasha, D.E., 42 Shashidhar, V.R., 208 Shatil, A., 426 Shavrukov, Y., 11, 18 Shaw, R.G., 106 Sheen, J., 65, 107–108, 203, 209, 230, 253, 263, 269, 274, 423, 449 Shelden, M.C., 56, 456 Shen, A., 320, 324–331, 333 Shen, B., 166 Shen, G., 419–420 Sheng, Q.H., 335 Shengqiang, Z., 306 Shen, Q., 220 Shen, Y.G., 126, 128 Shen, Y.-Y., 68, 213, 215, 218, 260–261 Sherman, T.D., 334 Sherwin, H.W., 322, 325, 330 Shetty, S.H., 125 Sheveleva, E., 107 Shevyakova, N.I., 185 Shibahara, T., 305 Shibasaka, M., 60, 426 Shibata, D., 205, 470 Shibata, N., 214 Shi, H.H., 18, 51, 55, 158, 169, 172, 177–178, 272, 301, 303, 418 Shi, J., 299, 303 Shi, K., 64, 454, 459 Shikanai, T., 55 Shi, M., 70, 74, 430, 461 Shimada, T., 76 Shimamoto, K., 388, 423 Shimazaki, K.-I., 228, 264, 266 Shimizu, H., 54–55, 68, 210–211, 254 Shimizu, K., 55 Shimizu, Y., 159–160, 163, 165, 181 Shimoda, T., 159–160, 163, 165, 181 Shimura, Y., 361 Shin, D.J., 70, 224, 273, 303–304, 306 Shin, J., 79 Shinmyo, A., 420 Shinn, P., 294 Shinozaki, K., 36, 68–69, 72, 77–79, 109, 115, 124, 169, 183, 201, 203, 205–207, 210–213, 216–221, 224–227, 229–232, 254, 262–264, 268, 270, 294–295, 298–300, 356, 386, 392–393, 395–396, 409–410, 420–421, 425, 431, 448–449, 451–452, 456, 458, 460–462, 466, 468, 470 Shin, R., 186, 461 Shinwari, Z.K., 386, 393 Shirasawa, K., 115–116 Shirley, N., 419
533
Shiroto, Y., 431 Shitole, M.G., 122 Shono, M., 170, 175 Shope, J.C., 64 Shorina, M., 185 Shou, H., 423 Showalter, A.M., 125, 128, 156 Shpak, E.D., 75 Shriram, V., 122 Shukla, M., 299 Shukla, V.K., 388, 456 Shulaev, V., 106, 108–110, 182, 300–301, 306, 337, 428, 461 Shuman, J.L., 108, 461 Shyy, J.Y.-J., 299 Siahpoosh, M.R., 65, 107, 109–110 Sibley, L.D., 277 Sieberer, T., 357, 363 Siebke, K., 63 Siefritz, F., 60, 455 Siegel, R.S., 59, 73, 264, 305 Siemens, J.A., 60 Si, H., 278, 281 Sijmons, P.C., 76 Silk, W.K., 36, 447 Silva, M.J., 65 Silveira, J.A.G., 65 Simon, M., 109 Simonneau, T., 69, 166, 456 Simon, P., 415 Simon, W.J., 336 Simpson, G.G., 378 Simpson, S.D., 460–461, 468 Sim, S., 465 Singh, A., 415, 468 Singh, J., 78 Singh, S., 59 Singh, U., 299, 303 Singh, V., 119, 126, 130 Sioson, A.A., 299 Sirault, X.R.R., 6, 20–21, 23, 25 Sirichandra, C., 55, 73, 218, 221, 229, 231, 262–263, 267–268, 277, 305, 450–451, 469 Siripornadulsil, S., 114, 122 Sivaguru, M., 42 Sivamani, E., 425 Sivaprakash, K.R., 183 Sjødin, A., 466, 469 Sjolander, K., 472 Skerrett, I.M., 166 Skirycx, A., 35–36 Skirycz, A., 470 Skoczowski, A., 54 Skogstrøm, O., 466, 469 Skoneczka, J., 277 Skriver, K., 453 Skrumsager Møller, I., 419 Slabas, A.R., 336
534
AUTHOR INDEX
Slafer, G.A., 408, 431 Slavich, P.G., 11 Slesak, I., 184 Sloan, D.B., 75 Smalle, J., 226, 229, 394 Smart, L.B., 456 Smeeton, R.C., 427, 471 Smigocki, A.C., 7 Smillie, I.R.A., 427, 471 Smirnoff, N., 114, 183, 296, 324–325, 327 Smith, A.R., 109 Smith, C., 467 Smith, J.A.C., 174–175, 325, 330, 333 Smith, K.S., 59 Smith, M. T., 116 Smith, S.J., 169, 185 Smith, S.M., 452, 468 Snedden, W.A., 174, 419, 453 Snell, C.R., 278 Soffer, D., 305 Sofo, A., 54 Sokabe, M., 356 Sokolchik, I., 272 Sokolik, A., 185 Solano, R., 219 Solomon, K.S., 384–386, 389 Solomon, M., 304 Somero, G.N., 107, 110, 113 Somers, D.E., 379, 385–386, 388, 396 Sommarin, M., 266, 451 Sonderegger, J., 109 Song, C.-P., 70, 219, 223–224, 267–268, 271, 273, 301, 305, 463, 471 Song, G.Q., 118 Song, H.R., 386–388, 391 Song, L., 227, 306 Song, S.I., 74, 117, 414–415, 421, 463, 472 Song, W., 459 Song, Y.H., 396 Song, Y.W., 70 Son, O., 352, 463 Soon, F.F., 72, 214 Sopory, S., 421 Soppe, W.J., 228, 388–389 Sorrells, M.E., 469 Soto, M. J., 116 Sotta, B., 55, 69, 203, 206, 252, 263 Souyris, I., 466 Spaner, D., 466 Spannagl, M., 462 Sparstad, T., 115 Spector, D., 228–229 Speer, M., 53 Sperry, J.S., 35 Spielmeyer, W., 3, 12–14, 17, 25, 53 Spollen, W.G., 252–253, 274, 364, 467 Spreitzer, R.J., 80 Springer, G.K., 253 Sridha, S., 272, 461
Sriprang, R., 218 Sritubtim, S., 70, 224, 273 Staal, M., 170, 174, 186 Stacy, R.A.P., 327 Stadelhofer, B., 121 Stadtman, E.R., 459 Stamati, K., 467 Stanca, A.M., 3 Stanca, M.A., 271 Standaert, E., 35, 40 Stange, A., 72, 231, 268, 457 Staxen, I., 451 Stedingk, V., 267 Steed, A., 466 Steele, K.A., 3, 11, 407–408, 466 Stefanelli, D., 81 Stefano, G., 44 Stegalkina, S., 299 Steiner-Lange, S., 55 Steinfath, M., 108 Stelter, W., 160, 181 Stelzer, R., 162–164, 169, 173 Stepanova, A.N., 294, 470 Steponkus, P.L., 353–354 Stetsenko, L.A., 185 Stettler, R., 55 Steudle, E., 38, 68, 158, 207–209 Stevanovic, B., 65, 328, 334, 354 Stevenson, B., 227, 295, 300 Stevenson, D.K., 294 Stevenson-Paulik, J., 452, 461, 468, 471 Stewart, C.R., 119–121 Stiller, I., 117 Stitt, M., 8, 67, 107–108, 352, 469 Stockinger, E.J., 392 Stoecker, M., 431 Stolc, V., 78 Stone, S.L., 226 Stoop, J.M.H., 412 Storer, A., 393 Storey, J.D., 467 Storey, R., 11, 153, 162–165, 169 Stracke, B., 42 Strader, L.C., 366 Strait, A.A., 396 Strasser, B., 391 Straume, M., 385, 392, 396 Strauss, S.H., 388 Strays, C.A., 384–385 Streeter, J.G., 119 Street, N.R., 466, 469 Street, T.O., 113 Strizhov, N., 111, 114, 120–123 Strøm, A.R., 115 Stubbs, K.A., 459 Sturla, L., 277–278, 291 Stushnoff, C., 412 Sua´rez-Lo´pez, P., 387–388, 390 Suarez, N., 178
AUTHOR INDEX Sua´rez, R., 414–415 Suarez-Rodriguez, M.C., 327, 329, 337 Subramanian, S., 227, 462 Subudhi, P.K., 466 Suefferheld, M., 412 Sugano, S.S., 76, 384, 386 Suge, H., 355–357 Sugimoto, E., 68, 210–211, 254 Sugiyama, N., 216, 221, 229–230, 262–264 Sugliani, M., 228 Su, H., 167, 169, 174, 182 Suh, S.J., 69, 256, 379, 386, 396 Suino-Powell, K.M., 72, 214 Su, J., 115, 411 Sukumar, P., 363–364 Sullivan, J.A., 389, 395 Sulpice, R., 8, 107, 352, 469 Su¨mer, A., 39, 42 Sumner, L.W., 108 Sunagawa, H., 159–160, 163, 165, 181, 184 Sun, D.Y., 455 Sun, F., 364, 462–463 Sung, D.Y., 108, 116 Sung, S., 385–386 Sung, Z.R., 455 Sun, J., 226 Sunkar, R., 4, 107, 228, 295, 298, 407, 450, 454, 458, 462–463, 467 Sun, Q., 462–463 Sun, S., 429 Sun, W.Q., 328, 458 Sun, Y.H., 413, 455, 462 Sun, Z., 114, 127 Su, P., 39 Surowka, E., 183 Surpin, M., 280 Susanne, F., 108–109 Susek, R.E., 261 Sussex, I.M., 112, 118 Sussman, M.R., 222, 448, 450, 469 Su, W.-A., 417, 426 Su, Z., 468 Suzuki, H., 109, 205, 336, 470 Suzuki, I., 336 Suzuki, K., 232 Suzuki, N., 38, 295–296, 304–306, 461, 468 Suzuki, S., 383 Suzuki, T., 336 Svistunenko, D., 185 Swanson, S.J., 362 Swartz, T.E., 365 Swarup, R., 357, 363–364 Swiezewski, S., 272 Syono, K., 362, 366 Syvertsen, J.P., 54 Szabados, L., 20, 55, 114, 121–123, 132 Szekely, G., 114, 121–123 Szigeti, Z., 325
535
Szoke, A., 111, 120 Szostkiewicz, I., 68, 211, 213–214, 218, 229, 256–260, 280 T Tabata, S., 69, 205, 356, 386, 393 Taconnat, L., 459 Tagliavia, C., 55, 263 Tago, Y., 383 Tahtiharju, S., 218 Taiz, L., 209 Tajima, T., 383, 388–390 Taji, T., 69, 109, 127, 130, 169, 183, 205, 421 Tajkhorshid, E., 456, 471 Takabayashi, J., 75, 218 Takabe, T., 39, 115–116, 417 Takagi, H., 120–121 Takahashi, A., 355, 359–361 Takahashi, E., 174 Takahashi, F., 72, 220, 263, 300 Takahashi, H., 69, 207, 231, 255, 266, 354–361, 364, 367 Takahashi, M., 72, 214, 259–260, 471 Takahashi, N., 355–356, 358–361, 364 Takahashi, R., 169 Takahashi, S., 25, 55, 72, 220, 263, 451–452 Takahashi, Y., 380 Takahasi, Y., 325, 331 Takano, M., 356–357 Takano, T., 59, 169 Takasaki, H., 425 Takeda, K., 70, 224, 273 Takeda, M., 109, 205, 470 Takeda, S., 468 Takeda, T., 70, 266 Takekawa, M., 396 Takio, S., 293, 296 Talame, V., 299, 452, 458, 467 Taleei, A., 465 Taleisnik, E., 182 Tal, M., 39 Tamai, A., 76 Tamaki, S., 388 Tamaoki, M., 70, 266 Tama´s, M. J., 298 Tameda, S., 363 Tamura, T., 298, 448 Tanaka, A., 170, 175, 419 Tanaka, H., 209 Tanaka, K., 305 Tanakamaru, S., 53 Tanaka, N., 336 Tanaka, S., 109 Tanaka, Y., 206, 209, 295, 300, 417 Tanaoka, Y., 39 Tan, B.C., 203, 206–207, 253, 274 Ta¨ngemo, C., 298
536
AUTHOR INDEX
Tang, G., 462 Tang, H.-J., 411, 447 Tang, N., 425, 427 Tang, W., 118 Tang, X., 462 Tang, Y.Y., 116, 221 Tang, Z.-C., 53, 426, 454, 463 Taniguchi, M., 72, 231, 268, 357 Taniguchi, Y., 357 Tanimoto, E., 42–43 Tanksley, S.D., 465 Tan, M.P., 303, 306 Tanokura, M., 232, 259–260 Tan, W., 114, 116 Tan, Y.F., 306, 470 Tao, W., 467 Tarczynski, M.C., 118 Tardieu, F., 278, 456, 472 Tari, I., 127, 129 Tasaka, M., 363 Tatematsu, K., 212 Tatsumi, H., 356 Tattersall, E.A., 109 Tattini, M., 14, 52 Tavakkoli, E., 22–23, 53 Tax, F.E., 224 Taylor, C.B., 65 Taylor, G., 466, 469 Taylor, I.B., 427, 471 Teakle, N.L., 10–11, 18, 418 Teasdale, R., 336 Teeri, T.H., 115 Teichmann, T., 54, 299, 470 Teige, M., 273 Tejera, N.A., 111, 116 Temguia, L., 253 Temple, B.R.S., 215 Tena, G., 423 Tenlen, J., 327, 329–330 Teplova, I., 252–253, 274 Tepperman, J.M., 386 Terashima, I., 60, 62 Termaat, A., 6, 23 Tester, M.A., 9–11, 14, 17–18, 24, 36, 38–39, 51, 55, 109, 153, 159, 161, 164, 166, 169, 171–172, 185–186, 301, 407–409, 412, 419, 446, 452, 468 Teulat, B., 466 Tezara, W., 51, 64 Tham, F.S., 214 Theobald, J., 55 Theobals, J., 263 Theodorou, G., 385 Theodoulou, F.L., 455 Thevelein, J.M., 297–298 Thibaud, J.-B., 69, 265, 453 Thi Lang, N., 465 Thimann, K.V., 355 Thimmapuram, J., 455, 467
Thines, D., 267 Thomas, A., 107–108 Thomas, B., 387 Thomashow, M.F., 109, 392, 420, 468 Thomas, J.C., 160, 181, 412 Thomas, M., 222 Thomas, S.M., 407 Thomas, T.L., 379, 455, 458 Thomas, W.T.B., 25 Thomine, S., 73, 230, 268, 301, 304–305 Thompson, A.J., 427, 471 Thompson, R., 336 Thompson, T.L., 157 Thomson, J.A., 326–329, 331, 333–334, 336, 455, 458 Thomson, W.W., 159 Thongjuea, S., 472 Thorne, E.T., 252, 274, 448 Thorpe, P.C., 328 Thorsen, M., 298 Thorsson, V., 330 Thyssen, N., 50 Tian, W., 115–116 Tian, X., 228, 462 Tian, Y., 11, 18, 429 Tiedemann, J., 225 Tierney, M. L., 125 Tietz, O., 363 Tilleman, S., 225–226 Tillemans, V., 229 Tillett, R.L., 109 Tina, G., 449 Tincopa, L.R., 110, 127, 130 Tingay, S., 379, 391 Tinker, N.A., 78 Tin, T., 50 Tipirdamaz, R., 109, 128 Tiriac, H., 221–222 Tisdall, J.M., 10–11 Tissue, D., 305 Tiwari, A., 59 Tjaden, J., 471 To, A., 203 Tobin, E.M., 379, 384, 386, 391, 396 Tobita, S., 66 Todaka, D., 460 Todd, C.D., 121 Tognetti, V.B., 459 Toki, S., 81 Tokutomi, S., 386 Toldi, O., 328 Tomas, M., 53, 58 Toma´s, M., 53, 58 Tomozoe, Y., 386, 388–391, 395 Tompa, P., 455 Tondelli, A., 3, 271 Tonelli, C., 69, 74, 448 Tongprasit, W., 78 Tonnet, M.L., 10
AUTHOR INDEX Torii, K.U., 75, 218 Toriyama, K., 431 Torje´k, O., 108 To¨rnroth-Horsefield, S., 456, 471 To¨ro¨k, K., 458 Torok, Z., 455 To¨ro¨nen, P., 472 Torrecillas, A., 54 Torregrosa, L., 453 Torres, M.A., 70, 268, 296, 304, 306 Torres-Ruiz, R.A., 360, 363 Tosens, T., 58 To, T.K., 294–295, 299 Toulmin, C., 407 Tournaire-Roux, C., 36 Tousch, D., 456 Town, C.D., 55 Toyoda, T., 229 Toyomasu, T., 207–209, 253, 274 Toyooka, K., 256 Toyota, M., 356 Traas, J., 35 Tracer, C., 115 Traina, S., 114, 122 Traini, M., 335 Tramnitz, B., 42 Tran, D., 264 Tran, L.S., 448, 460–461, 465, 468 Tran L-S, P., 356 Tranzer, P., 363 Traversi, M.L., 14, 52 Travers, S.E., 53, 56 Tregeale, J.M., 10–11 Trentmann, O., 471 Trethewey, R. N., 108 Trevisan, M., 366 Trewavas, A.J., 299, 356, 452 Trijatmiko, K.R., 422, 471 Troggio, M., 56 Troke, P.F., 19, 165 Truesdale, M.R., 328 Truman, W., 468 Tsaftaris, A.S., 271 Tsakas, S., 116 Tschaplinski, T.J., 80 Tseng, M.J., 305 Tsuboi, A., 166 Tsuboi, Y., 52 Tsuchida, H., 81 Tsuda, S., 367 Tsuge, T., 357 Tsujimura, T., 409 Tsutsui, K., 115–116 Tsvetkov, T., 114 Tuba, Z., 110, 119, 126, 128, 320, 325 Tuberosa, R., 12, 299, 464, 467, 472 Tubrosa, R., 452, 458 Tucci, M.L.S., 57 Tucker, J., 466, 469
537
Tumarkin, R., 277 Tumimbang, E., 405, 428 Tunnacliffe, A., 329, 424 Tuominen, H., 55, 70 Turchi, L., 467 Turkan, I., 114, 293, 296, 301, 303 Turk, B.E., 270 Turnbull, C., 388 Turner, N. C., 113 Turpin, D.H., 64 Tuteja, N., 295, 300, 303 Tuzet, A., 67 Tyagi, A.K., 115–116, 119, 126, 129 Tyerman, S.D., 10–11, 18, 56, 60, 166, 418, 426, 456 Tyler, M.I., 335 Tyree, M.T., 60, 455 Tytell, M., 276 U Uchacz, T., 276, 430–431 Uchimiya, H., 69, 231, 255, 266 Uchiyama, Y., 262 Udayakumar, M., 116, 422, 471 Udupa, S.M., 467 Udvardi, M., 65, 107, 109–110 Udvardi, M.K., 55 Ueda, A., 163, 178 Ueda, T., 360, 363 Uehlein, N., 60 Uemura, M., 353–354 Ueno, O., 159–160, 163, 165, 181 Ulanov, A., 167, 467 Ulat, V.J., 472 Ul Haq, T., 3, 11 Ullah, H., 73 Umezawa, T., 68, 72, 109, 201, 206–207, 216, 220–221, 225, 229–232, 262–264, 268, 270, 294–295, 299, 409–410, 431 Undurraga, S., 420, 451 Unesco Water Portal, 294 Ungar, I.A., 156, 178 Uno, Y., 163, 178, 225 Unver, T., 462, 467 Uozumi, N., 72, 166, 231–232, 268 Urano, K., 36, 109, 205, 466, 470 Urao, S., 456 Urao, T., 298, 356, 448, 461, 468 Urban, L., 65 Usai, C., 277–278, 291 Uyttewaal, M., 35 V Vadez, V., 411, 421 Vahisalu, T., 231, 266 Valencia-Cruz, G., 185
538
AUTHOR INDEX
Valerio, G., 231 Valero, G., 266 Valkoun, J., 115, 129 Vallabhaneni, R., 468 Valladares, F., 52, 320, 336 Valliyodan, B., 43, 467 Valon, C., 55, 73, 267, 277, 394, 457 Valot, B., 55, 73, 230–231, 268, 305, 450 Valpuesta, V., 459 Valupesta, V., 453 Valverde, F., 387–390 Van Aken, O., 459, 470 Van Breusegem, F., 35, 40, 182, 296, 301, 303, 305, 470 Van Camp, W., 218 Vandame, M., 65 van de Cotte, B., 459 van de Cotte, V., 458 Vandeleur, R.K., 56, 60, 456 Vandenbroucke, K., 459 van den Dries, N., 464 Vanderauwera, S., 182, 296, 301, 303 Vanderbeld, B., 36, 39 Van der Does, C., 298 Van Der Kelen, K., 35, 40 van der Lee, F.M., 76 Vandermeeren, C., 267 van der Merwe, M.J., 451 van der Straeten, D., 68, 276, 364 van der Willigen, C., 323, 325–326, 333 Van Deynze, A., 428 van Dun, K., 117, 413–414 van Elteren, J., 20 van Herwaarden, A.F., 3 van Hintum, T., 472 Vani, G., 411 Vanlerberge, G.C., 306 van Meeteren, U., 63 Van Montagu, M., 111, 120, 300–301 Vanneste, S., 209, 363 Van Nguyen, D., 460 van Staden, J., 65, 107, 110, 113–114, 116, 125, 427 Van Volkenburgh, E., 38 van Voorst, F., 298 Vargas, M., 12 Varotto, C., 55 Varshney, R.K., 55, 115, 129 Vartanian, N., 217, 262, 274, 365 Vasquez-Robinet, C., 299 Vass, I., 458 Vavasseur, A., 55, 69, 72, 74–75, 220, 230, 252–253, 262–263, 267–268, 305, 450, 457 Vaya´, J.L., 117 Vazzana, C., 109, 328 Veierskov, B., 267 Velasco, R., 56, 327 Veljovic-Jovanovic, S., 328, 334
Vendruscolo, E.C.G., 411 Venisse, J.S., 60 Venuprasad, R., 466 Vera-Estrella, R., 163–164, 167–170, 173–176, 181–182, 186 Vera, P., 327, 461 Verbruggen, N., 111, 120, 122–124, 132, 300–301, 411, 458 Verdeil, J.L., 453 Verhey, S.D., 220, 230, 269 Verma, D.P.S., 110–111, 113–114, 120–124, 411 Vernieri, P., 60 Vernon, D.M., 160, 181 Verrier, P.J., 279 Verslues, P.E., 23, 123, 220, 227, 264, 296, 303 Vertucci, C.W., 320–321, 324, 328–329 Verwoerd, T. C., 117 Very, A.A, 69 Very, A.-A., 179–180, 186, 265 Vezzulli, S., 56 Viant, M. R., 108 Vicente-Carbajosa, J., 115, 124, 225 Vicre´-Gibouin, M., 322, 353 Vicre´, M., 321–323 Vidal, G., 264 Vidal, S., 20, 55 Vie´gas, R.A., 65 Vieira, L.G.E., 411 Vierling, E., 328, 458 Vierstra, R.D., 226, 229 Vieten, A., 360, 363 Vigh, L., 276 Villa, M., 466 Villani, M.C., 57 Villarroel, R., 111, 120 Vincent, C., 388 Vincent, D., 109 Vince-Prue, D., 387 Vinocur, B., 23, 53–56, 299, 410, 470 Vinocur, B.J., 426 Viola, R., 56 Vlad, F., 55, 73, 218, 221, 229–230, 262–263, 267, 270, 277, 450–451, 469 Voelker, C., 70, 73 Voesenek, L.A.C.J., 52 Voetberg, G., 119 Vogel, G., 111, 117 Vogel, J.P., 470 Voisin, R., 109 Volkman, B.F., 214 Volkov, V., 164, 167–169, 171–172 Volokita, M., 39 von Bothmer, R., 11 von Caemmerer, S., 8, 57, 59, 67, 81, 452, 468 von Korff, M., 467 von Stedingk, E., 42
AUTHOR INDEX Voogd, E., 117 Vooijs, R., 119, 122 Vorosmarty, C.J., 408 Vranova, E., 218 Vuorela, H.J., 327 Vuylsteke, M., 69, 74 W Wachter, R., 253 Wada, M., 365 Wada, S., 355 Waditee, R., 39 Wagner, A., 298 Wagner, D.R., 379–380, 384–386, 389, 391 Wagner, R.L., 230, 269 Wain, R.L., 364 Waisel, Y., 179 Waldron, L., 468 Walia, H., 405, 428 Walker, J.C., 76 Walker, R.R., 10–11 Walker-Simmons, K., 222 Walker-Simmons, M.K., 220, 230, 269 Wallace, A., 456 Wallach, R., 426 Walsh, B.J., 335 Walter, A., 36 Walter, M.H., 212 Walters, C., 55, 320–322, 324, 328–329 Walton, L.J., 329, 424 Wanchana, S., 472 Wandrey, M., 55 Waner, D., 57, 59 Wang, A., 70, 72–73, 214, 230, 264, 305 Wang, B.S., 163–164, 167–169, 174–175, 183–184, 466 Wang, C.Q., 183–184, 452–453, 458 Wang, D., 216, 226, 422 Wang, F.-Z., 114, 127, 417 Wang, G.K., 223 Wang, G.-P., 413 Wang, G.-Y., 419 Wang, H.B., 183–184 Wang, H.-C., 76, 299, 305, 327, 389, 448 Wang, H.W., 471 Wang, J.L., 72, 184, 214, 259–260, 305, 416, 468 Wang, J.Y., 42, 275 Wang, K., 423, 472 Wang, L.L., 268, 323, 330, 389–390, 412 Wang, L.W., 125, 128 Wang, M.C., 468–469 Wang, P.T., 22, 24, 70, 78, 219, 268, 271, 468 Wang, Q.-B., 417, 420 Wang, R.-G., 268, 275, 465 Wang, S.H., 183–184, 454, 463 Wang, S.M., 163, 169
539
Wang, W.-Q., 413, 428, 463 Wang, W.X., 462 Wang, X.-C., 109, 115, 124, 213, 215–216, 218–220, 222, 268, 294, 299, 352, 357, 392, 396, 419, 452 Wang, X.-F., 68, 260–261, 280 Wang, X.-L., 68, 260–261 Wang, X.-P., 421 Wang, X.-Q., 73, 263 Wang, Y.B., 55 Wang, Y.-F., 221–223, 231, 256, 263, 266, 276, 306, 364, 430–431, 454, 456, 461, 463, 471 Wang, Y.L., 160, 181, 467 Wang, Y.R., 76 Wang, Z.L., 55, 59, 82, 158, 169, 177–178, 327, 330–331, 333–334, 336 Wang, Z.Y., 17, 379, 391, 456 Wan, J., 36, 39, 276, 430–431 Wanke, D., 358, 466 Wankhade, U., 281 Warner, D.C., 423, 431, 461 Warren, C.R., 57–59, 64, 82 Warren, D., 306 Washitani-Nemoto, S., 409 Wasilewska, A., 55, 57, 68, 73, 267, 277 Watanabe, K., 421 Waterman, D., 337 Waters, D. L., 115 Waters, M.T., 78, 468 Watkinson, J.I., 299 Watson, J.D., 34 Watson, L.T., 299 Watson, M.B., 220, 263 Watson, M.C., 157 Watt, M., 3, 9, 12–14, 19–20, 25, 53, 56 Webb, A.A.R., 217, 262 Webb, M.A., 322 Webb, M.S., 353–354 Weber, A.P., 109 Weckwerth, W., 108, 469 Weder, B.D., 69, 73, 256, 457, 471 Weers, B.D., 299 Wegner, L.H., 155, 172 Wehmeyerand, N., 458 Wehmeyer, N., 328 Wei, A., 112, 118, 132 Wei, F.J., 452–453, 458 Weig, A., 456 Weigel, D., 228, 250, 371, 388, 463 Weigel, P., 111 Wei, H., 472 Weijers, D., 363 Wei, J.G., 183–184 Wei, L., 462 Weiler, E.W., 38, 68, 207–209, 256 Weilmunster, A., 253 Weiner, J.J., 72, 214 Weinl, S., 222–223, 358, 466
540
AUTHOR INDEX
Wein, S., 453 Weis, A.E., 465 Wei, S.J., 184 Weisshaar, B., 225 Weissman, R., 44 Wei, W., 112, 118, 132 Wei, Z., 116 Welin, B., 115 Wellmann, C., 73, 222, 231–232, 268, 457 Welti, R., 109, 268, 452 Weltmeier, F., 115, 124 Wenkel, S., 73, 389 Went, F.W., 355 Wen, X., 116 Weretilnyk, E.A., 77, 111, 299, 447, 458, 467 Wery, J., 466 Wesselman, H., 172 Westall, K.L., 323, 326–327, 334 Westergren, T., 451 Westgate, M.E., 447 West, J.D., 63 Wettlaufer, S., 324 Wettschureck, N., 261 Weynard, M., 267 Wharmby, C., 153 Wheatley, K., 386–388, 390 Wheaton, A., 25 Wheeler, K.P., 177 Wheeler, L.M., 320 Wheeler, N., 467 Whelan, J., 306, 470 Whitecross, M.I., 11 Whitehead, D., 358, 466 Whitelam, G.C., 76, 385 Whitlow, T.H., 352, 354 Whitney, S.M., 80 Whitsitt, M.S., 351, 354 Whittaker, A., 109, 325–326, 328, 333, 458 Whitty, B., 77, 299, 467 Wichert, K., 212, 253 Wided, M., 109 Widmer, C.K., 463 Widodo, 126, 129 Wiemken, A., 111, 116–117 Wienkoop, S., 469 Wigger, J., 253 Wilhelm, K.S., 420 Wilkins, H., 364 Wilkins, O., 468 Wilkinson, S., 68, 207–209, 304, 427 Willcocks, D.A., 164, 170, 172 Willcut, B., 462 Willekens, H., 218 Williams, J.P., 419 Williams, K.L., 335 Williams, L.A., 226 Williamson, J.D., 412 Williamson, L., 55, 263
Williams, R.D., 328 Williams, T.D., 109 Willmann, R., 224, 273 Willmitzer, L., 108, 179 Wilmoth, J.C., 363 Wilson, I.W., 13, 25 Wilson, P.B., 452, 468 Winge, P., 115 Wingler, A., 111, 116–117 Wingsle, G., 55, 70 Winicov, I., 125 Wininger, S., 60, 352, 426 Wise, M.J., 329 Wise, R.R., 62 Wisman, E., 363 Wisniewska, J., 357, 363 Wistrom, C.A., 328 Wiswedel, S., 323, 325, 337, 464 Witcombe, J.R., 407–408 Witters, E., 54, 225–226, 470 Wittinghofer, A., 267 Witucka-Wall, H., 108 Wohlbach, D.J., 448 Wojciechowski, T., 25 Wolff, P., 362 Wolters, H., 360, 363 Wolverton, C., 362 Womersley, C., 328 Wong, C.E., 77, 299, 467 Wong, H.L., 388 Wong, S.C., 62, 66 Wood, A.J., 129, 320, 327 Woodrow, I.E., 66 Woodward, F.I., 57, 76 Woo, N.S., 452, 468 Woo, Y., 457 Wormit, A., 471 Wornik, S., 109 Wraith, J.M., 425 Wright, B.J., 164, 170, 172 Wright, J.B., 362 Wright, L., 386–388 Wright, R.J., 466 Wu, C.A., 228, 462 Wu, D., 72, 214, 259–260 Wu, F.Q., 68, 213, 215–216, 218, 222, 260–261, 280 Wu, G., 416 Wu, J.J., 423, 452–453, 458, 461 Wu, J.M., 461, 471 Wu, J.R., 335 Wu, K., 272, 461 Wulfetange, K., 265 Wullschleger, S.D., 80 Wunnenberg, P., 180 Wurgler-Murphy, S.M., 297 Wu, R.H., 330 Wu, R.J., 117, 323, 411, 414, 431 Wurtzel, E.T., 468
AUTHOR INDEX Wusirika, R., 299 Wu, S.-J., 18, 51, 294, 303, 418, 459 Wu, W.H., 68, 213, 215, 223, 427, 452–453, 458–459 Wu, X., 226, 431 Wu, Y.J., 42–43, 299, 352, 392, 396, 420, 448, 460 Wu, Y.P., 76 Wu, Z.-Y., 215, 260 Wyatt, S.E., 299, 303 Wyn Jones, R.G., 153, 162–163, 165, 169 Wysocki, R., 298
X Xia, G.-M., 419, 469 Xia, L., 110, 130 Xia, M., 421 Xiang, C.-B., 427, 461 Xiang, F., 462 Xiang, Y., 423 Xiao, B.-Z., 424–425, 427, 460, 468 Xiao, G., 115–116 Xiao, H., 462 Xiao, S., 305 Xia, Q.C., 335 Xia, T., 170, 175 Xia, Y.F., 455 Xie, C., 267 Xie, H., 209, 299, 471 Xie, K., 424 Xie, Q., 226, 460, 463, 471 Xie, W., 424 Xie, X.D., 55, 263 Xiloyannis, C., 54, 64 Xing, Y., 224, 273, 462 Xin, M., 462–463 Xin, Q., 216, 261, 280 Xiong, L.-Z., 25, 69, 223, 227, 275, 295, 300, 423–425, 427, 449, 460, 462, 465, 468 Xuan, Y., 223 Xu, C.-Y., 183–184, 419 Xu, D.-Q., 411 Xue, G.-P., 419 Xue, S.W., 53, 73, 82, 264, 305 Xue, T., 216 Xue, X., 121 Xue, Y., 299, 459 Xue, Z.-Y., 419 Xu, J., 223, 428 Xu, P., 461 Xu, Q., 453 Xu, W., 227, 299, 459, 462 Xu, X.J., 184 Xu, Y.-H., 68, 72, 213–215, 218, 222, 260–261 Xu, Z.Z., 54–55, 352, 463
541
Y Yabuuchi, H., 68, 210–211, 254 Yadav, R.S., 466 Yadav, V., 415 Yadegari, R., 224 Yakir, D., 59, 466 Yakubov, B., 298, 448 Yamada, K., 109, 166 Yamada, S., 158, 160, 163, 181, 456 Yamagishi, K., 205–206 Yamagoe, S., 232 Yamaguchi, M., 43 Yamaguchi-Shinozaki, K., 68–69, 77, 79, 115, 124, 203, 205, 212, 216, 220–221, 224–226, 229–230, 232, 259–260, 262–264, 270, 294–295, 298–300, 356, 386, 392–393, 409–410, 420–421, 425, 431, 448–449, 451, 456, 458, 460–461, 468, 470 Yamaguchi, T., 152, 166 Yamaguchi, Y., 298, 448 Yamamoto, A., 39, 225, 230, 269 Yamamoto, R., 42–43 Yamamoto, S., 220, 225, 230, 269 Yamamoto, Y., 256 Yamanaka, S., 421 Yamashino, T., 383, 386, 393 Yamashita, M., 355, 357 Yamazaki, T., 349 Yamazaki, Y., 336, 356 Yan, C., 72, 214, 259–260 Yancey, P.H., 107, 110, 113 Yan, F., 42 Yang, A., 412–413 Yang, A.F., 419 Yang, B., 468 Yang, C., 226, 460, 463, 471 Yang, G., 305 Yang, H.Q., 366, 389–390, 420, 451 Yang, J.H., 228, 300 Yang, M.S., 184, 228 Yang, P., 226 Yang, S.L., 36, 39, 119 Yang, W., 327, 331, 333–334, 336 Yang, X.H., 114, 116, 224, 363, 411 Yang, Y.Z., 59, 68, 73, 211, 213–214, 218, 221–222, 229, 256–260, 264, 267, 305, 423 Yang, Z., 116 Yan, J.Q., 305, 416, 419 Yan, L., 216, 261, 280 Yan, M., 268 Yan, N., 72, 214, 259–260 Yano, M., 380 Yano, R., 212 Yanovsky, M.J., 383, 387–388 Yao, J., 424, 460, 468 Yao, Y., 462–463
542
AUTHOR INDEX
Yates, J.R., 70, 72, 214, 230 Yazici, I., 296 Ye, C.J., 165 Yeh, K.C., 366 Ye, L., 472 Yen, H.C.E., 160, 181 Yensen, N.P., 181 Yeo, A.R., 18–19, 158, 163–165, 176–177, 179, 182 Yeo, E.T., 117 Yephremov, A., 459 Ye, Q., 426 Yesbergenova, Z., 305 Yin, B., 460 Yin, D., 422 Ying, J., 276, 430–431 Ying, K., 299 Yin, H.B., 174 Yin, P., 72, 214, 259–260 Yip, J.Y.H., 306 Yiu, J.C., 305 Yoine, M., 227 Yoklic, M., 156 Yokoi, S., 388 Yokotani, N., 468 Yokota, S., 115–116 Yokota, Y., 115 Yonamine, I., 420 Yong, E., 214 Yoo, C.Y., 225, 429 Yoo, J.H., 123 Yoo, J.-Y., 69, 255 Yoon, E.K., 228 Yoon, K.A., 121 Yoon, U.H., 472 Yoshiba, Y., 111, 119–120, 123 Yoshida, K.T., 112, 118, 420 Yoshida, R., 72, 220, 224–225, 230, 263, 270, 386, 388–391, 395–396, 449 Yoshida, T., 72, 214, 216–218, 221, 259–260, 263, 270, 468, 471 Yoshida, Y., 75, 218 Yoshiwara, K., 68, 109, 299 You, J., 424 Young, B.D., 327 Young, I.M., 25 Young, J.C., 55, 70, 379 Young, M.W., 379 Yousfi, N., 126, 129 Yuan, X., 72, 214, 259–260 Yuasa, T., 224, 449 Yue, Y., 68, 213, 215 Yu, H., 461 Yu, J.Q., 64, 454, 459 Yu, J.W., 389, 395 Yu, L.X., 299 Yu, M., 420 Yumoto, F., 232 Yun, D.-J., 167, 272, 301
Yun, H.S., 364 Yu, O., 43 Yurin, V., 185 Yu, S., 59 Yusufov, A.G., 176 Yuwansiri, R., 116 Yu, X.-C., 68, 213, 215, 218, 222, 260–261, 389–390, 426 Yu, Z., 299 Z Zabirnyk, O., 121 Zagotta, M.T., 379 Zamir, D., 109 Zanetti, M.E., 467 Zarrouk, O., 56 Zeevaart, J.A.D., 203, 205, 305 Zeiger, E., 66, 180, 209 Zeinali, H., 465 Zeller, G., 228, 463 Zeng, B., 114, 127 Zeng, C., 463 Zeng, Q., 215, 278, 327 Zeng, R., 335 Zepeda-Jazo, I., 169 Zhai, Q., 226 Zhang, A., 301, 304–305 Zhang, B.H., 462 Zhang, C.Q., 55, 158, 169, 177–178, 223, 303, 459 Zhang, D.-P., 215, 260, 462 Zhang, F.C., 174 Zhang, G.-L., 428 Zhang, G.P., 169 Zhang, H.R., 70, 77 Zhang, H.-S., 55, 158, 169, 174, 177–178, 305, 366, 411–413, 416–417, 419–420, 428–429, 461, 471 Zhang, H.W., 79 Zhang, H.-X., 114, 116, 174, 419 Zhang, J.H., 43, 114, 116, 208–209, 224, 226, 273, 300–301, 303, 364, 412–413, 462, 468 Zhang, J.L., 163, 169 Zhang, J.R., 419 Zhang, J.S., 365, 416, 448, 471 Zhang, J.Y., 55 Zhang, J.Z., 276, 295, 303 Zhang, K., 412 Zhang, L., 39, 115–116, 268, 301, 305, 335, 460 Zhang, M.-X., 305, 461, 471 Zhang, P.G., 229, 428 Zhang, Q.-F., 55, 158, 167, 169, 177–178, 183–184, 268, 424, 427, 460, 468 Zhang, R., 419 Zhang, S.B., 53, 216, 452 Zhang, S.J., 183–184
AUTHOR INDEX Zhang, S.Q., 76, 455 Zhang, S.W., 455 Zhang, W.K., 218, 268, 364, 413, 416, 448, 463, 467, 471 Zhang, X.-F., 59 Zhang, X.-J., 420 Zhang, Y.C., 117, 226, 268, 389–390, 395, 414, 420–421, 455, 459–460, 463, 467–468, 471 Zhang, Y.I., 64 Zhang, Z.J., 79 Zhang, Z.L., 79, 114, 122, 214, 428–429, 462 Zhao, B., 462 Zhao, C.S., 167, 169, 174, 182 Zhao, F.-Y., 174, 267, 417, 419–420 Zhao, H.Y., 78, 358, 389 Zhao, J., 218, 267, 301, 305–306, 419, 465, 468 Zhao, K.F., 165 Zhao, M.-R., 413 Zhao, Q., 226, 460, 463, 471 Zhao, R., 42, 216, 222, 261, 267, 280 Zhao, W., 108 Zhao, X.L., 70 Zhao, Y.-X., 68, 72, 114, 174, 211, 213–214, 218, 229–230, 256–260, 364, 412–413, 419–420 Zhao, Z.-X., 215, 260 Zheng, C.C., 216, 228, 462 Zheng, J., 468 Zheng, M., 323 Zheng, N., 226, 463, 471 Zheng, Q.A., 110 Zheng, W., 454, 463 Zheng, X., 425 Zheng, Y., 463 Zhi, D.-Y., 419 Zhifang, G., 118 Zhi, Y., 72, 214, 471 Zhong, Y., 216, 472 Zhou, G.S., 54–55 Zhou, H.E., 109 Zhou, H.L., 365 Zhou, L., 203, 209, 253, 274 Zhou, M.X., 169 Zhou, T., 294 Zhou, X.E., 72, 214, 467–468 Zhou, X.F., 70, 77
543
Zhou, Y.H., 64, 70, 454, 459 Zhou, Y.J., 184 Zhu, C., 114, 127 Zhu, D., 303, 306 Zhu, H., 268 Zhu, J.H., 55, 158, 169, 177–178 Zhu, J.J., 455 Zhu, J.K., 18, 51, 55, 66, 69–70, 75–77, 107, 110, 115, 123, 169, 172, 183, 213, 218, 220–223, 227–228, 230, 263–264, 267, 269, 271–272, 276, 294–296, 298–301, 303, 418, 459–463, 467, 471 Zhu, J.-K., 272 Zhu, J.M., 42 Zhu, L., 420 Zhu, M.-Z., 17, 70, 74, 430, 461 Zhu, S.-Y., 68, 213, 215, 218, 222, 260–261 Zhu, T., 109, 294, 299, 352, 392, 396 Zhu, X.G., 55, 80 Zhu, Y., 272, 301 Ziemak, M.J., 116 Zikihara, K., 386 Zimmer, I., 459 Zimmerman, J., 294 Zimmermann, U., 181, 455 Zingarelli, L., 174–175 Zingaretti, S.M., 467 Zipfel, C., 258 Zivy, M., 55, 73, 230–231, 268, 305, 450 Zocchi, E., 277–278, 291 Zolla, G., 392, 396 Zolman, B.K., 273 Zoran, M.J., 379 Zo¨rb, C., 39, 42 Zorn, M., 109 Zou, H.F., 471 Zou, J.J., 452–453, 458 Zsigmond, L., 114, 121–123 Zuber, H., 336 Zuily-Fodil, Y., 354, 451 Zulfugarov, I.S., 79 zur Nieden, U., 253 Zuther, E., 109, 126 Zwerger, K., 273 Zwiazek, J.J., 60 Zyl, L.V., 299
SUBJECT INDEX
A ABA. See Abscisic acid ABA receptors chloroplast membrane Scatchard plot analysis, 260–261 wild-type protein GUN5, 261 heterotrimeric G proteins description, 261 GTG proteins, 262 subunits and GPCR, 261–262 PYR/PYL/RCAR HSQC, 258 mutant protein, 260 PP2Cs, 258–259 pyrabactin, 256–258 PYR1 and CL2 loop, 259–260 PYR–PP2C interaction, 258 Ser112, 260 START proteins, 256 strcuture, molecular mechanisms, 257 Vicia guard cells, 256 ABA-responsive element (ABRE) Arabidopsis HAB1, 272 cis-regulatory elements, 275–276 downstream target genes, 269 promoter analyses, 270 transcriptome, 280 ABC transporters. See ATP-binding cassette transporters ABI2 gene, 217 ABI5 interacting protein (AFP) definition, 226 proteolysis, 226 yeast two-hybrid screen, 225 Abiotic stress defined, 34 hormonal homeostasis and, 427–429 miRNA network, 228 P5CS1, 121 plant adaptations, 408–409 ROS, 300 ROS accumulation, 114 stomata aperture, 74 ABRE. See ABA-responsive element Abscisic acid (ABA) biosynthesis and signalling, 68 gravitropism regulation biosynthesis, 364 stress tolerance, 365 guard cells, 67–68 hydrotropism
regulation, 358–359 synthesis and signalling, 359 negative regulators, 74 receptors, 68, 71 signalling, 72–73 stomatal aperture, 73 treatments, 61 vascular tissues, 68 water shortage, 71 Anion channels, 265–266 Antioxidant systems ascorbate synthesis, 327 de novo, 327 enzymes role, ROS detoxification, 326–327 novel and housekeeping, 326 polyphenols and phenolic antioxidant enzymes, 327–328 Arabidopsis-relative model systems (ARMS), 110 Arabidopsis thaliana, 108, 252, 262, 272. See also Early flowering 3 (ELF3) mutant, Arabidopsis ARF. See Auxin response factor ATP-binding cassette (ABC) transporters AtABCB14, 255, 270 AtABCG25, 254, 279 AtABCG40, 254–255 description, 253–254 Auxin response factor (ARF), 363 B Boea hygrometrica, 323, 329–330 C CO. See Constans Constans (CO) expression level, 388, 390 flowering pathway regulation, 387–388 Constitutive photomorphogenesis 1 (COP1) ELF3–GI pathway, 390 ELF3 role, 389 COP1. See Constitutive photomorphogenesis 1 Crassulacean acid metabolism (CAM) genes, 78 phosphoenolpyruvate carboxylase, 78 water-limited environments, 78 Cyclic nucleotide-gated channels (CNGCs), 167
546
SUBJECT INDEX
D Desiccation tolerance (DT) in higher plants definition, 320 mechanical stress and damage minimization arabinose polymers, 322 Craterostigma wilmsii, 322 GRP, 323 vacuolar content, resurrection plants, 323–324 water loss, 321–322 metabolic stresses and protection mechanism antioxidant systems, 326–328 ROS production, 324–325 subcellular milieu stabilization, 328–330 metabolomics, 337 proteomics, 330–336 ‘resurrection plants’, 320–321 water’s role, 321 Drought adaptive responses, ABA anion channels, 265–266 downstream control Atgpx3 mutation, 269 H2O2 signal, 268 in vitro phosphatase activity, 268–269 Xenopus oocyte, 268 potassium (Kþ) channels cytosolic signals, 264 inward-rectifying channels, 264–265 S6 gating domain, 265 Shaker-type, 264–265 P-type proton pumps Actuator domain, 267 calcium-dependent kinase PKS5, 267 Hþ-ATPase, 266–267 pumping activity, 266 Drought and salinity carbon fixation under environmental stress atmosphere limitations, 80 carboxylation rate, 79 C4 pathway, 81 negative impacts, 80 photorespiratory rates, 81 Rubisco specificity factor, 80 stomatal conductance, 80 cell dehydration, 51 controlled conditions acclimation responses, 52–53 antagonistic response, 52 leaf water loss, 52 natural growing environment, 53 soil solution and matrix, 53 description, 50–51 leaves under water deficits environmental variables, 66 leaf water potential, 67
stomatal conductance, 66–67 well-watered conditions, 67 limitations, 51 model plants Arabidopsis mutants, 55 environmental stress-responsive genes, 55 limitations, 56 model legumes, 55–56 proline-related signalling pathways, 55 stress-tolerance mechanisms, 54 osmosensors (see Osmosensors, osmotic stress) photosynthesis related genes activating/ repressing gene expression, 77 adverse environmental conditions, 77 CAB proteins, 78 phytochrome interacting factors (PIFs), 79 transcription factors, 77–78 TSRF1, 79 water-limited environments, 78 plasticity and resilience, 51 recovery responses average stomatal conductance, 54 de novo synthesis, 54 instability, photosynthetic membranes, 53 leaf water potential, 53–54 stress duration, 54 signalling components antioxidative, 305–306 cellular homeostasis, 300 osmotic stress, 300 plant stress responses, 299–300 protein–protein reactions, 299 ROS, 300–305 stress-responsive gene expression, 299 soil water deficit and salinity, 50 stomatal aperture regulation ABA-induced signalling transduction, 72–73 ABA perception, 68–72 abiotic stress tolerance, 67 transcription factors, 73–75 stomatal development Arabidopsis, 75 ERECTA family, 75 leaf gas exchange, 76 MMC and GMC, 75 signalling pathway, 76 stress, plant responses adaptations to abiotic stress, 408–409 salinity stress, 408 water deficit, 408 tolerance mechanisms leaf morphology and root system architecture, 8–9
SUBJECT INDEX plant responses, 7 root and leaf growth, 6 salt specific differences, 10–11 stomatal conductance and leaf expansion, 7–8 turgor maintenance and osmotic adjustments, 9 tolerant crop plants ionic balance, 418–420 mitigating oxidative damage, 415–417 osmoregulation, 410–415 regulatory and signalling genes, 420–431 water and saline stress biochemical and metabolic limitations, 64–66 diffusive limitations, 56–64 Drought and salt tolerance (DST), 74, 416, 430, 461 Drought stress antioxidants anthocyanin production, 459 ROS accumulation, 458 Arabidopsis mutant, 459–460 bioinformatics and databases, 471–472 compatible solutes, 454 conventional breeding, 465 domestication, modern crops, 447 Green Revolution, 446 growth responses, 447–448 methyl jasmonate (MeJA) levels, 460 model organisms Craterostigma plantagineum, 464 seed germplasm banks, 465 omics studies proteomics and metabolomics, 469–470 transcriptomics, 466–469 post-transcriptional regulation cereal species, 463 isoprenylcysteine methylesterase (ICME), 464 microRNAs (miRNAs), 462–463 SUMOylation, 463–464 ubiquitination, 463 protective proteins aquaporins, 455–456 heat shock proteins, 457–458 ion channels, 456–457 LEA, 454–455 QTLS and marker-assisted breeding intergenic and gene–environment interactions, 466 linkage maps, 466 polygenic nature, trait, 465 semi-dwarfism, 446–447 signalling pathways MAPK, 449–450 perception, 448
547
phosphatases, 450–451 phospholipid signalling, 451–452 salicylic acid and nitric oxide, 453–454 secondary messengers and calcium, 452–453 SNF-1-like kinases, 450 transduction, 448–449 tolerance, 447 transcriptional regulation ABA-dependent and -independent pathways, 460 maize, 461 NAC genes, 460 nonethylene receptor histidine kinases, 461 stomatal closure, 461–462 transgenic approaches A. thaliana, 470–471 functional approaches, 471 E Early flowering 3 (ELF3) mutant, Arabidopsis biochemical functions, 379 biological clocks, 378–379 flowering time control COP1 and GI, 389 GI, CO and FT expression, 388 input, oscillator and output of clock, 388–389 loss and gain-of-function, 387 photoperiodicity, 387–388 suppressors and enhancers identification, 389–390 gene expression, 396 genes, 381–382 light signalling input pathway, clock, 384–385 interaction, PHYB, 385 molecular hierarchy, 383, 384 PHYB-independent roles, 385–386 MAPKs, 395–396 molecular mechanisms, 380 non-Arabidopsis plant species, 380, 383 novel nuclear protein, 380 photoreceptors, F-box proteins and transcription factors, 396 polyglutamine tracts, 383 protein–protein interactions, 394–395 Q repeats, 380 short-day and long-day conditions, 379 stress tolerance ambient temperature, 391 gated induction, DREB/CBF, 392–393 microarray analysis, 392 PIF7 functions, signalling, 393–394 PRR9, PRR7 and PRR5, 393
548
SUBJECT INDEX
Early flowering 3 (ELF3) mutant, Arabidopsis (cont.)
seed dormancy, 394 signal transducers and transcription factors, 391 TOC1/PRR1 component, 394 viability, 391–392 Epidermal bladder cells (EBCs), 160 Extreme environments, stress tolerance accumulate nitrogenous compounds, 128 b-alanine betaine, 128 genotypes, 125–127 importance, 128–128 optimal growth conditions, 130 stress tolerance, 125 sugar alcohols and proline, 129–130 transcript profiling, 129 F Flowering locus T (FT) expression level, 388 flowering pathway regulation, 387–398 FT. See Flowering locus T G GI. See Gigantea Gigantea (GI) COP1–ELF3 pathway, 390 ELF3 role, 389 expression level, 388 flowering pathway regulation, 387–388 interaction, FKF1 and ZTL, 379 Glycine-rich protein (GRP), 323 G-protein-coupled receptors (GPCRs) ABA-binding ability, 213 developmental processes and stress responses, 215 type protein, 215 Gravitropism and drought avoidance ARF and AUX/IAA protein, 363 Cholodny-Went hypothesis, 362 columella and lateral cells, 361–362 mechano-sensitive ion channels activation, 362 PIN auxin transporters, 363 regulation ABA biosynthesis, 364 aux1 mutants, 365 role of ethylene, 364–365 osmotic stress, 363–364 GRP. See Glycine-rich protein Guard cells, ion transport electrophysiology halophyte vs. non-halophyte Aster species, 180 KIR channels, 179–180 osmotic effect, 180
patchclamp investigation, 179 ionic relations, 179 stomata control gas exchange properties, 177–178 halophytes vs. glycophytes, 177 leaf water content, 178 water use efficiency, 178 Guard mother cell (GMC), 75 H Halophytes, ion transport definition, 153 evolution and diversity Arabidopsis roots, 154–155 glycophyte species, 153 high-affinity Kþ uptake, 154 physiological mechanisms, 154 salt tolerance, 154 species, 155 tolerate monovalent ion concentrations, 153 guard cells electrophysiology, 179–180 ionic relations, 179 stomata control, 177–178 leaf succulency, 158 leaves, unloading and ion transport mesophyll, 173–174 pinocytosis, 176–177 vacuolar sequestration, 174–176 vacuole, ion retention, 177 oxidative signalling and damage repair antioxidant systems and control, 182–183 stress signalling and tolerance, 183–186 plant ionic relations inorganic ion accumulation and osmotic adjustment, 165 organic osmolytes, 165–166 tissue-specific compartmentation, 162–164 radial ion transport root epidermis, 166–170 root vacuoles, 170–171 root structure anatomical features, 157 Casparian bands, 158 water retention, 158 salinity tolerance, crop breeding, 152–153 salt bladders and glands, 158–162 saltwater management animal feeding systems, 157 200-day growing cycle, 157 poor-quality water, 156 Salicornia, 156–157 seawater irrigation, 156 xylem ion loading, 171–172
SUBJECT INDEX Heteronuclear single quantum coherence (HSQC), 258 High osmolarity glycerol (HOG ) hyperosmotic stress, 297 pathway activation, 297 HSQC. See Heteronuclear single quantum coherence Hydrotropism and drought avoidance Arabidopsis roots ABA and water stress, 358–359 DNA microarray technology, 358 gnom alleles, 360–361 miz1 and miz2 mutant, 359–360 nhr1 mutant, 359 waving, 361 described, 354–355 hydrostimulation signal transmission maize, 356 phospholipase D proteins, 357 role, calcium ions, 356–357 hydrotropic root bending auxin role, 357–358 species specificity determination, 358 miz1 gene, 355 physiological studies, 355 sensing hydrostimulation Cholodny-Went hypothesis, 355 mechano-sensitive ion channel, 356 root tip removal, 355–356 I Introgression line (IL) populations, 108 K katE gene, 416, 417 L Late embryogenesis-abundant (LEA) proteins Boea hygrometrica, 329–330 description, 454 groups, 455 indole-3-acetic acid (IAA), homeostasis, 455 properties, 329 tobacco and Arabidopsis, 330 Lockhart equation, 36 M MAPKs. See Mitogen-activated protein kinases Marker-assisted breeding, 465–466 Meristemoid mother cell (MMC), 75 Metabolomics, 337
549
Mitogen-activated protein kinases (MAPKs) ABA signalling, 224 cascades and clock components, 395 description, 272–273 dual-specificity protein phosphatases, 273 eukaryotes, 224 role, signal transduction pathways, 395–396 TILLING mutants, 273 MIZU-KUSSEI1 (miz1) mutant, 359–360 MIZU-KUSSEI2 (miz2) mutant, 359–360 Molecular biology, drought antioxidants anthocyanin, 459 ROS accumulation, 458 Arabidopsis mutant, 459 compatible solutes, 454 growth responses Arabidopsis thaliana, 448 mild osmotic stress, 447 methyl jasmonate (MeJA) levels, 460 modern crops, domestication, 447 protective proteins aquaporins, 455–456 heat shock proteins, 457–458 ion channels, 456–457 LEA, 454–455 signalling pathways MAPK, 449–450 phosphatases, 450–451 phospholipid signalling, 451–452 salicylic acid and nitric oxide, 453–454 secondary messengers and calcium, 452–453 signal perception, 448 SNF-1-like kinases, 450 transduction, 448–449 tolerance, 447 Molecular mechanisms, ABA adaptive responses, land plants, 251 Arabidopsis thaliana, 252 circulation, 253 coding capacity, Arabidopsis, 250–251 components, signalling, 279 drought adaptive responses anion channels, 265–266 potassium (Kþ) channels, 264–265 P-type proton pumps, 266–267 epigenetics Arabidopsis PP2C, HAB1, 272 histone variant and deacetylase, 271–272 fluridone ability, 281 gene expression and chromatin modelling, SNRKS ABRE, 270–271 b-ZIP proteins and transcription factors, 270
550
SUBJECT INDEX
Molecular mechanisms, ABA (cont.)
rice cell cultures, 269–270 TaABF, 269 guard cells transport membrane ABCG25 expression, 254 anion transporters, 255–256 Arabidopsis mutant Atmrp5, 256 AtABCB14, 255 AtABCG40, 254–255 described, ABC transporters, 253–254 NBFs and TMDs, 254 human health improvement ABA–cyclic ADP-ribose–Ca2þ, 277 AIN-93-G-based rodent diet, 277 biosynthesis and signal transduction pathways, 276–277 LANCL2, 278 PPAR , 278 xenohormesis, 276 42-kDa protein, 280 MAPKs, 272–273 photosynthetic rates, CO2, 251 as positive and negative regulator root elongation, Arabidopsis, 252–253 vegetative growth, 252 PYR signalling complex clade A PP2Cs, 262–263 in vitro reconstruction, 263 kinase-encoding loci, 263–264 Vicia faba guard cells, 263 receptors chloroplast membrane, 260–261 heterotrimeric G proteins, 261–262 PYR/PYL/RCAR, 256–260 root growth AAO3 gene, 274 cis-regulatory elements, 275–276 designer roots, 274 Green Revolution, 273–274 hair development, 274–275 lateral root development (lrd)2, 275 mannitol, 275 mutations, 274 root growth pattern, 280–281 steps, signalling, 251–252 transpiration and photosynthesis, 278–279 transporters and transcriptome, 279–280 N Nax1 gene, 17 9-cis-epoxycarotenoid dioxygenase (NCED) ABA biosynthetic pathway, 205 description, 205 gene expression level, 206 identification, 203 No hydrotropic response1 (nhr1) mutant, 359
O Osmoprotective compounds adaptation to extreme environments, 125–130 environmental conditions, 110 functions, 113 glycine betaine (GB) accumulation, stress tolerance, 115–116 choline monooxygenase (CMO), 115 codA9 gene, 116 heat tolerance, 116 osmotic adjustment, 110 in plants, 110–112 polyalcohols accumulation, 117–118 halophytic plants, 119 metabolic pathways, 119 myo-inositol, 118 phosphorylation, 118 proline abiotic and biotic stresses, 119 accumulation, 122 arginine, 121 biosynthetic pathway, 122 chloroplast, 120 drought and heat tolerance, 122–123 importance, 122 mitochondrial electron transport chain, 121 prokaryotic and eukaryotic pathways, 120 regulation, 123 rich proteins and stress response, 124–125 ROS signals, 121 protein stabilisation, 113 radical oxygen production, 114 ROS accumulation, 114 transcript profiling, 115 trehalose accumulation, 116–117 desiccation-tolerant organisms, 116 function, 117 importance, 117 universal protecting effect, 113 Osmoregulation abiotic stress, 410–411 biosynthesis and accumulation, 410 glycine betaine (GB), 412–413 mannitol, 412 osmotin genes, 415 proline accumulation, 411 transpiration efficiency (TE), 411 trehalose alfalfa, synthesis and accumulation, 414–415
SUBJECT INDEX biological structure stabilization, 413–414 transgenic rice plants, 414 water and salinity stresses, 415 Osmosensors, osmotic stress cytokinin response 1 (Cre1), 298 glycerol accumulation, 297–298 HOG pathway, 298 protein–lipid interactions, 298–299 two-component regulatory system, 297 water and osmotic stresses, 299 Osmotin genes, 415 Oxidative damage, crop plants antioxidant capacity, 416 drought and salt stress, 415 photosynthetic electron transport, 415–416 ROS-scavenging genes, 417 signalling pathways, 417 stomatal density, 416 P Peroxisome proliferator–activator receptor
(PPAR ), 278 Phototropism and drought avoidance Arabidopsis roots, 365 auxin-dependent asymmetric growth, 366 ecological function, 366 signal transduction mechanisms, 365–366 PhyB. See Phytochrome B Phytochrome B (PhyB) ELF3 independent roles, 385–386 interaction, 385, 394–395 mutations, 396 Phytochrome-interacting factor 4 (PIF4), 76 Phytochrome-interacting factor 7 (PIF7), 393–394 Phytochrome-interacting factors (PIFs), 79 PINFORMED (PIN) auxin transporters, 363 mRNA expression, 364–365 Plant adaptations, salt and water stress commonalities and differences genotypes, 5–6 leaf death rate, 4 photosynthetic leaf area, 4 salinity stress components, 5 two-phase effect, 5 drought and salinity tolerance mechanisms leaf morphology and root system architecture, 8–9 plant responses, 7 root and leaf growth, 6 salt specific differences, 9–11 stomatal conductance and leaf expansion, 7–8
551
turgor maintenance and osmotic adjustments, 9 growth studies and experimental design hydroponics, 21 modern vs. old cultivars, 21 salinity, 22 salt-specific effects, 22–24 soil drying, 20–21 phenotyping infrared thermography, 25 plant growth rate, 24 reverse genetic approaches, 25 root function, 25 selection techniques, 24 spectroscopic techniques, 25 QTLS drought resistance, 12–14, 19–20 gene discovery, 20 quantitative measurement, 12 salinity, 14 salt-specific traits, 14–18 Plant breeding, drought resistance leaf architecture traits, 14 measurable adaptive characters, 12 root vigour and architecture, 13 temperate cereals, 12–13 water supply, 12 Plant growth inhibition abiotic stress, defined, 34 apoplastic changes, 35 cellular parameters, 36 colloid stress, 44 defined, 35 ‘post-genomic’ contributions, 34 salinity stress concentration-dependent uptake, 38 salt treatment, 39 inheritable epigenetic changes, 39–40 negative water potentials, 6 osmotic mechanism, 37–39 respiratory energy-use efficiency, 40 ROS accumulation, 38 sea water infiltration, 37 traditional breeding approaches, 39 soil water reserves, 35 water stress cell wall proteins, 41–42 cytoplasmic water potential, 35–36 growth inhibition, 40–41 Hþ ATPase and wall acidification, 42 in vitro extensibility, 43 leaf growth and elongation zone, 41, 42 proton-pumping ATPases, 41 root growth inhibition, 42–43 symptoms, 40 Plant ionic relations, halophytes inorganic ion accumulation and osmotic adjustment, 165
552
SUBJECT INDEX
Plant ionic relations, halophytes (cont.)
organic osmolytes compatible solutes, 165–166 glycine-betaine (GB) and proline, 165 plant adaptive responses, 166 root epidermal cells, 166 tissue-specific compartmentation bladder-less species, 163 gradients, 163 Kþ concentrations, 164 monocotyledonous species, 162 osmotic adjustment, 162 SK/Na selectivity, 164 sodium and potassium content, 162–164 Plant responses, ABA biosynthesis and catabolism AAO catalyses, 203, 205 ABA 80 -hydroxylase, 206–207 hydroxylation and conjugation, 205 metabolic sites, 207 molecular basis, 203 NCED, 205–206 pathways, plants, 203–204 core signalling pathway autophosphorylation, 230 direct dephosphorylation, 231 global negative regulators, 229 kinase activation loop, 230 PYR/PYL/RCAR–PP2C–SnRK2, 230 signal transduction network, 231–232 gene expression, 212 intercellular signalling, transport and localisation apoplast, short-distance transport, 209–211 genes and factors, 207–208 leaf stomatal conductance, 207 molecular basis, 208 organelles, subcellular transport, 211–212 vasculature, long-distance transport, 208–209 model system, 212 physiological roles, 202–203 phytohormones, defined, 202 protein kinases Ca2þ dependent protein kinase, 221–222 CIPK/PKS/SnRK3, 222–223 eukaryotes, 219 MAPK, 224 RLK, 224 signalling pathways, 219 SnRK2, 219–221 protein phosphatases, 216–219 receptors defined, 213
GPCRs, 213, 215 Mg-chelatase H subunit, 215–216 PYR/PYL/RCAR family, 213–214 regulation through protein degradation, 226 responsive gene expression AFP identification, 225 bZIP-type transcription factors, 225 phosphorylation, 225 transcriptional factors and ciselements, 225 RNA metabolism binding proteins, 228 components, 227 degradation process, 227 hyl1 mutant, 227–228 splicing factors, 228–229 transcriptome analysis, 229 Polyethylene glycol (PEG), 6 PPAR . See Peroxisome proliferator– activator receptor PP2C genes, 216 ProDH gene, 130 Proline rich proteins (PRP), 124 Protective proteins aquaporins Arabidopsis MIPs and PIPs, 456 V. vinifera roots, 456 water flow changes, 455 heat shock proteins, 457–458 ion channels anion efflux, 457 Ca(2þ)-independent OST1, 457 stomatal opening and closure, 456–457 LEA (see Late embryogenesis-abundant proteins) Protein phosphatases, ABA abi2–1 mutant, 217 abi1–1 proteins, 217 biochemical properties, 218 eukaryotes, 216 homology to ABA1 (HAB1), 217 interacting factors, 218 loss-off-function mutations, 217–218 phosphatase activity, 218–219 PP2Cs, 216 signalling, 219 Proteomics, resurrection plants leaf tissues, 330–331 subcellular approaches dehydration-induced nuclear proteins, 335 2D SDS-PAGE, 336 non-model organisms, 335–336 vs. whole approaches, 334–335 vs. transcriptomics, 330 whole approaches dehydration-induced proteins, 331, 332
SUBJECT INDEX detoxification and protection, 334 energy metabolism, 334 photosynthesis-related genes, 333–334 RWC mechanism, 331, 333 upregulated proteins, 333 Xerophyta viscosa, 331 Q QTLs. See Quantitative trait loci Quantitative trait loci (QTLs) analysis, 12 breeding, 12 mapping and identification, 466 Nax1 and Nax2, 17 polyglutamine-repeat tracts, 380 RIL and IL populations, A. thaliana, 108 R Radial ion transport root epidermis direct patch-clamp experiments, 168 glycophyte roots, 166 plasma membrane transport systems, 167 potassium retention, 169 T. halophila root cells, 168 tissue-specific expression, 167 voltage gating and roots ability, 169 root vacuoles osmotic adjustment, 170 single channel conductance and selectivity, 171 Thellungiella root cells, 170 tonoplast channel characteristics, 170–171 Raffinose family oligosaccharides (RFOs), 328 Reactive oxygen species (ROS) ABA and stress NADPH oxidase activity, 304–305 stomatal conductance, 305 generation and levels, 38 ‘housekeeping’ antioxidants, 324 lipid peroxidation, 415–416 oxidative stress, 415 photosynthesis regulation homoiochlorophylly, 325 poikilochlorophylly, 325 production, 416–417 regulating mechanisms, 423 resurrection plants, 324 root proton exportation capacity, 419 scavenging genes, 412, 417 signalling abiotic stresses, 300 accumulation, 303
553
antioxidant defence system, 301 Arabidopsis guard cells, 301 enzymatic and non-enzymatic molecules, 300–301 ionic and osmotic stresses, 304 NADPH oxidase inhibitors, 303–304 saline soils, 301 SOS, 301–302 transduction pathway, 301 signalling upstream, 416 water loss and electron transport, 324 Recombinant inbred line (RIL) populations, 107 Regulatory and signalling genes aquaporins, 426 DREB/CBF, 420–422 hormonal homeostasis and abiotic stress, 427–429 LEA gene expression, 424–425 NAC proteins, 424 nuclear factor Y-B subunit, 423–424 protein kinase, 422–423 stomatal response to stress, 429–430 transcription factors, 430–431 Relative water content (RWC) subcellular milieu stabilization, 328 whole proteomic approaches de novo proteins, 331 mechanisms, 331, 333 RFOs. See Raffinose family oligosaccharides RNA metabolism binding proteins, 228 components, 227 degradation process, 227 hyl1 mutant, 227–228 splicing factors, 228–229 transcriptome analysis, 229 Root tropism in drought avoidance gravitropism ARF and AUX/IAA protein, 363 Cholodny-Went hypothesis, 362–363 columella and lateral cells, 361–362 mechano-sensitive ion channels activation, 362 PIN auxin transporters, 363 regulation, 363–365 hydrotropism, 354–361 negative phototropism and positive hydrotropism, 350–351 phototropism, 365–366 physiological and molecular mechanisms, 350 plant responses, water stress aquaporin degradation, 352 cell damage, 352–353 desiccation and freezing tolerance, 353–354
554
SUBJECT INDEX
Root tropism in drought avoidance (cont.)
environmental factors, 351 germination time control, 353 growth inhibition, 351–352 permeability, 352 potential measurement, 351 RWC. See Relative water content S Salinity stress concentration-dependent uptake, 38 salt treatment, 39 inheritable epigenetic changes, 39–40 negative water potentials, 6 osmotic mechanism, 37–39 respiratory energy-use efficiency, 40 ROS accumulation, 38 sea water infiltration, 37 traditional breeding approaches, 39 Salt bladders and glands Chenopodium quinoa, 160–161 EBCs, 160 epidermal cells, 158 functional characterisation, 160 graminoids, 159–160 leaf cutinisation, 162 mangroves, 162 Naþ transport pathways, 160–161 tissue-specific expression, 161 two-celled excretory structures, 159–160 types, 159 Salt tolerance crop breeding dominance and additive effects, 152 gene transcription level, 153 HKT genes, 14 Naþ and Kþ homeostasis, 14, 17 Naþ/Hþ antiporter controls, 18 Nax1 and Nax2, 17 traits, wheat and barley, 15–16 Severe drought conditions avoidance and tolerance, plants desiccation and freezing tolerance, 353–354 germination time control, 353 plant cell damage cellular macromolecules, 353 intramembranous particles, 352–353 lamellar and hexagonal II structures, 353 water potential, 352 SNF-1-related kinase (SnRK1), 414 SnRK2s ABA signalling, 223 activation loop, 230
activation mechanism, 221 AMPK, 219 co-immunoprecipitation assay, 230 downstream factors, 230 global ABA responses, 229 guard cells, 220 membrane proteins, 231 plant-specific protein kinase, 220 signal transduction network, 232 upstream protein kinases, 225 Split-ubiquitin two-hybrid system (SUS), 262 Steroidogenic acute regulatory related lipid transfer (START) proteins, 256 Stress tolerance, protective compounds osmoprotective compounds adaptation to extreme environments, 125–130 amino acids, 119–125 environmental conditions, 110 functions, 113 glycine betaine (GB), 115–116 osmotic adjustment, 110 in plants, 110–112 polyalcohols, 117–119 protein stabilisation, 113 radical oxygen production, 114 ROS accumulation, 114 transcript profiling, 115 trehalose, 116–117 universal protecting effect, 113 osmotic stress abiotic stress tolerance, 109–110 adverse environmental conditions, 107 biomass and primary metabolism, 108 decipher plant stress response, 110 halophytic extremophiles, 109 lipid molecular profiles, 109 metabolic composition, 107–108 physico-chemical pressure, 107 Thellungiella salsuginea, 110 thermotolerance, 108 transcriptomic and proteomic approaches, 108 Subcellular milieu stabilization hydrophobic effect, water, 328 LEA proteins Boea hygrometrica, 329–330 properties, 329 tobacco and Arabidopsis, 330 sugars sucrose and galactinol synthase transcripts, 329 sucrose and RFOs accumulation, 328 vitrification, 328–329 SUS. See Split-ubiquitin two-hybrid system
555
SUBJECT INDEX T Timing of cab expression 1 (TOC1), 216, 379, 386, 393, 394, 396 TOC1. See Timing of cab expression 1 Toxoplasma gondii, 277, 281 Trehalose-6-phosphate synthase (TPS1) gene, 414 W Water and saline stress biochemical and metabolic limitations ionic imbalance, 65 light harvesting and electron transport components, 64 non-photochemical quenching, 66 Rubisco activity, 64–65 starch hydrolysis, 65 stomatal closure, 65 diffusive limitations aquaporins role, 60 carbonic anhydrase role, 59 conductance components, 56–57 impaired photobiochemistry, 57 leaf structure, 58 mesophyll resistance, defined, 57 metabolic component, 58–59 stomata guard cells, 57
stomatal and photosynthetic patchiness, 60–64 water–water cycle, 57 Water stress cell wall proteins, 41–42 cytoplasmic water potential, 35–36 growth inhibition, 40–41 Hþ ATPase and wall acidification, 42 in vitro extensibility, 43 leaf growth and elongation zone, 41, 42 proton-pumping ATPases, 41 root growth inhibition, 42–43 symptoms, 40
X Xylem ion loading, halophyte glycophytes salinity tolerance, 171 NaCl treatment, 172 Naþ concentration, 171–172 transport mechanisms, 172
Z Zeitlupe (ZTL), 396 ZTL. See Zeitlupe
