Transgenic Crop Plants: Volume 2: Utilization and Biosafety

Transgenic Crop Plants

Chittaranjan Kole Charles H. Michler Albert G. Abbott Timothy C. Hall l

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Editors

Transgenic Crop Plants Volume 2: Utilization and Biosafety

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Editors Prof. Chittaranjan Kole Department of Genetics & Biochemistry Clemson University Clemson, SC 29634, USA [email protected]

Prof. Albert G. Abbott Department of Genetics & Biochemistry Clemson University Clemson, SC 29634, USA [email protected]

Prof. Charles H. Michler Director Hardwood Tree Improvement and Regeneration Center at Purdue University NSF I/UCRC Center for Tree Genetics West Lafayette, IN, USA [email protected]

Prof. Timothy C. Hall Institute of Developmental & Molecular Biology Department of Biology Texas A&M University College Station, TX, USA [email protected]

ISBN: 978-3-642-04811-1 e-ISBN: 978-3-642-04812-8 DOI 10.1007/978-3-642-04812-8 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009939124 # Springer-Verlag Berlin Heidelberg 2010 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Transgenic Plants – known also as Biotech Plants, Genetically Engineered Plants, or Genetically Modified Plants – have emerged amazingly fast as a boon for science and society. They have already played and will continue to play a significant role in agriculture, medicine, ecology, and environment. The increasing demands for food, feed, fuel, fiber, furniture, perfumes, minerals, vitamins, antibiotics, narcotics, and many health-related drugs and chemicals necessitate the development and cultivation of transgenic plants with augmented or suppressed trait(s). From a single transgenic plant (Flavr Savr tomato with a longer shelf-life) introduced for commercialization in 1994, we have now 13 transgenic crops covering 800 million ha in 25 countries of six continents. Interestingly, the 13.3 million farmers growing transgenic crops globally include 12.3 million (90%) small and resource poor farmers from 12 developing countries. Increasing popularity of transgenic plants is well evidenced from an annual increase of about 10% measured in hectares but actually of 15% in “trait hectares.” Considering the urgent requirement of transgenic plants and wide acceptance by the farmers, research works of transgenic plants are now being conducted on 57 crops in 63 countries. Transgenic plants have been developed in over 100 plant species and they are going to cover the fields, orchards, plantations, forests, and even the seas in the near future. These plants have been tailored with incorporation of useful alien genes for several desirable traits including many with “stacked traits” and also with silencing of genes controlling some undesirable traits. Development, applications and socio-political implications of transgenic plants are immensely important fields now in education, research, and industries. Plant transgenics has deservedly been included in the course curricula in most, if not all, leading universities and academic institutes all over the world, and therefore reference books on transgenic plants with a class-room approach are essential for teaching, research, and extension. There are some elegant reviews on the transgenic plants or plant groups (including a 10-volume series “Compendium of Transgenic Crop Plants” edited by two of the present team of editors C. Kole and T.C. Hall published by Wiley-Blackwell in 2008) and on many individual tools and

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techniques of genetic transformation in plants. All these reviews could surely serve well the purpose for individual crop plants or particular methodologies. Since transgenic plant development and utilization is studied, taught, and practiced by students, teachers, and scientists of over a dozen disciplines under basic science, agriculture, medicine, and humanities at public and private sectors, introductory reference books with lucid deliberations on the concepts, tools, and strategies to develop and utilize transgenic plants and their global impacts could be highly useful for a broad section of readers. Deployment of transgenic crop plants are discussed, debated, regulated, and sponsored by people of diverse layers of the society, including social activists, policy makers, and staff of regulatory and funding agencies. They also require lucid deliberations on the deployment, regulations, and legal implications of practicing plant transgenics. More importantly, depiction of the positive and realistic picture of the transgenic plants should and could facilitate mitigation of the negative propaganda against transgenic plants and thereby reinforce moral and financial support from all individuals and platforms of the society. Global population is increasing annually by 70 millions and is estimated to grow to eight billion by 2025. This huge populace, particularly its large section from the developing countries, will suffer due to hunger, malnutrition, and chemical pollution unless we produce more and more transgenic plants, particularly with stacked traits. Compulsion to meet the requirements of this growing population on earth and the proven innocuous nature of transgenic plants tested and testified for the last 13 years could substantiate the imperative necessity of embracing transgenics. Traditional and molecular breeding practiced over the last century has provided enormous number of improved varieties in economic crops and trees including wheat and rice varieties that fostered the “green revolution.” However, these crop improvement tools depend solely on the desirable genes available naturally, creatable by mutation in a particular economic species, or their shuffling for desired recombinations. Transgenic breeding has opened a novel avenue to incorporate useful alien genes from not only other cross-incompatible species and genera of the plant kingdom, but also from members of the prokaryotes including bacteria, fungi, and viruses, and even from higher animals including mice and humans. An array of plant genetic engineering achievements starting from the development of insect resistance cotton by transforming the cry genes from the bacteria Bacillus thuringiensis to the present-day molecular pharming that enables the expression of interferon- gene from human in tobacco evidence for this pan-specific gene transfer. Human and animal safety is another general concern related to transgenic food or feed. However, there is no reliable scientific documentation of these health hazards even after 13 years of cultivation of transgenic plants and consumption of about 1 trillion meals containing transgenic ingredients. Utilization of transgenic plants has reduced the pesticide applications by 359,000 tons that would otherwise affect human and animal health besides causing air, water, and soil pollution and also mitigated the chance of consumption of dead microbes and insects along with foods or feeds.

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Gene flow from transgenic crop species to their cross-compatible wild relatives is a genuine concern and therefore required testing of a transgenic crop plant before deployment followed by comprehensive survey of the area for presence of interfertile wild and weedy plants before introduction of a transgenic crop are being seriously conducted. Addition of novel genotypes with transsgenes in the germplasms is increasing the biological diversity rather than depleting it. Using the genetically engineered plants has also eliminated greenhouse gas emission of 10 million metric tons through fuel savings. In fact, 1.8 billion liters of diesel have been saved because of reduced tillage and plowing owing only to herbicide-resistant transgenic crops. Many transgenics are now being used for soil reclamation. Above all, cultivation of transgenic crops has returned $44 billion of net income to the farmers. Perhaps, these are the reasons that 25 Nobel Laureates and 3,000-plus eminent scientists appreciated the merits and safety and also endorsed transgenic crops as a powerful and safe way to improve agriculture and environment besides the safety of genetically modified foods. Many international and national organizations have also endorsed health and environmental safety of transgenic plants; these include Royal Society (UK), National Academy of Sciences (USA), World Health Organization, Food and Agriculture Organization (UN), European Commission, French Academy of Medicine and American medical Association, to name a few. Production, contributions, and socio-political implications of biotech plants are naturally important disciplines now in education, research, and industries and therefore introductory reference books are required for students, scientists, industries, and also for social activists and policy makers. The two book volumes on “Transgenic Crop Plants” will hopefully fill this gap. These two book volumes have several unique features that deserve mention. The outlines of the chapters for these two books are formulated to address the requirements of a broad section of readers. Students and scholars of all levels will obtain a lot of valuable reading material required for their courses and researches. Scientists will get information on concepts, strategies, and clues useful for their researches. Seed companies and industries will get information on potential resources of plant materials and expertise for their own R&D activities. In brief, the contents of this series have been designed to fulfill the demands of students, teachers, scientists, and industry people, for small to large libraries. Students, faculties, or scientists involved in various subjects will be benefited from this series; biotechnology, bioinformatics, molecular biology, molecular genetics, plant breeding, biochemistry, ecology, environmental science, bioengineering, chemical engineering, genetic engineering, biomedical engineering, pharmaceutical science, agronomy, horticulture, forestry, entomology, pathology, nematology, virology, just to name a few. It had been our proud privilege to edit the 23 chapters of these two books those were contributed by 71 scientists from 14 countries and the list of authors include one of the pioneers of plant transgenics, Prof. Timothy C. Hall (one of the editors also); some senior scientists who have themselves edited books on plant transgenics; and many scientists who have written elegant reviews on invitation for quality books and leading journals. We believe these two books will hopefully

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serve the purposes of the broad audience who are studying, teaching, practicing, supporting, funding, and also those who are debating for or against plant transgenics. The first volume dedicated to “Principles and Development” elucidates the basic concepts, tools, strategies, and methodologies of genetic engineering, while the second volume on “Applications and Safety” enumerates the utilization of transgenic crop plants for various purposes of agriculture, industry, ecology, and environment, and also genomics research. This volume also deliberates comprehensively on the legal and regulatory aspects; complies to the major concerns; and finally justifies the compulsion of practicing plant transgenics. Glimpses on the contents of this volume (Volume 2: Transgenic Crop Plants: Applications and Safety) will perhaps substantiate its usefulness. This volume enumerates the application of transgenic technologies in crop plants for particular objectives in the first ten chapters. Biotic stress resistant, specifically insect resistant, transgenics have been developed and commercialized in several crops. An example with Bt-expressing cotton and maize alone, with current market share of about $3.26 billion substantiates their success and popularity (Chap.1). Abiotic stresses, particularly drought, salinity, and temperature extremes, have always been difficult to manipulate. Still success stories are pouring in recently from works mainly in cereals and vegetables (Chap.2). Herbicide-resistant transgenic plants (in cotton and canola) were first deregulated in 1995 and in 2008 more than 80% of the transgenic plants grown globally possess a transgenic trait for herbicide resistance. Chapter 3 details the present and emerging herbicide-resistant transgenic plants. Although the first transgenic trait was developmental, shelf-life in tomato to be precise, transgenics research for these traits are yet to make significant commercial headway but started producing encouraging results (Chaps.4 and 5). Deployment of transgenic plants for biofuel, pharmaceuticals, and other bioproducts has been enunciated in three chapters (Chaps.6, 7, and 9). Transgenic plants have been labeled as a culprit for potential threats to ecology and environment by a few groups of social activists. Chapter 8 addresses these weird concerns with suitable examples of utilization of transgenic plants for phytoremediation, biomonitoring, and the production of bioplastics and biopolymers for amelioration of ecology and environment. Plant genomics has emerged fast within the last three decades and facilitated fine-scale view of the plant genes and genomes. Transgenic plants have provided enormous resources for functional genomics studies and expected to play their roles as more plants systems and genes are targeted (Chap. 10). Scientists practicing transgenics are no less aware of the potential risks of genetic engineering than the few people with antagonistic views. Neither are the regulatory agencies at institutional, state, national, and international level regulatory agencies unaware of the steps to be involved for inspection, monitoring, and approval of transgenic plants for commercial use. Chapter 11 delineates all these aspects with examples from US and other continents and countries. Any original innovation or effort deserves recognition and also an incentive. The scope of patenting and intellectual property rights for materials owned and generated and methodologies implemented have been appreciated and enforced legally. These aspects related to transgenic crop plants have been discussed in Chap.12.

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The concluding chapter (Chap.13) briefs the contributions and concerns with the compliances and compulsion of practicing plant transgenics for science and society. We thank all the 41 scientists from nine countries for their elegant and lucid contributions to this volume and also for their sustained support through revision, updating and fine-tuning their chapters. We also acknowledge for the recent statistics that have been accessed from the web sites of Monsanto Company on “Conversations about Plant Biotechnology” and “International Service for the Acquisition of Agri-Biotech Applications on ISAAA Brief 39-2008: Executive Summary” and used them in this preface and elsewhere in the volume. We enjoyed a lot of our Clemson–Purdue–Texas A&M triangular interaction, constant consultations, and dialogs while editing this book, and also our working with the editorial staff of Springer, particularly Dr. Sabine Schwarz who had been supportive since inception till publication of this book. We will look forward to suggestions from all corners for future improvement of the content and approach of this book volume. Chittaranjan Kole, Clemson, SC Charles H. Michler, West Lafayette, IN Albert G. Abbott, Clemson, SC Timothy C. Hall, College Station, TX

Contents

1

Transgenic Crop Plants for Resistance to Biotic Stress . . . . . . . . . . . . . . . 1 N. Ferry and A.M.R. Gatehouse

2

Transgenic Plants for Abiotic Stress Resistance . . . . . . . . . . . . . . . . . . . . . . 67 Margaret C. Jewell, Bradley C. Campbell, and Ian D. Godwin

3

Transgenic Crops for Herbicide Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Stephen O. Duke and Antonio L. Cerdeira

4

Understanding and Manipulation of the Flowering Network and the Perfection of Seed Quality . . . . . . . . . . . . . . . . . . . . . . . . . 167 Stephen L. Goldman, Sairam Rudrabhatla, Michael G. Muszynski, Paul Scott, Diaa Al-Abed, and Shobha D. Potlakayala

5

Biotechnological Interventions to Improve Plant Developmental Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Avtar K. Handa, Alka Srivastava, Zhiping Deng, Joel Gaffe, Ajay Arora, Martı´n-Ernesto Tiznado-Herna´ndez, Ravinder K. Goyal, Anish Malladi, Pradeep S. Negi, and Autar K. Mattoo

6

Transgenics for Biofuel Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Anjanabha Bhattacharya, Pawan Kumar, and Rippy Singh

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Plant Produced Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Jared Q. Gerlach, Michelle Kilcoyne, Peter McKeown, Charles Spillane, and Lokesh Joshi

8

Biotech Crops for Ecology and Environment . . . . . . . . . . . . . . . . . . . . . . . . 301 Saikat Kumar Basu, Franc¸ois Eudes, and Igor Kovalchuk

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9

Algal Biotechnology: An Emerging Resource with Diverse Application and Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Cunningham Stephen and Joshi Lokesh

10

Transgenic Crops and Functional Genomics . . . . . . . . . . . . . . . . . . . . . . . . . 359 Narayana M. Upadhyaya, Andy Pereira, and John M. Watson

11

Deployment: Regulations and Steps for Commercialization . . . . . . . . 391 Kelly D. Chenault Chamberlin

12

Patent and Intellectual Property Rights Issues . . . . . . . . . . . . . . . . . . . . . . . 411 Jim M. Dunwell

13

Transgenic Crop Plants: Contributions, Concerns, and Compulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Brian R. Shmaefsky

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

Contributors

Diaa Al-Abed Edenspace Systems Corporation, Manhattan, KS 66502, USA, [email protected] Ajay Arora Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi 110012, India Saikat Kumar Basu Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4; Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB, Canada T1J 4B1 Anjanabha Bhattacharya National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA, [email protected] Bradley C. Campbell School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia Antonio L. Cerdeira Brazilian Department of Agriculture, Agricultural Research Service, EMBRAPA/Environment, C.P. 69, Jaguariuna-SP-13820000, Brazil Kelly D. Chenault Chamberlin USDA-ARS, Wheat, Peanut, and Other Field Crops Unit, 1301 N. Western, Stillwater, OK 74075, USA, kelly.Chamberlint@ars. usda.gov Zhiping Deng Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305, USA Stephen O. Duke Agricultural Research Service, United States Department of Agriculture, P. O. Box 8048, University, MS 38677, USA, [email protected]

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Contributors

Jim M Dunwell University of Reading, Whiteknights, Reading RG6 6AS, UK, [email protected] Franc¸ois Eudes Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB, Canada T1J 4B1 N. Ferry School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK, [email protected] Joel Gaffe Laboratoire Adaptation et Pathoge´nie des Microorganismes, LAPM, UMR 5163 CNRS-UJF, Institut Jean Roget BP 170, 38042 Grenoble cedex 9, France A.M.R. Gatehouse School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK Jared Q. Gerlach Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Ian D. Godwin School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia, [email protected] Stephen L. Goldman Department of Environmental Sciences, The University of Toledo, Toledo, OH 43606, USA, [email protected] Ravinder K. Goyal Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8W 3P6 Avtar K. Handa Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA, [email protected] Margaret C. Jewell School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia Lokesh Joshi Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland, [email protected] Michelle Kilcoyne Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland

Contributors

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Igor Kovalchuk Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4, [email protected] Pawan Kumar National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA Joshi Lokesh Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland, [email protected] Anish Malladi 30602, USA

Department of Horticulture, University of Georgia, Athens, GA

Autar K. Mattoo Sustainable Agricultural Systems Laboratory, The Henry A. Wallace Beltsville Agric Research Center, Beltsville, MD 20705-2350, USA Peter McKeown Genetics and Biotechnology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland Michael G. Muszynski Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA, [email protected] Pradeep S. Negi Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA Andy Pereira 24061, USA

Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA

Shobha D. Potlakayala Penn State Milton S. Hershey College of Medicine, Hershey, PA 17033, USA, [email protected] Sairam Rudrabhatla Environmental Engineering, College of Science, Engineering and Technology, Penn State University, Middletown, PA 17057, USA, [email protected] Paul Scott Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA, [email protected]; Department of Agronomy, USDA-ARS, Iowa State University, Ames, IA 50011, USA, paul.scott@ ars.usda.gov Brian R. Shmaefsky Lone Star College – Kingwood, HSB 202V, 20,000 Kingwood Drive, Kingwood, TX 77339-3801, USA, Brian.R.Shmaefsky@ lonestar.edu

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Contributors

Rippy Singh National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA Charles Spillane Genetics and Biotechnology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland Alka Srivastava Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA Cunningham Stephen Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Martı´n-Ernesto Tiznado-Herna´ndez Fisiologı´a y Biologı´a Molecular de Plantas, Coordinacio´n de Tecnologı´a de Alimentos de Origen Vegetal, Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C., Hermosillo, Sonora, Me´xico Narayana M. Upadhyaya CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 260, Australia, [email protected] John M. Watson Australia

CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 260,

Abbreviations

1-FFT 1-MCP 1-SST 2,4-D 2D-PAGE 4C3H 4CL 6G-FFT 6-SFT AAFC ABA ABRE Ac ACC AChE ACP ADP ae1 AHK2/3 AL-PCD ALS AMGT AMPA ANVISA AOS AOX ap AP1 AP2

Fructan:fructan 1-fructosyltransferase 1-Methylcyclopropene Sucrose:sucrose 1-fructosyltransferase 2,4-Dichlorophenoxyacetic acid Two-dimensional polyacrylamide gel electrophoresis 4-Coumarate 3-hydroxylase 4-Hydroxycinnamoyl CoA ligase Fructan:fructan 6G-fructosyltransferase Sucrose:fructan 6-fructosyltransferase Agriculture and Agri-Food Canada Abscisic acid ABA responsive element Activator gene 1-Aminocyclopropane-1-carboxylate Acetylcholinesterase Acyl-carrier protein Adenosine di-phosphate amylose extender gene Arabidopsis histidine kinase Apoptotic-like programmed cell death Acetolactate synthase Agrobacterium-mediated gene transfer Aminomethylphosphonic acid National Agency for Health and Surveillance of the Ministry of Health Allene oxide synthase Altenative oxidase apetalla gene APETALA1 gene APETALA2/ Apetela2 gene

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APHIS APX ARF arsC Asc at AtCKX1 Avr AZ BA BADH BAP BAR BBC BC BGI-RIS bla BMR BRS Bt bZIP CAD CAL CAMBIA CaMV CAT CBER CBF cbLCV CBS CCR CDC CDF cDNA CEL CEPA CFIA CFSAN CGH-1 CGIAR cGMP CHO CHO

Abbreviations

Animal and Plant Health Inspection Service Ascorbate peroxidase Auxin response factor Arsenate reductase Ascorbate antherless gene Arabidopsis thaliana cytokinin oxidase gene Avirulence Abscission zone Benzyladenine Betaine aldehyde dehydrogenase 6-Benzylaminopurine Bialaphos resistance gene British Broadcasting Corporation Biotech crop Beijing Genomics Institute- Rice Information System Beta-lactamase Brown midrib Biotechnology Regulatory Service Bacillus thuriengensis Basic leucine zipper Cinnamoyl alcohol dehydrogenase CAULIFLOWER gene Center for the Application of Molecular Biology to International Agriculture Cauliflower mosaic virus Chloramphenicol acetyltransferase/catalase Centre for Biologics Evaluation and Research CRT binding factor Cabbage leaf curl virus Columbia Broadcasting System Cinnamoyl CoA reductase Centers for Disease Control Cycling DOF Factor Complementary-DNA Cellulase Canadian Environmental Protection Act Canadian Food Inspection Agency Centre for Food Safety and Applied Nutrition Cardenolide 16’-O-glucohydrolase Consultative Group on International Agricultural Research Current GMP Chinese hamster ovary Choline dehydrogenase

Abbreviations

chs CIGB CMO CMS CMS CNN CNR CO ConA CONABIA conz1 COR COR CP CpTI CRE CRIIGEN CRT Cry CTNBio CVM CV-N D2GT2A dab DDB DDT DEFRA DET DGDG DHAsc dlf1 DNMA DRE DREB driPTGS Ds DsE DsG DSL dzr1 EA EBV EC EDB

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chalcone synthase gene Cuban Centre for Biotechnology and Genetic Engineering Choline monooxygenase Cytoplasmic male sterility Cellular membrane stability Cable News Network Colorless non-ripening gene Constans gene Concanavalin A National Advisory Commission on Agricultural Biotechnology constans of Zea mays1 gene Cold responsive Cold responsive gene Chloroplast Cowpea trypsin inhibitor Cytokinin response Comite´ de Recherche et d’Information Inde´pendantes sur le Ge´nie C-Repeat Crystal National Technical Commission on Biosafety Centre for Vetinary Medicine Cyanovirin Diacylglycerol acyltransferase 2A delayed abscission gene Damaged DNA binding protein Dichlorodiphenyltrichloroethane Department of Environment, Food and Rural Affairs Detiolated gene Digalactosyldiacylglycerol Dehydroascorbate delayed flowering1 gene Directorate of Agricultural Markets Dehydration responsive element DRE binding protein Direct repeat-induced PTGS Dissociation gene Enhancer trap Ds Gene trap Ds Domestic Substance List delta zein regulator1 gene Environmental assessment Epstein-barr virus European Commission Ethylene dibromide

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EFSA EFSA EIN EIS EMEA EMS En EPA EPA EPSP EPSPS ER ERE ERS EST ETC ETH EU F F1 FAD1 FAD3 FAO FD FDA FFDCA FIFRA fl2 FONSI FPPA Fr FST FT FT-ICR-MS Fuc FucT FUL Fx G3P GA/ GA3 Gal GalNAc GalT GAT

Abbreviations

European Food Safety Authority European Food Standards Agency Ethylene-insensitive gene Environmental Impact Statement European Agency for Evaluation of Medicinal Products Ethylmethane sulfonate Enhancer transposon Eicosapentaenoic Environmental Protection Agency Enolpyruvyl-shikimate-3-phosphate 5-Enolpyruvyl-shikimate-3-phosphate synthase Endoplasmic reticulum Ethylene responsive element Economic Research Service Expressed sequence tag Electron transport chain Ethylene European Union Florigenic signal Fraction 1 anti-phagocytic capsular envelope protein Flavin adenine dinucleotide Omega-3 fatty acid desaturase Food and Agriculture Organization of the United Nations FLOWERING LOCUS D gene Food and Drug Administration Federal Food, Drug and Cosmetic Act Federal Insecticide, Fungicide and Rodenticide Act floury-2 gene Finding of no significant impact Federal Plant Pest Act Fertility restorer gene Flanking sequence tag Flowering Locus T/Flowering Transition gene Fourier-transform ion cyclotron mass spectrometry Fucose Fucosyltransferase FRUITFUL gene Fucoxantine Glycerol-3-phosphate Gibberellic acid Galactose N-Acetylgalactosamine Galactosyltransferase Glyphosate N-acetyltransferase

Abbreviations

GC GCase GCS GDP GE GFLV GFP GI gigz1 GlcNAc GlyBet GM GMHT GMO GMP GMP GMPO GMS GNA GOI GOX GPAT GPX GR GRC GS1 GSH GSSG GST GTN GUS HBcAg HBsAg HBV HCMV hEPO hGM-CSF HIV HMX HR HRC HS HSP HSV

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Glutathione synthetase Glucosyl-N-acylspingosineglycohydrolase g-Glutamylcysteine synthase Gross domestic product Genetic engineering/Genetically engineered Grapevine fanleaf virus Green fluorescent protein Gigantea gene gigantea of Zea mays1 gene N-Acetylglucosamine Glycine betaine Genetically modified Genetically modified herbicide tolerant Genetically modified organism Genetically modified plant Good manufacturing practise Genetically modified plant organism Genic male sterility Galanthus nivalis agglutinin Gene of interest Glyphosate oxidoreductase Glycerol-3-phosphate acyltransferase Glutathione peroxidase Glutathione reductase/ Glyphosate resistant Glyphosate resistant Crop Glutamine synthase gene 1 Glutathione/ Glutamate synthase Glutathione disulfide Glutathione S-transferase Glycerol trinitrate ß-Glucuronidase Hepatitis B core antigen Hepatitis B surface antigen Hepatitis B virus Human cytomegalovirus Human erythropoietin Human granulocyte-macrophage colony-stimulating factor Human immunodeficiency virus Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine Hypersensitive response/ Herbicide resistant Herbicide resistant crop Heat shock Heat shock protein Herpes simplex virus

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HT/Ht I i.p. iAc IBA ICTSD id1 ida IDD IFN IgA IL-12 IMI IPM IPP IPR IPT IR IRGSP ISAAA ISIS ISR JA LB LD LEA Leu LFY LOG LOX1/2/3 lpa1 LPS LRR LTB Lys MAb MAPK MGDG MHBsAg Mi MIP miRNA MPSS mRNA

Abbreviations

Herbicide tolerant/tolerance Inhibitor transposon Intraperitoneal Immobile Ac transposon Indole-3-butyric acid International Center for Trade and Sustainable Development indeterminate1 gene inflorescence deficient in abscission gene ID-domain Interferon Immunoglobulin A Interleukin-12 Imidazolinone Integrated pest management Isopentyl diphosphate Intellectual Property Rights Isopentenyl transferase/ Isopentyl transferase Insect resistant/resistance International Rice Genome Sequencing Project International Service of AgriBiotech Applications Institute for Science and International Security Induced systemic resistance Jasmonic acid Left border of T-DNA Long-day Late embryogenesis abundant Leucine Leafy gene Lonely guy gene Lipoxygenase gene Lysophosphatidic acid receptor Lipopolysaccharide Leucine rich repeats Heat-labile enterotoxin, subunit B Lysine Monoclonal antibody Mitogen-activated protein kinase Monogalactosyldiacylglycerol Middle HBsAg Meloidogyne incognita resistance gene Major intrinsic protein Micro-RNA Massively parallel signature sequencing Messenger-RNA

Abbreviations

MS MS MST MT MTP MTT Mu MuIL-12 MV MVL NAA NAM NAS NAS NBS NCBI NDV NEPA Neu5Ac NIH NIL NMR NO NOI NoV NR NST NUE o2 OECD OFB OMT ORF Ori OSTP PAHs PAL PAMPs PAT PC PCB PCD PCR PCS

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Mass spectrometry Murashige and Skoog (medium) Members of the Landless Rural Workers Movement Metallothionein Metal-tolerance protein Multi-tasking transgenics Mutator transposon Murine IL-12 Methyl viologen Microcystis viridis lectin a-Napthalene acetic acid Napthaleneacetamide National Academy of Sciences Nicotinamine synthase Nucleotide binding site National Center for Biotechnology Information Newcastle disease virus National Environmental Policy Act 5-N-Acetyl-D-neuraminic acid National Institute of Health Near-isogenic line Nuclear magnetic resonance Nitric oxide Notice of Intent Norovirus Nitrate reductase Nac secondary wall thickening promoter factor Nitrogen use efficiency opaque-2 gene Organization for Economic Cooperation and Development Office of Food Biotechnology O-Methyl transferase Open reading frame Origin of replication Office of Science and Technology Policy Polycyclic aromatic hydrocarbon Phenyalanine ammonia lyase Pathogen associated molecular patterns Phosphinothricin-acetyltransferance Phytochelatin Polychlorinated biphenyl Programmed cell death Polymerase chain reaction Phytochlelatin synthase

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PEG PETN PG PG PHB pi PiP PIP PL PLC PLD PLE PME PMP PNT PPO PR Pro PS PSI PSII PTGS Put PVP PVX PyMSP4/5 QPM QTL RAP-DB rasiRNA RB RB rDNA RDX Rf RFLP R-gene rGSII rhCVFVIII rhEPO rhIF RHS RID1 RIL

Abbreviations

Polyethylene glycol Pentaerythritol tetranitrate Polygalacturonase Phosphatidylglycerol Polyhydroxybutyrate pistillata gene Plant Incorporated Protectant Plasma membrane intrinsic protein Pectate lyase Phospholipase C Phospholipase D Phospholipid cleaving enzyme Pectin methylesterase Plant-made pharmaceutical Plant with novel trait Polyphenol oxidase Pathogenesis-related Proline Phytosiderophores Photosystem 1 Photosystem 2 Post-transcriptional gene silencing Putrescine Plant Variety Protection Potato virus X Murine P. yoelii merozoite surface protein 4/5 Quality protein maize Quantitative trait loci Rice Annotation Project-Database Repeat-associated siRNA Non-toxin B-chain from ricin Right border of T-DNA Recombinant-DNA Hexahydro-1,3,5-trinitro-1,3,5 triazine Restorer of fertility gene Restriction fragment length polymorphism Resistance-gene Recombinant Griffonia simplicifolia lectin II Recombinant human clotting factor VIII Recombinant human erythropoietin Recombinant human intrinsic factor Royal Horticultural Society Rice Indeterminate1 gene Recombinant inbred line

Abbreviations

rin Rip RNAi ROIs ROS RT-PCR RWC s.c. SA SA SAGE SAGPyA SAM SAM SAR scFv scN SD SE SENASA Ser SFI1 sh2 siRNA SIV SL SMT soc SOC1 SOD SOliD Spd Spm Spm SQDG ssRNA STP su1 SVN TA TAC TAGI tasiRNA tb1

xxv

ripening inhibitor gene Ribosome inactivating protein RNA-interference Reactive oxygen intermediates Reactive oxygen species Reverse transcriptase-PCR Relative water content Subcutaneously Salicylic acid Splice acceptor Serial analyses of gene expression Secretariat of Agriculture, Livestock, Fisheries and Food S-Adenosylmethionine Shoot apical meristem Systemic acquired resistance Single chain variable fragment Soyacystatin N Short-day Substantial equivalence/ equivalent National Agri-food Health and Quality Service Serine Segestria florentina venom peptide shrunken2 gene Short/Small interfering RNA Simian immunodeficiency virus Selenocysteine lyase Seleno-cysteine methyl transferase suppessor of overexpression of constans gene Suppressor of Overexpression of Constans1 gene Superoxide dismutase Supported Oligo Ligation Detection Spermidine Spermine Suppressor-Mutator transposon Sulfoquinovosyldiacylglycerol Single-stranded RNA Signal transduction pathway sugary1 gene Scytovirin Transcriptional activator Tiller angle control gene The Arabidopsis Genome Initiative Transacting siRNA teosinte branched1 gene

xxvi

TCE TCOH TCP T-DNA TDZ TET TETRYL TF TFL Thr ti TILLING TIP TMV TNT Trp TRV TSCA UAS uf UN UNCTAD US USAID USDA USPTO UV VB Vgt1 Vgt2 VIGS VIP VLP VRO WHO WT WUE XTH Xyl XylT YCF1 YFP ZCN zfl1

Abbreviations

2.4,6-Trichloroethylene Chloral and trichoethanol 2,4,6-Tricholorophenol Transferred-DNA Thidiazuron Transiently expressed transposase N-Methyl-N, 2, 4, 6-tetranitroaniline Transcription factor Terminal Flower gene Threonine Trypsin inhibitor allele Targeting induced local lesions in genomes Tonoplast intrinsic protein Tobacco mosaic virus Trinitrotoluene Tryptophan Tobacco rattle virus Toxic Substances Control Act Upstream activator sequence uniflora gene United Nations United Nations Conference on Trade and Development United States US Agency for International Development United States Department of Agriculture United States Patent and Trademark Office Ultraviolet Vector backbone Vegetative to generative transition1 gene Vegetative to generative transition2 gene Virus-induced gene silencing Vegetative Insecticidal Protein Virus-like particle Variety Registration Office World Health Organisation Wild type Water use efficiency Xyloglucan endotransglucosylase/hydrolase Xylose Xylosyltransferase Yeast vacuolar glutathione Cd transporter Yellow fluorescent protein Zea CENTRORADIALIS gene Zea FLO/LFY1 gene

Abbreviations

zfl2 ZFN ZMM4/5 ZmRap2 g-GCS o3

xxvii

Zea FLO/LFY2 gene Zinc-finger nuclease Zea mays FULL1-like gene Zea mays related to AP2 gene g-Glutamyl cysteine synthetase Omega 3

Chapter 1

Transgenic Crop Plants for Resistance to Biotic Stress N. Ferry and A.M.R. Gatehouse

1.1

Introduction

We couldn’t feed today’s world with yesterday’s agriculture and we won’t be able to feed tomorrow’s world with today’s. – Lord Robert May, President of the Royal Society, March 2002

The human population is everexpanding; conservative estimates predict that the population will reach ten billion by 2050 (United Nations Population Division), and the ability to provide enough food is becoming increasingly difficult (Chrispeels and Sadava 2003). The planet has a finite quantity of land available to agriculture and the need for increasing global food production has led to increasing exploitation of previously uncultivated land for agriculture; as a result wilderness, wetland, forest and other pristine environments have been, and are being, encroached upon (Ferry and Gatehouse 2009). The minimization of losses to biotic stress caused by agricultural pests would go some way to optimizing the yield on land currently under cultivation. For nearly 50 years, mainstream science has told us that this would be impossible without chemical pesticides (Pimental 1997). The global pesticide market is in excess of $30 billion per year (Levine 2007); despite this, approximately 40% of all crops are lost directly to pest damage (Fig. 1.1). These figures are simplified rough estimates; in reality crop losses to biotic stress are extremely difficult to quantify and vary by crop, year, and region.

N. Ferry (*) School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_1, # Springer-Verlag Berlin Heidelberg 2010

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Fig. 1.1 The world agricultural cake

1.2 1.2.1

Biotic Stress due to Insect Pests Crop Losses due to Insect Pests

[A]nimals annually consume an amount of produce that sets calculation at defiance; and, indeed, if an approximation could be made to the quantity thus destroyed, the world would remain skeptical of the result obtained, considering it too marvelous to be received as truth. – John Curtis, 1860

Arthropods are the most widespread and diverse group of animals, with an estimated 4–6 million species worldwide (Novotny et al. 2002). While only a small percentage of arthropods are classified as pests, they cause major devastation of crops, destroying around 14% of the world annual crop production, contributing to 20% of losses of stored grains and causing around US$100 billion of damage each year (Nicholson 2007). Herbivorous insects and mites are a major threat to food production for human consumption. Larval forms of lepidopterans are considered the most destructive insects, with about 40% of all insecticides directed against heliothine species (Brooks and Hines 1999). However, many species within the orders Acrina, Coleoptera, Diptera, Hemiptera, Orthoptera and Thysanoptera are also considered agricultural pests with significant economic impact (Fig. 1.2). Insect pests may cause direct damage by feeding on crop plants in the field or by infesting stored products and so competing with humans for plants as a food resource. Some cause indirect damage, especially the sap-feeding (sucking) insects by transmitting viral diseases or secondary microbial infections of crop plants.

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Fig. 1.2 Phylloxera, a sap-sucking pest of grape, almost devastated the European wine industry in the nineteenth century

1.2.1.1

The Phylloxera Plague

In the late nineteenth century, a phylloxera epidemic destroyed most of the vineyards for wine grapes in Europe, most notably in France. Grape phylloxera (Daktulosphaira vitifoliae, family Phylloxeridae) is a pest of commercial grapevines worldwide, originally native to eastern North America. These minute, pale yellow sap-sucking insects feed on the roots of grapevines. In Vitis vinifera, the resulting deformations and secondary fungal infections can damage roots, gradually cutting off the flow of nutrients and water to the vine. Phylloxera was inadvertently introduced to Europe in the 1860s. The European wine grape V. vinifera was highly susceptible to the pest and the epidemic devastated most of the European winegrowing industry. Some estimates hold that between two-thirds and nine-tenths of all European vineyards were destroyed. Native American grapes Vitis labrusca are naturally Phylloxera-resistant. The grafting of European grape vines onto resistant grape rootstock is the preferred method to cope with the pest problem even today (http://www.calwineries.com). Thus, phylloxera provides a clear example of how a single insect pest can nearly devastate a whole industry. Innumerable examples exist of insect pests that are highly injurious to agricultural production. The most notable for their destructive capacity being the

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Fig. 1.3 Monument to the cotton boll weevil Source: Wikimedia Commons

migratory locust (Locusta migratoria), Colorado potato beetle (Leptinotarsa decemlineata), boll weevil (Anthonomus grandis), Japanese beetle (Popillia japonica), and aphids, which are among the most destructive pests on earth as vectors of plant viruses (many species in ten families of the Aphidoidea), and the western corn rootworm (Diabrotica virgifera virgifera), also called the billion dollar bug because of its economic impact in the US alone. Curiously, one of these pests, the cotton boll weevil, responsible for neardestruction of the cotton industry in North America, is also ultimately responsible for subsequent diversification of agriculture in many regions, thus warranting a monument in the town of Enterprise, Alabama, in profound appreciation of its role in bringing to an end the state’s dependence on a poverty crop (Fig. 1.3). The global challenge facing agriculture is to secure large and high-quality crop yields and to make agricultural production environmentally sustainable. Control of insect pests would go some way towards achieving this goal.

1.2.2

Insecticides

Insecticides have been, and still are, a highly effective method to control pests quickly when they threaten to destroy crops. The chemical nature of the

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insecticides used has evolved over time. In early farming practices, inorganic chemicals were used for insect control; however, with the advances in synthetic organic chemistry that followed the two world wars the synthetic insecticides were born. In the 1940s, the neurotoxic organochlorine, DDT, was the pesticide of choice, but following its indiscriminate use it was reported to bio-accumulate in the food chain where it affected the fertility of higher organisms – such as birds. Rachel Carson first highlighted this in the book Silent Spring published in 1962; while her presumptions have since been proven to be wrong, the book was nevertheless an important signature event in the birth of the environmental movement. This pesticide was subsequently replaced by the comparatively safer organophosphate and carbamate-based pesticides (both acetylcholinesterase inhibitors) and many of these were replaced in turn by the even safer pyrethroid-based pesticides (axonic poisons). Synthetic pyrethroids continue to be used today despite the fact that they are broad-spectrum pesticides. The major limiting factor on the insecticide strategy is the occurrence of resistance in insect populations. In fact, resistance to insecticides has now been reported in more than 500 species (Nicholson 2007). Furthermore, resistance has evolved to every major class of chemical. The underlying causes of insecticide resistance are manyfold. Owing to wide usage and narrow target range, arthropods have been put under a high degree of selection pressure (Feyereisen 1995). Insecticide resistance may be characterized by: (a) Metabolic detoxification (upregulation of esterases, glutathione-S-transferases, and monoxygenases) (b) Decreased target site sensitivity (via mutation of the target receptor) (c) Sequestration or lowered insecticide availability In addition, cross-resistance to different classes of chemicals has occurred because of the fact that many insecticides target a limited number of sites in the insect nervous system (Raymond-Delpech et al. 2005). The five target sites in insects comprise: nicotinic acetylcholine receptors (e.g., imidacloprid), voltagegated sodium channels (e.g., DDT, pyrethroids), g-aminobutyric acid receptors (e.g., fipronil), glutamate receptors (e.g., avermectins), and acetylcholinesterase (AChE) (e.g., organophosphates and carbamates). The world insecticide market is dominated by compounds that inhibit the enzyme AChE. Together, AChE inhibitors and insecticides acting on the voltage-gated sodium channel, in particular the pyrethroids, account for approximately 70% of the world market (Nauen et al. 2001). Unfortunately, as insecticide target sites are conserved between invertebrates and vertebrates, insecticides have undesirable nontarget effects and unacceptable ecological impacts. Insecticides are implicated in the poisoning of nontarget insects, other arthropods, marine life, birds, and humans (Fletcher et al. 2000). The poisoning of nontarget organisms has obvious implications for biodiversity.

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Integrated Pest Management and Organic Agriculture

In parallel to the development of modern insecticides, the specific microbial toxins produced by the soil-dwelling bacterium Bacillus thuringiensis (Bt) are increasingly being adopted as biopesticides. In fact, microbial sprays of Bt are used in organic agriculture. This shift is due in part to a demand for increased safety both for humans and for the environment. Organic agriculture is a form of agriculture that relies on crop rotation, green manure, compost, biological pest control, and mechanical cultivation to maintain soil productivity and control pests, excluding or strictly limiting the use of synthetic fertilizers and synthetic pesticides, plant growth regulators, and genetically modified organisms (Directorate General for Agriculture and Rural Development of the European Commission). Integrated pest management (IPM) has also been proposed as a sustainable control system for insects. Several control systems are combined, including the judicious application of chemicals and biopesticides, use of trap crops, biological control, rotation, good husbandry and cultural control to manage all the pests of a particular crop (Gatehouse and Gatehouse 1999). Increasing crop varietal resistance is critical to both IPM and organic agriculture. It is unfortunate that ultimately neither organic agriculture nor IPM will be able to feed the world. In order to feed an increasing world population, more food must be produced in the future and on either the same amount, or less land (Ferry and Gatehouse 2009). Neither of these farming methods will be as productive as will be necessary to meet increased demands (Amman 2009).

1.2.4

Transgenic (Genetically Modified) Crops

Genetically modified (GM) maize and cotton varieties that express insecticidal proteins derived from B. thuringiensis (Bt) have become an important component in agriculture worldwide. At present, 20.3 million hectares of land is planted with insect-protected transgenic Bt cotton and maize (James 2007), with economic benefits from Bt cotton estimated at US$9.6 billion and maize US$3.6 billion (James 2007). Significantly, Phipps and Park (2002) showed that on a global basis GM technology has reduced pesticide use. These authors estimated that pesticide use was reduced by a total of 22.3 million kg of formulated product in 2000 alone.

1.2.4.1

Bacillus thuringiensis Toxins

B. thuringiensis (Bt) is a soil-dwelling bacterium of major agronomic and scientific interest. Whilst the subspecies of this bacterium colonize and kill a large variety of

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Table 1.1 Insecticidal properties of Bt toxins Insect order Cry protein Lepidoptera Cry1A, Cry1B, Cry1C, Cry1E, Cry1F, Cry1I, Cry1J, Cry1K, Cry2A, Cry9A, Cry9I, Cry15A Coleoptera Cry1I, Cry3A, Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, Cry8C, Cry14A, Cry23A Diptera Cry2A, Cry4A, Cry10A, Cry11A, Cry11B, Cry16A, Cry19A, Cry20A, Cry21A Hymenoptera Cry22A Nematodes Cry5A, Cry6A, Cry6B, Cry12A, Cry13A, Cry14A Liver fluke Cry5A

host insects, each strain tends to be highly specific. Toxins for insects in the orders Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes), Coleoptera (beetles and weevils), and Hymenoptera (wasps and bees) (Table 1.1) have been identified (de Maagd et al. 2001), but interestingly none with activity towards Homoptera (sap suckers) have, as yet, been identified, although a few with activity against nematodes have been isolated (Gatehouse et al. 2002). Further, there is little evidence of effective Bt toxins against many of the major storage insect pests. Bt toxins (also referred to as d-endotoxins; Cry proteins) exert their pathological effects by forming lytic pores in the cell membrane of the insect gut. On ingestion, they are solubilized and proteolytically cleaved in the midgut to remove the C-terminal region, thus generating an “activated” 65–70 kDa toxin. The active toxin molecule binds to a specific high-affinity receptor in the insect midgut epithelial cells. Following binding, the pore-forming domain, consisting of a-helices, inserts into the membrane; this results in cell death by colloid osmotic lysis, followed by death of the insect (de Maagd et al. 2001). A number of putative receptors in the insect gut have been identified and include aminopeptidase N proteins (Knight et al. 1994; Sangadala et al. 1994; Gill et al. 1995; Luo et al. 1997), cadherin-like proteins (Vadlamudi et al. 1995; Nagamatsu et al. 1998; Gahan et al. 2001) and glycolipids (Denolf 1996). Transgenic plants expressing Bt toxins were first reported in 1987 (Vaeck et al. 1987) and following this initial study numerous crop species have been transformed with genes encoding a range of different Cry proteins targeted towards different pest species. Since bacterial cry genes (genes encoding Bt toxins) are rich in A/T content compared to plant genes, both the full-length and truncated versions of these cry genes have had to undergo considerable modification of codon usage and removal of polyadenylation sites before successful expression in plants (de Maagd et al. 1999). Crops expressing Bt toxins were first commercialized in the mid-1990s, with the introduction of Bt potato and cotton. Currently, 20.3 million hectares of land is planted with Bt cotton and maize (James 2007). Bt Maize Lepidopteran pests such as European corn borer Ostrinia nubilalis, fall armyworm Spodoptera frugiperda, and corn earworm Helicoverpa zea perennially cause leaf

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and ear damage to corn. The Bt concept was particularly attractive for maize, since it made it possible to combat European corn borer larvae hidden inside the stem of the plant for the first time. Bt maize has now been grown on a large scale for over a decade, particularly in the US. In 2007, insect-resistant Bt maize was grown on 21% of the total maize cultivation area, and Bt maize with a combination of insect and herbicide resistance was grown on a further 28% (James 2007). Various Bt maize varieties are also authorized in the EU. In 2007, there was notable cultivation of Bt maize primarily in Spain, where it was grown on around 75,000 hectares (Ortego et al. 2009). Transgenic corn hybrids expressing the insecticidal protein Cry1Ab from B. thuringiensis (Bt) var. kurstaki were originally developed to control European corn borer, and offer the potential for reducing losses by fall armyworm and corn earworm. Several events of transgenic Bt corn have been developed with different modes of toxin expression (Ostlie et al. 1997). Amongst the most promising events were Bt11 expressing the cry1Ab gene from B. thuringiensis subsp. kurstaki (Novartis Seeds) and MON810 expressing a truncated form of the cry1Ab gene from B. thuringiensis subsp. kurstaki HD-1 (Monsanto Co.). In both events, the endotoxins are expressed in vegetative and reproductive structures throughout the season (Armstrong et al. 1995; Williams and Davis 1997). Crops containing either of these events are collectively referred to as having “YieldGard technology.” Furthermore, a modified cry9C gene from B. thuringiensis subsp. tolworthi strain BTS02618A is expressed in maize (tradename StarLink – marketed by Aventis CropScience). StarLink corn has only been approved in the US for livestock feed use. In recent years, there has been increasing focus on another maize pest, this time a Coleopteran (beetle); the western corn rootworm. Western corn rootworm is one of the most devastating corn rootworm species in North America. Its larvae are root pests and can destroy significant percentages of corn if left untreated. In the US, current estimates show that 30 million acres (120,000 km) of corn (out of 80 million grown) are infested with corn rootworms. The United States Department of Agriculture (USDA) estimates that corn rootworms cause US$1 billion in lost revenue each year. To make matters worse, this pest is extending its geographical range all of the time – including spreading throughout Europe. Bt maize which is resistant to the western corn rootworm has been authorized in the US since 2003 and has been grown on a large scale since. YieldGard Rootworm uses event MON 863 and expresses the Cry3Bb1 protein from B. thuringiensis (subsp. kumamotoensis) in the plant, protecting the plant against root feeding from the western and northern corn rootworm larvae. Products containing both YieldGard Corn Borer (MON810) and YieldGard Rootworm (MON 863) are marketed under the trade name YieldGard Plus (http://www.agbios.com). Corn rootworm-resistant maize is also produced by expression of the cry34Ab1 and cry35Ab1 genes from B. thuringiensis strain PS149B1 (DOW AgroSciences LLC and Pioneer Hi-Bred International, Inc.).

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Bt Cotton Cotton fibers used in textiles around the world come from the seed hairs of Gossypium hirsutum. Cotton develops in closed, green capsules known as bolls that burst open when ripe, revealing the white, fluffy fibers. But cotton is more than just a fiber for textiles. It is also an important source of raw materials used in animal feed and for various processed food ingredients, including cottonseed oil, proteinrich cottonseed meal (mostly used as animal feed) and even leftover fibers can be used as food additives. Lepidopteran, particularly heliothine, pests can have an enormously damaging effect on a cotton crop and controlling these insects in conventional farming involves treatment with a number of insecticide sprays. In 1996, Bollgard1 cotton (Monsanto) was the first Bt cotton to be marketed in the US. Bollgard cotton produces the Cry1Ac toxin from B. thuringiensis (subsp. kurstaki), which has excellent activity on tobacco budworm and pink bollworm. These two insects are extremely important as both are difficult and expensive to control with traditional insecticides and the damage caused by them directly impacts on the harvestable plant organ, the cotton bolls themselves. Bollgard II1 was introduced in 2003, representing the next generation of Bt cottons. Bollgard II contains Cry1Ac plus a second gene from the Bt bacteria which encodes the production of Cry 2Ab (also subsp. kurstaki). WideStrike (a Trademark of DowAgrosciences) was registered for use in 2004, and like Bollgard II, it expresses two Bt toxins but this time Cry1Ac and Cry1F were used in combination. Both Bollgard II and WideStrike have better activity on a wider range of caterpillar pests than the original Bollgard technology. GM Bt cotton has become widespread, covering a total of 15 million hectares in 2007, or 43% of the world’s cotton. Most GM cotton is grown in the US and China, but it can also be found in India, South Africa, Australia, Argentina, Mexico, and Columbia (http://www.agbios.com). Currently, 20% of the cotton grown commercially in China expresses Cry1Ac in combination with a plant protease inhibitor, cowpea trypsin inhibitor (CpTI) (He et al. 2009).

Bt Potato Potato (Solanum tuberosum L.) is a major world food crop. Potato is exceeded only by wheat, rice, and maize in terms of world production for human consumption (Ross 1986). Many commercial potato varieties are highly susceptible to damage by the Colorado potato beetle. In 1999, 93% of the 1.1 million potato acres grown in the US were treated with a total of 2.6 million pounds of insecticide (http://www. usda.gov/). To date, few traditionally bred varieties have been produced with resistance to this major pest. Unfortunately, many of the pesticides currently used are broad-spectrum pesticides, killing not only the target pest but most of its natural enemies as well. The Cry3A d-endotoxin from B. thuringiensis Berliner subsp. tenebrionis is toxic to coleopterans, particularly chrysomelids (Krieg et al. 1983;

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Bauer 1990; MacIntosh et al. 1990). It is insecticidal against the Colorado potato beetle, L. decemlineata (Ferro and Gerlernter 1989). In 1995, Bt.Cry3A (NewLeafTM) potato became the first Bt-crop to be commercialized, although they are currently withdrawn from the US market. 1.2.4.2

Evolution of Resistance in Pest Populations

Perhaps one of the most important issues surrounding the cultivation of Bt crops relates to the evolution of target pest resistance, which could limit the life span of the technology. In the case of Bt toxins, this is a major concern for the organic farming community, since the potential for insect populations to evolve resistance to Bt will not only limit the effectiveness of Bt-expressing crops but also Bt-based biopesticides. Bt resistance in insect pests has been reported to develop within 4–5 generations in the laboratory (Stone et al. 1989). To date, the mechanism of resistance to Cry toxins in insects has been most commonly ascribed to the loss or inactivation of specific toxin-binding sites on midgut cell membranes (Ferre´ and Van Rie 2002). Other resistance mechanisms that have been proposed include a defect in the toxin activation by midgut proteases (Oppert et al. 1994; Sayyed et al. 2001), or an increased repair and/or replacement rate of Cry-damaged midgut cells by stem cells (Forcada et al. 1999). Studies have also revealed evidence for novel resistance mechanisms based on active defensive responses (Rahman et al. 2004; Ma et al. 2005). When one considers the ability of insects to evolve resistance to chemical pesticides (French-Constant 2004), the development of field resistance is inevitable and in fact was recently reported to have already occurred (Tabashnik and Carrie`re 2009). Analysis of monitoring data shows that some field populations of H. zea have evolved resistance to Cry1Ac, the toxin produced by first-generation Bt cotton (Tabashnik et al. 2008). Nonetheless, resistance of H. zea to Cry1Ac has not caused widespread crop failures in the field for several reasons (Tabashnik et al. 2008). First, the documented resistance is spatially limited. Second, from the outset, insecticide sprays have been used to improve the control of H. zea on Bt cotton because Cry1Ac alone is not sufficiently effective to manage this pest. Finally, GM cotton producing two Bt toxins (Cry2Ab and Cry1Ac) was planted on more than one million ha in the US in 2006; control of H. zea by Cry2Ab would limit problems associated with resistance to Cry1Ac (Jackson et al. 2004). Considerable effort has been devoted to delaying the onset of evolution of resistance, e.g., the use of refugia has been required/recommended in most regions growing Bt-crops depending upon the country in question (Tabashnik and Carrie`re 2009). Gene-stacking and integrated pest management should be combined to control this problem. 1.2.4.3

Unexpected Benefits

Interestingly, the expression of Bt has resulted in improved crop quality as a consequence of decreased levels of Fusarium infestation and fumonisin mycotoxin

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production as a direct result of reduced levels of insect pest damage. This benefit is particularly important in food crops such as maize (Glaser and Matten 2003).

1.2.4.4

A Global Technology

Of the global total of 12 million biotech farmers in 2007, over 90% were small and resource-poor farmers from developing countries; the balance of one million were large farmers from both industrialized countries such as Canada and developing countries such as Argentina. Of the 11 million small farmers, most were Bt cotton farmers; 7.1 million in China (Bt cotton), 3.8 million in India (Bt cotton), and the balance of 100,000 in the Philippines (GM maize), South Africa (GM cotton, maize and soybeans often grown by subsistence women farmers) and the other eight developing countries which grew GM crops in 2007. This modest uptake by subsistence farmers contributes towards the Millennium Development Goals of reducing poverty by 50% by 2015 and is a very important development (James 2007).

1.2.5

Other Sources of Insecticidal Molecules

The concept of employing genes encoding Bt toxins to produce insect-resistant transgenic plants arises from the successful use of Bt-based biopesticides. A number of other strategies for protecting crops from insect pests actually exploit endogenous resistance mechanisms (Harborne 1998; Gatehouse 2002a, b). Genes encoding such defensive proteins are obvious candidates for enhancing crop resistance to insect pests.

1.2.5.1

Enzyme Inhibitors

Interfering with digestion, and thus affecting the nutritional status of the insect, is a strategy widely employed by plants for defense, and has been extensively investigated as a means of producing insect-resistant crops (Gatehouse 2002a, b). Insectdigestive proteases tend to fall into four mechanistic classes (serine, cysteine, aspartic or metallo proteases – depending on the enzyme-active site residue). Numerous studies since the 1970s have confirmed the insecticidal properties of a broad range of protease inhibitors from both plant and animal sources (Jouanin et al. 1998; Gatehouse 2002a, b). Proof of concept for exploiting such molecules for crop protection was first demonstrated with expression of a serine protease inhibitor from cowpea (CpTI), which was shown to significantly reduce insect growth and survival (Hilder et al. 1987). These studies were subsequently extended to include a greater range of target pests (Gatehouse et al. 1994; Graham et al. 1995; Xu et al. 1996), and a broader range of inhibitors and plant species, including economically

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important pest species, particularly lepidopterans (Broadway 1997; De Leo et al. 2001). Since many economically important coleopteran pests predominantly utilize cysteine proteases for protein digestion, inhibitors for this class of enzyme (cystatins) have also been investigated as a means for controlling pests from this order. Oryzacystatin, a cysteine protease inhibitor isolated from rice seeds, is effective towards both coleopteran insects and nematodes when expressed in transgenic plants (Leple et al. 1995; Urwin et al. 1995; Pannetier et al. 1997). Similarly, the cysteine/aspartic protease inhibitor equistatin, from sea anemone, is also toxic to several economically important coleopteran pests, including the Colorado potato beetle (Outchkourov et al. 2003). More recent studies have included the stacking of different families of inhibitors to increase the spectrum of activity (Abdeen et al. 2005). A major limitation, however, to this strategy for the control of insect pests arises from the ability of some lepidopteran and coleopteran species to respond and adapt to ingestion of protease inhibitors by either overexpressing native gut proteases, or producing novel proteases that are insensitive to inhibition (Bown et al. 1997; Jongsma and Bolter 1997). Thus, detailed knowledge about the enzyme–inhibitor interactions, both at the molecular and biochemical levels, together with detailed knowledge on the response of insects to exposure to such proteins is essential to effectively exploit this strategy. The concept of inhibiting protein digestion as a means of controlling insect pests has been extended to the inhibition of carbohydrate digestion. For example, inhibitors of a-amylase have been expressed in transgenic plants and shown to confer resistance to bruchid beetles (Shade et al. 1994; Schroeder et al. 1995).

1.2.5.2

Lectins

Lectins, found throughout the plant and animal kingdoms, form a large and diverse group of proteins identified by a common property of specific binding to carbohydrate residues, either as free sugars, or more commonly, as part of oligo- or polysaccharides. Many physiological roles have been attributed to plant lectins, including defense against pests and pathogens (Chrispeels and Raikhel 1991; Peumans and Vandamme 1995). Although some lectins are toxic to mammals, and are thus not suitable candidates for transfer to crops for enhanced levels of protection, this is by no means universal. Many lectins are not toxic to mammals, yet are effective against insects from several different orders (Gatehouse et al. 1995), including homopteran pests such as hoppers and aphids (Powell et al. 1995; Sauvion et al. 1996; Gatehouse et al. 1997). This finding has generated significant interest, not least since no Bts effective against this pest order have been identified to date. One such lectin is the snowdrop lectin (Galanthus nivalis agglutinin; GNA). Both constitutive and phloem-specific (Rss1 promoter) expression of GNA in rice is an effective means of significantly reducing survival of rice brown planthopper (Nilaparvata lugens), and green

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leafhopper (Nephotettix virescens) both serious economic pests of rice (Rao et al. 1998; Foissac et al. 2000; Tinjuangjun et al. 2000). GNA has been expressed in combination with other genes encoding insecticidal proteins, including the cry genes (Maqbool et al. 2001). Although lectins such as GNA, and ConA are not as effective against aphids as they are against hoppers, they nonetheless have significant effects on aphid fecundity when expressed in potato (Down et al. 1996; Gatehouse et al. 1997, 1999) and wheat (Stoger et al. 1999). The precise mode of action of lectins in insects is not fully understood although binding to gut epithelial cells appears to be a prerequisite for toxicity. In the case of rice brown planthopper, GNA not only binds to the luminal surface of the midgut epithelial cells, but also accumulates in the fat bodies, ovarioles and throughout the haemolymph, suggesting that the lectin is able to cross the midgut epithelial barrier and pass into the insect’s circulatory system, resulting in a systemic toxic effect (Powell et al. 1998; Du et al. 2000). As with protease inhibitors, the levels of protection conferred by the expression of lectins in transgenic plants are generally not high enough to be considered commercially viable. However, the absence of genes with proven high insecticidal activity against homopteran pests may well mean that transgenic crops with partial resistance may still find acceptance in agriculture, especially if expressed with other genes that confer partial resistance, or if introduced into partially resistant genetic backgrounds.

1.2.5.3

A Brief Aside; Plant Parasitic Nematodes

Plant parasitic nematodes include several groups causing severe crop losses. The most common genera are: Aphelenchoides (foliar nematodes), Meloidogyne (rootknot nematodes), Heterodera, Globodera (cyst nematodes) such as the potato cyst nematode, Nacobbus, Pratylenchus (lesion nematodes), Ditylenchus, Xiphinema, Longidorus, and Trichodorus. Several phytoparasitic nematode species cause histological damages to roots, including the formation of visible galls (Meloidogyne). Some nematode species transmit plant viruses through their feeding activity on roots. One of them is Xiphinema index, vector of GFLV (grapevine fanleaf virus), an important disease of grapes. Bt toxins, lectins (Burrows et al. 1999) and protease inhibitors have shown some promise for control, particularly the expression of cystatins (Cowgill et al. 2002). For a recent review of the topic, the reader is referred to Fuller et al. (2008).

1.2.5.4

Other Sources of Insecticidal Molecules

Generating insecticidal transgenic crops harboring genes from nonconventional sources is an extremely active area, with amongst others, foreign genes from plants (e.g., enzymes inhibitors and novel lectins) and animal sources including insects

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(e.g., biotin-binding proteins, neurohormones, venoms and enzyme inhibitors) being a major focus (Ferry et al. 2006). The development of second-generation transgenic plants with greater durable resistance might result from the expression of multiple insecticidal genes such as the Vip (vegetative insecticidal proteins) produced by B. thuringiensis during its vegetative growth. The benefit of such an approach is a broader insect target range than conventional Bt proteins and the proposed expectation to control current Bt resistant pests due to the low levels of homology between the domains of the two proteins classes (Christou et al. 2006). With Bt toxins as the classical reference, toxins from other insect pathogens provide a potential repository of novel insecticidal compounds. Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes, which are lethal to a wide range of insects (Chattopadhyay et al. 2004). Photorhabdus toxin expression in Arabidopsis caused significant insect mortality (see for review Ferry et al. 2006). Thus, toxins from other insect pathogens are also opening up new routes to pest control using transgenic-based strategies. Interesting recent developments include the use of novel proteins from insect biological control agents and insect hormones to generate transgenic crops. A teratocyte secretory protein from a hymenopteran endoparasitoid (a parasitic wasp often used in biocontrol programs) has been expressed in transgenic tobacco and shown to increase resistance to lepidopteran pests (Maiti et al. 2003). Similar protection has also been achieved with insect peptide hormones (Tortiglione et al. 2003). Interestingly, they replaced the tomato systemin peptide region of prosystemin (a plant signaling molecule) with the insect peptide and showed that this resulted in the production of biologically active insecticidal peptides. Reliance on the expression of a single gene product for pest control is a relatively short-term strategy that parallels the use of exogenously applied chemical pesticides. Thus, pyramiding (stacking) of genes encoding different Bt toxins has been developed as a method for preventing the onset of evolution of pest resistance, and for conferring greater levels of pest control (Boulter et al. 1990; Maqbool et al. 2001; Zhao et al. 2003). This strategy has now been adopted in commercially available crops (see above). For example, corn lines have recently been developed (Moellenbeck et al. 2001; Ostlie 2001) coexpressing two d-endotoxins from Bt for resistance to corn rootworm. Hybrid proteins have also been developed to enhance and extend the activity of Bt toxins. The use of a single Bt toxin in a crop is limited in that many insects attack a single crop and toxins generally show very high specificity towards a single pest species. Therefore, toxins have been engineered to modify their receptor recognition and pore formation. Each toxin consists of three domains. Domain I is involved in membrane insertion and pore formation. Domains II and III are both involved in receptor recognition and binding. Additionally, a role for domain III in pore function has been found. This approach has proved successful in both enhancing activity (Karlova et al. 2005) and extending host range (Singh et al. 2004). Such hybrid/fusion proteins offer an alternative/ complementary strategy to address potential limitations in conventional transgenic insect pest control.

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Transgenic Crop Plants for Resistance to Biotic Stress

1.2.5.5

15

Transgenic Plants Expressing Fusion Proteins

The concept of “gene stacking” has recently been extended to the development and use of fusion proteins. Such proteins not only provide a means of increasing durability, but also provide a “vehicle” for more effective targeting of insecticidal molecules, including peptides. It thus offers an alternative/complementary strategy to address potential limitations in conventional transgenic insect pest control. For example, recognition of binding sites in the insect gut is an important factor determining the toxicity of Bt. Enhancing toxin-binding capabilities should thus extend host range and delay resistance in pest populations. Bt is believed to bind primarily to aminopeptidase N or cadherin membrane proteins, while the generation of a fusion protein with the nontoxic B-chain from ricin (RB) was shown to extend the binding of Bt to include specific glycoproteins. Transgenic plants expressing the Bt fused RB demonstrated that the addition of the RBbinding domain provided a wider repertoire of receptor sites within target species and significantly enhanced the levels of toxicity of Bt. For example, survival of the armyworm Spodoptera littoralis, a species of insect not sensitive to Bt, was reduced by ~90% when feeding on transgenic maize expressing the fusion, compared to plants expressing either Bt Cry1Ac alone, or the RB-binding domain. Expression of the fusion protein resulted in the insect becoming susceptible to Bt (Mehlo et al. 2005). This strategy has shown great potential beyond just extending the toxicity of Bt. Zhu-Salzman et al. (2003) have generated fusion proteins with anchor regions to other insecticidal proteins to the insect gut epithelium. Using the legume lectin rGSII, they proposed a system to combat the ability of certain insect species to activate protease inhibitor-insensitive proteolytic enzymes. The soybean cysteine protease inhibitor soyacystatin N (scN) was covalently linked to the GlcNAc-specific legume lectin using a naturally occurring linker region from the potato multicystatin. In this instance, the fusion protein not only has a novel binding ability that is proposed to initiate a concentration effect by localizing the inhibitor at the anterior of the gut, but the fused lectin moiety additionally offers a degree of protection to the insecticidal moiety by blocking the access of scN-insensitive proteases, thereby preventing proteolytic destruction of the cystatin. Not only do fusion proteins have potential for use in transgenic crops, but also to improve the efficacy of biopesticide-based sprays. Neuropeptides potentially offer a high degree of biological activity, and thus provide an attractive alternative pest management strategy. There are major drawbacks to their use, particularly as topical sprays. They are unlikely to be rapidly absorbed through the insect cuticle to their site of action, and are prone to proteolysis and rapid degradation in the environment. Should they survive the application process and are then taken up by the insect, they are then unlikely to survive the conditions of the insect gut or be delivered to the correct targets within the insect. The discovery that snowdrop lectin (GNA) remains stable and active within the insect gut after ingestion, and that it is able to cross the gut epithelium, provided an opportunity for its use as a “carrier molecule” to deliver other peptides to the circulatory system of target insect

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species. This strategy effectively delivered the insect neuropeptide hormone, allatostatin, to the haemolymph of the tomato moth Lacanobia oleracea (Fitches et al. 2002). Subsequent expression of the fusion protein in potato further provided proof of concept for the efficacy of fusion proteins as a means of delivery. The results demonstrated significant reduction in mean larval weight when compared to the controls. GNA can be used to deliver insecticidal peptides isolated from the venom of the spider Segestria florentina (SFI1) to the haemolymph of L. oleracea (Fitches et al. 2004). Neither the GNA nor the SFI1 moieties alone were acutely toxic; however, the SFI1/GNA fusion was insecticidal to first stage larvae, causing 100% mortality after 6 days. This spider venom neurotoxin is believed to irreversibly block the presynaptic neuromuscular junctures. Such venom toxins show high degrees of specificity and thus lend themselves to environmentally benign pest management strategies.

1.2.6

Manipulation of Plant Endogenous Defenses

Alternative strategies for protecting crops from insect pests, that are not dependent on the expression of single or stacked genes, seek to exploit the induced endogenous resistance mechanisms exhibited by plants to most insect herbivores. For further information, the reader is referred to Chap. 10 of this volume. Such induced defenses are exemplified by the wounding response, first identified as the local and systemic synthesis of proteinase inhibitors (PIs), which block insect digestion in response to plant damage (Gatehouse 2002a, b). Many transgenic strategies have attempted to exploit the potential overexpression of plant PIs to protect crops from pest damage (Jouanin et al. 1998) but these have relied on the transfer of a single PI gene, and many insects have been able to adapt to this. More recent research has shown that induced defenses also involve the plant’s ability to produce toxic or repellent secondary metabolites as direct defenses, and volatile molecules, which play an important role in indirect defense (Kessler and Baldwin 2002). Insect herbivores activate induced defenses both locally and systemically via signaling pathways involving systemin, jasmonate, oligogalacturonic acid and hydrogen peroxide (Fig. 1.4). Ecologists have long understood that plants exhibit multi-mechanistic resistance towards herbivores, but the molecular mechanisms underpinning these complicated responses have remained elusive (Baldwin et al. 2001). However, recent studies investigating the plant’s herbivore-induced transcriptome, using microarrays and differential display technologies, have provided novel insights into plant–insect interactions. The jasmonic acid cascade plays a central role in transcript accumulation in plants exposed to herbivory (Hermsmeier et al. 2001). A single microarraybased study revealed that the model plant Arabidopsis undergoes changes in levels of over 700 mRNAs during the defense response (Schenk et al. 2000). In contrast, only 100 mRNAs were upregulated by spider mite (Tetranicus urticae) infestation in lima bean (Phaseolus lunatus), although a further 200 mRNAs were upregulated

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17

Herbivory physical forces local peptide hormone release eg. prosystemin

insect dervived elicitors

ethylene

systemin plant-plant interactions

pest deterrent/ attraction of natural enemies

receptor binding ABA linolenic acid SA

Voltiles e.g. -green leaf volatiles -C10, C15 terpenoids -indole

octadecanoid pathway methyl jasmonate jasmonic acid auxin, SA (-) ethylene, ABA (+) polygalactouronidase oligalacturonic acid vascular bundle

H202

Insecticidal compounds e.g. Protease Inhbitors

late genes (defence)

Cell wall

NAPDH oxidase

plant-plant interactions (systemic induction of Pls)

early genes signalling

mesophyll cells

Fig. 1.4 The generalized plant-wounding response. Generalized overview of the plant wounding response, and signalling molecules which can modulate it, showing the pathways necessary for both local and systemic induction of insecticidal proteins. (Adapted from: Ferry et al. 2004)

in an indirect response mediated by feeding-induced volatile signal molecules (Arimura et al. 2000). Deciphering of the signals regulating herbivore-responsive gene expression will afford many opportunities to manipulate the response. Signaling molecules such as salicylic acid, jasmonic acid and ethylene do not activate defenses independently by linear cascades, but rather establish complex interactions that determine specific responses. Knowledge of these interactions can be exploited in the rational design of transgenic plants with increased insect resistance (Rojo et al. 2003; De Vos et al. 2005; Giri et al. 2006).

1.2.6.1

A Special Case: Sap-Feeding Insects

While most herbivorous insects cause extensive damage to plant tissues when feeding, many insects of the order Homoptera feed from the contents of vascular tissues by inserting a stylet between the overlying cells, thus limiting cell damage and minimizing induction of a wounding response. In contrast to wounding, plant responses following attack by these insects have been shown to be typical of pathogen attack, with examples of gene-for-gene interactions being known (Walling 2000;

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Moran et al. 2002). However, these pathogen-induced pathways can induce expression of many of the genes upregulated by wounding because of pathway cross-talk. Moran and Thompson (2001) demonstrated that phloem feeding by the green peach aphid (Myzus persicae) on Arabidopsis induced expression of genes associated with salicylic acid (SA) responses to pathogens, as well as a gene involved in the jasmonic acid-mediated response pathway. These results suggest stimulation of response pathways involved in both pathogen and herbivore responses. Microarray data has identified genes involved in oxidative stress, calcium-dependent signaling, pathogenesis-related responses, and signaling as key components of the induced response (Moran et al. 2002). It may be that transgenic strategies that activate such signaling cascades could enhance plant resistance to these problematic pests. 1.2.6.2

Indirect Defense (Volatile Production)

The role of plant volatiles in indirect defense has been described as “top-down” defense (Baldwin et al. 2001). Some volatiles appear to be common to many different plant species, including C6 aldehydes, alcohols and esters (green leaf volatiles), C10 and C15 terpenoids, and indole, whereas others are specific to a particular plant species. Many volatiles are preformed and act in herbivore deterrence; furthermore, the wounding response also includes the formation of volatile compounds. Top-down control of herbivore populations is achieved by attracting predators and parasitoids to the feeding herbivore, mediated by these volatile organic compounds (VOCs). For example, genes involved in the biosythesis of the maize VOC bouquet are upregulated by insect feeding (Frey et al. 2000; Shen et al. 2000). In addition, herbivore oviposition has been shown to induce VOC emissions, which attract egg parasitoids (Hilker and Meiners 2002). Herbivoreinduced VOCs can also elicit production of defence-related transcripts in plants near the individual under attack (Arimura et al. 2000; Dicke et al. 2003). Exposure to herbivore-induced volatiles in lima bean results in transcription of genes involved in ethylene biosynthesis (Arimura et al. 2000). Manipulation of volatile biosynthesis can affect insect resistance. Transgenic potatoes in which production of hydroperoxide lyase (the enzyme involved in green leaf volatile biosynthesis) was reduced were found to support improved aphid performance and fecundity, suggesting toxicity of these volatiles to M. persicae (Vancanneyt et al. 2001). In a review of the topic Degenhardt et al. (2003) discuss the potential of modifying terpene emission with the aim of making crops more attractive to herbivore natural enemies. 1.2.6.3

Detoxification and Insect Modulation of the Wounding Response

However, insect pests are able to feed on plants despite their defenses, both constitutive and inducible. Many insects are able to detoxify potentially toxic secondary metabolites, using cytochrome P-450 monoxygenases and glutathioneS-transfereases. These enzymes are induced by exposure to toxic plant secondary

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19

compounds, for example, xanthotoxin (a furanocoumarin) induces P-450 expression in corn earworm (Li et al. 2000). More recently, Li et al. (2002) have shown that corn earworm uses signaling molecules from its plant host, jasmonate and salicylate, to activate four of its cytochrome P450 genes, thus making the induction of detoxifying enzymes rapid and specific. Recent strategies based on RNAi technology have shown that it is possible to overcome these insect responses (discussed later in this chapter).

1.2.7

RNAi

Disrupting gene function by the use of RNAi is a well-established technique in insect genetics based on delivery by injection into insect cells or tissues. The observation that RNAi could also be effective in reducing gene expression, measured by mRNA level, when fed to insects (Turner et al. 2006) has led to two recent articles in which transgenic plants producing double-stranded RNAs (dsRNAs) which are shown to exhibit partial resistance to insect pests. Transgenic maize producing dsRNA directed against V-type ATPase of corn rootworm showed suppression of mRNA in the insect and reduction in feeding damage compared to controls (Baum et al. 2007). Similarly, transgenic tobacco and Arabidopsis expressing dsRNA directed against a detoxification enzyme (Cytochrome P450 gene CYP6AE14) for the breakdown of gossypol (a defensive metabolite) in cotton bollworm caused the insect to become more sensitive to gossypol in the diet (Mao et al. 2007). This approach holds great promise for future development. It is also proving effective for nematode control (Bakhetia et al. 2005). For a recent review on RNAi-mediated crop protection against insect pests the reader is referred to Price and Gatehouse (2008). Advances in our understanding of induced responses in plants and their regulation, has refocused attention on potential exploitation of endogenous resistance mechanisms for crop protection. While plant resistance is an integral component of organic and IPM strategies, it does not afford similar levels of protection as those provided by the use of “direct” protective methods such as the expression of Bt toxins. The goal of the plant breeder, and now the biotechnologist, is to engineer durable multi-mechanistic resistance to insect pests in crops, and increased knowledge of induced defense mechanisms and their molecular control is likely to play an important role in realizing this aim.

1.2.8

Environmental Impact of IR Crops

Almost from the beginning of the production of transgenic crops there have been concerns over their use and introduction into the environment. There is international agreement that GM crops should be evaluated for their safety, including their environmental impact (Dale 2002). During the past 15–20 years, there have been

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extensive research programs of risk assessment, with several areas of major concern identified.

1.2.8.1

Impact on Nontarget Organisms

Assessing the consequences of pest control on nontarget organisms is an important precursor to their becoming adopted in agriculture. The expression of transgenes that confer enhanced levels of resistance to insect pests is of particular significance since it is aimed at manipulating the biology of organisms in a different trophic level to that of the plant. Potential risks to beneficial nontarget arthropods exist. Those groups most at risk include: nontarget Lepidoptera, beneficial insects (pollinators, natural enemies) and soil organisms. Exposure of nontarget Lepidoptera to insecticidal transgene products may occur through both direct consumption of transgenic plant tissues including via consumption of transgenic pollen; many nontarget Lepidoptera are rare butterflies having great conservation value. The case of the Monarch butterfly (Danaus plexippus), a conservation flagship species in the US, highlighted the need for ecological impact research. In a letter to Nature, Losey et al. (1999) claimed that both survival and consumption rates of Monarch larvae fed milkweed leaves (natural host) dusted with Bt pollen were significantly reduced, and that this would have profound implications for the conservation of this species. However, a series of ecologically based studies rigorously evaluated the impact of pollen from such crops on Monarchs and demonstrated that the commercial wide-scale growing of Bt-maize did not pose a significant risk to the Monarch population (Hellmich et al. 2001; Gatehouse et al. 2002). In fact, the initial experiments did not quantify the dose of pollen used, or indeed, if this was a realistic level likely to be encountered in the field; nevertheless, this work highlighted the importance of studying nontarget effects. In a separate field study Wraight et al. (2000) showed that Papilio polyxenes (black swallowtail) larvae were unaffected by pollen from Bt expressing maize event Mon810 at 0.5, 1, 2, 4 and 7 m from the transgenic field edge, highlighting the need for a case-by-case study of organisms considered to be at risk. In addition to the potential direct impacts of Bt toxins on susceptible target insects, as in the case of the Morarch butterfly, some Lepidoptera have been shown to have a reduced sensitivity to the lepidopteran-specific Bt toxins. For example, S. littoralis can survive on maize expressing Cry1Ab (Hilbeck et al. 1998) and thus present a route of exposure to the next trophic level. In the case of Bt Cry3Aa or Cry3Bb expressing potatoes or maize, some Lepidoptera may represent nontarget secondary pests, and whilst not directly affected by the transgene product themselves may again present a route of exposure to the next trophic level, as do other nontarget herbivores. Organisms such as those belonging to the orders Homoptera, Hemiptera, Thysanoptera, and Tetranychidae are not targeted by Bt toxins expressed in transgenic plants; however, they do utilize the Bt crop (Groot and Dicke 2002). The direct effect that this may have on these insects is dependent on the presence of Bt receptors in the first instance, and it is so far unclear whether such receptors are

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21

present in nontarget organisms (de Maagd et al. 2001). In addition, the fate of the toxin ingested by nontarget herbivores is unclear, since if it retains toxicity then this may have implications at the next trophic level. The impacts of insect-resistant transgenic crops at higher trophic levels have also been considered, where there are concerns over the risks to beneficial arthropod biodiversity (Schuler et al. 1999; Bell et al. 2001); in particular, predators and parasitoids, which play an important role in suppressing insect pest populations both in the field and under specialized cultivation systems (glasshouses). Natural enemies may ingest transgene products via feeding on herbivorous insects that have themselves ingested the toxin from the plant; such tritrophic interactions will be influenced by the susceptibility of the herbivore to the plant protection product. If, as in the case with Bt toxins, the prey item is susceptible to the toxin, then the predator will not come into contact with the toxin as the pest will effectively be controlled, and in target insects the toxin is bound to receptors in the midgut epithelium that are structurally rearranged and may lose their entomotoxicity (de Maagd et al. 2001). In nontarget insects (and resistant insects), the toxins do not bind and may thus retain biological activity. However, the overwhelming weight of evidence from independent laboratory and field studies show that Bt toxins have a limited ability to affect the next trophic level (reviewed in Sanvido et al. 2007; Ferry and Gatehouse 2009; Romeis et al. 2008). Pollinators represent another group of nontarget organisms highlighted as at risk from Bt toxins in GM crops. The current generation of transgenic crops produce Bt toxin in the pollen as well as in the vegetative tissues. Several studies have been conducted to determine toxicity of Bt toxins to pollinators (Vandenberg 1990; Sims 1995, 1997; Arpaia 1997; Malone and Pham-Delegue 2001); generally, they all conclude that neither the adults nor the larvae of bees were affected by Bt toxins. For a comprehensive review of the impact of transgenic crops on pollinators, the reader is referred to two recent reviews (Malone et al. 2008; Malone and Burgess 2009). Finally, nontarget species may come into contact with Bt toxins via the environment. Several studies have shown that Bt toxins released from transgenic plants bind to soil particles (Palm et al. 1996; Crecchio and Stotzky 1998; Saxena et al. 1999). Soil-dwelling and epigeic insects such as Collembola and Carabidae may thus be exposed to the toxins. Several studies (Saxena and Stotzky 2001; Ferry et al. 2007) show no differences in mortality or body mass of bacteria, fungi, protozoa, nematodes and earthworms or carabid beetles exposed to Bt. Exposure to the transgene products, however, does not necessarily imply a negative impact. Most studies to date have demonstrated that crops transformed for enhanced pest resistance have no deleterious effects on beneficial insects (reviewed in Ferry et al. 2003; Romeis et al. 2008).

1.2.9

Conclusions

Ultimately one must consider the impact of transgenic crops and specifically Bt toxins in comparison to other pest control strategies such as conventional crop

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protection using insecticides. While pesticides have no doubt brought vast yield improvements, they have well-documented undesirable nontarget effects (Devine and Furlong 2007). It is worth remembering that whilst potential risks do exist to the environment from the cultivation of GM crops, their potential to decrease reliance on external inputs (less insecticide sprays) and to increase the availability of genetic resources available to breeders is great (Ferry and Gatehouse 2009).

1.3 1.3.1

Biotic Stress due to Weeds Crop Losses due to Weeds

Weeds can compete with productive crops or pasture, or convert productive land into unusable scrub. Weeds are also often poisonous, distasteful, produce burrs, or thorns that interfere with the use and management of desirable plants by contaminating harvests or excluding livestock. Weeds tend to thrive at the expense of the more refined edible or ornamental crops. They provide competition for space, nutrients, water and light, although how seriously they will affect a crop depends on a number of factors. Some crops have greater resistance to competition than others, for example smaller, slower-growing seedlings are more likely to be overwhelmed than those that are larger and more vigorous. Weeds also differ in their competitive abilities, and this can vary according to specific conditions and the time of year. Tall-growing vigorous weeds such as fat hen (Chenopodium album) can have the most pronounced effects on adjacent crops. Chickweed (Stellaria media), a low-growing plant, can happily coexist with a tall crop during the summer, but plants that have overwintered will grow rapidly in early spring and may swamp crops. Simply put, weeds are any plant growing in an area where it is not wanted. Of over 250,000 plant species in the world, only a few hundred are troublesome weeds. Although no single characteristic clearly defines a weed, two attributes are common in the worst weeds: competitiveness and persistence. The world’s worst weeds are shown in Table 1.2 (Chrispeels and Sadava 2003).

1.3.2

Weed Management: Prevention, Control, and Eradication

As weeds are persistent, once they have established in a field they are extremely difficult to eradicate. One of the most successful attempts in the eradication of a weed is provided by witchweed (Striga sp.) in the US. Witchweed is a parasitic plant; its seeds germinate in response to a chemical, strigol, produced by the roots of the host plant. The germinated seedling attaches to the root through special haustoria, as this occurs underground infestation can go unnoticed, consequently serious

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Table 1.2 World’s worst weeds (from: Chrispeels and Sadava 2003) Plant family Weed examples Grass (Poaceae) Bermuda grass (Cynodon dactylon) Barnyard grass (Echinochloa crus-galli) Johnson grass (Sorghum halepense) Sedge (Cyperaceae) Purple nutsedge (Cyperus rotundus) Yellow nutsedge (Cyperus esculentus) Smallflower umberella sedge (Cyperus difformis) Sunflower (Asteraceae) Canada thistle (Cirsium arvense) Common cocklebur (Xanthium strumarium) Common dandelion (Taraxacum officinale) Buckwheat (Polygonaceae) Wild buckwheat (Polygonum convolvulus) Curly dock (Rumex crispus) Red sorrel (Rumex acetosella) Pigweed (Amaranthaceae) Smooth pigweed (Amaranthus hybridus)

Mustard (Brassicaceae)

Legume (Fabaceae)

Morning glory (Convolvulaceae)

Spurge (Euphorbiaceae)

Goosefoot (Chenopodiaceae)

Mallow (Malvaceae)

Nightshade (Solanaceae)

Spiny amaranth (Amaranthus sinosus) Redroot pigweed (Amaranthus retroflexus) Shepherd’s purse (Capsella bursa-pastoris) Hoary cress (Brassica draba) Wild mustard (Brassica kaber) Black medic (Medicago lupulina) Sensitive plant (Mimosa pudica) Kudzu (Pueraria lobata) Field bindweed (Convolvulvus arvensis) Field dodder (Cuscuta campestris) Swamp morning glory (Ipomoea aquatica) Leafy spurge (Euphorbia esula) Garden spurge (Euphorbia hirta) Spotted spurge (Euphorbia maculata) Common Lamb’s quarters (Chenopodium album) Russian thistle (Salsola iberica) Nettleleaf goosefoot (Chenopodium murale) Venice mallow (Hibiscus trionum) Velvetleaf (Abutilon theophrasti) Arrowleaf sida (Sida rhombifolia) Black nightshade (Solanum nigrum) Jimsonweed (Datura stramonium) Cutleaf groundcherry (Physalis angulata)

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Crop Wheat

None

Sunflower

Buckwheat

Grain amaranth

Canola

Soybean

Sweet potato

Cassava

Sugarbeet

Cotton

Potato

infestation leading to yield loss occurs before the parasitic plant even appears through the soil. Once the witchweed comes through the soil it becomes photosynthetically active, but still dependent on its host for water. Such parasitic weeds are extremely difficult to control and yield losses of approximately 50% can be expected under drought conditions. In the US, only quarantine of infected areas reduced witchweed infection from 150,000 hectares to a couple of 1,000 hectares, but this quarantine lasted for nearly 50 years (Chrispeels and Sadava 2003)!

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For this reason, preventative weed management is critical. This involves keeping weed seeds and vegetative propagules from getting to the field in the first place and is mainly achieved through seed cleaning and the purchase of certified clean seed. Good management practice and the cleaning of farm equipment moved between different areas are critical in preventative control. Ultimately weed control is achieved by mechanical, biological and predominately chemical means.

1.3.3

Herbicides

The economic importance of weeds is emphasized by the fact that the herbicides comprise as large a share of the world agrochemical market as all other pesticides combined, farmers spend an annual US$10 billion to control weeds in the US alone (Chrispeels and Sadava 2003). Herbicides have evolved over time, and herbicide use has seen a move towards more biodegradable chemicals that only need to be applied at a very low concentration of active ingredient. Thus, the new generation of herbicides are relatively environmentally benign. Many herbicides exploit the differences in plant physiology between the crop species and its weeds (usually the differences between monocots and dicots); they may be systemic or act on contact (Table 1.3). Major crops such as wheat, rice, and maize; and legumes such as soybean, common bean, and peanut come from the same plant families as their weeds, thus creating a control problem. The mode of action of common herbicides is varied (Naylor 2002). For example, inhibition of photosynthesis and light-dependent membrane destruction (acting on photosystems II and I, respectively) are the mode of action of the foliar-acting nonselective herbicides like atrazine, paraquat and diquat. Hormone herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) induce abnormal plant growth by interring with auxin regulation. The sulfonyl ureas, imidazolines and the environmentally benign, but nonselective glyphosate (acting on 5-enolpyruvylshikimate3-phosphate synthase) inhibit amino acid synthesis. Others include inhibitors of lipid synthesis, inhibition of cell division and pigment synthesis. The advantages of herbicides are clear – they control multiple weed species, control perennial weeds, cause no injury to the crop plant, and can readily be applied to large areas. Table 1.3 Herbicide modes of action Herbicide type Contact herbicides destroy only that plant tissue in contact with the chemical spray. Generally, these are the fastest-acting herbicides. They are ineffective on perennial plants that are able to regrow from roots or tubers Systemic herbicides are foliar-applied and are translocated through the plant and destroy a greater amount of the plant tissue. Modern herbicides such as glyphosate are designed to leave no harmful residue in the soil Soil-borne herbicides are applied to the soil and are taken up by the roots of the target plant Pre-emergent herbicides are applied to the soil and prevent germination or early growth of weed seeds

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Nonchemical weed control includes: cultural weed control practices such as intercropping and rotation; mechanical removal of weeds; and biological control, all of which are labor intensive and unpredictable. Chemical treatment is by far the most effective method, but it is not without problems.

1.3.3.1

Resistance to Herbicides

A common consequence of repeated use of herbicide application is the evolution of resistance in weed populations because of strong selection pressure. The first example of a problematic herbicide resistant weed was reported in the 1970s (Chrispeels and Sadava 2003) when common groundsel resistant to triazine was identified in the US. The mechanism of herbicide resistance is usually a change in the target site with a reduction in herbicide affinity – this may be achieved through a single point mutation and alteration of a single amino acid. By 2001, over 150 weed species had resistance to at least one herbicide. Some biotypes of weeds have resistance to herbicides with different modes of action.

Rigid Ryegrass When a weed evolves resistance to a particular herbicide, the farmer is forced to use an alternative control strategy. This often involves the use of another herbicide with a different mode of action. Subsequent selection pressure may lead to a weed population acquiring multiple herbicide resistance. In nearly all the cases so far, resistance to only one or two chemical families has been reported. Rigid ryegrass is a notable exception. In the most severe cases, biotypes exist that are resistant to nine chemical families with five different modes of action! Resistance is conferred by multiple mechanisms; both altered sites of action and the ability to metabolize particular herbicides, via nonspecific monoxygenases.

1.3.4

Transgenic Crops for Weed Control

The adoption of transgenic herbicide-resistant (HR) crops has made remarkable changes to global agriculture within the last decade. Currently, an estimated 114.3 million hectares of transgenic crops are planted throughout a variety of agroecosystems in 23 developing and industrial countries. Approximately, 90% of the land area with transgenic crops includes a trait for glyphosate resistance (GR) (Owen 2008). While there are a number of herbicide-tolerant genetically modified crops that have been developed for several herbicides with different modes of phytotoxic action, the primary influence in world agriculture are glyphosate-resistant crops

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(GRCs) (Duke 2005; Duke and Powles 2008). More specifically, of the 23 countries that grow plant glyphosate-resistant crops, the US, Canada, Argentina and Brazil are the countries that account for most hectares (Owen 2008). In these countries, the principle GRCs planted include maize (Zea mays), soybean (Glycine max), cotton (G. hirsutum), and canola (Brassica napus). For further information, the reader is referred to Chap. 3 of this volume.

1.3.4.1

Round-up1 Ready Crops

Glyphosate (Round-up1) is a highly effective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a branch point enzyme in aromatic amino acid biosynthesis. A naturally occurring EPSP synthase gene (cp4) was identified from Agrobacterium sp. strain CP4, whose protein product provided glyphosate tolerance (Fig. 1.5) in plants (Padgette et al. 1995). COO– COO–

SHKP

+

P O

OH

CH2

P O

OH

phosphoenolpyruvate (PEP)

GLYPHOSATE

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase)

shkG H3PO4

COO–

COO–

H3PO4 CH2

P O

O

OH 5-enolpyruvylshikimate-3-phosphate (EPSP )

CH2

shkH COO–

chorismate synthase

O

COO–

OH chorismate (CHA)

Fig. 1.5 Glyphosate; mode of action. Glyphosate kills plants by interfering with the synthesis of the amino acids phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate (SHKP) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (EPSP). EPSP is subsequently dephosphorylated to chorismate, an essential precursor in plants for the aromaticamino acids: phenylalanine, tyrosine and tryptophan. These amino acids are used as building blocks in peptides, and to produce secondary metabolites such as folates, ubiquinones and naphthoquinone

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Furthermore, glyphosate detoxification pathways are known in microbes involving glyphosate oxidoreductase (gox) genes (Jacob et al. 1988). These two genes confer glyphosate resistance in selected crops. Crops engineered for resistance to this broad-spectrum herbicide allow control of multiple weeds without injury to the main crop. The economic benefits to farmers of glyphosate-resistant crops are estimated at US$17.5 billion, mainly accrued from reduced sprays and lower inputs of labor. In addition, herbicide-tolerant crops reduce the amounts of active ingredient required for weed control; they also promote the usage of no/low till farming and thus lower fuel consumption with direct benefits to both soil structure and carbon emissions (James 2007).

1.3.4.2

Other Transgenic Herbicide-Tolerant Crops

Traits for resistance to three other classes of herbicides have been developed, but have not reached the same level of popularity as glyphosate resistance. Resistance to oxynil herbicides conferred by the BXN nitrilase from Klebsiella pneumoniae (subsp. ozaenae) (Stalker et al. 1988) was the first trait engineered in cotton (developed by Calgene, Davis, CA [now Monsanto]). Because glyphosate is less expensive and controls more weed species, interest in using the oxynil herbicides has waned and 2004 was the final year of BXN1 cotton sales. BXN canola was commercialized by Rhone-Poulenc Canada (now Bayer Crop Science, Monheim, Germany) and subsequently discontinued. Phosphinothricin acetyltransferase (PAT or BAR) detoxifies phosphinothricin- or bialaphos-based herbicides (glufosinate). The pat gene is native to Streptomyces viridichromogenes and bar is from S. hygroscopicus where they act in both the biosynthesis and detoxification of the tripeptide bialaphos (De Block et al. 1987). Like glyphosate, phosphinothricin herbicides control a broad spectrum of weed species and break down rapidly in the soil so that the problems with residual activity and environmental impact are greatly reduced. Bayer CropScience markets this trait as LibertyLink1 in several species. The pat and bar genes are also popular plant transformation markers in the research community. Finally, BASF (Ludwigshafen, Germany) markets nontransgenic CLEARFIELD1 imidazolinone-resistant canola, wheat, sunflower, corn, lentils, and rice, while DuPont (Wilmington, DE) markets nontransgenic STS1 soybeans with tolerance to sulfonylurea herbicides. A sulfonylurea-tolerant flax variety called CDC Triffid, developed by the University of Saskatchewan (Canada), was grown commercially in Canada in 2000 but is no longer offered. All these crops contain mutagenized versions of the acetohydroxyacid synthase, also called acetolactate synthase (ALS), which are not inhibited by imidazolinone and/or sulfonylurea herbicides (Devine and Preston 2000). Herbicides that inhibit ALS are considered low or very low use-rate herbicides with a good spectrum of weed control and are likely to remain an important part of weed resistance management programs (Castle et al. 2006).

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1.3.5

N. Ferry and A.M.R. Gatehouse

HT Canola

There are two GM traits for herbicide resistance in canola: glyphosate and glufosinate resistance. Resistance to glyphosate herbicides is conferred by the genes 5-enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4 epsps) (event GT73, Monsanto) that is targeted to the chloroplast, and a glyphosate oxidoreductasegene from Achromobacter sp. strain LBAA (gox v247) (both Roundup Ready1). Resistance to phosphinothricin herbicides is conferred by phosphinothricin-acetyltransferance (pat gene), and marketed as LibertyLink1 by AgrEvo (now Bayer CropScience). Herbicide-resistant canola (oilseed rape) allows post-emergence application of a single herbicide with a wide spectrum of activity. For effective control, it has to be applied before the weeds reach 10 cm height. This extends the potential time period for spraying (Benbrook 2004). The timing is more flexible and the application of a single herbicide simplifies weed control (Firbank and Forcella 2000). Since many herbicides allow post-emergence applications, HT crops do not generally provide a new option. However, post-emergence spraying with non-GMHT oilseed rape is confined to a short 3–5 week period after crop emergence (Pallutt and Hommel 1998). Thus, GMHT canola offers greater flexibility (Owen 1999). With HT oilseed rape, the intention is to reduce the amount of active ingredient of herbicide used and to rely preferably on one broad-spectrum herbicide only (http://www.canola-councildemo.org). The intent is also to reduce the number of spraying rounds, which helps reduce soil compaction and erosion (Madsen et al. 1999). During the first years of cultivating GMHT oilseed rape, most farmers had reduced active ingredient rates and applications (Champion et al. 2003). GMHT oilseed rape was usually sprayed only once, with an average active ingredient amount that was either lower (Champion et al. 2003) or not significantly different from conventional oilseed rape (Schu¨tte et al. 2004). However, in Canada (where HT canola is widely cultivated), application of herbicide was seen to actually increase (Canola Council of Canada 2001). In fact, after years of continued GMHT oilseed rape cultivation, secondary adverse effects on application rates were reported. This was due to weeds becoming herbicide tolerant on- and offsite (Devos et al. 2004; Hayes et al. 2004). HT oilseed rape volunteers occurred in subsequent rotations (Devos et al. 2004), and multiple tolerances of volunteers to herbicides were recorded because of seed impurities and seed banks after outcrossing events between fields (Hall et al. 2000). Outcrossing to weedy relatives also occurred (Daniels et al. 2005); these weedy relatives, however, have not yet been proved to be fit enough to persist on cultivated fields (Hails and Morley 2005). Nevertheless, improved weed suppression with HT oilseed rape in many agronomic respects has been demonstrated (Westwood 1997; Bohan et al. 2005), and ultimately the technology remains popular with farmers because of its simplicity and reduced costs (Canola Council of Canada 2001; Graef et al. 2007). Genes have always moved between the natural and agroecosystem, and to date – despite the formation of hybrids between HT canola and wild relatives in Canada (Gressel

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2008) – the technology has proven safe and effective (Cerdeira and Duke 2006; Darmency et al. 2007; James 2007; Gressel 2008).

1.3.6

HT Maize

C. album, Amaranthus retroflexus, Abutilon theophrasti and several annual grass species cause economic losses in maize, if left uncontrolled. The option to use glyphosate in these systems is extremely valuable because it is a broad-spectrum herbicide that provides excellent control of a wide range of weed species. Resistance to glyphosate-herbicides in maize is conferred by the epsps gene in event GA21 (the GA21 trait for glyphosate-resistant maize relies on a modified maize epsps gene) (Roundup Ready), developed by DeKalb (now Monsanto) in 1998. This is now being largely replaced by event NK603, Roundup Ready corn 2, with two epsps expression cassettes under the transcriptional regulatory control of the rice (Oryza sativa L.) actin 1 (P-Ract1) and the enhanced cauliflower mosaic virus 35S (P-e35S) promoters to impart fully constitutive expression. Maize has also been developed with resistance to phosphinothricin herbicides via transformation with the pat gene in events T14 and T25, developed by Aventis (now Bayer CropScience), and marketed as LibertyLink. Maize is an open-pollinated, wind-facilitated species and gene flow via pollen is well recognized (Haslberger 2001). Thus, the movement of GM traits is a significant consideration in maize production (Luan et al. 2001; Ma et al. 2004). Generally, the introgression of GR traits in seed maize can be managed successfully (
1% outcross) by establishing isolation distances of 200 m between fields (Ma et al. 2004). However, in typical maize production regions of the US, these isolation distances are not possible and GR trait introgression into non-GR fields is prevalent. The occurrence of the GR transgene in non-GM maize can have significant economic consequences, if the grower of the non-GM maize has a contract to provide a GM-free product. Furthermore, incidences of GM gene introgression in local landraces of maize in Oaxaca, Mexico have been reported (Quist 2001). The implications for transgene occurrence reflect concerns for the maintenance of the genetic resource of the landrace maize. However, the initial report of transgene introgression was followed by a second report that suggested that no transgenes existed in these landraces of maize (Ortiz-Garcia et al. 2005). Regardless, given the adoption of GR maize, the discovery of transgene introgression into landrace maize is likely in the longer term.

1.3.7

HT Cotton

Cotton is a slow-growing plant, and only a limited selection of herbicides can be used for weed control. These two factors sometimes make weed control difficult. Cotton is a semitropical, perennial plant, although it is grown as an annual crop.

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Its growth is especially slow in the northern cotton belt in the US, where it is often planted early into cool soils. Post-emergence broadleaf control is accomplished with herbicides that can injure cotton but that are applied in a directed spray that misses most of the cotton plant; thus, weed control in cotton is particularly difficult. Cotton with resistance to glyphosate herbicides has been developed based on the expression of the CP4 epsps in events MON1445/1698, Monsanto, Roundup Ready1. Resistance to phosphinothricin herbicides is based on the expression of the bar gene in LLCotton25, Bayer CropScience LibertyLink1. Herbicide-tolerant cotton expanded from around 2% of the cotton acreage in 1996 to 26% in 1998, and reached 46% in 2000 (James 2001). In 2008, adoption of GM cotton in the US with either or both herbicide tolerance and Bt reached 86% (James 2007). There are limited reports that cotton demonstrates introgression at low frequencies (Ellstand et al. 1999). Pollen movement in cotton is dependent on insects. Cotton is predominantly self-pollinated and natural outcrossing is typically quite low (Xanthopoulos and Kechagia 2000). Thus, there is a very low probability that the GR transgene would move into non-GR cotton cultivars via pollen flow. While gene flow via GR cotton seed can occur, the consequences of this are not thought to be important.

1.3.8

HT Soybean

Soybean engineered for resistance to glyphosate herbicides has been developed by Monsanto with the CP4 epsps gene, event GTS-40-3-2 and is marketed as Roundup Ready1. Glyphosate-resistant soybeans were one of the earliest transgenic crops brought to market and they have experienced rapid adoption to the point that over 85% of US soybeans and 56% of soybeans globally are now glyphosate-resistant (James 2007). Soybeans are an autogamous (self-fertilizing) species with limited opportunity for pollen-directed gene flow (Palmer et al. 2001). Spontaneous gene flow in cultivated soybeans ranges from 0.02 to 5% depending on distance and is facilitated by thrips (Thrips tabaci) and honeybees (Apis mellifera). While the movement of the GR transgene has been observed in soybeans, there are extremely limited opportunities for this occurrence and pollen-mediated gene flow in GR soybeans is essentially a nonissue (Abud et al. 2004; Owen 2005). However, gene flow by seed is highly probable and represents a significant economic problem (Swoboda 2002; Owen 2005).

1.3.9

Other GMHT Crops

In terms of commercial crops, glyphosate-resistant alfalfa had been developed and was launched in 2006 (James 2007).

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However, of special interest genetically engineered herbicide-resistant crops are being tested as a solution to the parasitic plants Orobanche spp. (broomrapes) and Striga spp. (witchweeds), which parasitize the roots of important crops, heavily reducing yields. Joel et al. (1997) showed that the use of three target-site resistances decimated Orobanche, demonstrating the potential of transgenic herbicide-resistant crops in the control of parasitic weeds. This is potentially of great importance as every year parasitic plant damage to crops accounts for an estimated US$7 billion in yield loss in sub-Saharan Africa, and affects the welfare and livelihood of over 100 million people. For this technology to have a great impact in Africa, crops adapted to African agroecologies need to be available to farmers.

1.3.10

Changes in Agronomic Practice

The adoption of GM Crops, and specifically glyphosate-resistant crops, has resulted in significant changes in agronomic practices. Most obviously, these changes include the increase glyphosate use at the cost of other herbicides and the manner and frequency in which glyphosate is used. In addition, the amount of tillage that is conducted for crop production has significantly changed (Young 2006; Service 2007a, b; Foresman and Glasgow 2008). This reduction in tillage has an important benefit of reducing the use of petroleum-based fuels as well as an implicit gain in time use efficiency by growers. A significant reduction in pesticide use has been attributed to the adoption of herbicide-tolerant (HT) crops (Sankula 2006). Furthermore, the benefits ascribed to herbicide-tolerant crops have dramatically changed the crop cultivars selected by growers and have hastened the development of new transgenic crops for commercial distribution worldwide (Duke 2005; Dill et al. 2008). Despite wide scale commercial plantings of GM soybean, canola, cotton and maize, several herbicide-resistant crops have been developed but have not been commercially introduced. Notably, sugarbeet (Beta vulgaris) cultivars that are resistant to glyphosate were deregulated in 1999, but not commercially offered until 2008 because of concerns about the acceptability of sugar refined from a GM crop (Duke 2005; Gianessi 2005). The development of GM rice (O. sativa) cultivars modified to be resistant to glufosinate herbicide proceeded from 1998 to 2001, but were withdrawn and commercial development terminated because of concerns about market acceptance of the GM rice (Gealy and Dilday 1997; Gealy et al. 2007). Similarly, wheat (Triticum aestivum) glyphosate-resistant cultivars were under development but the program was terminated in 2004 (Dill 2005). While the GR-based wheat production systems demonstrated excellent opportunities for improved weed management, concerns about the acceptance of the flour made from GR wheat cultivars as an export commodity in GM-adverse countries resulted in the decision to halt further development (Stokstad 2004; Howatt et al. 2006). In addition to concerns centered on consumer acceptability, several concerns over potential environmental impact have been raised. These are addressed below.

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1.3.11

N. Ferry and A.M.R. Gatehouse

Weeds, Gene Flow, Invasiveness, and Biodiversity Impacts

There have been significant concerns regarding the potential of GM crops, particularly glyphosate-resistant crops, to become major agricultural problems (by invasion, volunteerism) or to create “superweeds” by cross-pollination (Ellstrand 2003). 1.3.11.1

Gene Flow

While GM crops do have the potential to cross-pollinate other crops and wild relatives, there are four basic elements determining the likelihood and consequences of gene flow: first, the distance of pollen movement from the GM crop; second, the synchrony of flowering between crop and pollen recipient; third, sexual compatibility between crop and recipient; and fourth, ecology of the recipient species (Dale et al. 2002). Research has shown that pollination declines sharply with distance from the pollen source (Lutman 1999) and one could reduce the chances of GM pollen reaching other crops through the use of isolation or buffer zones, although pollen may travel further if plants are insect-pollinated. Ellstrand et al. (1999) reviewed the sexual compatibility of crops with weeds and feral species. For example, oilseed rape (canola), barley, wheat and beans can hybridize with weeds in some countries; however, in the UK, for example, the probability of hybridization with weeds is considered minimal for wheat, low for oilseed rape and barley, and high for sugarbeet. Although sugarbeet can readily hybridize, in the case of herbicide-tolerant varieties of sugarbeet the crop would be harvested before flowering and hence shed no pollen. Indeed, methods have been developed to block expression in the pollen of transgenic plants, including engineering of the chloroplast genome (Heifetz 2000) and transgene mitigation strategies (Gressel 2008). Transgene Mitigation Strategies Transgenic crops may interbreed with nearby weeds, increasing their competitiveness, and may themselves become a “volunteer” weed in the following crop. The desired transgene can be coupled in tandem with genes that would render hybrid offspring or volunteer weeds less able to compete with crops, weeds and wild species. Genes that prevent seed shatter or secondary dormancy, or that dwarf the recipient could all be useful for mitigation and may have value to the crop. Many such genes have been isolated in the past few years (Gressel 1999). Examples include: apomixis (asexual reproduction) as a fail-safe so that the seed is actually of vegetative origin and not from sexual pollination (Zemetra et al. 1998); chromosome-specific cytogenetic fail-safes (the risk of transgenic traits spreading into weeds can be reduced by orders of magnitude by using cytogenetic mapping to locate transgenes and releasing only those transgenic lines in which it is on a genome incompatible with local weeds (Gressel and Rotteveel 2000)); plastome-specific cytogenetic fail-safes (it is possible to introduce some traits into the chloroplast or

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mitochondrial genomes;thus, in species where these genomes are completely maternally inherited, the transgenes cannot move into other species (Daniell et al. 1998)); seed dormancy (the transgenic abolition of secondary dormancy is neutral to the crop but deleterious to weeds (Gressel 1999)); seed ripening and shattering (uniform ripening and anti-shattering genes are harmful to weeds but neutral for some crops (Schaller et al. 1998)), and dwarfing (dwarfing is disadvantageous for weeds, because they can no longer compete with the crop for light (Gressel 1999)). However, these strategies are not as of yet fully developed and in use, with the exception of plastid transformation. 1.3.11.2

Invasiveness

The potential exists for GM crops to become invasive. There has been a great deal of concern that such crops could persist in the wild and disperse from their cultivated habitat. However, recent studies (and experience from the field) have indicated that the ability of GMHT crops to invade and persist was actually no better than that of their conventional counterparts (Crawley et al. 2001). Finally, GM crops persisting in fields after harvest thus becoming a weed in a different crop may be dealt with in two ways; simple treatment with an appropriate herbicide or mitigation technologies that prevent the transgene being carried over to the next generation (Gressel 2008). 1.3.11.3

A Dose of Reality

In order to put these concerns into perspective, one must understand that flow from the agroecosystem to natural ecosystems has always occurred. Gene flow is a continuing process and is the source of biological diversity (Thies and Devare 2007). There has always been gene flow from commercial crops to relatives living in near proximity (Ferry and Gatehouse 2009). In reality, the vast majority of the major cultivated crops have no wild or weedy relatives outside of their centers of origin (Gressel 2008); however, some crops are grown in areas where gene flow may occur and appropriate measures must be taken in these areas.

1.3.12

Coexistence of GM and Non-GM Crops

A pervasive problem that exists with the production of GR crops is their coexistence with non-GM crops (Byrne and Fromherz 2003). The issue of coexistence includes three possibilities: (1) introgression of the trait via pollen (pollen drift), (2) containment of plant products during the production year (grain segregation), and (3) volunteer GRC plants in following years (Owen 2005). While GR crops and non-GM crops can coexist, growers must go to great lengths to accomplish segregation (Anonymous 2007). Grain segregation, while difficult to

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maintain, can be accomplished and will effectively minimize the impact of GR crops on non-GM crops. Given that the mixing of grain is equivalent to loss of intellectual property (IP), this can be costly to growers; thus, appropriate tactics to isolate GR crops from non-GM crops must be in place (Owen 2000). Controlling volunteer GR crops is also relatively easy, depending on the rotational crop, but does require diligence on the part of the grower (Owen 2005). The introgression of the HT trait via pollen movement is another possibility and management of this problem is considerably more difficult, particularly in open-pollinated crops such as maize (Luan et al. 2001; Palmer et al. 2001; Westgate et al. 2003; Abud et al. 2004). A number of factors affect the success of maize pollen movement and subsequent pollination, and generally, the greater the distance between the pollen source and the donor, the less likely is the introgression of the GR trait (Luan et al. 2001; Westgate et al. 2003). However, given the tolerance levels established for some GM traits in non-GM crops, the isolation distances required to mitigate the risks of gene flow are too large to be realistic (Matus-Cadiz et al. 2004). Other open-pollinated crops have also undergone a great deal of scrutiny (Charles 2007; Fisher 2007; Harriman 2007; Weise 2007). It is suggested that the issues of the coexistence of GRCs with non-GM crops will continue to be a concern as long as there are economic differences between the crop cultivar types (Hurburgh 2000; Ginder 2001; Hurburgh 2003). Nevertheless, coexistence guidelines and buffer zone (isolation zone) regulations are in existence (Boller et al. 2004).

1.3.13

Stacked Traits

Stacked products are a very important feature and future trend for GM crops, which meets the multiple needs of farmers and consumers; these are now increasingly deployed by ten countries – US, Canada, the Philippines, Australia, Mexico, South Africa, Honduras, Chile, Colombia, and Argentina (James 2007). In fact, 37% of all GM crops in the US in 2007 were stacked products containing two or three traits that delivered multiple benefits. Currently, there are a number of GM crops that have stacked herbicide resistance and Bt events (Owen 2006). In some events, the pat gene is used as a marker for the Bt – the pat gene also confers resistance for glufosinate herbicide. Interestingly, the Bt trait is combined with a GR hybrid, the resultant GM cultivar is resistant to both glyphosate and glufosinate. Monsanto has announced new transgenic maize cultivars that will combine several Bt events plus two herbicide resistance events. The Bt event functions ecologically differently compared to herbicide-resistant transgenes; the transgene for herbicide resistance can be considered benign to the weed population and has no impact until the herbicide is applied (Owen 2009). In contrast, Bt exerts continuous selection pressure on the target insect whether the insect population is at economic threshold or not. Furthermore, given the potential for the introgression of transgenes into near-relatives, Bt could potentially improve the fitness of compatible weed species (Snow and Pedro 1997). For example, the fitness of canola was increased by the inclusion of Bt (Steward et al. 1997); thus, gene flow

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was kept in check. However, there is no reason to believe that stacked traits will have any greater impact on nontargets than the crops with single traits, and for Bt non-target impacts have been shown to be minimal (Ferry and Gatehouse 2009).

1.3.14

Conclusions

Herbicide-resistant crops, specifically GR crops, have been globally adopted as the basis for the production of maize, soybean, cotton and canola (Dill et al. 2008). Their adoption provides economic benefits to agriculture and has major positive impacts on the environment; specifically, conservation tillage which can reduce soil erosion (Duke and Powles 2008; Shipitalo et al. 2008). However, growers must adopt measures to proactively sustain the technology (Mueller et al. 2005; Johnson and Gibson 2006; Sammons et al. 2007; Christoffoleti et al. 2008) and to steward GR crops to avoid the evolution of GR weeds (Duke and Powles 2008; Owen 2009). While there are tactics that are capable of mitigating some of the other concerns, including the risks of transgene introgression into near-relative plants (Snow and Pedro 1997; Gepts and Papa 2003), an improved process to assess the environmental risk of GR crop technologies and communicating those risks to the lay public should be in place (Owen 2009).

1.4 1.4.1

Biotic Stress due to Plant Pathogens Crop Losses due to Plant Pathogens

Plants are challenged constantly by many different potential pathogens (Table 1.4). There are hundreds of thousands of viral, bacterial, and fungal species in the world and thousands of these are pathogens that infect plants (Chrispeels and Sadava 2003). Any one pathogen can severely depress the yield of a given crop. Pathogens may reduce yield by causing tissue lesions; by reducing leaf, root, or seed growth; or by clogging up vascular tissue and causing wilt. Even in the absence of obvious symptoms, pathogens can still be a major metabolic drain that reduces productivity. They may also cause pre- or postharvest (stored products) damage to the harvested product. Table 1.4 Organisms that cause infectious disease in plants Types of plant pathogens Fungi Ascomycetes e.g., Fusarium, Verticillium Basidiomycetes e.g., Rhizoctonia, Puccinia (rust) Oomycetes e.g., Phytopthora (blight) Bacteria e.g., Xanthomonas, Pseudomonas Phytoplasmas and Spiroplasmas Viruses Viroids and virus-like organisms Nematodes, protozoa, and parasitic plants are also considered plant pathogens

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1.4.1.1

N. Ferry and A.M.R. Gatehouse

Current Disease Control Measures

Disease control in commercial crops is both economically important and environmentally controversial. Disease management includes: quarantine, cultural practices such as modification of the plant environment (e.g., soil management) to reduce pest numbers, host plant resistance, biological control and chemical control. Chemical control can be highly effective. The first fungicide was discovered in the 1880s when Bordeaux mixture (a mixture of copper sulfate and lime) was found to suppress grape downy mildew. Numerous compounds with antifungal or antibacterial activity have since been discovered, most often applied as sprays, dusts or seed coatings. Many older compounds are broad spectrum and toxic with the newer chemistries acting systemically with a narrower target range. Such chemicals are expensive and normally reserved for use on high-value fruit and vegetable crops (Ferry and Gatehouse 2009). They are expensive to manufacture and a great deal of investment must be put into human and ecological safety testing before a product can be released (Chrispeels and Sadava 2003). As with other chemical control strategies, pathogens can evolve resistance to these compounds. Thus, plant breeders have often relied on genetic disease resistance traits to manage pathogens of particular crops. In classical plant breeding, this has relied on crosses between elite crops and wild relatives (that are more genetically diverse) to introduce new disease resistance traits into the crops. Extensive backcrossing of the elite line is then required to eliminate the undesirable traits in the wild relative and thus makes traditional breeding a time-consuming process with a time of about 15 years required before a new resistant variety is available for release to growers. Nevertheless, such traditional plant breeding has had significant successes. For example, the development of F1 hybrid crops (derived from crossing two inbred lines) resulted in vastly improved yields (Chrispeels and Sadava 2003). Despite this enormous success, the F1 hybrids also serve as a warning as to the dangers of largescale monoculture and the need for effective and durable disease resistance. In 1970, the highly successful but genetically uniform F1 maize crops in the US were left devastated by a disease, southern corn leaf blight (Bipolaris maydis Nisik), which caused damage to 710 million bushels of maize (Ullstrup 1972). In extreme cases, crop disease can change political as well as agricultural landscapes!

1.4.1.2

The Irish Potato Famine

The Irish Potato Famine (the Great Hunger) started in 1845, lasted until 1849–1852 and led to the death of approximately one million people through starvation and disease; a further million are thought to have emigrated as a result of the famine. Some estimate that the population of Ireland was reduced by 25% (Kinealy 1995). The cause of the famine was a potato disease commonly known as late blight caused by the oomycete, Phytophthora infestans. Although blight ravaged potato crops throughout Europe, the impact and human cost in Ireland was high as a third of the

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population was entirely dependent on the potato for food; it was also exacerbated by a host of political, social and economic factors. The famine was a watershed in the history of Ireland. Its effects permanently changed the island’s demographic, political and cultural landscape. For both the native Irish and the resulting diaspora, the famine became a rallying point for nationalist movements. The fallout of the famine continued for decades afterwards and Ireland’s population still has not recovered to prefamine levels. 1.4.1.3

The Bengal Famine of 1943

The Bengal famine of 1943 occurred in British administered Bengal. It is estimated that around four million people died from starvation and malnutrition during the period. During the Second World War, the British had just suffered defeat in nearby Burma and British authorities feared a subsequent Japanese invasion of India by way of Bengal, so emergency measures were introduced to stockpile food for British soldiers and prevent access to supplies by the Japanese in case of an invasion. However, in the rice-growing season of 1942 weather conditions were exactly right to encourage an epidemic of the rice disease brown spot following a cyclone and flooding; brown spot in rice is caused by the fungus Helminthosporium oryzae. When food shortages became apparent, the Bengal government reacted to the crisis incompetently refusing to stop the export of food from Bengal to allied soldiers and failing to provide adequate famine relief in Bengal itself. Winston Churchill was Prime Minister at the time and while his involvement in the disaster, and indeed his knowledge of it remains unclear, it has been suggested that in response to an urgent request by the Secretary of State for India to release food stocks for India, Churchill responded with a telegram asking if food was so scarce, “why Gandhi hadn’t died yet” (Mishra 2007). Needless to say, whether this is completely true or is in fact an unfortunate misquote used out of context, the British Administration in India declined in popularity and there is little doubt that this incident fuelled feelings for Indian Independence. Given the vast array of diseases that threaten crops, and the scale of disaster that can ensue, it is a wonder that crops be produced at all?

1.4.2

Plant Defense Against Pathogens

Plants have evolved mechanisms to resist pathogen invasion that consist of different defense layers. Firstly, waxy coatings on epidermal cells provide a physical barrier; secondly, plants contain large amounts of preformed secondary metabolites that have antimicrobial activity (constitutive defenses including glucosides, saponins, alkaloids, antifungal proteins, antifeedants and enzyme inhibitors), these are effective in many cases – but pathogens have evolved enzymes capable of detoxifying these compounds. Often induced defenses are the plants’ last line of defense against pathogens and are sufficient to (partially) ward off invading microorganisms.

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Essentially, plants have two major types of induced disease resistance, basal defense and resistance (R) gene-mediated defense. All plants have basal defense, this is a general immune response to pathogens and other environmental stresses. R-gene-mediated defense is more specific and is only found in certain plant species. R-gene-mediated defense involves recognition of a specific pathogen effector by a plant ligand receptor. These pathogen effectors can suppress plant basal defense, making any plant without the R-gene defense susceptible. The ligand-effector recognition can result in a dramatic immune response such as cell death. In tomato, Solanum lycopersicum, the Mi gene, a member of a large family of R-genes, mediate resistance to potato aphids, whiteflies, and root-knot nematodes (Kaloshian and Walling 2005). Both types of plant defenses (R and basal) involve signaling via three major plant hormones: salicylic acid, jasmonic acid, and ethylene (ETH). In some instances, defense responses are induced distal to the site of infection and this is referred to as systemic acquired resistance (SAR). At least three nonspecific induced defense pathways are described which are triggered by these specific signaling molecules: (a) The salicylic acid (SA)-dependent pathway is induced by necrosis inducing pathogens and triggers systemic acquired resistance (SAR) (b) A second pathway is triggered by nonpathogenic rhizobacteria, it is dependent on jasmonic acid (JA) and ethylene (ETH) and constitutes induced systemic resistance (ISR) (c) JA and ETH regulate a third pathway that is effective against a different set of pathogens and not affected by ISR Most of the inducible defense-related genes are regulated by these signaling pathways (Delaney et al. 1994; Sticher et al. 1997; Van Loon 1997; Reymond and Farmer 1998; Knoester et al. 1998; Ananieva and Ananiev 1999). Defense gene regulation has been extensively studied and severa1 rapid processes characteristic of the hypersensitive response (HR) appear to involve primarily activation of preexisting components rather than changes in gene expression. One of these rapid processes is the striking release of reactive oxygen species.

1.4.2.1

Oxidative Burst

The oxidative burst, a rapid production of reactive oxygen species (ROS), is a welldocumented early plant response to biotic stress (e.g., Apel and Hirt 2004). ROS comprise radicals and other nonradical but reactive species derived from oxygen. Among them, the superoxide anion (O 2 ) and hydrogen peroxide (H2O2) exert various effects on cells, directly or in cooperation with other molecules. In excess, ROS pose a threat to important biomolecules and cell membranes. One of the consequences of ROS activity is oxidative damage of membrane integrity due to lipid peroxidation processes which may result in the generation of highly cytotoxic compounds. Glutathione-S-transferases (GSTs), induced upon pathogen attack, may detoxify lipid peroxides by conjugating them with glutathione (GSH). These

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enzymes can also catalyze the GSH-dependent reduction/inactivation of H2O2, forming glutathione disulfide (GSSG) and increasing GSH synthesis by feedback induction (Marrs 1996). On the other hand, numerous studies indicate an essential role of ROS in plant defense responses to biotic stress. In addition to direct antimicrobial activity and contribution to the strengthening of barriers against pathogens, recent reports point to H2O2 and O 2 as signal transduction agents activating defense pathways and as key mediators in cell death during hypersensitive response (HR) (Grant and Loake 2000). To maintain a balance between negative and beneficial functions of ROS, their levels are strictly controlled by a complex and flexible network of antioxidant systems (Mittler et al. 2004). The major enzymatic ROS-scavenging components of this network are superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Superoxide dismutases dismute O 2 to H2O2, an excess of which may be subsequently detoxified by CATs and/or APX. Ascorbate peroxidases, in contrast to CATs localized in peroxisomes, are present in almost all cellular compartments; they exhibit a high affinity to H2O2 and are considered to be responsible for the fine modulation of ROS level (Mittler 2002). Moreover, APX, in cooperation with two main low-molecular antioxidants, ascorbate (Asc) and glutathione (GSH), and enzymes for their regeneration, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase (GR), constitute the ascorbate–glutathione (Asc–GSH) cycle, believed to be the central part of the antioxidant network (Noctor and Foyer 1998). Ascorbate, present at high concentrations in all cellular compartments and capable of direct scavenging of O 2 and hydroxyl radicals, is considered to be the most powerful cell antioxidant (Noctor and Foyer 1998). Ascorbate and glutathione are the major cellular redox buffers, which together with their oxidized forms, dehydroascorbate (DHAsc) and glutathione disulfide (GSSG), enable cells to maintain a redox balance. Changes in the levels or redox state of ascorbate and glutathione pools as well as in H2O2 homeostasis, and thereby in cellular/compartment redox state, are considered to be pivotal signaling events influencing gene expression and modulating the plant defense response (Pastori and Foyer 2002; Foyer and Noctor 2005). Numerous genes are induced during the plant defense response and presumably these function in host plant pathogen defense. Induced defenses include the activation of phytoalexins (including terpenoids, glycosteroids and alkaloids) and PR proteins (including chitinase, b-1,3 glucanase and other antimicrobial proteins) and the production of reactive oxygen species (ROS) leading to a hypersensitive response (HR).

1.4.3

Utilizing Defense Mechanisms

Most conventional breeding strategies have relied on identifying major resistance genes (often in wild or ancestral plants) while fewer have concentrated on breeding for polygenic resistance. New opportunities and strategies to enhance disease

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resistance are afforded by genetic engineering of plants – these may follow the single gene model, or may involve manipulation of defense-activating mechanisms.

1.4.4

Transgenic Disease Resistance

Many transgenic-based approaches exist for conferring enhanced levels of disease resistance, as exemplified by Fig. 1.6, which provides a simplified model. 1.4.4.1

Expression of Single Genes

R-Genes A very effective defense mechanism in plants is gene-for-gene resistance, this induced resistance mechanism is based on the interaction between plant-derived Pathogen

ty

ici en g o th Pa tors c fa

(3) Pathogen mimicry

(1b) Induced defense

(2a) Detection of pathogen

(1a) Constitutive defense (2b) Defense regulation Host cell

Nucleus

Transgenic Strategies. 1a. Constitutive expression of antimicrobial factors, 1b. Pathogen induced expression of one or more genes, 2a. Altering recognition of the pathogen (e.g. R-genes) and 2b. altering downstream regulators, 3. Priming recognition of pathogens (genetic vaccination).

Fig. 1.6 Simplified model of transgenic strategies to enhance plant defense

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resistance (R) gene products and an avirulence (avr)-gene product (elicitor) produced by the pathogen. The interaction is generally very specific and results in the triggering of strong resistance responses including hypersensitive response (HR) and localized cell death at the point of infection. There is no common structure between avirulence gene products, except that most are secreted proteins. Because there would be no evolutionary advantage to a pathogen keeping a protein that only serves to be recognized by the plant, it is believed that the products of avr genes, sometimes known as effector proteins, play an important role in virulence in genetically susceptible hosts. The guard hypothesis model proposes that the R proteins interact, or guard, a protein known as the guardee which is the target of the Avr protein. When it detects interference with the guardee protein, it activates resistance. R-gene specificity (recognizing certain avr gene products) is believed to be conferred by the leucine-rich repeats (LLRs). LRRs are multiple, serial repeats of a motif of approximately 24 amino acids in length, with leucines or other hydrophobic residues at regular intervals. LRRs are involved in protein–protein interactions, and the greatest variation amongst resistance genes occurs in the LRR domain. LRR swapping experiments between resistance genes in flax rust resulted in the specificity of the resistance gene for the avirulence gene changing (Bergelson et al. 2001). Gene-for-gene resistance thus provides obvious targets to engineer disease resistance in transgenic plants. However, pathogens can often breakdown R-gene-mediated resistance if the corresponding avr gene mutates becoming inactivated. Pathogen adaptation is also seen in conventionally bred crops. The use of R-genes is limited by them conferring resistance to only a single pathogen or race of pathogen; however, they can provide effective (even broad spectrum) resistance if transformed by genetic engineering into new species or new genera of plant (Oldroyd and Staskawicz 1998). For example, Rxo1, an R-gene derived from maize, a nonhost of the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola, was successfully transformed into rice and shown to confer resistance against this pathogen (Zhao et al. 2005). Interspecies differences do radically influence R-gene function making it preferable to use R-genes from related species (Ayliffe and Lagudah 2004); however, the transgenic approach circumvents the tedious and time-consuming backcrossing required to introduce a single trait via traditional crop breeding.

Constitutive Expression of Inducible Antimicrobial Factors Several examples of transgenic strategies to enhance plant constitutive defenses are described below. This is an extension of the single gene paradigm that has worked so well for insect-resistant transgenic plants. Expression of Chitinases Chitinases are glycosyl hydrolases catalyzing the degradation of chitin, an insoluble linear b-1,4-linked polymer of N-acetylglucosamine. They are produced by a wide

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range of organisms from microbes to insects and mammals, including plants (Kasprzewska 2003). Plants produce chitinases as defense against chitin-containing fungal pathogens by inhibiting spore germination, germ tube elongation and degrading hyphal tips (Khan et al. 2008). Transgenic plants expressing chitinase ChiC genes have already been produced in several species. In carrot, the tobacco class I ChiC gene has shown resistance to Botrytis cinerea (Punja and Raharjo 1996). Transgenic tobacco transformed with bean class I ChiC exhibited enhanced resistance to Rhizoctonia solani (Broglie et al. 1991) and Alternaria alternata (Lan et al. 2000). The rice chitinase gene (RCC2) exhibited increased resistance to Sphaerotheca humuli in transgenic strawberry (Asao et al. 1997). Peanut (Arachis hypogae) plants transformed with tobacco ChiC gene were resistant to a fungal pathogen (Cercospora arachidicola), the causal organism of leafspot disease (Rohini and Rao 2001). Potato has been transformed with the Streptomyces griseus ChiC gene and resistance to Alternaria solani (causal agent of early blight) demonstrated (Kahn et al. 2008) and cotransfer and expression of ChiC, glucanase, and bialaphos resistance (bar) genes in creeping bent grass conferred resistance to the fungal pathogens Sclerotinia homoeocarpa and R. solani (Wang et al. 2003). Expression of Defensins Defensins are the best characterized cysteine-rich antimicrobial proteins in plants (Broekaert et al. 1995). All known members of this family have four disulphide bridges and are folded in a globular structure that includes three b-strands and an a-helix (Segura et al. 1998). Inhibition of fungal growth by defensins seems to occur by permeabilization of the plasma membrane through binding to a putative receptor (De Samblanx et al. 1997). Genes encoding plant defensins are developmentally regulated, with predominant expression in the outer cell layers, and are induced in response to pathogen infection and stress (Segura et al. 1998). Enhanced tolerance to the fungus Alternaria longipes is observed in transgenic tobacco overexpressing a radish defensin (Rs-AFP2) (Chiang and Hadwinger 1991). In support of this role, Manners et al. (1998) show that the promoter of the plant defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal pathogens and responds to methyl jasmonate, but not to salicylic acid. Expression of Oxalate Oxidase and H2O2 -Generating Enzymes Rapid generation of superoxide and accumulation of H2O2 is a characteristic early feature of the hypersensitive response following perception of pathogen avirulence signals. In germinating barley and wheat seeds as well as in barley leaves challenged with powdery mildew, oxalate oxidase activity has been identified as a generator of H2O2 (Lane et al. 1993; Zhou et al. 1998). Oxalate oxidase utilizes oxalic acid and oxygen as substrates producing H2O2 and CO2. For certain necrotrophic fungi, such as Sclerotinia sclerotiorum, oxalic acid is a major pathogenicity determinant, significantly altering environmental pH (Cessna et al. 2000).

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Pathogen-inducible oxalate oxidase both acts as a generator of H2O2, killing the invading pathogen, and simultaneously detoxifies the acid, which is phytotoxic at high concentrations. Sunflower plants expressing a wheat oxalate oxidase accumulate enhanced levels of salicylic acid and PR1 even in the absence of pathogen infection and display improved tolerance to Sclerotinia infection (Hu et al. 2003). This effect is most likely due to the production of H2O2 from endogenous oxalic acid. Amine oxidases are a class of enzymes mainly found in the plant apoplast that act on a variety of amine substrates, including mono-, di- and polyamines and release the corresponding aldehyde as well as NH3 and H2O2. Amines are present in the plant apoplast and accumulate in response to environmental stress (Bolwell et al. 1999). Thus, in plants several systems are available that can produce H2O2 following pathogen attack. The importance of H2O2 in plant defense has clearly been shown by several groups who have reported increased pathogen resistance in transgenic plants by introducing either H2O2-generating systems or by inhibiting H2O2-degrading systems. The expression of a fungal glucose oxidase resulted in enhanced resistance to P. infestans and Erwinia carotovora in potato (Wu et al. 1995). Similarly, the prevention of H2O2 breakdown resulted in higher levels of H2O2 and increased disease resistance (Chamnongpol et al. 1998). Whether this improved pathogen tolerance is due to the direct antimicrobial effect of H2O2, or due to the fact that the plant defense system is induced by the increased levels of H2O2, was, in either case, not investigated. Inducible Expression of Single Antimicrobial Factors Stilbene Synthase Production of stilbene, a phytoalexin, is a well-characterized defense reaction in grape vine. Stilbene plays an important role in resistance to fungal and bacterial infection in plants. It strongly inhibits the growth of fungi and sprout of spores. Stilbene synthase gene (Vst1) is responsible for the synthesis of resveratrol (trans3,40 5-trihydroxystilbene) when plants are challenged by fungal diseases. Thus, introduction of stilbene synthase genes into transgenic plants can be used to induce synthesis of phytoalexins. Vst1 has been transferred into many plants to enhance fungal resistance including common spring wheat using biolistic transformation, where plantlets with mild resistance to powdery mildew were identified (Leckband and Lo¨rz 1998). Other crops transformed to produce stilbene include: tomato (Thomzik et al. 1997); apple (Szankowski et al. 2003); and Vst1 overexpressed in its native grape (Fan et al. 2008). 1.4.4.2

Activation of Expression of Multiple Genes

Polygenic Resistance There are several types of disease resistance in terms of the effects of genes on the plant and pathogen. However, in terms of the number of genes involved, there are

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two general types of resistance. The first (as described above for R-genes) is called major-gene or single-gene resistance. This type of resistance is well defined and more easily measured; it is sometimes called qualitative resistance because plants are either resistant or susceptible, without intermediate levels. The second type, called polygenic resistance, involves several or many genes. This type of resistance is harder to define; exactly which genes are involved may be unknown. It usually is effective against all races of a pathogen. This type is often called quantitative, because there are intermediate levels ranging from resistant to susceptible. It is also harder to measure than major-gene or single-gene resistance. Often polygenic resistance does not give a plant as high a level of resistance as major gene resistance. Polygenic durable resistance to multiple diseases of a crop would be highly desirable. In nature, most host plant resistance is based on multiple genes and a diverse set of resistant factors. This diverse, polygenic resistance system helps to prevent plant-feeding microorganisms from overcoming the resistance in the host plant. At present, polygenic resistance may be bred into a crop via quantitative trait loci (QTL) analysis, and the use of molecular markers in molecular breeding approaches; promising transgenic strategies (as discussed below) activate polygenic resistance via elicitor-induced resistance and activation of multiple genes.

Detection of Pathogens; Expression of Elicitors and Defense Activators Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide binding site (NBS), and a putative amino-terminal signaling domain, termed NBS–LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the hypersensitive response (HR). It is thought that this halts pathogen growth. In the absence of specific recognition, a basal defense response also occurs, which is apparently driven by pathogen-associated molecular patterns (PAMPS), such as flagellin and lipopolysaccharides (LPS) elicitors of defense responses (Belkhadir et al. 2004). The basal defense response overlaps significantly with R-proteinmediated defense, but is temporally slower and of lower amplitude. However, Takakura et al. 2008 show that expression of a bacterial flagellin gene (N1141) in transgenic rice triggers disease resistance and enhances resistance against blast (Magnaporthe grisea). Furthermore, a number of transgenic plants expressing NBS–LRR proteins under the control of the CaMV 35S promoter have been described (Mindrinos et al. 1994; Ellis et al. 1999; Stokes et al. 2002). For example, Grant et al. (2003) show that an Arabidopsis mutant, adr1 (activated disease resistance), contains both elevated levels of SA and ROIs, accumulates a number of defense-related gene transcripts and exhibits resistance against a number of microbial pathogens. ADR1 encodes a distinct NBS–LRR protein which possesses N-terminal kinase subdomains. Furthermore, transient expression of ADR1 is sufficient to engage defense-related gene expression and establish disease resistance

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in the absence of significant seed yield penalty. ADR1 plants showed striking resistance against both P. parasitica (Noco2) and another biotrophic pathogen, E. cichoracearum (UED1).

Pathogen Phytosensing A related strategy for crop defense involves engineered phytosensors indicating the presence of key plant pathogens to provide an important first line of defense (Mazarei et al. 2008). Plant defense mechanisms are highly regulated on the transcriptional level, and can be induced by chemical elicitors produced by pathogens. These elicitors have been shown to cause changes in gene expression, which initiate a whole plant response from a localized encounter with a pathogenic organism (Metraux et al. 2002). This is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in HR, SAR, ISR, and pathogen specific responses, any of which could be useful in building phytosensors. Cis-acting elements are conserved among plant species, which enables them to be used efficiently as synthetic inducible promoters. Employing synthetic promoters with potential inducible elements to engineer plants that can sense the presence of plant pathogens at the molecular level provides novel technologies for monitoring and increasing resistance to diseases (Gurr and Rushton 2005). Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or “phytosensors,” by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Mazarei et al. (2008) have employed cis-acting elements from promoter regions of pathogen-inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the cauliflower mosaic virus CaMV 35S promoter to increase basal level of GUS expression. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies analyzed the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants. The end goal of such studies is to engineer transgenic plants for the purpose of early pathogen detection.

1.4.4.3

Regulation of Inducible Defenses

The overexpression of regulatory genes provides another tool to activate plant defenses in response to pathogen attack. For example, transduction of the SA signal requires the function of NPR1 (also known as NIM1), a regulatory protein that was

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identified in Arabidopsis through genetic screens for SAR-compromized mutants (Cao et al. 1994; Delaney et al. 1995; Shah et al. 1997). Mutant npr1 plants accumulate normal levels of SA after pathogen infection but are impaired in their ability to express PR genes and to mount a SAR response. The NPR1 gene encodes a protein with a BTB/BOZ domain and an ankyrin-repeat domain (Cao et al. 1997). Upon induction of SAR, NPR1 is translocated to the nucleus (Kinkema et al. 2000) where it interacts with members of the TGA/OBF subclass of basic domain/leucine zipper (bZIP) transcription factors (Zhang et al. 1999; Despre´s et al. 2000; Zhou et al. 2000; Subramaniam et al. 2001; Fan and Dong 2002) that are involved in the SA-dependent activation of PR genes (Lebel et al. 1998; Niggeweg et al. 2000). Physical interaction between NPR1 and TGA transcription factors has been shown to be required for the binding activity of these factors to promoter elements that play a crucial role in the SA-mediated activation of PR genes (Despre´s et al. 2000; Fan and Dong 2002). In separate studies NPR1 overexpression and enhanced resistance are correlated with either elevated or earlier expression of PR transcripts (Cao et al. 1998). Genes with high sequence similarity to NPR1 are found in Arabidopsis, tobacco, tomato, rice and maize (Campbell et al. 2003). Overexpression of NPR1 in rice has been shown to enhance resistance to bacterial blight (X.o.o) (Chern et al. 2001).

1.4.5

Pathogen Mimicry and Virus Resistance

Numerous reports concern transgenic resistance to plant viruses (Fuchs and Gonsalves 2007) in which RNA-mediated gene silencing is the predominant strategy (viral RNA is degraded and viral DNA is deactivated by methylation). Most of these strategies can be categorized as pathogen mimicry. Transgenes constitutively expressed to provide RNA-mediated virus resistance fall into three main types: 1. Sense or antisense viral sequences 2. Inverted repeats/hairpin RNA of viral sequences, and 3. Sequences of engineered microRNAs targeted against the virus RNA-mediated resistance against viruses was first reported by Lindbo et al. (1993). Since the discovery of what is now generally called RNA silencing in plants, the same specific RNA degradation mechanism has been identified in nearly all eukaryotes (Baulcombe 2004). In plants, genetic and molecular analyses have revealed at least three natural pathways for RNA silencing: cytoplasmic short interfering RNA (siRNA) silencing; endogenous mRNA silencing by microRNA (miRNA); and the silencing associated with DNA methylation and suppression of transcription (Baulcombe 2004). All these pathways involve the cleavage of doublestranded RNA molecules into short 21–26 nuclotide RNAs, known as siRNAs and miRNAs (Baulcombe 2004). To date, it has primarily been the cytoplasmic siRNA silencing pathway that has been exploited by genetic engineering to confer resistance to plant viruses.

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The best documented examples of commercial transgenic resistance involve type (1) resistance, although the mechanism of resistance was not initially known. In the 1990s, the papaya industry in Hawaii suffered a 50% reduction in production because of papaya ringspot virus; the solution came through the expression of viral coat protein in transgenic plants (thought to be immune priming, analogous to vaccination).

1.4.5.1

Papaya Ringspot Virus

Papaya ringspot is a destructive disease (a potyvirus) characterized by yellowing and stunting of the crown of papaya trees, a mottling of the foliage, damage to younger leaves, water-soaked streaking of the petioles (stalks), and small darkened rings on the surface of fruit. Papaya ringspot virus is transmitted from infected papaya trees to healthy trees by the feeding action of various species of aphids, especially the green peach aphid and melon aphid. The virus is transmitted in a nonpersistent manner, meaning that the virus does not multiply within the aphid but is instead carried on its mouth parts and is transmitted from plant to plant while feeding. In 1995, American researchers developed a transgenic papaya resistant to the virus, by expressing a copy of a viral coat protein in the plant (Ling et al. 1991). It was field-tested in Hawaii where it was shown to be effective against the virus. The virus-resistant papaya is now widely used by commercial papaya producers in Hawaii. The presence of small RNA species, presumably siRNA, corresponding to regions of the viral coat protein gene was later shown to be present in transgenic lines resistant to PRSV; thus it is posttranscriptional gene silencing involved in the establishment of resistance (Krubphachaya et al. 2007). Pathogen-derived resistance has also been shown to be effective against maize streak virus (Shepherd et al. 2007), potato leaf roll virus (Vazquez Rovere et al. 2001) and tomato yellow leaf curl (Yang et al. 2004). An important drawback of RNA-based approaches to enhanced resistance is the high level of sequence specificity required for RNA degradation. Viruses containing >10% nucleotide divergence are insensitive to RNA degradation. Another drawback is the size of the transgene, commonly >300 bp, required to trigger efficient RNA silencing. However, Parizotto et al. (2004) has provided experimental evidence using genetically engineered microRNA (miRNA) to show that a 21 nucleotide sequence complementary to GFP mRNA was sufficient to trigger complete GFP silencing in transgenic plants. In the future, similar miRNA-derived strategies could provide resistance to a large number of plant viruses. However, the major technical limitation for technologies based on RNA silencing is that many important plant crop species are difficult or impossible to transform, precluding the constitutive expression of constructs directing production of double-stranded RNA. Moreover, public concerns over the potential ecological impact of virus-resistant transgenic plants have so far significantly limited their use (Fermin et al. 2004). For DNA viruses (particularly, Gemini viruses), transgenic resistance involves both RNA-mediated resistance and mutated viral proteins exerting negative effects on viral replication (Vanderschuren et al. 2007).

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Conclusions

While insect-resistant and herbicide-tolerant transgenic plants are the most widely grown GM crops, very few strategies using genetically engineered plants for disease resistance have had a similar impact (Collinge et al. 2008). Given the effort put into biotechnological strategies for disease resistance over the last two decades, why have so few crops expressing these traits become commercialized crops? The development of molecular markers, which are particularly useful in screening for disease resistance and facilitate conventional breeding, provide a higher commercial incentive for seed companies because of strong public opposition to GM crops. Often, transgenic disease resistant plants have only partial resistance, or resistance to rapid breakdown (single genes), again giving a low economic incentive to developers. At present, clear field results show that transgenic virus resistance is effective; however, there are no signs that commercial bacterial- or fungal-resistant crops will be introduced onto the market at any point soon.

1.5

Transgenic Crops for Resistance to Biotic Stress: Conclusions

Approximately 10.3 million farmers in 22 countries grew transgenic (genetically modified) crops in 2006. Yet this technology remains one of the most controversial agricultural issues of current times. Many consumer and environmental lobby groups believe that GM crops will bring very little benefit to growers and to the general public, and that they will have a deleterious effect on the environment. The human population is currently 6.1 billion and it is predicted to increase to 9–10 billion in the next 50 years (Fig. 1.7). This is at a time when food and fuel are competing for land (Fig. 1.8) and climate change threatens to compromise current resources. Population growth, changing diets, higher transport costs and a drive towards biofuels are forcing food prices up (Fig. 1.9). The UN’s Food and Agriculture Organization (FAO) stated that the food crisis had thrown an additional 75 million people into hunger and poverty in 2007 alone. World Population Growth (billion people) 1950

1975

2000

2025

2050

2.5bn

4.1bn

6.1bn

8.0bn

9.2bn

Fig. 1.7 World population growth

1 Transgenic Crop Plants for Resistance to Biotic Stress Fig. 1.8 Demand for biofuels

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Demand for Biofuels Rise in use of course grains 2005-7 (FAO/OECD).

Total: 80m tonnes

Biofuel use: 47m tonnes

Price Rises in a Single Year (Mar 2007-Mar 2008) Source: Bloomberg, FAO/ Jackson Son & Co 130% 87% 74% 31%

Corn

Rice

Soya

Wheat

Fig. 1.9 Price rises of major food crops (2007–2008)

It is, and will continue to be, a priority for agriculture to produce more crops on less land. The minimization of losses to biotic stress caused by agricultural pests would go some way to optimizing the yield on land currently under cultivation. Traditionally, agricultural production has kept pace, even outstripped human population growth; however, we currently face a set of unique challenges. One of the greatest dangers to agriculture is its vulnerability to global climate change. The expected impacts are for more frequent and severe drought and flooding, and shorter growing seasons. The performance of crops under stress will depend on their inherent genetic capacity and on the whole agroecosystem in which they are

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managed. It is for this reason that any efforts to increase the resilience of agriculture to climate change must involve the adoption of stress-resistant plants as well as more prudent management of crops, animals and the natural resources that sustain their production. Currently, we may be at the limit of the existing genetic resources available in our major crops (Gressel 2008). Thus, new genetic resources must be found and only new technologies will enable this.

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Chapter 2

Transgenic Plants for Abiotic Stress Resistance Margaret C. Jewell, Bradley C. Campbell, and Ian D. Godwin

2.1

Introduction

Modern agricultural crop production relies on the growth of a few of the world’s plant species selected for their superior qualities and suitability as food, animal feed, fiber or industrial end uses. Centuries of selection and, more recently, scientific breeding for adaptation to biotic and abiotic stresses have been necessary to improve yield, yield stability, and product quality in agricultural species. Nevertheless, abiotic stresses remain the greatest constraint to crop production. Worldwide, it has been estimated that approximately 70% of yield reduction is the direct result of abiotic stresses (Acquaah 2007). The ever increasing pressure put on agricultural land by burgeoning human populations has resulted in land degradation, a cultivation shift to more marginal areas and soil types, and heavier requirements for agricultural productivity per unit area. Additionally, climate change has exacerbated the frequency and severity of many abiotic stresses, particularly drought and high temperatures, with significant yield reductions reported in major cereal species such as wheat, maize, and barley (Lobell and Field 2007). In many parts of the world, rainfall has become less predictable, more intense, and, due to increasing temperatures, subject to higher evapotranspiration. For example, in the major grain growing areas of eastern Africa, the predominant rainy season is starting later and ending earlier (Segele and Lamb 2005) with longer dry spells in between (Seleshi and Camberlin 2006). Agricultural practices to improve crop productivity per unit area have, in many cases, accelerated the rate of land degradation, with particularly marked effects in irrigated areas. Irrigation has led to salinity across large tracts of agricultural land,

I. D. Godwin (*) School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, Qld 4072, Australia e-mail: [email protected]

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with cases, such as in India, where it has reportedly led to the loss of seven million hectares from cultivation (Martinez-Beltran and Manzur 2005). In Australia, almost eight million hectares of land are under threat of dryland salinity (Munns et al. 2002). Higher yields are also only sustainable with higher nutrient use, and the heavy demand for fertilizers has caused rising costs for farmers worldwide. The environmental and economic consequences of increased nutrient use have been widely reported. For sustainability of crop production, there is a need to reduce the environmental footprint of food and fiber production, and nutrient use efficient crops are highly sought after. Transgenic approaches are one of the many tools available for modern plant improvement programs. Gene discovery and functional genomics projects have revealed multitudinous mechanisms and gene families, which confer improved productivity and adaptation to abiotic stresses. These gene families can be manipulated into novel combinations, expressed ectopically, or transferred to species in which they do not naturally occur or vary. Hence, the ability to transform the major crop species with genes from any biological source (plant, animal, microbial) is an extremely powerful tool for molecular plant breeding. Transgenic plants can be used as sources of new cultivars (or their germ plasm as new sources of variation in breeding programs) and they are also extremely useful as proof-of-concept tools to dissect and characterize the activity and interplay of gene networks for abiotic stress resistance. In this chapter, we will outline the major yield-limiting abiotic stresses faced by crop plants: drought, salinity, cold, nutrient deficiency, and metal toxicity. Within each section, we will then cite specific examples of transgenic crop approaches to overcome these stresses and also discuss a number of conserved plant stress response mechanisms, which have been demonstrated to confer better adaptation to a number of different abiotic stresses.

2.2

Water Scarcity and Agriculture

Drought is the most significant environmental stress on world agricultural production (Tuberosa and Salvi 2006; Cattivelli et al. 2008) and enormous effort is being made by plant scientists to improve crop yields in the face of decreasing water availability. During the twentieth century, the world’s population tripled from approximately 1.65 to 5.98 billion and population projections of 8.91 and 9.75 billion are expected to occur by 2050 and 2150, respectively. Developing countries in Africa and Asia account for approximately 80% of this growth and, with an estimated 800 million people in these countries already undernourished, the Food and Agriculture Organization (FAO) of the United Nations predicts that a 60% increase in world food production over the next two decades is required in order to sustain these populations. Agriculture accounts for approximately 70% of global water use and irrigation accounts for up to 90% of total water withdrawals in arid nations (World Water

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Council 2008; FAO 2009a). Approximately, 40% of all crops produced in developing countries are grown on irrigated arable land, which accounts for only 20% of the total arable land in these nations (FAO 2009c). The water withdrawal requirement for irrigation is expected to increase by 14% in developing countries by 2030 and strategies to decrease this demand by developing crops that require less irrigation will, therefore, play a vital role in maintaining world food supply. The area of plant drought tolerance research and improvement encompasses an enormous range of environmental, genetic, metabolic, and physiological considerations and an exhaustive discussion of all available avenues for developing droughtresistant crop varieties is beyond the scope of this chapter. Rather, this section attempts to provide an overview of some of the genetic mechanisms that have been manipulated in order to develop transgenic crops with improved drought tolerance and focuses on research that has involved long-term and field-based drought stress treatments performed on agronomic and horticultural crop species at yield determining plant life stages.

2.2.1

Improving Drought Tolerance in Agricultural Crops

All plants require water to complete their life cycle, with most plant cells consisting of at least 70% water on a fresh weight basis. When insufficient water is available, plant water status is disrupted, which causes imbalances in osmotic and ionic homeostasis, loss of cell turgidity, and damage to functional and structural cellular proteins and membranes. Consequently, water-stressed plants wilt, lose photosynthetic capacity, and are unable to sequester assimilates into the appropriate plant organs. Severe drought conditions result in reduced yield and plant death. The overall aim of genetically improving crops for drought resistance is to develop plants able to obtain water and use it to produce sufficient yields for human needs under drought conditions. While advances have been made in developing crops that are genetically improved with traits such as herbicide and pesticide resistance, attempts to improve plant drought resistance have been hindered by the complexity of plant drought resistance mechanisms at the whole plant, cellular, metabolic, and genetic levels. Interactions between these mechanisms and the complex nature of drought itself, adds another layer of intricacy to this problem.

2.2.2

Complexity of Drought and Plant Responses to Drought Stress

Drought (nonavailability of water for crop growth) and water deficit (insufficient plant water status) are variable, complex, and recurring features in most parts of the

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world. Even in areas with very high precipitation, many crops are likely to experience a certain level of water deficit at some stage during the growing cycle. Elucidation of plant drought resistance and response mechanisms has been compounded by the variable levels and forms of drought. Drought can be spatially and temporally variable; terminal, short-term, or sporadic; severe, moderate, or minor; and can occur at rates ranging from very sudden to gradual. In addition, the effects of drought and water deficit on crop productivity vary for different crops, macro- and microenvironments across a single field, plant life stages, and the plant material to be harvested. Furthermore, the effects of drought on crop productivity are often compounded by associated stresses such as heat, salt, and nutrient stress.

2.2.3

Plant Drought Resistance and Response

Plant drought resistance mechanisms can be broadly grouped into avoidance or tolerance mechanisms. Drought avoidance mechanisms are associated with physiological whole-plant mechanisms such as canopy resistance and leaf area reduction (which decrease radiation adsorption and transpiration), stomatal closure and cuticular wax formation (which reduce water loss), and adjustments to sink-source allocations through altering root depth and density, root hair development, and root hydraulic conductance (Beard and Sifers 1997; Rivero et al. 2007). Drought tolerance mechanisms are generally those that occur at the cellular and metabolic level. These mechanisms are primarily involved in turgor maintenance, protoplasmic resistance, and dormancy (Beard and Sifers 1997). Plants respond to water-limiting conditions by altering the expression of a complex array of genes (Fig. 2.1) and, although elucidation of biochemical pathways associated with many of these genes has been the focus of an enormous amount of research over the last two to three decades, the mechanisms by which these genes and their products interact remains relatively poorly understood. Abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes that adversely affect plant growth and productivity (Wang et al. 2001). Drought, salinity, extreme temperatures, and oxidative stress are often interconnected, and may induce similar cellular damage. For example, drought and/or salinization are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell (Serrano et al. 1999; Zhu 2001). Oxidative stress, which frequently accompanies high temperature, salinity, or drought stress, may cause denaturation of functional and structural proteins (Smirnoff 1998). As a consequence, these diverse environmental stresses often activate similar cell signaling pathways (Knight 2000; Shinozaki and Yamaguchi-Shinozaki 2000; Zhu 2001, 2002) and cellular responses, such as the production of stress proteins, upregulation of antioxidants and accumulation of compatible solutes (Vierling and Kimpel 1992; Zhu et al. 1997; Cushman and Bohnert 2000; Wang et al. 2003b).

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Signal sensing, perception, transduction

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Fig. 2.1 Plant responses to abiotic stress

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The Genetic Basis of Drought Tolerance

Expression studies have shown that drought-specific genes can be grouped into three major categories: (1) Genes involved in signal transduction pathways (STPs) and transcriptional control; (2) Genes with membrane and protein protection functions; and (3) Genes assisting with water and ion uptake and transport (Vierling 1991; Ingram and Bartels 1996; Smirnoff 1998; Shinozaki and Yamaguchi-Shinozaki 2000). Plants adapt to drought conditions by tightly regulating specific sets of these genes in response to drought stress signals, which vary depending on factors such as the severity of drought conditions, other environmental factors, and the plant species (Wang et al. 2003b). To date, successes in genetic improvement of drought resistance have involved manipulation of a single or a few genes involved in signaling/regulatory pathways or that encode enzymes involved in these pathways (such as osmolytes/compatible solutes, antioxidants, molecular chaperones/osmoprotectants, and water and ion transporters; Wang et al. 2003b). The disadvantage of this is that there are numerous interacting genes involved, and efforts to improve crop drought tolerance through manipulation of one or a few of them is often associated with other, often undesirable, pleiotropic and phenotypic alterations (Wang et al. 2003b). These complex considerations, when coupled with the complexity of drought and the plant–environment interactions occurring at all levels of plant response to water deficit, illustrate that the task plant researchers are faced with in engineering drought resistant crops is dauntingly multi-faceted and extremely difficult.

2.2.5

Engineering Improved Drought Avoidance in Crops

Most transformation studies to improve plant drought resistance have produced transformants that display a variety of both tolerance and avoidance traits. An exception was demonstrated by Rivero et al. (2007) who manipulated a leaf senescence gene. Leaf senescence is an avoidance strategy and is accelerated in drought-sensitive plants to decrease canopy size. In crop plants, accelerated senescence is often associated with reduced yield and is thought to be the result of an inappropriately activated cell death program. Therefore, suppression of droughtinduced leaf senescence in tobacco plants was investigated as a tool to enhance drought resistance. Transgenic plants were developed by expressing isopentyl transferase (IPT), a key enzyme in the biosynthesis of cytokinin (a leaf senescence inhibitor) under the control of the senescence-associated receptor protein kinase promoter (PSARK). The SARK gene, which is induced during late maturation and drought and decreased during senescence development, encodes a maturation/sensescence-dependent protein kinase. Although transgenic plants wilted under a 15-day glasshouse drought stress treatment, senescence did not occur and a reduced

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photosynthetic capacity was maintained. Following rewatering, transgenic plants recovered full leaf turgor and resumed growth and maximum photosynthetic capacity, while the control plants were unable to recover from the drought stress. Water use efficiency (WUE) of the transgenic plants was also markedly higher than wild-type (WT) plants, and was two to three times higher after rewatering than before the drought treatment. An experiment to assess whether the transgenic plants could produce significant yields under water-limiting conditions determined that biomass and seed yield were not as affected in transgenic plants than in WT controls, although this result was not significant (Rivero et al. 2007). Other drought avoidance/whole-plant traits that have been investigated include stay-green and cuticular biosynthesis. Stay-green is a variable and quantitative trait, which generally refers to delayed senescence. It has not yet been used to successfully produce transgenic plants with increased drought resistance in the field. Cuticular biosynthesis was investigated by transgenic expression of AtMYB41, which encodes an R2R3-MYB transcription factor (TF) in Arabidopsis. AtMYB41 is expressed at high levels in response to drought, abscisic acid (ABA; Sect. 2.2.6.1), and salt treatments, and was demonstrated to have a role in cell expansion and cuticle deposition. The transformation of Arabidopsis with AtMYB41 was associated with undesirable pleiotropic phenotypes including dwarfism, enhanced sensitivity to desiccation, and enhanced permeability of leaf surfaces (Cominelli et al. 2008).

2.2.6

Improving Plant Drought Tolerance

2.2.6.1

Absicisic Acid and Transcriptional Regulation

The plant hormone ABA regulates the plant’s adaptive response to environmental stresses such as drought, salinity, and chilling via diverse physiological and developmental processes. ABA has functional roles ranging from seed maturation processes to lateral root development (McCourt and Creelman 2008; Wasilewska et al. 2008). Under abiotic stress, ABA induces stomatal closure, reduces water loss via transpiration, and induces gene expression (Chandler and Robertson 1994). Gene expression and biochemical studies into ABA synthesis in Arabidopsis and some other model plants have largely elucidated the basic ABA biosynthetic pathway (Schwartz et al. 2003) and many of the key enzymes involved in ABA synthesis have been investigated transgenically in relation to improving plant drought tolerance. For example, transgenic Arabidopsis plants constitutively overexpressing the zeaxanthin epoxidase gene, AtZEP, which encodes an enzyme required for an initial step in ABA synthesis from isopentyl diphosphate (IPP) and b-carotene (Schwartz et al. 2003) showed increased tolerance to drought and salinity stress. The increased drought stress tolerance was attributed to increased leaf and lateral root development, longer primary roots, higher fresh weight, and increased survival compared with control plants following drought treatment.

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Furthermore, compared with WT plants, the rate of water loss was lower, the levels of ABA were higher, the expression of stress responsive genes such as Rd29a was much higher, and stomatal aperture was smaller under salt and/or drought stress. Overall, the increased stress resistance was attributed to increased ABA levels in response to osmotic stress, which resulted in enhanced expression of ABAresponsive stress-related genes (Park et al. 2008). Many of the drought stress response pathways that have been identified to date appear to be under transcriptional regulation and ABA plays a key role in this process (Fig. 2.2). Transcriptional regulation involves interaction between TFs and specific cis-acting elements located within or near the promoter region upstream of expressed genes. Figure 2.2 shows links between responses to low temperature and dehydration stress at the transcriptional level. It can be seen that ABA is involved in both types of abiotic stress. Many transcriptional responses to drought stress have been well characterized and are classified as being ABA-dependent, ABA-independent, or both. ABA is Low Temperature

Dehydration

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CBF1, 2, 3 / DREB1a, b, c DREB2

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bZIP

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DREB2 AREB/ABF

CRT/DRE (Rd29a, Cor15a)

ABRE (Rd29a, Rd29b)

MYCR/MYBR (Rd22, AtADH1)

Fig. 2.2 Plant transcriptional processes induced by dehydration and low temperature stress. Displayed are transcription factors (rounded rectangles) both ABA-dependent (shaded) and ABA-independent (unshaded), posttranscriptional modification such as phosphorylation (elipses), transcription factor binding sites and representative promotors (rectangles), possible regulation points (dotted arrows), and possible cross-talk (bidirectional arrows)

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often used in stress and stress acclimation studies because it is produced in response to stress. ABA induces expression of many genes that are also induced by drought, cold, and salinity when applied exogenously (Shinozaki and Yamaguchi-Shinozaki 1996). There are two types of ABA-dependent transcription. The “direct” pathway involves cis-acting ABA-responsive elements (ABREs), which are directly activated by binding with TFs such as basic-domain leucine zipper (bZIP)-type DNAbinding proteins (Shinozaki and Yamaguchi-Shinozaki 1996; Kobayashi et al. 2008). Alternatively, the “indirect” ABA-dependent transcription pathway involves other cis-acting elements, such as MYC and MYB. These elements are activated through binding with ABA- or drought-inducible TFs, such as basic helix–loop– helix (bHLH)-related protein AtMYC2 and an MYB-related protein, AtMYB2 (Abe et al. 2003). An example of the indirect pathway can be seen in the expression of rd22 from Arabidopsis (Shinozaki and Yamaguchi-Shinozaki 1996). Some genes are induced by drought stress but are not expressed in response to exogenous ABA applications and these genes are the product of ABA-independent STPs. One such gene is rd29a (also known as lti78 and cor78). YamaguchiShinozaki and Shinozaki (Yamaguchi-Shinozaki and Shinozaki 1994) identified a dehydration-responsive element (DRE) in the promoter region of rd29a and the DRE-binding (DREB) protein transcription pathway has since been explored for its important roles in drought, cold, and salinity stress (Shinozaki and YamaguchiShinozaki 1996; Qin et al. 2007). Several C-repeat (CRT) binding factor (CBF)/ DREB proteins have now been identified from the promoter regions of other stressinducible Arabidopsis genes, such as cor15a, kin1, cor6.6 and cor47/rd17, and the CBF/DREB pathway has been shown to be conserved across species (Benedict et al. 2006; Pasquali et al. 2008). CBF/DREB1 and DREB2, belong to the ethyleneresponsive element/apetela 2 (ERE/AP2) TF family; their expression is induced by cold or drought stress and both activate expression of genes possessing a CRT/DRE cis-element (Stockinger et al. 1997; Liu et al. 1998). Likewise, DREB2A positively regulates expression of many abiotic stress-related genes possessing DRE sequences in their 5’-upstream regions. DREB2A overexpression in Arabidopsis confers significant drought tolerance in transgenic plants (Sakuma et al. 2006a, b). DREB genes have been used in transformation of several crops, including wheat and rice, in attempts to increase drought tolerance (Chen et al. 2008; Kobayashi et al. 2008). Although DREs are cis-acting elements that were first thought to activate ABA-independent stress-responsive gene expression, some are also implicated in ABA-dependent expression (Shinozaki and Yamaguchi-Shinozaki 2000). CBF4 is an apparent homolog of the CBF/DREB1 proteins that is thought to be a critical regulator of gene expression in drought stress signal transduction. The action of CBF4 is thought to be through its binding with CRT/DRE elements in promoter regions of drought- and cold-inducible genes (Haake et al. 2002). CBF4 gene expression has been shown to be upregulated in response to drought and ABA; however, constitutive expression of CBF4 was found to result in expression of cold- and drought-induced genes under nonstress conditions and this was associated with retarded growth, shorter petioles, darker green leaves, and delayed time to

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flowering in Arabidopsis seedlings (Haake et al. 2002). Another study showed that CBF4 expression was induced by salt, but not by drought, cold, or ABA (Sakuma et al. 2002). Similar observations, and observations of higher levels of soluble sugars and proline, have been recorded during many CBF overexpression studies, which suggest that the use of constitutively expressed CBF/DREB genes may not be applicable to the development of crops with improved drought tolerance. It is thought that the use of stress-inducible promoters that have low expression levels under non-stress conditions could be used in conjunction with CBF genes to alleviate the retarded growth observed in CBF overexpression studies (Zhang et al. 2004). Many studies have illustrated the potential of manipulating CBF/DREB genes to confer improved drought tolerance. For example, overexpression of CBF1/ DREB1B from Arabidopsis was able to improve tolerance to water-deficit stress in tomato. Furthermore, when driven by three copies of an ABA-responsive complex (ABRC1) from the barley HAV22 gene, transgenic tomato plants expressing CBF1 exhibited enhanced tolerance to chilling, water deficit, and salt stress, and maintained normal growth and yield under normal growing conditions when compared with control plants (Lee et al. 2003a). Other studies have also found that expression of CBF/DREB genes under stress-inducible promoters result in transgenic plants that do not express detectable levels of these genes under non-stress conditions, minimizing growth retardation and other adverse effects (Al-Abed et al. 2007). The CRT/DRE motif also acts as one of the binding sites for the ERF family of TFs (Trujillo et al. 2008). A novel ERF from sugarcane, SodERF3, was found to enhance salt and drought tolerance when overexpressed in tobacco plants. Under drought treatment, transgenic plants were significantly taller than controls and were able to flower under an extended growth period. Furthermore, the absence of observable differences in height, number of leaves, leaf area, leaf weight, and stalk weight between transgenic and control plants illustrates that this gene has potential for engineering drought stress tolerance in plants (Trujillo et al. 2008). Other TFs involved in mediation of ABA-dependent and ABA-independent signal transduction and gene expression include NAC, WRKY, RING finger, and zinc-finger TFs (Seki et al. 2003; Zhang et al. 2004; Chen et al. 2006). Nelson et al. (2007) showed that constitutive expression of a TF from the nuclear factor (NF-Y) family, AtNF-YB1, which belongs to the CCAAT-binding TF family, improved performance of Arabidopsis under drought conditions. Consequently, an orthologous maize TF gene, ZmNF-YB2, was constitutively expressed in maize. Transgenic lines were exposed to both glasshouse-based and field-based drought stress treatments. Transgenic lines exhibited less wilting and faster recovery and re-established growth more rapidly than WT (on average) under glasshouse-based drought treatment. Transgenic lines subjected to field-based drought stress at the late vegetative stage exhibited superior health, higher chlorophyll indices and photosynthetic rates, lower leaf temperatures, higher stomatal conductance, and less yield reduction than WT plants. Furthermore, under favorable conditions, transgenic plants were greener, flowered 1–3 days earlier, and had slightly compressed internodes. Most importantly, the stress adaptation response contributed to a yield advantage in

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transgenic maize grown within drought environments, suggesting that ZmNF-YB2 has a realistic application for use in commercial agriculture under severe waterlimiting conditions (Nelson et al. 2007). Another TF that has been manipulated in order to increase plant drought tolerance is the HARDY (HRD) gene, which has been linked to increased transpiration efficiency related to stomatal adjustment. HRD is an AP2/ERF-like TF isolated from hrd-dominant (hrd-D) Arabidopsis mutants, which displayed vigorous rooting and dark green leaves that were smaller and thicker than WT plants. Karaba et al. (2007) isolated the HRD gene and constitutively expressed it in Arabidopsis under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The thicker leaves, higher root density, and increased root strength were associated with abundant chloroplasts, increased secondary and tertiary root initiation and proliferation, and extra corticle cell layers and more compact, stele bearing vascular tissue, respectively. Furthermore, the mutants survived longer periods of drought stress and could reach full maturity under high levels of salt stress. HRD was also constitutively expressed in rice and transgenic plants displayed no reduction in growth, seed yield, or germination, but had significantly increased leaf canopy with more tillers under normal greenhouse conditions compared with WT controls. Under drought stress, the transgenic plants were of deeper green color (attributable to increased number of bundle sheath cells), displayed distinctive drought tolerance and lower stomatal conductance, had higher net carbon assimilation and photosynthetic rates, and possessed higher root biomass (Karaba et al. 2007). Recently, a novel drought-tolerant gene, HDG11, which encodes a protein from the homeodomain (HD)-START TF family (also known as the Class IV HD-leucine zipper TF family) was identified in Arabidopsis and was found to confer drought resistance via enhanced root growth and decreased stomatal density when constitutively overexpressed in transgenic tobacco (Yu et al. 2008). The constitutive expression of the gene was not associated with retarded growth or any other observable deleterious phenotypic effects and, the gene was also shown to transactivate a number of other genes involved in the drought stress response including ERECTA (Sect. 2.2.6.2.1; Yu et al. 2008). SNAC1 from rice has also been shown to have trans-activation activity. NAC TFs comprise a large gene family with proteins exhibiting a highly conserved N-terminal DNA-binding domain and a diversified C-terminal domain. NAC was derived from the names of the first three described proteins containing the DNA-binding domain, namely, NAM (no apical meristem), ATAF1-2, and CUC2 (cup-shaped cotyledon; Souer et al. 1996; Aida et al. 1997). NAC is a plant-specific TF family with diverse roles in development and stress regulation. When constitutively overexpressed in rice, SNAC1 was found to significantly improve plant resistance to severe drought stress during reproductive and vegetative growth and was not associated with any negative phenotypic effects or yield penalty (Hu et al. 2006). Transgenic plants were more sensitive to ABA and closed more stomata than WT plants but maintained continual photosynthetic activity. There was no difference between root morphologies of transgenic and WT plants indicating that the improved drought resistance was not related to increased root-water uptake.

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The WRKY superfamily of plant TFs has a conserved sequence (WRKYGQK) at their N-terminal ends (Wu et al. 2008b). Transgenic rice seedlings, expressing OsWRKY11 under the control of a rice heat shock protein (HSP) promoter, HSP101, were shown to survive longer and lose less water under a short, severe drought treatment, than WT plants (Wu et al. 2008b). A TFIIIA-type zinc-finger protein gene, ZFP252, was also found to confer improved drought stress resistance in rice. Young transgenic rice plants overexpressing ZFP252 survived longer, displayed less relative electrolyte leakage, and accumulated more compatible osmolytes than WT plants or plants with ZFP252 knocked out during a 14-day period of drought stress (Xu et al. 2008a). A salt- and drought-induced RING-finger protein, SDIR1, was found to confer enhanced drought tolerance to tobacco and rice (Zhang et al. 2008b). Arabidopsis E3 ligase SDIR1 is a positive regulator in ABA signal transduction. Tobacco and rice plants constitutively overexpressing the SDIR1 gene displayed less leaf wilting and rolling, longer survival, and improved recovery under drought conditions than control plants. The mechanism of drought tolerance was thought to be due to decreased stomatal aperture, which increased transpiration efficiency of transgenic plants. Some genes have been shown to suppress expression of drought-response transcription pathways. For example, Jiang et al. (2008) recently characterized SAZ, an Arabidopsis gene from the SUPERMAN (SUP) family of plant-specific zinc-finger genes, which encode proteins containing single C2H2-type zinc-finger motif with a conserved short amino acid sequence and a class II ERF-associated amphiphilic repression (EAR) motif-like TF domain at the carboxy-terminal region. SAZ was found to be rapidly downregulated in response to drought and other abiotic stresses and SAZ gene knockouts resulted in elevated expression of the ABA-responsive genes rd29B and rab18 under stressed and unstressed conditions. This shows that gene knockouts and gene silencing may also be applicable to the development of crops with improved drought resistance.

2.2.6.2

Signal Sensing, Perception, and Transduction

Prior to transcriptional activation of genes, drought stress signals are received and messages conveyed to the appropriate components of the downstream pathway (Xiong and Ishitani 2006). In general, STPs involve perception of stress by specific receptor molecules, which vary in identity, structure, perception, signal relay mechanism, and location within the cell (Xiong and Ishitani 2006). Plant stress STPs often involve secondary messengers, which may modify signals (often via reversible protein phosphorylation) prior to conveying them from receptor molecules to the activators of the appropriate gene expression pathway (Xiong and Ishitani 2006). Other molecules may also be involved in stress STPs and the functions of these include recruitment and assembly of signaling complexes, targeting of signaling molecules, and regulation of signaling molecule lifespan (Xiong and Ishitani 2006).

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The major molecules involved in drought stress signal sensing, perception, and transduction include receptor molecules/osmosensors, phospholipid-cleaving enzymes (PLEs), reactive oxygen species (ROS), mitogen-activated protein kinases (MAPK), and Ca2+ sensors.

Receptor Molecules/Osmosensors Receptor molecules/osmosensors are the initial stress signal perceivers and they convey the signal to the appropriate molecule to initiate STPs. On the basis of analyses of plants and other species, receptor molecules are thought to include receptor-like kinases, two-component receptors, receptor tyrosine kinases, G-protein-coupled receptors, iontropic channel-related receptors, histidine kinases, and nuclear hormone receptors. Receptor molecules that have been identified to date in plants include: ROP10, a small G protein from the ROP family of Rho GTPases, that negatively regulates ABA response in Arabidopsis (Zheng et al. 2002); ATHK1, a putative homolog of the yeast SLN1, which is a functional histidine kinase feeding into the HOG MAPK pathway (Urao et al. 1999; Reiser et al. 2003); NtC7, a receptor-like membrane protein from tobacco (Tamura et al. 2003); and Cre1, a putative cytokinin sensor and histidine kinase from Arabidopsis (Reiser et al. 2003). The ERECTA gene from Arabidopsis is a putative leucine-rich repeat receptorlike kinase (LRR/RLK). It was the first gene to be shown to act on the coordination between transpiration and photosynthesis (Masle et al. 2005). ERECTA was analyzed by its transgenic expression in null-mutants and was shown to have roles in lowering stomatal conductance, controlling leaf photosynthesis and organogenesis, modulation of cell expansion, cell division, cell–cell contact, cell–cell and tissue–tissue signaling, cell proliferation, and inflorescence differentiation. Owing to the range of traits attributed to ERECTA expression, ERECTA is thought to act as a master gene in transpiration regulation (Masle et al. 2005). No known studies have yet involved transgenic expression of ERECTA in economic crops; however, initial studies suggest that this gene may be useful in the design of crops with improved transpiration efficiencies, reduced stomatal limitations, and increased yield potentials.

Phospholipid-Cleaving Enzymes PLEs degrade phospholipid membranes, catalyzing the release of lipid and lipidderived secondary messengers (Chapman 1998; Sang et al. 2001). Phospholipases C (PLC) and D (PLD) are both involved in ABA-mediated signal transduction and drought stress tolerance perception in plants. Phosphatidic acid (PtdOH), a product of the PLC and PLD pathways, is also important in the signaling process (Bartels et al. 2007; Wang et al. 2008a).

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Wang et al. (2008a) successfully produced maize plants constitutively overexpressing ZmPLC1, a phospholipase catalyzing the hydrolysis of 4,5-bisphosphate to form diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, the products of which are the second messengers Ca2+ and PtdOH, respectively. This pathway is important in a wide variety of abiotic stress-responsive processes. Transgenic maize plants carrying the ZmPLC1 gene were shown to have increased photosynthetic activity, reduced anthesis to silking interval (ASI; an indicator of maize yield potential), better recovery, and higher grain yield than WT plants when subjected to 21 days of drought stress at the ten-leaf stage. Because there was no significant difference between stomatal conductances of WT and transgenic plants, the higher photosynthetic rate was attributed to better photochemical activity rather than the improved guard cell signaling. This has also been demonstrated in other studies (Staxen et al. 1999; Hunt et al. 2003; Mills et al. 2004). Sang et al. (2001) showed that overexpression of PLD results in enhanced sensitivity of transgenic tobacco and plays a key role in controlling stomatal movements and plant response to water stress.

Reactive Oxygen Species ROS are generated in plants as photoreaction and cellular oxidation byproducts under normal conditions and can cause cellular damage under water deficit when they accumulate to toxic levels. Some of these species also have important roles in early stress response through activation of cellular defense mechanisms and mitigation of cellular damage. While plant mechanisms must be in place to detoxify high levels of ROS that occur under drought, low levels of these beneficial ROS must also be maintained. Those ROS known to have important signaling roles in plant stress STPs include nitric oxide (NO) and hydrogen peroxide (H2O2).

Mitogen-Activated Protein Kinases MAPKs are enzymes that catalyze reversible phosphorylations, important for relaying signals. They function via cascades, which involve sequential phosphorylation of a kinase by its upstream kinase (Xiong and Ishitani 2006). Recently, the MKK2 pathway was identified in Arabidopsis as having involvement in cold and osmotic stress signal transduction. An example of a MAPK having specific involvement in drought and salt stress is the p44MMK4 kinase from alfalfa (Medicago sativa; Jonak et al. 1996). Phosphatases involved in the sequential phosphorylation of MAPKs and other protein kinases are also important for stress signaling. For example, the ABI1 and AB12 proteins from Arabidopsis have been shown to act in a negative regulatory feedback loop of the ABA signaling pathway (Merlot et al. 2001). Some MAPK and MAPKK proteins have also been shown to activate the Rd29a stress pathway in Arabidopsis (Hua et al. 2006). Other protein kinases involved in stress signaling include calcium-dependent protein kinases (CDPKs),

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kinases from the SNF1 family of protein kinases, and serine-threonine-type protein kinases (Xiong and Ishitani 2006; Bartels et al. 2007). Ca2+ Sensors Ca2+ sensors are important for coupling extracellular signaling to intercellular responses and comprise calmodulin (CaM) and CaM-related proteins (Sneddon and Fromm 1998; Sneddon and Fromm 2001), calcineurin B-like proteins (CBL; also known as SCaBP/SOS3-like calcium-binding proteins; Kudla et al. 1999), and CDPKs (Harmon et al. 2000). Ca2+ sensors that have been attributed with roles in drought tolerance in plants include the CBL1 gene (Kudla et al. 1999) and the AtCAMBP25 protein (Perruc et al. 2004) from Arabidopsis. 2.2.6.3

Stress-Responsive Mechanisms

The outcome of stress signal perception, transduction, and transcriptional up- or downregulation of genes is the production of molecules with various plant protection, repair, and stabilization functions. These molecules can be broadly grouped into five functional groups: (1) detoxification; (2) chaperoning; (3) late embryogenesis abundant (LEA) protein functions; (4) osmoprotection; and (5) water and ion movement. Detoxification To prevent stress injury, cellular ROS need to remain at nontoxic levels under drought stress. Antioxidants involved in plant strategies to degrade ROS include: (1) enzymes such as catalase, superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase; and (2) nonenzymes such as ascorbate, glutathione, carotenoids, and anthocyanins (Wang et al. 2003b). Some proteins, osmolytes, and amphiphilic molecules also have antioxidative functionality (Bowler et al. 1992; Noctor and Foyer 1998). Chaperoning Chaperone functions involve specific stress-associated proteins, which are responsible for protein synthesis, targeting, maturation and degradation, and function in protein and membrane stabilization, and protein renaturation. HSPs, which can be divided into five conserved families, have been shown to have particularly important stress-related chaperone functions in plants (Hendrick and Hartl 1993; Boston et al. 1996; Hartl 1996; Waters et al. 1996; Torok et al. 2001). HSPs, which are induced by heat, have been implicated in plant cell protection mechanisms under drought stress. Protein denaturation occurs under drought stress because decreased

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cellular volume increases the likelihood of degradative molecular interactions (Cho and Hong 2006). HSPs maintain or repair companion protein structure and target incorrectly aggregated and non-native proteins for degradation and removal from cells (Cho and Hong 2006). One such protein, NtHSP70-1, was constitutively overexpressed in tobacco to ascertain its role in plant drought response and tolerance (Cho and Hong 2006). The drought tolerance of transgenic seedlings was increased and their optimum water content was maintained after progressive drought stress (Cho and Hong 2006). Few other studies have involved transforming plants with HSPs; however, HSP24 from Trichederma harzianum was found to confer significantly higher resistance to salt, drought, and heat stress when constitutively expressed in Saccharomyces cerevisiae (Liming et al. 2008).

Late Embryogenesis Abundant Protein Functions LEA proteins are produced in response to dehydration stress and function in water status stabilization, protection of cytosolic structures, ion sequestration, protein renaturation, transport of nuclear targeted proteins, prevention of membrane leakage, and membrane and protein stabilization. LEA and LEA-type genes are found universally in plants. They accumulate in seeds during the late stages of embryogenesis and are associated with the acquisition of desiccation tolerance under drought, heat, cold, salt, and ABA stress (Sivamani et al. 2000; Bartels et al. 2007). They are also present in the biomass tissue of resurrection plants and are upregulated in many desiccation-sensitive plants in response to drought stress (Bartels et al. 2007). LEA proteins are divided into groups based on conserved sequence motifs (Zhang et al. 2000; Wise 2003). Five of these groups have been characterized at the molecular and structural level (Table 2.1); however, recent research indicates that additional groups of LEA and LEA-like proteins are still being identified (Park et al. 2003; Wang et al. 2006; March et al. 2007). Common Table 2.1 The five groups of LEA proteins LEA group Description Group 1 Contain a 20-amino acid motif and are represented by the wheat Em protein, for which gene homologs have been identified in a wide range of plant species Group 2 The most extensively studied group. They contain a lysine-rich 15-amino acid (dehydrins) motif (K-segment; EKKGIMDKIKEKLPG), which is predicted to form an amphipathic a-helix, a tract of contiguous serine residues and a conserved motif containing the consensus sequence DEYGNP in the N-terminal section of the protein Group 3 Contain a characteristic repeat motif of 11 amino acids, which have been predicted to form an amphipathic a-helix with possibilities for intra- and inter-molecular interactions Group 4 Have a conserved N-terminus, which is proposed to form a-helices and a diverse C-terminal region with a random coil structure Group 5 Contain more hydrophobic residues than the other groups, are insoluble after boiling, and are likely to adopt a globular structure Source: Bartels and Salamini (2001), Ramanjulu and Bartels (2002)

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features of LEA proteins generally include hydrophilicity (Garay-Arroyo et al. 2000; Park et al. 2003), heat stability (Close and Gallagher-Ludeman 1989; Ceccardi et al. 1994; Houde et al. 1995; Thomashow 1998, 1999), and transcriptionally regulated and ABA-responsive gene expression (Close and Gallagher-Ludeman 1989). It is generally assumed that they play a role in water-deficit tolerance and the possible functions of LEA proteins include binding and replacement of water (Dure 1993), ion sequestration (Bray 1993), maintenance of protein and membrane structure (Baker et al. 1988), molecular chaperones (Close 1996), membrane stabilization (Koag et al. 2003), and nuclear transport of specific molecules (Goday et al. 1994). One class of LEAs, the dehydrins, which have detergent and chaperone-like properties, stabilize membranes, proteins, and cellular compartments (Close 1996). LEA genes have been manipulated in many plants in order to increase drought resistance. For example, a wheat dehydrin, DHN-5, was ectopically overexpressed in Arabidopsis and transgenic plants displayed superior growth, seed germination rate, water retention, ion accumulation, more negative water potential, and higher proline contents than WT plants under salt and/or drought stress (Brini et al. 2007a). The barley (Hordeum vulgare L.) group 3 LEA gene, HVA1 was constitutively overexpressed in rice plants to increase drought tolerance. Transgenic plants displayed significantly increased tolerance to water deficit and salinity, which was associated with higher growth rates, delayed onset of stress damage symptoms, and improved recovery following stress removal (Xu et al. 1996). A more recent study involving the overexpression of this gene in rice showed that transgenic plants had significantly higher relative water content (RWC), improved turgor, less reduction in shoot and root growth, and improved cell membrane stability under prolonged drought conditions. It was found that HVA1 did not function as an osmolyte and that membrane protection was the mechanism, which inferred drought resistance in rice plants (Chandra Babu et al. 2004). HVA1 was also expressed in Basmati rice under control of either a constitutive rice promoter or a stress-inducible promoter. Transgenic plants exhibited increased stress tolerance in terms of cell integrity and growth, and it was found that inducible expression of HVA1 resulted in transgenic plants that were able to grow normally under nonstress conditions (Rohila et al. 2002). Transgenic wheat plants expressing HVA1 displayed more root fresh and dry weights, and shoot dry weight than WT plants under water-deficit conditions (Sivamani et al. 2000). Similarly, HVA1 overexpressing transgenic mulberry, Morus indica, exhibited improved cellular membrane stability, photosynthetic yield, less photo-oxidative damage, and superior WUE than WT plants under salt and drought stress (Lal et al. 2008). The discussion of expression of HVA1 in mulberry will be continued in Sect. 2.3.5. Other group 3 LEA genes that have been manipulated in order to improve plant drought tolerance include a Brassica napus group 3 LEA gene, which conferred improved salt and drought tolerance when constitutively expressed in Chinese cabbage (Park et al. 2005a), and TaLEA3 from wheat, which increased RWC, leaf water potential, and relative average growth rate of transgenic plants compared to WT plants under drought stress when constitutively overexpressed in the perennial grass Leymus chinensis (Wang et al. 2008b). Two group 4 LEA proteins,

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BhLEA1 and BhLEA2 from the resurrection plant Boea hygrometrica, conferred improved drought tolerance in transgenic tobacco. This was associated with plant cell protection and increased membrane and protein stability during dehydration (Liu et al.). A novel LEA gene from Tamarix androssowii also conferred increased drought tolerance when expressed in transgenic tobacco (Wang et al. 2006).

Osmoprotection Osmoprotection involves the upregulation of compatible solutes (osmolytes) that function primarily to maintain cell turgor, but are also involved in antioxidation and chaperoning through direct stabilization of membranes and/or proteins (Yancey et al. 1982; Bohnert and Jensen 1996; Lee et al. 1997; Hare et al. 1998; McNeil et al. 1999; Diamant et al. 2001). Compatible solutes are low molecular weight, highly soluble compounds that are usually nontoxic at high cellular concentrations. The three major groups of compatible solutes are amino acids (such as proline), quaternary amines (glycine betaine (GlyBet), polyamines, and dimethylsulfonioproprionate), and polyol/sugars (such as mannitol, galactinol, and trehalose; Wang et al. 2003b). Many genes involved in the synthesis of these osmoprotectants have been explored for their potential in engineering plant abiotic stress tolerance (Vinocur and Altman 2005). GlyBet and trehalose act as osmoprotectants by stabilizing quaternary structures of proteins and highly ordered states of membranes. Mannitol serves as a freeradical scavenger. Proline serves as a storage sink for carbon and nitrogen and a free-radical scavenger. It also stabilizes subcellular structures (membranes and proteins), and buffers cellular redox potential under stress. Many crops lack the ability to synthesize the special osmoprotectants that are naturally accumulated by stress tolerant organisms. It is believed that osmoregulation would be the best strategy for abiotic stress tolerance, especially if osmoregulatory genes could be triggered in response to drought, salinity, and high temperature. Therefore, a widely adopted strategy to develop stress-tolerant crops has been to engineer or overexpress certain osmolytes in plants (Bhatnagar-Mathur et al. 2008). GlyBet is a compatible solute that has been extensively studied for its role in drought stress response and increasing the levels of GlyBet in plants via genetic engineering has enhanced the drought tolerance of many model plants (Sakamoto and Alia 1998; Sakamoto and Murata 2000; Mohanty et al. 2002). A two-step enzymatic process accomplishes production of GlyBet in plants. The first step involves conversion of choline to betaine aldehyde by choline monoxygenase (CMO), a stromal enzyme with a Rieske-type (2Fe-2S) center (Brouquisse et al. 1989), and the second step involves betaine aldehyde dehydrogenase (BADH), a nuclear-encoded chloroplast stromal enzyme, which converts betaine aldehyde to GlyBet (Weigel et al. 1986). Quan et al. (2004) reported one of the first attempts to increase the GlyBet expression levels of maize by overexpressing the betA gene, which encodes choline dehydrogenase (CHO), another key enzyme in the choline–betaine aldehyde reaction (Zhang et al. 2008a). The study showed that

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transgenic maize plants were more drought tolerant than WT plants at three different life stages, including the ten-leaf-flowering stage, and also that yields of transgenic plants were less affected by drought stress than WT. Tobacco lacks GlyBet; however, it possess some BADH activity and the transfer of CMO is, therefore, a means of installing the GlyBet pathway in tobacco. Furthermore, because conversion of choline to GlyBet occurs in the chloroplast, it is also possible to use chloroplast genetic engineering to transfer CMO into GlyBet non-accumulators (Zhang et al. 2008a). Zhang et al. (2008a) transformed tobacco with a gene for CMO from beetroot via chloroplast genetic engineering and found that the transgenic plants accumulated GlyBet in leaves, roots, and seeds, and exhibited improved tolerance to toxic choline levels and salt and drought stress. GlyBet accumulation in the chloroplasts may be more effective than in other organelles, such as the nucleus, for abiotic stress protection because of protection and stabilization of chloroplast proteins, membrane, and photosynthesis systems under stress conditions (Zhang et al. 2008a). Lv et al. (2007) found that transgenic cotton plants constitutively overexpressing betA had increased RWCs, increased photosynthesis, better osmotic adjustment, decreased percentage of ion leakage, decreased lipid membrane peroxidation, and increased yield in response to drought stress at the seedling, squaring, and anthesis stages.

Water and Ion Movement Water and ions move through plants via transcellular and intracellular pathways. Aquaporins (major intrinsic proteins; MIPs), which are either tonoplast- (TIP) or plasma membrane- (PIP) localized, facilitate water, glycerol, small molecule, and gas transfer through membranes and, therefore, have a role in water homeostasis (Bartels et al. 2007). Active transport of solutes into the cell and cellular organelles, particularly the vacuole, is another means of cell turgor maintenance as increased solute potential facilitates the passive movement of water into cells and cellular compartments (Li et al. 2008). Successful attempts made in engineering plants expressing genes for enzymes involved in proton pumps that generate energy for tonoplast transport of solutes into vacuoles include the overexpression of the Arabidopsis H+-pyrophosphatase (H+-PPase; AVP1) in Arabidopsis (Gaxiola et al. 2001), upregulation of AVP1 in tomato (Park et al. 2005c), heterologous expression of the Thellungiella halophila vacuolar-H+-PPase (V-H+-PPase; TsVP) in tobacco (Gao et al. 2006), and overexpression of the wheat Na–H+ antiporter, TNHX1, and H+-PPase, TVP1, in Arabidopsis (Brini et al. 2007b; Li et al. 2008). In all the cases, the transgenic plants displayed superior drought and/or salinity resistance compared with WT plants with resistance being attributed to mechanisms such as increased vacuolar H+ to drive secondary uptake of ions into the vacuole and more enhanced development and robustness of root systems (Gaxiola et al. 2001; Li et al. 2005a; Park et al. 2005c; Gao et al. 2006; Brini et al. 2007a, b). Recently, Li et al. (2008) reported that

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heterologous expression of the potassium-dependent TsVP gene from the halophyte T. halophyta in maize under the control of the maize ubiquitin promoter could infer drought tolerance. Under drought stress, transgenic plants had a higher percentage of seed germination, better-developed root systems, more biomass, increased solute accumulation, less cell membrane damage, less growth retardation, shorter ASI, and much higher grain yields than WT plants. Attempts have also been made to improve drought tolerance of plants by altering the expression of aquaporins (Aharon et al. 2003; Porcel et al. 2005; Yu et al. 2005; Peng et al. 2006; Jang et al. 2007; Cui et al. 2008; Miyazawa et al. 2008; Zhang et al. 2008c). Aquaporins facilitate transport of water and other small solutes and ions across membranes via the apoplastic route (Aharon et al. 2003; Cui et al. 2008; Jang et al. 2007; Peng et al. 2006; Zhang et al. 2008c). Research into the role of aquaporins in plant drought tolerance has shown that various aquaporins function differently depending on the severity and type of stress. For example, some aquaporins, such as the Arabidopsis Rd28, and rice RWC3, are upregulated under drought stress and others, such as NtQP1 and AtPIP1, remain unchanged under drought stress (Cui et al. 2008). Additionally, some aquaporins genes, such as AtPIP1b have been shown to diminish the drought tolerance capability of some plants, while others, such as the Vicia faba PIP1, Panax ginseng PgTIP1, Brassica napus BnPIP1, and Brassica juncea BjPIP1, have been shown to improve drought tolerance (Aharon et al. 2003; Yu et al. 2005; Peng et al. 2006; Cui et al. 2008; Zhang et al. 2008c). There is also evidence that overexpression of aquaporins in some plants causes them to respond differently to different stresses. For example, Jang et al. (2007) found that Arabidopsis and tobacco plants overexpressing Arabiopsis PIP’s displayed enhanced water flow and improved germination under cold stress, but exhibited rapid water loss, retarded seedling growth, and inferior germination under drought conditions. It is therefore thought that different aquaporin isoforms are associated with different physiological processes and that plants respond to drought conditions either by increasing aquaporin expression, which facilitates water movement (especially into the tonoplast in order to maintain cell-turgor) or downregulating aquaporin expression to avoid excessive water loss (Aharon et al. 2003; Peng et al. 2006). Overexpression of aquaporins has also been implicated in conferring heavy metal tolerance to transgenic plants by alleviation of metal ion-induced water deficit and oxidative damage caused by metal ions (Zhang et al. 2008c).

2.3 2.3.1

Engineering Salt Tolerance in Plants Impacts of Salinity on Agricultural Production

The damaging effects of salt accumulation in agricultural soils have severely affected agricultural productivity in large swathes of arable land throughout the world. Salt-affected land accounts for more than 6% of the world’s total land area

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(FAO 2009c) and is distributed largely amongst coastal salt marshes or inland desert sands. These have primarily arisen naturally through mineral weathering (which leads to the release of soluble salts such as chlorides of calcium, magnesium and sodium, and, to a lesser extent, sulfates and carbonates) or wind and rain deposition of oceanic water (Szabolcs 1989; Munns and Tester 2008; FAO 2009b). Secondary salinization occurs when irrigation and tree clearing of agricultural land cause water tables to rise and concentrate salts in the root zone (Rengasamy 2006). Approximately 20% of the world’s irrigated land, from which one-third of the world’s food supply is produced, is presently affected by salinity (Ghassemi et al. 1995). With the expected increase in world population, the loss of arable land due to salinity presents a serious challenge to food sustainability and productivity. Removal of salts from the root zone (reclamation) is perhaps the most effective way to ameliorate the detrimental effects of salinity; however, this is a slow and expensive process. The use of plant breeding and genetic engineering technologies to alter the salt tolerance of crops will, therefore, play an important role in maintaining global food production in the future.

2.3.2

Improving Salinity Tolerance of Agricultural Crops

Plants have evolved a complex adaptive capacity to perceive and respond to salt stress. The existence of salt-tolerant flora (halophytes) and differences in salt tolerance between genotypes within the salt-sensitive plant species (glycophytes) give rise to the belief that salt tolerance has a genetic basis (Yamaguchi and Blumwald 2005). As for drought, efforts to improve the salt tolerance of crops have met with limited success because of the physiological and genetic complexity of the trait. Salinity tolerance is a multi-genic trait, with quantitative trait loci (QTL) identified in barley, wheat, soybean, citrus, rice, and tomato (Flowers and Flowers 2005; Jenks et al. 2007). Genetic approaches currently being used to improve salinity tolerance include the exploitation of functional genomics, bioinformatics, and natural genetic variations, either through direct selection in stressful environments or through the mapping of QTLs and subsequent marker-assisted selection (Yamaguchi and Blumwald 2005), or the generation of transgenic plants (Vij and Tyagi 2007).

2.3.3

Physiological Effects of Salinity on Plants and Salinity Tolerance Mechanisms

Salinity imposes a variety of stresses on plant tissues. Two of these are osmotic stress, which results from the relatively high soil solute concentrations, and ion

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cytotoxicity. The decreased rate of leaf growth that occurs after an increase in soil salinity is primarily due to the osmotic effect of the salt around the roots, which inhibits plant water uptake and causes leaf cells to lose water. However, this loss of cell volume and turgor is transient and reductions in cell elongation and also cell division lead to slower leaf appearance and smaller final size over the longer term (Bartels and Sunkar 2005; Munns and Tester 2008). Under prolonged salinity stress, inhibition of lateral shoot development becomes apparent within weeks and, within months, there are effects on reproductive development, such as early flowering and reduced floret number. Concomitantly, older leaves may die while the production of younger leaves continues. The cellular and metabolic processes involved are similar to those occurring in drought-affected plants and are responses to the osmotic effect of salt (Yeo et al. 1991; Munns and Tester 2008). Ion cytotoxicity occurs when salt accumulates to toxic concentrations in fully expanded leaves (which, unlike younger leaves, are unable to dilute high salt concentrations), causing leaf death. Replacement of K+ by Na+ in biochemical reactions leads to conformational changes and loss of protein function, as Na+ and Cl– ions penetrate hydration shells and interfere with noncovalent interactions between amino acids. If the rate of leaf death generated by ion cytotoxicity is greater than the rate at which new leaves are produced, the photosynthetic capacity of the plant will no longer be able to supply the carbohydrate requirement of young leaves, which further reduces their growth rate (Munns and Tester 2008). Halophytes, though taxonomically widespread, are relatively rare amongst the flowering plants and virtually all crop plants are glycophytes (Flowers and Flowers 2005). However, there is considerable variability in the tolerance of glycophytes to salt. Munns and Tester (2008), categorize salinity tolerance under three broad categories: (1) Tolerance to osmotic stress, which immediately reduces cell expansion in root tips and young leaves, and causes stomatal closure; (2) Na+ exclusion from leaf blades, which ensures that Na+ remains at nontoxic concentrations within leaves; and (3) Tissue tolerance to Na+ or Cl–, which requires compartmentalization of Na+ and Cl– at the cellular and intracellular level to avoid accumulation of toxic concentrations within the cytoplasm.

2.3.4

Salt Tolerance Using Transgenic Approaches

2.3.4.1

Osmoprotectants

Osmoprotectants were discussed previously (Sect. 2.2.6.3.4) in relation to their use in developing drought-tolerant crops and the transfer of GlyBet intermediates have improved the drought and salt tolerance of transgenic plants in many cases. Mohanty et al. (2002) demonstrated that Agrobacterium-mediated transformation of an elite indica rice cultivar to increase GlyBet synthesis through the incorporation of the codA gene, which encodes choline oxidase, was an effective way to

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improve salinity tolerance. Challenge studies performed with R1 plants by exposure to salt stress for one week, followed by a recovery period, revealed that in some cases more than 50% of the transgenic plants could survive salt stress and set seed whereas WT plants failed to recover. A more recent example of enhanced GlyBet synthesis experiments involved transformation of maize with the BADH gene, introduced by the pollen-tube pathway (Wu et al. 2008a). Transgenic lines were examined for tolerance to NaCl by induced salt stress and, after 15 days of treatment, most transgenic seedlings survived and grew well, whereas WT seedlings wilted and showed loss of chlorophyll. The amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities in response to environmental stresses (Kavi Kishore et al. 2005; Ashraf and Foolad 2007). In plants, the precursor for proline biosynthesis is l-glutamic acid. Two enzymes, pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), play major roles in the proline biosynthetic pathway (Delauney and Verma 1993; Ashraf and Foolad 2007). Su and Wu (2004) established that the rate of growth of transgenic rice plants expressing mothbean D1-pyrroline-5-carboxylate synthetase (p5cs) cDNA under either a constitutive or stress-inducible promoter led to the accumulation of p5cs mRNA and proline in third-generation (R2) transgenic rice seedlings. Significantly higher salinity and water-deficit stress tolerance of R2 seedlings were attributed to faster growth of shoots and roots in comparison with non-transformed plants. Stressinducible expression of the p5cs transgene showed significant advantages over constitutive expression in increasing the biomass production of transgenic rice grown in soil under stress conditions. The osmoprotectant role of proline has been verified in other plants such as potato, where salt tolerance, measured by comparing tuber yield of transgenic lines cultivated in a greenhouse and watered with saline water to that of plants watered with normal tap water, had a less significant effect on tuber yield of transgenic plants than WT (Hmida-Sayari et al. 2005). Polyamines, including spermidine (Spd, a triamine), spermine (Spm, a tetramine), and their obligate precursor putrescine (Put, a diamine), are aliphatic amines widely present in living organisms. The polyamine biosynthetic pathway is depicted in Fig. 2.3. Recently, it has been demonstrated that plant polyamines are involved in the acquisition of tolerance to such stresses as high and low temperatures, salinity, hyperosmosis, hypoxia, and atmospheric pollutants (Liu et al. 2007). Furthermore, genetic transformation of several plant species with polyamine biosynthetic genes encoding arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), or Spd synthase (SPDS) led to improved environmental stress tolerance (Liu et al. 2007). He et al. (2008) tested transgenic apple engineered with (SPDS)-overexpressing transgenic European pear (Pyrus communis L. “Ballad”) for changes in enzymatic and nonenzymatic antioxidant capacity in response to NaCl or mannitol stress. Their research revealed that transgenic plants accumulated more Spd than WT. The transgenic line contained higher antioxidant enzyme activities (less malondialdehyde and H2O2) than the WT, implying that it suffered from less injury and enhanced enzymatic and nonenzymatic antioxidant capacity (He et al. 2008).

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N

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Fig. 2.3 Polyamine biosynthesis

Mannitol is a primary photosynthetic product that is associated with exceptional salt tolerance. In celery, mannitol metabolism is clearly altered by salt stress, with several lines of evidence indicating a connection between mannitol and salt tolerance, including increases in the capacity for mannitol biosynthesis and accumulation and decreases in catabolism (Williamson et al. 2002). Sickler et al. (2007) showed that Arabidopsis plants transformed with celery’s mannose6-phosphate reductase (M6PR) gene produced mannitol and grew normally in the absence of stress. However, in the presence of salt stress, daily analysis of the increase in growth (fresh and dry weight, leaf number, leaf area per plant, and specific leaf weight) over a 12-day period showed less effect of salt on transformants than WT plants. The daily energy use efficiency for photochemistry by photosystem 2 (PSII) was also measured and demonstrated that, unlike transformed plants, which were not affected, WT plants treated with 100 mM NaCl displayed a reduction in PSII yield after 6 days with a 50% loss in yield after 12 days. Similarly, under atmospheric levels of CO2, growth with 200 mM NaCl caused an increase in sub-stomatal levels of CO2 in WT plants but not in transformants. Trehalose is a disaccharide sugar widely distributed in bacteria, fungi, insects, plants and invertebrate animals. In microbes and yeast, trehalose is produced from glucose by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), and functions in sugar storage, metabolic regulation, and protection against abiotic stress (Strom and Kaasen 1993; Wiemken 1990). Trehalose acts as a compatible solute, protecting membranes and proteins and conferring desiccation

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tolerance on cells in the absence of water (Crowe et al. 1984). Ge et al. (2008) illustrated the protective role of trehalose in higher plants. Expression analysis demonstrated that OsTPP1 isolated and cloned from rice, was initiated and transiently upregulated after salt, osmotic, and ABA treatments but slowly upregulated under cold stress. OsTPP1 overexpression in rice enhanced salt and cold stress tolerance. Tolerance of transgenic plants to abiotic stress was examined by observing 2-week-old seedlings exposed to salt. Following one week of exposure, seedlings exhibited salt-induced damage symptoms such as wilted leaves. However, after prolonged salt treatment, transgenic lines were more vigorous and displayed increased leaf greenness and viability over control plants. Generally, two broad themes have emerged from the results of attempts to engineer overexpression of osmoprotectants. The first is that metabolic limitations have been encountered in generating absolute levels of target osmolytes, especially when compared with salt-tolerant halophytes and the second is that the degree to which transformed plants are able to tolerate salinity stress is not necessarily correlative with the levels of osmoprotectants attained.

2.3.4.2

Transporter Genes

Mechanisms that confer salt tolerance vary with the plant species; however, the ability to maintain low cytosolic Na+ is thought to be one of the key determinants of plant salt tolerance (Tester and Davenport 2003). Salt “inclusion” and “exclusion” are recognized as different mechanisms by which higher plants tolerate salinity. The functional removal of Na+ from the cytoplasm of plant cells and the maintenance of low cytosolic Na+ concentrations under salinity conditions (Blumwald et al. 2000) is accomplished by either pumping Na+ out of cells (plasma membrane antiporter) or into vacuoles (vacuolar antiporter) in exchange for H+. Na+/H+ antiporter activity is driven by the electrochemical gradient of protons (H+) generated by the H+ pumps (H+-ATPase) in the plasma membrane or the tonoplast (Chinnusamy and Zhu 2003; Tester and Davenport 2003). In Arabidopsis, active exclusion of Na+ is mediated by the plasma membrane-localized Na+/H+ antiporter, AtSOS1 (Shi et al. 2003). In contrast, the sequestration of excess Na+ into the vacuole is mediated by the vacuolar membrane-localized Na+/H+ antiporter, AtNHX1 (Gaxiola et al. 1999; Shi et al. 2008). In a similar way, overexpression of the S. cerevisiae HAL1 gene (Gaxiola et al. 1992) conferred salt tolerance in yeast by increasing intracellular K+ and decreasing Na+ levels. The successful use of transporter genes has been demonstrated in several plants. He et al. (2005) created transgenic cotton plants expressing AtNHX1 and found that transgenic plants generated more biomass and produced more fibers under salt stress in a greenhouse. It was suggested that the increased fiber yield was due to superior photosynthetic performance and higher nitrogen assimilation rates observed in the transgenic plants compared with WT. Interestingly, the researchers demonstrated that field-grown irrigated AtNHX1-expressing cotton plants produced higher fiber yields (fiber plus seeds) than WT, with an average increase of more

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than 25% per line. Furthermore, the fibers produced by transgenic plants were generally more uniform, stronger, and longer than those of WT. Similarly, Chen et al. (2007) engineered maize plants overexpressing the rice OsNHX1 gene. Transformants accumulated more biomass under greenhouse-based salt stress. Higher Na+ and K+ content was observed in transgenic leaves than in WT when treated with 100–200 mM NaCl, while the osmotic potential and the proline content in transgenic leaves was lower than in WT. Salt stress field trials revealed that the transgenic maize plants produced higher grain yields than WT plants at the vegetative growth stage. Biochemical studies suggest that Na+/H+ exchangers in the plasma membrane of plant cells contribute to cellular sodium homeostasis by transporting Na+ ions out of the cell (Qiu et al. 2002). SOS1 encodes a plasma membrane Na+/H+ exchanger in Arabidopsis (Qiu et al. 2002) and the important role of the plasma membrane Na+/H+ exchangers for plant salt tolerance was supported by the finding that overexpression of SOS1 improved plant salt tolerance (Shi et al. 2003). Zhao et al. (2006) demonstrated that expressing the plasma membrane Na+/H+ antiporter SOD2 from yeast (Schizosaccharomyces pombe) in transgenic rice also increased salt tolerance. Transgenic plants accumulated more K+, Ca2+, and Mg2+ and less Na+ in their shoots compared with non-transformed controls. Moreover, measurements on isolated plasma membrane vesicles derived from the SOD2 transgenic rice plant roots showed that the vesicles had enhanced P-ATPase hydrolytic activity as well as being able to maintain higher levels of photosynthesis and root proton exportation capacity. Martinez-Atienza et al. (2007) identified an AtSOS1 homolog, OsSOS1, in rice, which demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (S. cerevisiae) cells and reduced their net cellular Na+ content. OsSOS1 was also shown to suppress the salt sensitivity of an sos1-1 mutant of Arabidopsis. In relation to the introduction of genes that modulate cation transport systems, many researchers have sought to employ the overexpression of the S. cerevisiae HAL1 gene, which has conferred salt tolerance in yeast by facilitating intracellular K+ accumulation and decreasing intracellular Na+ (Gaxiola et al. 1992; Rios et al. 1997). Rus et al. (2001) established ectopic expression of HAL1 in transgenic tomato plants, and showed that transformants were able to minimize the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K+ contents in leaf calli and leaves in the presence of salt, which indicates that, similar to the yeast gene, plant HAL1 functions by facilitating K+/Na+ selectivity under salt stress. Ellul et al. (2003), utilizing an optimized Agrobacterium-mediated gene transfer protocol, developed HAL1-expressing watermelon (Citrullus lanatus). Salt tolerance of transgenic plants was confirmed in a semi-hydroponic system on the basis of the higher growth performance of transgenic lines compared to control plants. The halotolerance observed supports the potential usefulness of the HAL1 gene as a molecular tool for genetic engineering salt-stress protection in other crop species.

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Detoxifying Genes

The mechanisms of plant detoxification of ROS under drought stress were introduced in Sects. 2.2.6.2 and 2.2.6.2.3. As an antioxidant enzyme, glutathione peroxidase (GPX) reduces hydroperoxides in the presence of glutathione to protect cells from oxidative damage, including lipid peroxidation (Maiorino et al. 1995). Gaber et al. (2006) generated transgenic Arabidopsis plants overexpressing GPX2 genes in cytosol (AcGPX2) and chloroplasts (ApGPX2). The activities toward a-linolenic acid hydroperoxide in ApGPX2- and AcGPX2-expressing plants were 6.5–11.5 and 8.2–16.3 nmol min–1 mg protein–1, respectively, while no activity was detected in the WT plants. Both transgenic lines showed enhanced tolerance to oxidative damage caused by the treatment with H2O2, Fe ions, or methylviologen (MV) and environmental stress conditions, such as chilling with high light intensity, high salinity or drought. The degree of tolerance of the transgenic plants to all types of stress was correlated with the levels of lipid peroxide suppressed by the overexpression of the GPX-2 genes. SOD is the first enzyme in the enzymatic antioxidative pathway and halophytic plants, such as mangroves, reported to have a high level of SOD activity. SOD plays a major role in defending mangrove species against severe abiotic stresses. Prashanth et al. (2008) further characterized the Sod1 cDNA (a cDNA encoding a cytosolic copper/zinc SOD from the mangrove plant Avicennia marina) by transforming it into rice. Transgenic plants were more tolerant to MV-mediated oxidative stress in comparison to WT and withstood salinity stress of 150 mM of NaCl for a period of 8 days while WT plants wilted at the end of the hydroponic stress treatment. Pot-grown transgenic plants tolerated salinity stress better than the WT when irrigated with saline water. In plant cells, APXs are directly involved in catalyzing the reduction of H2O2 to water, which is facilitated by specific electron donation by ascorbic acid. APXs are ubiquitous in plant cells and are localized in chloroplasts (Takahiro et al. 1995), peroxisomes (Shi et al. 2001), and cytosol (Caldwell et al. 1998). Xu et al. (2008b) transformed Arabidopsis plants with a pAPX gene from barley (HvAPX1). The transgenic line was found to be more tolerant to salt stress than the WT. There were no significant differences in Na+, K+, Ca2+, and Mg2+ contents and the ratio of K+ to Na+ between pAPX3 and WT plants, which indicated that salt tolerance in transgenic plants was not due to the maintenance and re-establishment of cellular ion homeostasis. However, the degree of H2O2 and lipid peroxidation (measured as the levels of malondialdehyde) accumulation under salt stress was higher in the WT than in transgenic plants. The mechanism of salt tolerance in transgenic plants was explained by a reduction of oxidative stress injury. Apart from catalase and various peroxidases and peroxiredoxins (Dietz 2003), four enzymes, APX, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase (GR), are involved in the ascorbate-glutathione cycle, a pathway that allows the scavenging of superoxide radicals and H2O2 (Asada 1999). Most of the ascorbate-glutathione cycle enzymes are located in the stroma, cytosol, mitochondria, and peroxisomes (Jimenez et al. 1998). APX and GR, the first and

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last enzymes in this cycle, respectively, are responsible for H2O2 detoxification in green leaves (Foyer et al. 1994). GR has a central role in maintaining the reduced glutathione (GSH) pool during stress (Pastori et al. 2000). Lee and Jo (2004) introduced BcGR1, a Chinese cabbage gene that encodes cytosolic GR into tobacco plants via Agrobacterium-mediated transformation. Homozygous lines containing BcGR1 were generated and tested for their acquisition of increased tolerance to oxidative stress. When ten-day old transgenic tobacco seedlings were treated with 5 to 20 mM MV, they showed significantly increased tolerance compared to WT seedlings. The most drastic difference was observed at a concentration of 10 mM MV. In addition, when leaf discs were subjected to MV, the transgenic plants were less damaged than the WT with regard to their electrical conductivity and chlorophyll content.

2.3.5

Late Embryogenesis Abundant (LEA) Proteins

The LEA proteins were introduced in Sect. 2.2.6.3.3 in relation to their use in improving plant drought tolerance. These proteins have also been used in engineering salt-tolerant crops. Park et al. (2005b) introduced a B. napus LEA protein gene, ME-leaN4 (Wakui and Takahata 2002) into lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation. Transgenic lettuce demonstrated enhanced growth ability compared with WT plants under salt- and water-deficit stress. After 10-day growth under hydroponic 100 mM NaCl conditions, average plant length and fresh weight of transgenic lettuce were higher than those of WT and the increased tolerance was also reflected by delayed leaf wilting caused by water-deficit stress. Brini et al. (2007a) analyzed the effect of ectopic expression of dehydrin (Dhn-5; Table 2.1) cDNA in Arabidopsis under salt and osmotic stress. When compared to WT plants, the Dhn-5-expressing transgenic plants exhibited stronger growth under high concentrations of NaCl or water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in WT than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in WT plants and the transgenic lines had higher proline contents and lower water loss rates under water stress. Na+ and K+ also accumulated to a greater extent in the leaves of the transgenic plants. Lal et al. (2008) reported the effects of overexpression of the HVA1 gene in mulberry under a constitutive promoter. HVA1 is a group 3 LEA (Table 2.1) isolated from barley aleurone layers and has been found to be inducible by ABA. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of HVA1 in conferring tolerance. Using leaf discs as explants, growth performance under salt-stress and water-deficit conditions were carried out from 8- to 10-month-old transgenic plants. Leaf discs of uniform size were cut and used for simulated salt and water-deficit stress treatments for different durations. After the stress treatments, leaf discs were analyzed for proline content,

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photosynthetic yield, RWC, and cellular membrane stability (CMS). Transgenic plants showed better CMS, photosynthetic yield, less photooxidative damage, and better WUE as compared with the nontransgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants showed a manyfold increase in proline concentration over WT plants and, under water-deficit conditions, proline accumulated only in the nontransgenic plants. Results also indicated that the production of HVA1 proteins enhanced the performance of transgenic mulberry by protecting plasma and chloroplast membrane stability under abiotic stress.

2.3.6

Transcription Factors

The importance of TFs in plant stress response was discussed in Sect. 2.2.5. In addition to binding to cis-acting elements in the promoters of environmental stressresponsive genes, TFs can activate and repress gene expression through interactions with other TFs, thus playing a central role in plant response to environmental stresses (Chen and Zhu 2004). It is generally accepted that activation or ectopic expression of a specific TF can result in expression of many functional genes related to stresses. CBF/DREBs are key regulatory factors that function primarily in freezing tolerance by activating a network of target genes (Fowler and Thomashow 2002; Maruyama et al. 2004). Oh et al. (2007) isolated a barley gene, HvCBF4, whose expression is induced by low temperature stress. Transgenic overexpression of HvCBF4 in rice resulted in an increase in tolerance to drought, high-salinity, and low temperature stresses without stunting growth. Interestingly, under low temperature conditions, the maximum photochemical efficiency of PSII in the dark-adapted state in HvCBF4 plants was higher by 20 and 10% than that in nontransgenic and CBF3/DREB1A-expressing plants, respectively. Using the 60K Rice Whole Genome microarray, 15 rice genes were identified that were activated by HvCBF4. When compared with 12 target rice genes of CBF3/DREB1A, 5 genes were common to both HvCBF4 and CBF3/DREB1A, and 10 and 7 genes were specific to HvCBF4 and CBF3/DREB1A, respectively. Results suggested that CBF/ DREBs of barley acted differently from those of Arabidopsis in transgenic rice. The NAC family of TFs (Sect. 2.2.6.1) has applicability for generating salttolerant crops. Hu et al. (2008) characterized a stress-responsive NAC gene (SNAC2) isolated from upland rice for its role in stress tolerance. Northern blot and SNAC2 promoter activity analyses demonstrated that SNAC2 expression was induced by drought, salinity, cold, wounding and ABA treatment. The SNAC2 gene was overexpressed in japonica rice to test the effect on improving stress tolerance. To test salinity tolerance, germinated positive transgenic and WT seeds were transplanted on Murashige and Skoog (MS) medium containing 150 mM NaCl and the normal MS medium without NaCl as a control. Under saline conditions, transgenic seedlings grew faster and their shoots were significantly longer than WT after 20 days. However, there was no difference in root length or root numbers

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between transgenic and WT seedlings grown under saline conditions and no difference in growth performance was observed between transgenic and WT seedlings in the normal MS medium. Hu et al. (2008) also evaluated germination ability of transgenic lines harboring SNAC2 under salt-stress conditions. After 4 days of germination on the medium containing 150 mM NaCl, only 40% of WT seeds were poorly germinated, whereas more than 70% of transgenic seeds germinated efficiently. In the MS medium, more than 90% of both transgenic and WT seeds germinated well and there was no significant difference in germination rates, suggesting that overexpression of SNAC2 does not affect seed germination under normal conditions. The significantly higher germination rate of transgenic seeds than that of WT under saline conditions further supported the improved salt tolerance of SNAC2-overexpressing plants. DNA chip profiling analysis of the transgenic plants revealed many upregulated genes related to stress response and adaptation such as peroxidase, ornithine aminotransferase, heavy metal-associated protein, sodium/hydrogen exchanger, HSP, GDSL-like lipase, and phenylalanine ammonia lyase. This data suggests that SNAC2 is a novel stress-responsive NAC TF that possesses potential utility in improving stress tolerance of rice. The TFIIIA-type zinc-finger proteins, first discovered in Xenopus, represent an important class of eukaryotic TFs (Miller et al. 1985). More than 40 TFIIIA-type zinc-finger protein genes have been identified from various plants, including petunia, soybean, Arabidopsis, and rice (Kim et al. 2001; Sugano et al. 2003; Mittler et al. 2006; Huang and Zhang 2007) and these genes have been shown to be induced by various abiotic stresses. Xu et al. (2008a) have recently reported the functional analysis of ZFP252 (a salt and drought stress responsive TFIIIA-type zinc-finger protein gene from rice), using gain- and loss-of-function strategies. They discovered that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes, and enhanced rice tolerance to salt and drought stresses compared with ZFP252 antisense and nontransgenic plants. Their findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced drought and salt tolerance (Xu et al. 2008a).

2.3.6.1

Signal Transduction Genes

Plant salt-stress-response genes are involved in many plant cellular processes, including physiological metabolism, cell defense, energy production and transportation, ion transfer and balance, and cell growth and division. These genes function through certain coordination mechanisms to maintain the normal growth of plants under salt stress. As discussed previously, components of the STP may also be shared by various stress factors such as drought, salt, and cold (Shinozaki and Yamaguchi-Shinozaki 1999). Signal molecules, H2O2 and NO, are involved in the ABA-induced stomatal closure and gene expression and activities of antioxidant enzymes (Zhang et al. 2006, 2007a). ABA-induced H2O2 production mediates NO generation, which in

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turn activates MAPK and results in upregulation of the expression and activities of antioxidant enzymes (Zhang et al. 2007a). The importance of ABA in plant environmental stress responses was discussed in Sect. 2.2.6.1. The oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycartenoid dioxygenase (NCED) is the key regulatory step of ABA biosynthesis in higher plants. Overexpression of SgNCED1 in transgenic tobacco plants resulted in 51–77% more accumulation of ABA in leaves (Zhang et al. 2008d). Transgenic tobacco plants were shown to have decreased stomatal conductance and transpiration and photosynthetic rates and increased activities of SOD, catalase, and APX activities. H2O2 and NO in leaves were also induced in the transgenic plants. Compared with WT, the transgenic plants displayed improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl induced salinity stress. It was suggested that the ABA-induced H2O2 and NO generation upregulates stomatal closure and antioxidant enzymes and, therefore, increases drought and salinity tolerance in transgenic plants. Salt stress is known to trigger a rapid and transient increase of free calcium concentration in plant cells (Knight 2000; Pauly et al. 2000). As such, Ca2+ signaling processes are one of the earliest events in salt signaling and may play an essential role in the ion homeostasis and salt tolerance in plants ( Zhu 2003; Reddy and Reddy 2004). CBLs represent a unique family of calcium sensors in plants and function as a positive regulator in the salt-stress-response pathway. Extensive studies have progressed toward understanding of Arabidopsis CBLs, yet knowledge of their functions in other plant species is still quite limited. Wang et al. (2007a) have reported the cloning and functional characterization of ZmCBL4, a novel CBL gene from maize. ZmCBL4 encodes a putative homolog of the Arabidopsis CBL4/SOS3 protein. Under normal conditions, ZmCBL4 was shown to be expressed differentially at a low level in various organs of maize plants and its expression was regulated by NaCl, LiCl, ABA and PEG treatments. Expression of 35S::ZmCBL4 not only complemented the salt hypersensitivity in an Arabidopsis sos3 mutant, but also enhanced the salt tolerance in Arabidopsis WT plants at the germination and seedling stages. ZmCBL4-expressing Arabidopsis lines accumulated less Na+ and Li+ as compared with WT plants. Wang et al. (2007a) concluded that the maize CBL gene functions in salt-stress-elicited calcium signaling and thus in maize salinity tolerance.

2.4 2.4.1

Engineering Cold Tolerance in Plants Impacts of Cold Stress on Agricultural Production

Agricultural borders for crop species are defined geographically by occurrences of low temperatures and frost, which cause severe yield losses in marginal areas. Approximately two-thirds of the world’s landmass is annually subjected to temperatures below freezing and half to temperatures below –20 C.

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Most crops of tropical origin, as well as many of subtropical origin, are sensitive to chilling temperatures. Amongst the major world food crops, maize and rice are sensitive to chilling temperatures and yield loss or crop failure of these species can occur at temperatures below 10 C. Many other crops, such as soybean, cotton, tomato, and banana, are injured at temperatures below 10–15 C (Lynch 1990). The temperature below which chill injury can occur varies with species and regions of origin and ranges from 0 to 4 C for temperate fruits, 8 C for subtropical fruits, and about 12 C for tropical fruits (Lyons 1973). Cold acclimation, also known as cold hardening, describes an increase in tolerance over time to cold temperatures and cellular desiccation in response to conditions such as cold temperature, short photoperiods, and mild drought and results from changes in gene expression and physiology (Xin and Browse 2000; Kalberer et al. 2006). Most temperate plants can cold-acclimate and acquire tolerance to extracellular ice formation in their vegetative tissues. Winter-habit plants such as winter wheat, barley, oat, rye, and oilseed rape have a vernalization requirement, which allows them to survive freezing stress as seedlings during winter. However, after vernalization and at the end of the vegetative phase, the cold acclimation ability of winter cereals gradually decreases, making them sensitive to freezing injuries (Fowler et al. 1996; Chinnusamy et al. 2007). Therefore, it is not surprising that the impacts of cold stress on plant life have been comprehensively studied. Many attempts have been made to improve cold resistance of important crop plants; however, progress in achieving frost hardiness of plants either by classical breeding or by gene transfer is difficult because of the fact that cold resistance is not a quality conferred by the product of one gene, but, as for most abiotic stress tolerance mechanisms, is quantitative in nature (Mahajan and Tuteja 2005).

2.4.2

Physiological Effects of Cold Stress on Plants and Cold Tolerance Mechanisms

The symptoms of chilling-induced stress injury in cold-sensitive plants are variable and generally manifest within 48 to 72 h of stress exposure. Observed phenotypic symptoms in response to chilling stress include reduced leaf expansion, wilting, chlorosis, and necrosis (Mahajan and Tuteja 2005). Chilling also severely inhibits plant reproductive development, with species such as rice displaying sterility when exposed to chilling temperatures during anthesis (Jiang et al. 2002). The extent of plant damage caused by exposure to low temperature depends on factors such as the developmental stage, the duration and severity of the frost, the rates of cooling (and rewarming), and whether ice formation takes place intra- or extra-cellularly (Beck et al. 2004). Chilling stress (<20 C) is a direct result of low temperature effects on cellular macromolecules, which leads to slowing of metabolism, solidification of cell

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membranes, and loss of membrane functions. This kind of damage results primarily from loss of function of biomembranes associated with decreased fluidity and inactivation or deceleration of membrane-bound ion pumps. Absorbance of light energy, which occurs independently of temperature, results in oxidative stress if metabolism cannot keep pace with the excitation of the photosynthetic components. Freezing stress (<0 C), which causes extracellular ice-crystal formation, freeze-induced dehydration, and concentration of cell sap, has major mechanical impacts on cell walls and plasma membranes and leads to cell rupture (Margesin et al. 2007). Generally, freezing results in loss of membrane integrity and solute leakage. The integrity of intracellular organelles is also disrupted under freezing stress and leads to loss of compartmentalization and reduction and impaired photosynthesis, protein assembly, and general metabolic processes. The primary environmental factors responsible for triggering increased tolerance against freezing are collectively known as “cold acclimation” (Mahajan and Tuteja 2005). Frost resistance can be achieved by two main mechanisms: (1) avoidance of ice formation in tissues; or (2) tolerance of apoplastic extracellular ice. An individual plant may employ both mechanisms of frost resistance in different tissues (Sakai and Larcher 1987; Margesin et al. 2007). A key function of cold acclimation is to stabilize membranes against freezing injury through mechanisms such as adjustment of lipid composition and accumulation of protective sugars, hydrophilic and LEA proteins, and antioxidants (Thomashow 1999).

2.4.3

Cold Tolerance Using Transgenic Approaches

2.4.3.1

ROS Detoxifying Substances

The mechanisms of ROS accumulation and detoxification were discussed in Sects. 2.2.6.2 and 2.3.4.3. The decrease in the amount of unsaturated fatty acids during lipid peroxidation elevates membrane viscosity, promotes lipid transition from a liquid crystalline phase to a gel phase, raises proton permeability of membranes, diminishes membrane electric conductance, and causes inactivation of membrane-localized enzymes. The adaptation of plant cells to low temperature is based on their ability to maintain saturation of fatty acids in membrane lipids, thus modifying membrane fluidity (Szalontai et al. 2003). Demin et al. (2008) examined the role of D12-acyl-lipid desaturase in plant resistance to hypothermia-induced oxidative stress. The study focused on modulation of free-radical processes occurring at low temperature in leaf cells of potato plants transformed with the gene for D12-acyl-lipid desaturase from cyanobacterium. Plants were grown in vitro on MS agar medium containing 2% sucrose. During hypothermia (–9 C), the rate of O2– generation and H2O2 concentration decreased significantly. In addition, the contents of both primary products (conjugated dienes and trienes) and secondary products (malonic dialdehyde) of lipid

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peroxidation were lower in transformed plant leaves than in leaves of WT plants. It was hypothesized that insertion of D12-acyl-lipid desaturase into the plant genome stabilizes the composition and physical properties of biomembranes by promoting polyunsaturation of fatty acids, which averts the accelerated generation of O2– and suppresses lipid peroxidation during hypothermia. Evidence suggests that ROS are the cause of photosystem 1 (PSI) inactivation in chilling-sensitive plants during cold stress in the light, and that H2O2 accumulation is a major factor leading to the decline in PSI activity (Sonoike 1996). Cotton is considered to be a chilling-sensitive species; its photosynthetic performance and ability to rapidly recover photosynthetic activity following chilling in the light diminish considerably with time of exposure (Koniger and Winter 1993). Kornyeyev et al. (2003) compared the photosynthetic performance between leaf discs of WT cotton and transgenic cotton overexpressing chloroplast stroma-based APX+ plants during exposure to 10 C and 500 mmol photons m–2 s–1. They showed that APX+ leaves did not exhibit as large an increase in cellular H2O2 as WT leaves shortly after the imposition of the chilling treatment. In addition, APX+ leaves exhibited slightly but significantly less PSI and PSII photoinhibition. The electron transport chain (ETC) of plant mitochondria has also been researched in relation to production of freeze-tolerant transgenic plants. Plant ETCs have unique features compared with other eukaryotes, including the ubiquitous presence of a terminal alternative oxidase (AOX) that competes for electrons with the standard cytochrome (Cyt) pathway (Finnegan et al. 2004). The maintenance of ETC redox balance is critical because electron input in excess of ETC capacity can be responsible for the production of ROS, particularly O2– and H2O2. It has been proposed that AOX may be an important factor in allowing plants to tolerate chilling-induced ROS damage to membrane phospholipids (Purvis and Shewfelt 1993). Evidence suggests that the AOX pathway of plant mitochondria uncouples respiration from mitochondrial ATP production and may ameliorate plant performance under stressful environmental conditions, such as cold temperatures, by preventing excess accumulation of ROS (Wagner et al. 1998). Fiorani et al. (2005) tested this model in whole tissues by growing AtAOX1a-transformed Arabidopsis plants at 12 C. Twenty-one days after sowing, antisense mutants showed (on average) a 27% reduction in leaf area and 25% smaller rosettes versus 30% increased leaf area and 33% larger rosette size for overexpressing lines compared with WT plants. These results demonstrate that AOX activity plays a role in shoot acclimation to low temperature in Arabidopsis.

2.4.3.2

Membrane Modifications

Chilling-resistant plants have a greater abundance of unsaturated fatty acids than chilling-sensitive plants and it has been shown that the proportion of unsaturated fatty acids increases during acclimation to cold temperature (Palta et al. 1993). This modification allows membranes to remain fluid by lowering the temperature at which the membrane lipids experience a gradual phase change from fluid to

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semi-crystalline. Thus, desaturation of fatty acids provides protection against damage from chilling temperatures (Khodakovskaya et al. 2006). Khodakovskaya et al. (2006) developed a cold-inducible genetic construct cloned using a chloroplastspecific omega-3-fatty acid desaturase gene (FAD7) under the control of a coldinducible promoter (cor15a) from Arabidopsis and expressed it in young tobacco plants. When seedlings were exposed to low-temperature (0.5, 2, or 3.5 C) for up to 44 days, survival within independent cor15a-FAD7 transgenic lines (40.2%–96%) was far superior to the WT (6.7%–10.2%). In addition, the major trienoic fatty acid species remained stable in cold-induced cor15a-FAD7 plants under prolonged cold storage while the levels of hexadecatrienoic acid (16:3) and octadecatrienoic acid (18:3) declined in WT plants under the same conditions (79 and 20.7%, respectively). Electron microscopy showed that chloroplast membrane ultrastructure in cor15a-FAD7 transgenic plants was unaffected by prolonged exposure to cold temperatures. In contrast, WT plants experienced a loss of granal stacking and disorganization of the thylakoid membrane under the same conditions. In higher plants, the most abundant lipids of thylakoid membranes are glycolipids (monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)). PG is the only phospholipid in thylakoid membranes. The chilling resistance of higher plants is apparently closely correlated with the level of cis-unsaturated fatty acids in PG from chloroplast membranes (Nishida and Murata 1996). Chilling-resistant plants contain a large proportion of cis-unsaturated fatty acids at the sn-1 position of PG and there are few cis-unsaturated fatty acids in chilling-sensitive plants. The sn-2 position is occupied mainly by saturated and trans-unsaturated fatty acids (Bertrams and Heinz 1981) and hence the content of cis-unsaturated fatty acids at the sn-1 position of PG determines plant chilling resistance. The dominant factor that determines the level of cis-unsaturated fatty acids in PG is the substrate selectivity of glycerol-3-phosphate (G3P) acyltransferase (GPAT: EC2.3.1.15) in chloroplasts, which catalyzes the first step of glycerolipid biosynthesis by transferring the acyl group of acyl-(acyl carrier protein; ACP) to the sn-1 position of G3P to yield 1-acylglycerol-3-phosphate (lysophosphatidate; LPA; (Roughan and Slack 1982)). GPAT from chilling-resistant plants prefers oleoyl-ACP (18:1-ACP) to palmitoyl-ACP (16:0-ACP) as a substrate resulting in a high level of cis-unsaturated fatty acids in PG. The enzymes from chilling-sensitive plants hardly distinguish 18:1-ACP from 16:0-ACP resulting in a low level of cis-unsaturated fatty acids at the sn-1 position of PG (Weber et al. 1991). In this way, fatty acids remain saturated, which renders the plants sensitive to chilling stress. Sui et al. (2007) isolated a tomato GPAT gene (LeGPAT), which despite the chilling sensitivity of tomato exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in the thylakoid membrane. Chilling treatment (4 C for 4 h) induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than WT plants under optimal

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conditions. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membranes, which was beneficial for the recovery of chilling-induced PSI photoinhibition in tomato.

2.4.3.3

LEA and Chaperoning Modifications

Several proteins are expressed in plants upon exposure to low temperature. These are either located in the cytosol or secreted to the apoplast. They have various putative functions, including cryoprotection, altered lipid metabolism, protein protection, desiccation tolerance, and sugar metabolism (Hiilovaara-Teijo and Palva 1999; Margesin et al. 2007). During cold acclimation, several stress proteins that may function as chaperones and membrane stabilizers during freeze dehydration are expressed in the cytosol (Puhakainen et al. 2004). Of the many low temperature-responsive genes characterized to date, several have been predicted to encode proteins with the characteristics of the dehydrin class of LEA proteins (Table 2.1). Houde et al. (2004) reported an improvement of the selection procedure and the transfer of the wheat Wcor410a acidic dehydrin gene to strawberry. The WCOR410 protein was expressed in transgenic strawberry at a level comparable with that in cold-acclimated wheat. Freezing tests showed that cold-acclimated transgenic strawberry leaves had a 5 C improvement in freezing tolerance over WT leaves or transformed leaves not expressing the WCOR410 protein. However, no difference in freezing tolerance was found between the different plants under non-acclimated conditions, suggesting that the WCOR410 protein needs to be activated by another factor induced during cold acclimation. The data demonstrated that WCOR410 protein prevents membrane injury and greatly improves freezing tolerance in leaves of transgenic strawberry. HSPs (Sect. 2.2.6.1) accumulate in response to low temperature. The HS response in plants has been extensively investigated (Waters et al. 1996). Plants synthesize predominantly small (15–30 kDa) HSPs (sHSPs) during the heat-shock response, and it has been suggested that the accumulation of sHSPs is correlated with thermotolerance (Vierling 1991). Guo et al. (2007), characterized a sweet pepper cDNA clone, CaHSP26 encoding the chloroplast (CP)-sHSP, with regard to its sequence, response to various temperatures, and function in transgenic tobacco plants. Expression of the CaHSP26 gene showed that the mRNA accumulation of CaHSP26 was induced by heat stress. Higher transcript levels were observed when sweet pepper leaves were treated at 42 C for 3 h. However, the expression of the CaHSP26 gene was not induced by chilling stress (4 C) in the absence of HS and the transcripts were detected at 48 h at 4 C after HS while not at 25 C. The photochemical efficiency of PSII (Fv/Fm) and the oxidizable P700 in transgenic tobacco overexpressing CaHSP26 were higher than that in WT tobacco during chilling stress under low irradiance. These results suggest that the CP sHSP protein plays an important role in the protection of PSII and PSI during chilling stress under low irradiance.

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Osmoprotectants/Compatible Solutes

Long-term acclimation to the cold and winter survival in herbaceous plants is strongly correlated with the recovery of photosynthesis at low temperature and the maintenance of soluble carbohydrate reserves (Stitt and Hurry 2002; Strand et al. 2003), namely, through upregulation of sucrose biosynthesis. Strand et al. (2003) tested this hypothesis by comparing the acclimation responses of WT Arabidopsis with transgenic plants overexpressing sucrose phosphate synthase (over-sps) or with antisense repression of either cytosolic fructose-1,6-bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants overexpressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT plants. The improved photosynthetic performance by the overexpressing sps plants was associated with an increase in freezing tolerance relative to WT (–9.1 and –7.2 C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (–5.2 and –5.8 C for antifbp and antisps, respectively). Similarly, metabolic engineering for the biosynthesis of fructans is a potential strategy for increasing waterstress tolerance. Fructans are a class of water-soluble fructose polymers based on sucrose, which accumulate in many bacterial and plant species serving as an important storage carbohydrate and protecting plants against water deficit caused by low matrix potential, salinity, or low temperatures (Spollen and Nelson 1994). In plants, fructans are synthesized in vacuoles from sucrose by the action of two or more different fructosyltransferases, including sucrose:sucrose 1-fructosyltransferase (1-SST), sucrose:fructan 6-fructosyltransferase (6-SFT), fructan:fructan 1-fructosyltransferase (1-FFT), and fructan:fructan 6G-fructosyltransferase (6G-FFT; Vijn and Smeekens 1999). Kawakami et al. (2008) used rice, which is highly sensitive to chilling temperatures and is not able to synthesize fructans, to study the effect of fructan biosynthesis against water stress. Two wheat fructan-synthesizing enzymes, 1-SST, encoded by wft2, or 6-SFT, encoded by wft1, were introduced into rice plants. The transgenic seedlings with wft2 accumulated significantly higher concentrations of oligo- and polysaccharides than nontransgenic rice seedlings, and exhibited enhanced chilling tolerance (11-day exposure to 5 C). The oligo- and polysaccharide concentrations of seedlings expressing wft1 were visibly lower than those of lines expressing wft2, and no correlation between oligo- and polysaccharide concentrations and chilling tolerance was detected in wft1-expressing rice lines. The results suggest that transgenic rice lines expressing wheat-derived fructosyltransferase genes accumulated large amounts of fructans in mature leaf blades and exhibited enhanced chilling tolerance at the seedling stage.

2.4.3.5

Transcription Factors

Cold acclimation in plants is a complex process involving changes in the expression of numerous cold-responsive (COR) genes (Chinnusamy et al. 2006). This results in

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modification of plant cell structural, biochemical, and photosynthetic properties that facilitate an increase in the plant’s freezing stress tolerance. Regulatory factors influencing expression of COR genes and/or freezing tolerance have been identified over the last decade (Chinnusamy et al. 2006). The cold-induced CBF transcriptional regulatory factor CBF1-3 controls the cold-responsive expression of a major regulon of COR genes that increases plant freezing tolerance (van Buskirk and Thomashow 2006). Ectopic expression of CBF transgenes under warm conditions activates a suite of COR genes without cold stimulus, increasing plant freezing tolerance and inducing biochemical and structural alterations normally observed during exposure to cold. Pino et al. (2008) studied the effect of ectopic AtCBF overexpression on physiological alterations that occur during cold exposure in frost-sensitive (Solanum tuberosum) and frost-tolerant (S. commersonii) potato. Relative to WT plants, ectopic AtCBF1 overexpression enhanced expression of COR genes without a cold stimulus in both species and imparted a significant increase in freezing tolerance gain in both species (2 C in S. tuberosum and up to 4 C in S. commersonii). Transgenic S. commersonii displayed improved cold acclimation potential, whereas transgenic S. tuberosum was still incapable of cold acclimation. During cold treatment, leaves of WT S. commersonii showed significant thickening resulting from palisade cell lengthening and intercellular space enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity induced these same leaf alterations in the absence of cold in both species. In transgenic S. commersonii, AtCBF1 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to WT, transgenic S. commersonii leaves were darker green, had higher chlorophyll and lower anthocyanin levels, greater stomatal numbers, and displayed greater photosynthetic capacity, suggesting higher productivity potential. These results suggest that an endogenous CBF pathway is involved in many of the structural, biochemical, and physiological alterations associated with cold acclimation in potato. The cuticle is one of the most important barriers for all terrestrial plants and mitigates damage to above-ground biomass caused by low humidity and other biotic and abiotic stresses (Jenks and Ashworth 1999). The important physiological and ecological functions of the cuticle (which include control of transpiration and leaching and facilitation of foliar penetration of pesticides) are related to the presence of cuticular waxes that are embedded in or deposited on the cutin matrix (Kunst and Samuels 2003). Increased accumulation of cuticular waxes in leaves has been achieved through the overexpression of TF genes such as WIN1/SHN1 in Arabidopsis (Broun et al. 2004) and WXP1 in alfalfa (Zhang et al. 2005). Elevated leaf cuticular wax deposition led to significant improvement in drought tolerance of the transgenic plants (Broun et al. 2004; Zhang et al. 2005). Zhang et al. (2007b) reports the functional characterization of two putative ERF TF genes WXP1 and its paralog WXP2 from Medicago truncatula. Transgenic expression of WXP1 and WXP2 in Arabidopsis led to significantly increased cuticular wax deposition on leaves of 4- and 6-week-old transgenic plants. Differences in the accumulation of various wax components, as well as their chain length distributions, were found in the WXP1 and WXP2 plants. Analysis of fresh weight loss from detached leaves

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revealed that the transgenic leaves retained more water than controls. Both WXP1 and WXP2 transgenic plants showed significantly enhanced whole-plant drought tolerance. Analysis of freezing tolerance at the whole-plant level and measurement of electrolyte leakage from detached leaves revealed that the WXP1 plants had increased freezing tolerance while the WXP2 plants were more sensitive to low temperature when compared to the control. Transgenic expression of WXP1 had no obvious effects on plant growth and development; however, the expression of WXP2 led to slower plant growth. These results indicate that WXP1 is a useful candidate gene for improving plant drought and freezing tolerance by genetic transformation.

2.5

Nutrient Deficiency and Nutrient Use Efficiency

In all, plants require 17 essential elements, 14 of which are taken up in inorganic forms by the roots. The absence or paucity of any one of these essential elements will commonly lead to plant death or inability to complete its life cycle. In the presence of nutrient deficiencies, even at asymptomatic levels, crop performance, yield, and quality are frequently compromised. Hence, fertilizer and other soil ameliorations are essential to ensuring food security and the sustainability of agriculture, and these add a major economic and potentially environmental burden to crop production. The ability to improve and enhance the efficiency of nutrient uptake and utilization in major crop plants has become a major objective in modern plant improvement. Interestingly, different species have evolved differential mechanisms for improving their ability to scavenge essential elements at low concentrations in the soil. In this section, we will discuss the ways in which biotechnological intervention can be applied to improve the ability of crop plants to survive and produce yield in nutrient-poor environments. The discussion will focus on the most limiting nutrient, nitrogen, with some discussion of phosphorus and iron, which are particularly susceptible to soil pH variation.

2.5.1

Nitrogen

Nitrogen (N) is quantitatively the most important nutrient that plants obtain from the soil (Paungfoo-Lonhienne et al. 2008), with an estimated 1011 tons of N fertilizer applied annually worldwide (Lea and Azevedo 2006). The total cost of this agricultural input exceeds US $50 billion. In general, however, plants are only able to acquire 30–40% of this applied N, with significant losses occurring through leaching, denitrification, and the volatilizationz of ammonium to the atmosphere (Lea and Azevedo 2006). Nitrogen assimilation is the process by which inorganic N forms (typically nitrate) are reduced to ammonium, and then converted into organic forms (amino

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acids) for transportation and use by the plant. Nitrate is the main form of N taken up by plants and is reduced in the plant through the combined action of the enzymes, nitrate reductase (NR) and nitrite reductase. It has long been believed that this step is the major control point in nitrate assimilation and it has been shown that expressing a constitutive NR as opposed to a fully regulated NR demonstrates accumulation of high concentrations of asparagine and glutamine (Good et al. 2004; Lea and Azevedo 2006). Inorganic N is then converted into organic amino acids by the GS-GOGAT cycle, and these amino acids are used to transport N around the plant. The major N transport amino acids are glutamate, glutamine, aspartate, and asparagine, with differences in importance among different phyla and also environmental conditions. Ammonium is incorporated into glutamine through the enzyme glutamine synthetase (GS). This places the ammonium molecule initially into the amide position of the glutamine molecule from where the N is relocated to the a-amino position of glutamate through the action of glutamate synthase (GOGAT) (Glass et al. 2002; Good et al. 2004; Lea and Azevedo 2006). The GS-GOGAT has been considered a primary target for the manipulation of nitrogen use efficiency (NUE). NUE is usually described as unit biomass per unit of N present in the plant or the yield of N in the plant per unit of available N in the soil (Bushoven and Hull 2001; Lea and Azevedo 2006). NUE is dependent on physiological traits such as uptake and assimilation of N (Good et al. 2004). While there are other processes that use N in plants, it is these two traits that control NUE. The ability to uptake and transport N throughout the plant is, along with photosynthetic efficiency and WUE, one of the major determinants of plant survival, growth, and yield. In addition, the mobilization of organic N into fruits and seeds is a major determinant of yield and product quality, especially in cereal and legume grains, where the protein content determines the nutritional and end-use qualities. N nutrients are powerful signaling molecules within the plant (Vidal and Gutierrez 2008). Nitrate controls the expression of thousands of genes involved in many plant processes, with some genes responding to nanomolar concentrations (Wang et al. 2003a, 2007b). This includes the nitrate transporter molecules (for an introduction to transporter genes, see the description in Sect. 2.3.4.2). The nitrate transporters, NRT1.2 and NRT2.1, are not only involved with N uptake, but they are also sensor molecules, and have been shown to increase N uptake rate at low rhizosphere concentration. It is known that root branching occurs at higher frequency in regions of the rhizosphere with local N-enrichment (Scott Russell 1977). The transporter NRT1.1 is involved in signaling, which leads to colonization of nitrate-rich patches in the soil by promoting localized root proliferation in concert with ANR1, a MADS-box TF gene not yet fully functionally characterized (Remans et al. 2006). Both these genes have regulatory sequences which direct reporter gene expression in root primordia and root tips. Amino acid synthesis and transport genes have been manipulated to alter the NUE in numerous plants. For example, the overexpression of a bacterial asparagine synthetase gene in the leaves of lettuce was shown to affect N status (Giannino

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et al. 2008). Early vegetative growth rate of these transgenic lettuce plants was approximately 1.3-fold higher for the first 35 days post-germination, accompanied by higher leaf number and leaf area. However, these plants also attained reproductive phase earlier, which was not advantageous for a leaf vegetable. Similar effects have been reported in transgenic oilseed rape (Seiffert et al. 2004) and Lotus (Vincent et al. 1997). The transgenics had approximately twofold more aspartate and asparagine than WT, and interestingly also had elevated levels of glutamine with no effect on glutamate concentration. Leaf assays also revealed a lower level of free nitrate in the transgenic lettuce lines, a phenomenon previously reported in lettuce overexpressing a nitrate reductase gene (Curtis et al. 1999). The authors suggested that these plants may be suitable for breeding new quick-harvest lettuce varieties with lower N fertilizer requirement, or enhanced NUE.

2.5.2

Phosphorus

Worldwide, it is estimated that 5.7 billion hectares of land lack sufficient quantities of plant-available Phosphorus (P) (Batjes 1997). P deprivation in plants has been widely studied, as P becomes limiting to plant productivity with falling soil pH, and tends to bind very tightly with soil. In mildly acid soils with pH < 6, soil P rapidly becomes immobile and forms insoluble compounds, commonly with Fe and Al. Even in soils with abundant P, usually only about 1% of the soil P is actually in a readily available, soluble form, and over 90% is generally bound tightly to soil particles in organic and inorganic forms, which require mineralization before they become plant available. P uptake and transporter genes are generally regarded as high or low affinity, with the high affinity genes becoming more important for scavenging nutrient from the soil as P becomes limiting. Many Australian soils are particularly P-deficient and, as a result, many native Australian plants have evolved different mechanisms to overcome this with high-affinity P transporters, association with vesiculararbuscular mycorrhiza, the secretion of organic acids into the rhizosphere, and the formation of proteoid roots (Lambers et al. 2006). Attempts to overexpress high affinity P transporters have met with mixed success. Overexpression of high-affinity transporter genes in barley did not have any effect on P uptake or plant productivity and growth (Rae et al. 2004), despite having demonstrated that these genes increased P uptake in transgenic rice callus cultures. However, the overexpression of homologous high-affinity P transporter genes in rice enabled the plants to accumulate twice as much P as WT rice, and the resulting phenotype produced more tillers (Seo et al. 2008). Further research has also demonstrated that many plant phosphate transporters have complex interactions with vesicular arbuscular mycorrhizae (Glassop et al. 2007). Plants have been demonstrated to alter the rhizosphere with specific exudates, commonly organic acids or enzymes, to improve the availability of nutrients such as phosphate. The exudation into the rhizosphere of acid phoaphatase has led to

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improved biomass and P accumulation in Arabidopis (Xiao et al. 2007) and rice (Park et al. 2007). Similar results have been achieved with the ectopic expression of phytase in root tissues of potato (Hong et al. 2008).

2.5.3

Iron

Iron is not only an essential plant nutrient, but its deficiency, known as anemia, also represents the world’s most frequent and debilitating human mineral nutrient deficiency (Wintergerst et al. 2007). Hence, improving the ability of plants to uptake, translocate and store iron in bioavailable forms is not only a plant health and productivity issue, it is a major human and animal nutritional target. Plants have the ability to sense low-iron conditions, which induces a coordinated response. The predominant form of iron in aerobic soils is Fe(III), and plants must possess strategies to utilize this form. Most plants use what has become widely known as Strategy I, whereby they acidify the rhizosphere with the induction of a specific proton pump which solubilizes more Fe, and then, by the production of Fe chelate reductase, convert Fe(III) to Fe(II), which is then transported into the cell by a membrane-bound Fe(II) transporter. Grasses and cereals, however, utilize Strategy II, which involves the production and secretion of a specialized group of chemicals, known as phytosiderophores (PS) into the rhizosphere. PS are a form of mugineic acids, which form strong chelates with Fe(III), and help to solubilize them for plant uptake by specialized protein forms, known as the Yellow Stripe proteins (von Wiren et al. 1994; Curie et al. 2001), which are actually Fe(III) transporter genes. Interestingly, rice is a special case and appears to be able to utlilize both strategies of iron uptake. Unusually, among the grasses rice has an efficient Fe(II) uptake system and does not produce much PS (Mori et al. 1991). Rice has been engineered to produce larger quantities of PS, and the transgenic lines were more tolerant to Fe-deficient soils (Suzuki et al. 2008). Other successful approaches to improve Fe uptake of transgenic rice in alkaline soils have included the overexpression of Fe chelate reductase genes (Ishimaru et al. 2007) and barley nicotianamine amino transferase genes (Takahashi et al. 2001).

2.6 2.6.1

Engineering Metal Toxicity Tolerance in Plants Heavy Metal Contaminated Soil

Soils naturally consist of varying high levels of heavy metals; however, concentrations in soils are greatly increased where humans have extracted them and used them for industrial purposes (Greger 1999; Harmsen 2002; UN-Oceans 2008).

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Heavy metals, especially those that are present from weathering of parent rock, are usually bound or immobile in soils and their removal via leaching or plant accumulation is slow and inefficient. As a consequence, metals accumulate in soils (Haygarth and Jones 1992). Heavy metals usually exist as cations under biological conditions and form complexes with soil sediments and colloids, which are made up of negatively charged organic substances and inorganic clay particles. Formation of this complex is a slow process and, because unbound metals are bioavailable and may deleteriously affect agricultural products or leach into groundwater, areas where human activities have led to elevated soil metal content are a socioeconomic as well as an environmental concern (Greger 1999; Harmsen 2002). Common heavy metal soil pollutants include arsenic, cadmium, chromium, copper, nickel, lead, and mercury (UN-Oceans 2008). Cd and Hg are particularly concerning because they are widespread and have no known function in human metabolism (Nordberg et al. 2002; UN-Oceans 2008).

2.6.2

Engineering Heavy Metal Tolerance in Plants

Few known studies have focused on engineering heavy metal tolerance in plants. It is assumed that this is due to the complex interactions of stress factors, which mean that many studies focusing on improving other types of abiotic stress, such as osmotic and low temperature stress, include analysis of improved tolerance to one or a few heavy metals. For example, Zhang et al. (2008c) isolated an aquaporin gene BjPIP1 from the heavy metal hyperaccumulator Indian mustard, which is upregulated in leaves under drought, salt, low temperature, and heavy metal stress. Constitutive expression of BjPIP1 in tobacco decreased water loss rate, transpiration rate, and stomatal conductance of transgenic plants compared to WT under osmotic stress. On exposure to Cd, transgenic plants displayed enhanced Cd resistance of root growth and lowered transpiration rates and stomatal conductance, increased activities of antioxidative enzymes, lower levels of electrolyte leakage, and lower malondialdehyde content. The study suggested that the increased heavy metal resistance was conferred by maintaining reasonable water status in transgenic plants. Other proteins that have been implicated in conferring heavy metal tolerance include LEA proteins and cation-efflux transport proteins (Zhang et al. 2007c). When Koh et al. (2006) introduced a yeast Cd factor (YCF1), which sequesters glutathione chelates of heavy metals and xenobiotics into vacuoles, into Arabidopsis in order to improve heavy metal tolerance, transgenic plants were found to have improved salt tolerance as well (Zhang et al. 2007c). While the use of genetic engineering to develop crops with improved heavy metal tolerance remains relatively unexplored, a related field is the use of genetic engineering to develop plants with increased ability to uptake and accumulate metals in order to remove the soil contamination in a process known as phytoremediation. Many of the plant characteristics required to confer metal accumulation ability also confer heavy metal tolerance and, as such, plants engineered with enhanced phytormediation

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capabilities inevitably possess enhanced heavy metal tolerance as well. Unlike engineering plant heavy metal tolerance, this field has been the focus of many studies over the last decade. Therefore, the remainder of this chapter will briefly discuss the use of transgenic technology to improve the phytoremediation capabilities of plants.

2.6.3

Soil Heavy Metal Management and Remediation Options

There are many management and remediation options available to prevent entry of soil heavy metals into the human food chain via agricultural crops. These include: prevention, agronomic management, and physical remediation. These options, while contributing to reductions in human exposure to some heavy metals, can be unrealistic, labor intensive, and prohibitively expensive (McLaughlin et al. 1999; Ensley 2000; Glass 2000; Clarkson 2002; Madden et al. 2002; Prasad 2002; Robinson et al. 2003; Song et al. 2003).

2.6.3.1

Phytoremediation: Engineering Crops to Increase Heavy Metal Uptake

Phytoremediation is a biological technology, which utilizes the physiology of plants to extract and detoxify soil and water pollutants (Kramer and Chardonnens 2000; Rosselli et al. 2003). The use of plants for contaminated land reclamation is based on the existence of metal hyperaccumulator plants, which are capable of growing on metal contaminated soils and accumulating extremely high levels of heavy metals in their harvestable tissue (Robinson et al. 2003). The major metal tolerance mechanisms identified in heavy metal accumulator plants include: (1) cell wall metal ion binding; (2) inhibition of metal ion transport across plasma membranes; (3) active metal ion efflux from cells; (4) chelation and detoxification of metal ions; and (5) compartmentalisation of metal ions in organelles (Bargagli 1998). The several types of phytoremediation include: phytostabilization (the use of plants to immobilize metals in the environment to prevent them from leaching into groundwater (Prasad 2002)), phytodegradation (the use of plants to degrade contaminants in the root zone), phytoextraction, and phytovolatization. Phytoextraction and phytovolatilisation are particularly important for metal phytoremediation. Phytoextractor plants remove metal ions from soils and store them in above-ground tissue using the photosynthetically driven processes of soil-ion extraction and translocation. Normal harvesting processes enable easy removal of contaminants and their responsible disposal. Phytovolatilization is similar to phytoextraction but, rather than be stored in biomass, extracted elements are converted into less toxic, volatile forms via plant metabolic pathways and released into the atmosphere (Kramer and Chardonnens 2000; Robinson et al. 2003).

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Phytovolatilization of Soils Contaminated with Hg

Relatively low exposure to Hg and mercurial compounds can cause severe detrimental physiological effects in all biological organisms. Atmospheric Hg increased from approximately 2  106 kg to 4  106 kg over the twentieth century because of Hg use in the chemical, medical, paper, mining, and defense industries (Bizily et al. 2000). Environmental Hg is present as Hg(II), elemental Hg (Hg(0)), and organomercurial compounds (R-Hg+) such as CH3–Hg+. Toxicity symptoms between these forms differ; CH3–Hg+ is the most dangerous because it is lethal at doses two to three orders of magnitude lower than Hg(0) or Hg(II), diffuses passively through biological membranes, is highly reactive with biological compounds, has a long retention time in the body, and is biomagnified through the food chain (Rugh et al. 2000). There are no known naturally occurring plants that are able to detoxify or hyperaccumulate Hg (Pilon-Smits and Pilon 2002). Therefore, a bacterial Hg volatilization pathway has been used in the development of Hg phytoremediators (Pilon-Smits and Pilon 2002). Bacterial colonies that metabolically convert Hg(II) and R-Hg+ compounds into the less toxic Hg(0) have been discovered inhabiting Hg-contaminated sites. The volatilization pathway is conferred by the presence of the mer-operon, which encodes a set of genes involved in the detection, mobilization, and enzymatic detoxification of R-Hg+ compounds via two main reactions, which are catalyzed by organomercurial lyase (MerB) and mercuric ion reductase (MerA) (Rugh et al. 1998). A modified form of the MerA gene, merA9, was transferred into Arabidopsis and tobacco and transgenic plants were found to be resistant to 50 mm Hg(II) (Rugh et al. 2000). Expression of the same gene in the forest species, Liriodendron tulipifera (yellow poplar) resulted in transgenic plants, which thrived in solution containing 50 mm Hg(II) and volatized Hg(0) at a rate tenfold higher than WT controls (Kramer and Chardonnens 2000). MerA has also been expressed in the eastern cottonwood (Populus deltoids) with similar success. The transformation of Arabidopsis with constructs of both the merA and merB genes resulted in transgenic plants able to tolerate media containing 40-fold higher CH3– Hg+ levels than WT plants and were able to volatilize Hg at a rate severalfold higher than WTs (Bizily et al. 2000). Tobacco was also transformed with MerA and MerB via the chloroplast genome and transgenic plants grew well with root Hg concentrations up to 2,000 mg g–1, accumulated R-Hg+ compounds and inorganic mercurials to levels surpassing soil concentrations and displayed a 100-fold increase in the efficiency of shoot Hg accumulation over WT plants (Hussein et al. 2007). MerA and MerB have also been expressed simultaneously in transgenic poplars and eastern cottonwood trees (Lyyra et al. 2007; Young et al. 2007). Transgenic poplars were tolerant to 50 mM HgCl2 and 2 mM CH3HgCl in culture; however, a high level of variation in Hg-tolerance was observed in transgenic plants (Young et al. 2007). Transgenic eastern cottonwood trees were highly resistant to R-Hg+ compounds and detoxified them two to three times more rapidly than controls (Lyyra et al. 2007).

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Phytoextraction of Soils Contaminated with Cd

Plants able to tolerate high Cd levels do so via exclusion (reducing metal uptake by cell wall binding) or intracellular compartmentalization (binding of metals with detoxifying ligands within the cell and the sequestration of these complexes in organelles where the metals cannot interact with the plants metabolic processes (Robinson et al. 1994)). The two major heavy metal detoxifying ligands in plant cells are the metallothioneins (MTs) and the phytochelatins (PCs; Cobbett and Goldsborough 2000). Class I and II MTs are low molecular weight metal-binding products of mRNA translation. PCs are represented by the class III MTs and are products of enzymatic reactions (Cobbett and Goldsborough 2002).

2.6.5.1

Metallothioneins

It has been difficult to analyze plant metallothionein (MT) proteins because they are unstable under aerobic conditions; however, isolated and cloned mammalian MT genes have been assessed for their ability to increase metal tolerance when expressed in transgenic organisms. Table 2.2 lists the findings of some of these studies. These results, obtained in laboratory trials, demonstrate that expression of mammalian MTs in plants can provide protection against toxic effects of heavy metal ions such as Zn2+, Cd2+, and Hg(II); but it has not yet been possible to achieve similar results in the field (Kramer and Chardonnens 2000).

2.6.5.2

Phytochelatins

Phytochelatins (PCs) were first discovered in plants and are structurally related to GSH, which is thought to act as the substrate for PC synthesis by PC synthase (PCS). Various methods have been employed to enhance the effectiveness of PCassisted metal detoxification. Indian mustard was engineered to express the E. coli GSH synthesis gene, gsh2, and transgenic plants displayed increased GSH and PC levels and a 25% increase in shoot Cd concentration (Zhu et al. 1999). The enzyme that catalyzes synthesis of the GSH precursor, g-glutamylcysteine synthase (GCS), was also expressed in B. juncea and shoot Cd concentrations were increased by 40–90% in the transformed plants (Kramer and Chardonnens 2000). A variety of PC genes have been isolated from different species and overexpressed in endogenous or exogenous species to determine their potential for Cd phytoremediation. The most extensively studied PCS gene is AtPCS1 from Arabidopsis, which has been found to play roles in increased tolerance and/or accumulation of Cd and As (Gasic and Korban 2007b). However, depending on the expression level, transfer of PCS genes have also been associated with decreased Cd accumulation and Cd hypersensitivity (Lee et al. 2003b, c; Li et al.

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Table 2.2 Genetic transfer of MT genes for improved phytoremediation capabilities of plants Transformed Transgene(s) Findings References species Eschericia Human MT genes Increased metal adsorption Kramer and coli Chardonnens (2000) Ralstonia Mouse MTs genes Enhanced Cd immobilizing ability Valls et al. eutrophia when expressed on cell surface (2000) Tobacco Mammalian MT Root-shoot Cd transport is reduced Kramer and gene Chardonnens (2000) Brassica Yeast MT gene, Increased leaf Cd accumulating Kramer and oleracea CUP1 ability Chardonnens (2000) Tobacco Construct containing Up to 90% increased Cd Macek et al. CUP1 and an accumulation in harvestable (2002) additional metalparts without any visible binding domain difference in growth characteristics Sunflower CUP1 Enhanced Cd tolerance when Watanabe et al. expressed at the callus stage (2005) Tonkovska et al. Tobacco Arabidopsis MT Expression strongly induced by (2003) gene, MT2aI Cu2+, Zn2+, and Cd2+ suggesting the promoter has specificity for heavy metal stress Tobacco Construct encoding a 45–75% increase in Cd Pavlikova et al. poyhistidine accumulation in harvestable (2004a, b) cluster with a plant parts and increased yeast MT resistance to Cd-induced stress Arabidopsis Garlic MT gene Stronger Cd tolerance and higher Zhang et al. Cd accumulation (2006) Zhigang et al. Arabidopsis Brassica juncea MT Increased tolerance to Cu2+ and Cd2+ based on shoot growth and (2006) gene, BjMT2, chlorophyll content and decreased root growth in the absence of heavy metal exposure Tobacco Silene vulgaris L. Signigicantly increased Cd Gorinova et al. MT gene accumulation in roots and (2007) leaves Sun et al. (2007) Arabidopsis Brassica rapa MT Chloroplast target of gene resulted gene in detoxification of Cd and H2O2

2004, 2005b; Gasic and Korban 2007a). Other studies have shown that AtPCS1 can lead to increased Cd tolerance and accumulation in roots but not translocation of Cd into harvestable tissue (Pomponi et al. 2006). PCS genes from Cyndon dactylon (CdPCS1) and wheat (TaPCS1) have also been transformed into tobacco and have led to increases in leaf accumulation of Cd and other heavy metals (Li et al. 2006; Martinez et al. 2006). Tobacco transformed with TaPCS1 was grown in the field to

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assess its Cd-accumulating ability and it was found that transgenic plants could accumulate more heavy metals and 100 times more biomass on contaminated soils than the hyperaccumulator Thlaspi caerlescens (Martinez et al. 2006). Another approach was employed by Song et al. (2003) and was aimed at increasing the efficiency of metal transport across the tonoplast. In this study, YCF1 from S. cerevisiae was overexpressed in Arabidopsis and transformed plants displayed increased tolerance to Cd and Pb and improved vacuolar Cd sequestration. Many studies have also shown that dual- or multiple-gene expression using constructs with combinations of PC synthesis genes such as GCS, GSH synthase, ATP sulfurylase, and serine acetyltranferase may be the most promising route toward the development of a useful Cd phytoremediator and a phytoremediator useful for removal of mixtures of heavy metals from soils (Bennett et al. 2003; Wawrzynski et al. 2006; Guo et al. 2008; Reisinger et al. 2008).

2.7

Conclusion and Future Directions

This review has briefly summarized some of the stress resistance mechanisms that have been targeted for manipulation in the endeavor to develop crop plants with improved resistance to drought, salinity, cold, nutrient deficiencies, and metal toxicities. In the majority of cases, there are many knowledge gaps that need to be filled prior to development and release of these crops. Water-limiting environments are the most widespread form of stress, and also the least amenable to rapid advances in resistance. Reasons for this include the incomplete knowledge regarding plant drought responses, lack of field trials and drought stress treatments which are truly reflective of climatic conditions, the lack of site and crop specificity of drought tolerance studies, and the lack of integration of disciplines. The last century of breeding effort and crop physiology studies have led to increases in the economic yield of most major crop species and have elucidated many traits that are associated with plant adaptability to drought-prone environments (such as small plant size, reduced leaf area, early maturity, and prolonged stomatal closure). However, many attempts to improve drought tolerance through breeding have been associated with reduced yield potential (Cattivelli et al. 2008). Nevertheless, enormous advances should have been make in the understanding of the physiological and molecular responses of plants to water deficit through breeding and physiological studies, and scientists in these areas will continue to have a fundamental role in the development of transgenic drought-resistant crops. One of the greatest limitations in drought stress tolerance breeding has been the fact that drought takes many varied forms. Depending on the crop and the season, water stress may be experienced in early vegetative stages, during transition to flowering and even post-anthesis. In some cases, this may be a terminal stress or, more commonly, a recurring event broken by sporadic rainfall precipitating a recovery of the whole plant, sometimes with reduced yield potential as a result of conservative plant cell survival responses.

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It is entirely possible that the development of transgenic crops with improved abiotic stress tolerance may lead to unforeseen outcomes, both positive and negative. The development of “super varieties” that offer promising solutions to the problems of water deficit or salinity stress, may also give rise to problems associated with high-input agriculture in monoculture situations. Additionally, this review has shown that there are a plethora of genetic components that may be manipulated in order to confer improved abiotic stress tolerance of crops and that there is, therefore, unlikely to be a single gene that will result in a suitable “droughttolerant” or “salt-tolerant” variety for all conditions. Consequently, it may be beneficial to trial novel technologies such as overexpression of multiple genes known to be involved in the adaptation to stress and introgression of these genes in random combinations into many cultivars of the one crop prior to field release. This may increase the stability of these genes and allow specific environments to “naturally select” the transgenes that are most suitable for their particular climatic conditions in the same way that increased natural genetic diversity does so in natural populations. Salt tolerance is a physiologically complex trait and halophytes and less tolerant plants show a wide range of adaptations to salt stress. Attempts to enhance tolerance have involved conventional breeding programs, the use of in vitro selection, physiological trait pooling, interspecific hybridization, the use of halophytes as alternative crops, the use of marker-aided selection, and the use of transgenic plants. The assessment of salt tolerance in transgenic experiments as described above has mostly been carried out using a limited number of seedlings or mature plants in laboratory experiments. In most cases, experiments were carried out in greenhouse conditions where the plants were not exposed to conditions that prevail in high-salinity soils (e.g., alkaline soil pH, high diurnal temperatures, low humidity, presence of sodic salts, and elevated concentrations of selenium and/or boron (Yamaguchi and Blumwald 2005)). Therefore, the salt tolerance of plants needs to be evaluated under field conditions and, more importantly, salt tolerance needs to be evaluated as a function of yield. The evaluation of field performance under salt stress is difficult because of the variability of salt levels in field conditions (Daniells et al. 2001) and the potential for interactions with other environmental factors, including soil fertility, temperature, light intensity and water loss due to transpiration. Evaluating tolerance is further complicated because of variation in sensitivity to salt during plant life cycles. According to Flowers and Flowers (2005), conventional breeding programs have rarely delivered enhanced salt tolerance, while wide crossing to achieve salt tolerance generally reduces yield to unacceptably low levels (Flowers and Flowers 2005). There has been success using physiological criteria as the basis of selection for rice (Dedolph and Hettel 1997) and such an approach has been advocated for wheat (Munns et al. 2002). According to Flowers and Flowers (2005) recent analysis has shown that, while it is possible to produce a wide range of transgenic plants where some aspect of a trait relating to salt tolerance is altered, none or few transgenic plants have been tested in the field and few claims for success meet minimal criteria required to demonstrate enhanced tolerance (Flowers 2004).

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While adaptation to stress under natural conditions has some ecological advantages, the metabolic and energy costs may sometimes mask and limit its benefit to agriculture and result in yield penalty. An ideal genetically modified crop should possess a highly regulated stress-response capability that does not affect crop performance when stress is absent. In this respect, conventional breeding and selection techniques will continue to make a contribution (Wang et al. 2001). As a result of this, transgenic approaches to plant improvement are best regarded as a means of widening genetic variation. Transgenic plants will nevertheless continue to be extremely useful tools in basic plant science research, and will lead to improved understanding of the gene networks and molecular physiology of plant responses to abiotic stresses.

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Chapter 3

Transgenic Crops for Herbicide Resistance Stephen O. Duke and Antonio L. Cerdeira

3.1

Introduction

A year after the introduction of the first commercial transgenic crop (Flavr Savr™ tomato with a longer shelf life) in 1994, transgenic, herbicide-resistant crops (HRCs) were introduced (Table 3.1) with the introduction of bromoxynil- (3,5dibromo-4-hydroxybenzonitrile) resistant cotton and glufosinate- [2-amino-4(hydroxymethylphosphinyl)butanoic acid] resistant canola. Bromoxynil resistance had little market penetration during the years when it was available. The next year, 1996, marked the introduction of the first glyphosate- [N-(phosphonomethyl) glycine] resistant (GR) crop (soybean). Other GR and glufosinate-resistant crops were introduced in the subsequent years. GR crops now represent well over 80% of all transgenic crops grown worldwide (James 2008). Accordingly, this chapter will deal primarily with GR crops. Several reviews (e.g., Duke 2005; Duke and Cerdeira 2005; Cerdeira and Duke 2006) and two books (McClean and Evans 1995; Duke 1996) are available on the topic of HRCs, but this rapidly evolving topic requires timely updates. Before the advent of transgenic crops, there was both controversy and optimism about their potential impact on farming, human health, and the environment (e.g., Goldberg et al. 1990; Duke et al. 1991). We have now (early 2009) had 14 years of experience with HRCs over vast areas in many parts of the world, providing a wealth of information on the utilization of this technology, as well as the impacts of HRCs on the environment. This review will deal, in part, with these topics as they apply to HRCs

S.O. Duke (*) Agricultural Research Service, United States Department of Agriculture, University of Mississippi, MS 38677, USA e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_3, # Springer‐Verlag Berlin Heidelberg 2010

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3.2 3.2.1

Present HRCS and Their Impact Commercially Available HRCs

In 2008, after 14 years of HRCs, there are only nine different HRCs being grown in the US (Table 3.1), and only a few of these are grown in other countries. From 1995 until 2000, one or two new HRCs were introduced to the market every year, after which the number of introductions has dwindled, with only a single new HRC in occasional subsequent years. The adoption rate of GR soybean was rapid in the US (Fig. 3.1), currently representing more than 90% of the area planted in soybean. The adoption rate of GR soybean in Argentina was even more rapid, reaching almost 90% adoption within 4 years of introduction (Penna and Lema 2003). Adoption of this HRC has also been rapid in other parts of South America. Both cotton and maize have varieties that are either stand-alone GR varieties or varieties that combine GR and transgenic Bt (Bacillus thuriengensis toxin) traits for insect resistance. In both the crops, there are also stand-alone Bt toxin varieties. To generate the data in Fig. 3.1, adoption rates of the two types of GR varieties must be added. GR cotton adoption was initially similar to that of soybean, but it has stabilized at about 70% (Fig. 3.1), partly because of the adoption of glufosinateresistant cotton in places where it fits the weed problems better than GR cotton. The economics for GR maize was not quite as good as with existing weed management methods when it was first introduced, but its adoption in the US is now rising rapidly and has almost caught up with that of cotton (Fig. 3.1). Relatively little GR canola is grown in the US, but about 90% of the canola grown in Canada was GR canola in 2006 (Dill et al. 2008). Of the canola grown in the US, 62% was GR

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Fig. 3.1 US adoption rate of glyphosate-resistant crops. Data from: http://www.ers.usda.gov/Data/ BiotechCrops

and 31% was glufosinate-resistant in 2005 (Sankula 2006). After a false start in 1999, GR sugar beet was reintroduced in 2008, with an unprecedented adoption rate of about 60% for the initial year of availability and an anticipated 95% adoption rate in 2009 (Thomas Schwarz, Beet Sugar Development Foundation, pers. comm). The adoption rate was limited by the availability of transgenic seed. GR alfalfa was introduced and well accepted by farmers in 2005, but deregulation was challenged in court by organic alfalfa growers in 2007, resulting in the removal of the product from the market. Glufosinate-resistant crops are also available (Table 3.1), but they have garnered a much smaller fraction of the HRC market. Their biggest market penetration is with canola in the US. Glufosinate-resistant cotton has been adopted at high rates in the US state of Texas. Partly owing to the evolution of GR weeds, glufosinateresistant crop adoption is increasing. Crops with both GR and glufosinate resistance are being made available. The economics for the biotechnology industry with HRCs is also good. HRCs offer profits from both a “technology fee” added to seed costs and for the purchase of the herbicide. No other type of transgenic trait offers this opportunity for dual profits from the seed and a chemical upon which the value of the gene is dependent. There has been some consideration of linking expression of transgenic traits to a chemical inducer of transgene expression (e.g., Jepson et al. 1998), but farmers would be unlikely to pay much for such a chemical, and the cost of applying the

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inducer would probably skew the economics away from such a strategy, unless the value of the trait was large.

3.2.2

Herbicide Resistance in HRCs

Plants can be made resistant to herbicides or other phytotoxins by a number of mechanisms. The molecular target site of the herbicide can be modified so that it no longer binds it and is thereby resistant. One or more herbicide-inactivating or herbicide-degrading enzymes can be introduced to or increased in a plant. The plant can be altered to have a mechanism that prevents the herbicide from reaching the molecular target site (increased sequestration, or decreased uptake or translocation). All three mechanisms have been described in weeds that have evolved resistance to herbicides. Metabolic inactivation or degradation is the principle mechanism in most cases of natural crop resistance to selective herbicides. The first two approaches have been useful in producing commercial HRCs. Glyphosate’s molecular target site is an enzyme of the aromatic amino acid pathway (the shikimate pathway). This enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), is highly sensitive to glyphosate (Duke 1988), and there are apparently no other good inhibitors of the enzyme. All plant EPSPS is sensitive to glyphosate, making it a nonselective herbicide that can be used to kill almost all weed species. Some fungi and bacteria also have EPSPS, and there are bacterial versions of the enzyme that are very resistant to glyphosate. A gene encoding Agrobacterium sp. EPSPS, the CP4 gene, has been used as a transgene for almost all GR crops (Cerderia and Duke 2007). In some maize varieties, a maize EPSPS gene modified by site-directed mutagenesis has been used as the transgene in GA21 varieties (Dill 2005). In canola, a gene from the soil bacterium Ochrobactrum antropi that encodes a glyphosate-degrading enzyme (glyphosate oxidoreductase, GOX) has been used with the CP4 EPSPS. This enzyme catalyzes the degradation of glyphosate to aminomethylphosphonic acid (AMPA) and glyoxylate, both relatively innocuous compounds. The CP4 EPSPS alone makes soybean approximately 50-fold less sensitive to glyphosate (Nandula et al. 2007) (Fig. 3.2). CP4 and GOX genes together provide about the same level of resistance to canola (Nandula et al. 2007). Despite a generally high resistance factor, there can be problems if the transgene promoter is not sufficiently active in reproductive tissue, as appeared to be case in some of the first GR cotton varieties (Pline et al. 2002a,b; Pline-Srnic 2005; Yasuor et al. 2006; Dill et al. 2008). This problem was solved by using a promoter that is stronger in reproductive tissues. A similar, but lesser problem, has been reported in some maize varieties (Thomas et al. 2004). Under some environmental circumstances, glyphosate can cause leaf damage in GR soybean (e.g., Reddy and Zablotowicz 2003), although this is a rare occurrence from which the crop eventually recovers. The degradation product of glyphosate, AMPA, is a weak phytotoxin (Hoagland 1980). Reddy et al. (2004) correlated

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Fig. 3.2 Response of glyphosate-resistant (GR) Asgrow 4603RR and nonGR HBKC 5025 soybean in the one- to two-trifoliolate leaf (22-day-old, 45-cm tall) growth stage to glyphosatepotassium 3 weeks after treatment. Mean values of nine replications are plotted. From Nandula et al. (2007)

mild phytotoxicity in glyphosate-resistant (GR) soybeans to high levels of AMPA formed from high-glyphosate-dose applications. Applications of AMPA alone that resulted in the same endogenous AMPA concentrations caused the same level of phytotoxicity. Papers have been published supporting the view that glyphosate impairs growth of GR crops under some conditions because of effects on micronutrient status (e.g., Jolley et al. 2004; Bott et al. 2008). Glyphosate chelates divalent metal cations (Duke 1988), which could limit their availability when the glyphosate levels are very high and the divalent metal cation concentration is very low. Conversely, binding of Ca2+, Mn2+, or Fe2+ can reduce the efficacy of glyphosate (e.g., Schonherr and Schreiber 2004; Bernards et al. 2005). The pH can strongly influence how tightly each cation species is chelated to glyphosate. There have been exceedingly few reports of nutrient deficiencies in the huge areas in which GR crops are grown. Considering the very disproportionate abundance between the number of molecules of glyphosate that would reach the soil and the number of divalent cations available for binding, it seems implausible that glyphosate would have a significant effect on the availability of essential minerals in GR crops. However, if a plant had a borderline mineral deficiency, chelation in vivo might cause a problem. The proper experiments have not been conducted to determine if this possibility could cause a significant problem in the field. Ozturk et al. (2007) reported that glyphosate inhibits ferric reductase in plant roots, suggesting that this effect would cause

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iron deficiency in GR crops. However, this work was carried out with glyphosatesensitive sunflower and the assay was an in vivo assay performed hours after treatment with high concentrations of glyphosate so that there was no way to determine if the effects were direct or indirect. These studies should be repeated with GR crops to answer this question. Root-associated microbes are involved in plant mineral nutrition (e.g., Jakobsen et al. 2002). Some of these microbes are glyphosate-sensitive (e.g., Moorman et al. 1992). Some of the glyphosate applied to foliage is exuded by roots (e.g., Coupland and Caseley 1979; Laitinen et al. 2007). Thus, mineral nutrition could be altered by adverse effects on root-associated microflora. However, considering that nutrient deficiency problems have not been a commonly reported problem with GR crops after more than 10 years of adoption over vast areas, the potential mineral nutrition problems reported by only very few laboratories are of questionable impact in the field. Glyphosate is toxic to Bradyrhizobium japonicum (Moorman et al. 1992). Several studies have indicated that there is potential for reduced nitrogen fixation in GR soybeans, but yield reductions due to such an effect have not been documented in the field when glyphosate is used at the label rate (Zablotowicz and Reddy 2004, 2007). The no longer used bromoxynil-resistant crops owed their resistance to a transgene of microbial origin (Klebsiella ozaenae) that converts the benzonitrile to a nonphytotoxic benzoic acid derivative (Stalker et al. 1996). This gave the crops a more than tenfold level of resistance. Bromoxynil is an older category of selective herbicide that inhibits photosynthesis by binding the D1 protein of photosystem II (Devine et al. 1993). Glufosinate is a synthetic version of the natural product, phosphinothricin. It is not accepted by organic farmers, partly because it is chemically synthesized as a racemic mixture, and the D enantiomer of the racemic mixture is not a natural compound. It acts by inhibition of glutamine synthetase, thereby causing accumulation of toxic levels of ammonium ion and indirectly stopping photosynthesis (Lydon and Duke 1999). It is considered a broad-spectrum herbicide, but is not quite as effective in some situations as glyphosate. One of the microbes that produce phosphinothricin, Streptomyces hygroscopicus, has an enzyme that inactivates phosphinothricin and glufosinate by acylating it. The gene (bar) encoding this enzyme, phosphinothricin N-acetyltransferase (PAT), is used as a transgene for glufosinate-resistant crops (Vasil 1996). In addition to the HRC use, the bar gene has been used extensively as a selectable marker.

3.2.3

Impacts on Weed Management

Nothing has had more impact on weed management in such a short time period as GR crops, except perhaps the introduction of synthetic, selective herbicides. Several factors have contributed to the strong acceptance of GR technology. A strong

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argument can be made that glyphosate is the most effective and useful herbicide available (Duke and Powles 2008b). It is a slow-acting, highly translocated, foliarly applied product that kills weeds by inhibiting a molecular target site that is ubiquitous to all plants, EPSPS (Duke et al. 2003a). Its slow action allows translocation to meristematic tissues, ensuring that all growing points, including subterranean ones, are killed. It has very good environmental safety characteristics (see Sect. 3.2.5) in that it is highly nontoxic, does not easily move to ground or surface water, and has a relatively short half-life in soil. Before GR crops, it could only be used in places without crops or with methods that avoided crop contact. GR crops opened the door to widespread utilization of this exceptional herbicide directly on field crops. In addition to the generally profound economic advantages to using GR crop/ glyphosate technology (Gianessi 2005, 2008; Clewis and Wilcut 2007), this technology simplifies weed management (Bonny 2008). The farmer can often rely on only glyphosate applied once or twice during a growing season for weed control, rather than using a complicated strategy of both soil-incorporated and foliarly applied herbicides, involving multiple herbicides with different molecular target sites. Since glyphosate is used only as a postemergence herbicide, the farmer can wait to see what kind of weed problem emergences before spraying. Tillage can often be reduced or eliminated, creating both environmental (see below) and economic benefits. The benefits of this technology are not farm size dependent. A small farmer who farms only on weekends might derive more benefits, as this technology is more forgiving than traditional methods of weed management. Thus, one does not have to hire pest management consultants for prescription recommendations in order to obtain excellent weed control at a reasonable cost. This technology is also useful to farmers who grow multiple GRCs, in that they can apply one herbicide to multiple crops. Prior to GRCs, a particular herbicide could rarely be used on more than one crop. Initially, weed management with GR crop technology was excellent, as indicated by the rapid adoption. But, the specter of the evolution of GR weeds is jeopardizing reliance on this weed control method (Powles 2008a). Although Bradshaw et al. (1997) gave reasons why the evolution of GR resistance was implausible, the first report of an evolved GR weed occurred the same year as their paper (Heap 1997). Since then, the number of cases of evolved GR resistance has grown at a steady pace all over the world (Table 3.2, Fig. 3.3), and about half of the cases have occurred in GR crops, where the selection pressure is intense (Fig. 3.3). In most of the other cases, resistance evolved in vineyards or orchards in which glyphosate was sprayed several times a year for several consecutive years. The levels of evolved resistance of weeds to glyphosate are much lower than that of GR crops, usually in the range of two- to tenfold. The most common mechanism of resistance in these evolved biotypes is reduced translocation (e.g., Lolium spp., Conyza spp.) (Preston and Wakelin 2008), although mutations in EPSPS that provide marginal resistance have also occurred in Eleucine indica (Baerson et al. 2002) and some populations of Lolium spp. (Wakelin and Preston 2006; Perez-Jones et al. 2007). In at least some evolved GR Conyza, increased numbers of EPSPS transcripts may also contribute to resistance (Dinelli et al. 2008).

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S.O. Duke and A.L. Cerdeira Table 3.2 Occurrence of evolved GR weeds by species, year, and country Species Year Location Amaranthus palmeri 2005 USA Amaranthus rudis 2005 USA Ambrosia artemisifolia 2004 USA Ambrosia trifida 2004 USA. Conyza bonariensis 2003 South Africa 2004 Spain 2005 Brazil 2006 Columbia 2007 USA Conyza canadensis 2000 USA 2005 Brazil 2006 China, Spain 2007 Czech Republic Digitaria insularia 2006 Paraguay 2008 Brazil Echinochloa colona 2007 Australia Eleusine indica 1997 Malaysia 2006 Colombia Euphorbia heterophylla 2006 Brazil Lolium multiflorum 2001 Chile 2003 Brazil 2004 USA 2006 Spain 2007 Argentina Lolium rigidum 1996 Australia 1998 USA 2001 South Africa 2005 France 2006 Spain Parthenium hysterophorus 2005 Columbia Plantago lanceolata 2003 South Africa Sorghum halepense 2005 Argentina 2007 USA Urochloa panicoides 2008 Australia Source: From the International Survey of Herbicide-Resistant Weeds: http:// www.weedscience.org/In.asp and Vila-Aiub et al. (2008)

Nature abhors a vacuum. In addition to the evolution of GR weeds, when farmers rely on glyphosate year after year, other species of weeds can fill the ecological niches vacated by the species that are easily managed with glyphosate. The process of weed species shifts in GR crop fields has been documented (e.g., Reddy 2004; Owen 2008). Some of these species have a low level of natural (not evolved) resistance, and others can avoid glyphosate by germinating later in the growing season or having a broad germination pattern throughout the season. One potential mechanism of some species that are naturally resistant is enhanced conversion of glyphosate to AMPA (Reddy et al. 2008). The advent of evolved GR weeds and shifts to naturally GR or glyphosateavoiding weeds has made the GR crop/glyphosate weed management package less

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Fig. 3.3 Incidence of species that have evolved resistance to glyphosate by year

effective in some locales. Application rates and number of applications of glyphosate in GR crops have increased. Herbicides, in addition to glyphosate, are being used with GR crops. New herbicide-resistant crops are being developed to have resistance to glyphosate, plus resistance to another herbicide or class of herbicides that might be effective on weeds that are not controlled by glyphosate. Other options for coping with GR weeds and weed shifts are resistance management strategies that involve approaches such as alternation of herbicides, utilization of alternative/or residual herbicides in conjunction with glyphosate, and use of cultivation (e.g., Gustafson 2008; Neve 2008; Werth et al. 2008). The effects of HRCs on weed management is in a constant state of flux because of weed dynamics, economics, and changes in the availability of both old and new weed management tools. GR crops have been a valuable asset for weed management and will probably continue to be, even as GR weeds evolve and weed species shift. The effective longevity of this technology will depend, in part, on how wisely it is used (Powles 2008a).

3.2.4

Impacts on Food and Feed

One of the concerns of those opposing transgenic crops is that the transgene will alter the quality and/or safety of the consumable part of the plant. This could occur through the protein from the transgene being toxic, through a metabolic product of

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the enzyme encoded by the transgene being toxic, through pleiotropic effects of the transgene, through alteration of expression of nontransgenic genes by the position of the transgene in the genome or through more indirect effects. For regulatory approval, transgenic crops are scrutinized to a far greater level than conventional crops using analytical, nutritional, and toxicological methods (Atherton 2002; Malarkey 2003; Ko¨nig et al. 2004), although some have proposed that even more extensive tests be done by metabolomic, proteomic, and transcriptomic analysis to detect potential unintended effects of the transgene and its insertion on food safety and quality (Kuiper et al. 2002; Cellini et al. 2004). In a safety evaluation for the CP4 EPSPS enzyme introduced into soybean to provide glyphosate resistance, Harrison et al. (1996) found the protein to be: (1) nontoxic to mice when consumed at doses thousands of times higher than potential human exposure; (2) readily degraded by digestive fluids; and (3) not structurally or functionally related to any known protein allergens or toxins, based on amino acid sequence homology searches. The potential allergenic properties of the protein products of transgenes must be determined before approval. These data are provided to regulatory agencies, but publications on this topic are scarce. However, there are a few published studies showing no allergenic properties of transgene products associated with HRCs. Sten et al. (2004) in a study with soybean-sensitized patients, found that the allergenicity of 10 GR and eight nonGR soybean cultivars were not different. Chang et al. (2003) found no significant allergenicity to rats of the CP4 EPSPS gene product conferring glyphosate resistance. On the basis of both an analysis of published literature and experimental studies, Herouet et al. (2005) concluded that there is a reasonable certainty of no harm resulting from the inclusion of the gene for glufosinate resistance in human food or in animal feed. Most of the tests have simply examined HRCs for equivalence in food quality to nonHRCs. Health Canada’s review of the information, presented in support of the food use of refined oil from glufosinate-resistant canola line HCN92, concluded that such refined oil does not raise concerns related to safety. Health Canada is of the opinion that refined oil from canola line HCN92 is as safe and nutritious as refined oil from the current commercial varieties (http://www.hc-sc.gc.ca/foodaliment/mh-dm/ofb-bba/nfi-ani/e_nf7web00.html). The nutritional properties of glufosinate-resistant sugar beets and maize grains were found to be essentially equivalent to nontransgenic cultivars in feeding studies with swine and ruminants (Daenicke et al. 2000; Bohme et al. 2001); similar results have been produced with glufosinate-resistant rice in swine feeding studies (Cromwell et al. 2005). Studies with GR maize line GA21 evaluated the compositional and nutritional safety of maize line GA21 compared to that of conventional maize (Sidhu et al. 2000). Compositional analyzes were conducted to measure proximate, fiber, amino acid, fatty acid, and mineral contents of grain and proximate, fiber, and mineral contents of forage collected from 16 field sites over two growing seasons. No significant differences were found. Similarly, Tutel’ian et al. (2001) found no compositional differences between conventional maize and maize line GA21. The nutritional safety of maize line GA21 was also evaluated by Sidhu et al. (2000) in a

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poultry feeding study. Results from the poultry feeding study showed that there were no differences in growth, feed efficiency, adjusted feed efficiency, and fat pad weights among chickens fed with GA21 grain or with parental control grain. These data taken together demonstrate that GR GA21 maize is as safe and nutritious as conventional maize for food and feed use. The occurrence of mycotoxins, specifically aflatoxin, contamination is of concern with maize cultivation in warm climates subject to preharvest moisture stress. Studies by Reddy et al. (2007) indicated increased incidence of aflatoxin contamination in GR compared to conventional maize in 1 year out of four. But, in 1 year out of four, fumonisin levels were higher in nonGR maize than in GR maize. The observed effects were thought to be due to differences in how the crops were grown, rather than due to glyphosate or the transgene. Several other studies have found no substantial difference in the nutrient content of GR and nontransgenic crops. These studies include maize (Ridley et al. 2002; Autran et al. 2003), soybean (Padgette et al. 1996), wheat (Obert et al. 2004), and cotton (Nida et al. 1996). In the Autran et al. (2003) study, the characteristics of glyphosate- and glufosinate-resistant maize in different foods (e.g., beer, hominy, oil, grits) were compared and found not to be substantially different from the respective, nontransgenic parental lines. Lappe et al. (1999) reported reductions of isoflavone levels on GR soybean varieties in the absence of glyphosate (i.e., a pleiotropic effect of the CP4 gene). However, this study was not done by comparing isogenic lines. Padgette et al. (1996) found no effects of the transgene on isoflavone content of soybean. Glyphosate targets the shikimate pathway (Duke et al. 2003a), and the estrogenic isoflavones of soybeans are products of this pathway. Glyphosate resistance from the CP4 EPSPS gene is not always complete (Pline et al. 2002a,b), and glyphosate preferentially translocates to metabolic sinks such as seeds (Duke 1988). Therefore, we reasoned that at relatively high and late applications of glyphosate to GR soybeans, a reduction of the content of these compounds could occur. In a wellreplicated field study at two sites, hundreds of kilometers apart, no significant effects of glyphosate on isoflavones were found at the highest and latest legal application rates (Duke et al. 2003b). Table 3.3 summarizes most of the published results of animal feeding studies with GR crops. All the studies support the view that food from GR crops is substantially equivalent to nontransgenic crops. In addition to these studies, no evidence of the CP4 gene or its protein product could be detected in pork from swine fed with GR soybean meal (Jennings et al. 2003). No effects on GR soybeans could be found on the immune system of mice (Teshima et al. 2000). HRCs, being highly resistant to the herbicide to which they have been made resistant, could also contain levels of the herbicide or its metabolite(s) that exceed the legal tolerance levels. Surprisingly, little has been published on herbicide residues in HRC foods. Most of what we know is from studies with nonHRC crops. However, herbicide residue data must be supplied for regulatory approval of HRCs. Glyphosate acid and its salts are moderately toxic compounds in EPA toxicity class II. Glyphosate (either the anion or the isopropylamine salt) is practically

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Table 3.3 Some results of animal feeding studies with glyphosate-resistant crops Crop Animal Result Reference Maize Rat No effect Hammond et al. (2004) No effect Healy et al. (2008) Maize Swine No effect Hyun et al. (2004) Maize Cattle No effect Erickson et al. (2003) Maize Dairy cattle No effect Donkin et al. (2003) No effect Ipharraguerre et al. (2003) No effect Grant et al. (2003) Maize Poultry No effect Sidhu et al. 2000 Soybean Rat No effect Zhu et al. 2004 No effect Hammond et al. (1996) No effect Appenzeller et al. (2008) Soybean Mice No effect Brake and Evenson (2004) Soybean Swine No effect Cromwell et al. (2002) Soybean Dairy cattle No effect Hammond et al. (1996) Soybean Catfish No effect Hammond et al. 1996 Soybean Poultry No effect Hammond et al. (1996) No effect Taylor et al. (2007) Canola Rainbow trout No effect Brown et al. (2003) Canola Poultry No effect Taylor et al. (2004) Alfalfa cattle No effect Combs and Hartnell (2008) Sugarbeet Sheep No effect Hartnell et al. (2005)

nontoxic by ingestion, with a reported acute oral LD50 of >5,000 mg kg
1 in the rat (Vencill 2002). The trimethylsulfonium salt of glyphosate is more toxic, with an oral LD50 of about 705 mg kg
1. It is not a restricted use pesticide and is a bestselling weed killer for home use. Animals do not contain the herbicide molecular target site (EPSPS) of glyphosate. Occasional reports of severe effects of ingestion of formulated glyphosate occur (e.g., Stella and Ryan 2004); however, the glyphosate molecule itself is considered one of the most toxicologically benign herbicides available. Williams et al. (2000) extensively reviewed the toxicology literature on glyphosate and its metabolites and concluded that under present and expected conditions of use, glyphosate does not pose a significant health risk to humans. In a testing program to detect whether GR soybeans had been sprayed with glyphosate or not, Lorenzatti et al. (2004) found glyphosate and AMPA in green, immature seeds. Both glyphosate and its degradation product, AMPA, were found in mature, harvested seeds of different GR soybean varieties grown in widely separated geographical regions (Duke et al. 2003b). Even though the glyphosate applications were at legal, but at relatively high rates and late timing, the residues were within the established tolerance levels. We were surprised to find higher AMPA than glyphosate levels since at that time plants were thought to degrade glyphosate very little, if at all (Duke 1988; Duke et al. 2003a). Subsequently, we found that some legume plant species readily convert glyphosate to AMPA (Reddy et al. 2008). In a study conducted earlier, but published later (Arregui et al. 2004), similar levels of glyphosate were found in harvested seed of GR soybeans, but these

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scientists found much lower AMPA levels. We have found no publications on glyphosate residues in GR crops other than soybean. Glufosinate is not a restricted use pesticide and is sold for home weed control in the USA. Its acute oral toxicity in rats is an LD50 of ca. 2.2 g kg
1. Glufosinate chemically resembles glutamine, a molecule used to transmit nerve impulses in the brain. Ebert et al. (1990) concluded in an extensive review that glufosinate is safe under conditions of recommended use. Similarly, Hack et al. (1994) also concluded from their studies that glufosinate is unlikely to cause health effects of either users or consumers when used as directed, although the herbicide target site, glutamine synthetase, is also found in animals. In a study to determine if glufosinate applied to glufosinate-resistant maize and canola could lead to an increase in herbicide residues or to the formation of new metabolites, Ruhland et al. (2004) found that L-glufosinate was in the form of known metabolites and the parent compound in both maize and canola. The highest content was in the leaves, and the lowest in the grains. No levels were found above the established tolerance levels. A last, but understudied, aspect of food quality and HRCs is their influence on contamination of food with poisonous weed seeds. Weed seeds can be the sources of toxic compounds (e.g., Powell et al. 1990). HRCs are generally more weed-free than conventional crops, resulting in less foreign matter, including weed seeds, in the harvested product (Canola Council of Canada 2001; Shaw and Bray 2003). Therefore, there is less likelihood of significant contamination of harvested food with toxic weed seeds in HRCs than with conventional crops.

3.2.5

Environmental Impacts

There are numerous possible environmental effects of HRCs. These can be either positive or negative. They can be associated with the transgene or with the herbicide to which the transgene is linked. But, ultimately, potential environmental impacts of HRCs must be compared with the impacts of the technologies that they replace. All the published studies and analyses of this type have found that the environmental benefits of substituting HRCs for conventional crops are usually substantial (e.g., Wauchope et al. 2002; Nelson and Bullock 2003; Bennett et al. 2004; Amman 2005; Brimner et al. 2005; Brookes and Barfoot 2006; Cerdeira and Duke 2007; Cerdeira et al. 2007; Kleter et al. 2007, 2008; Devos et al. 2008; Gardner and Nelson 2008; Shiptalo et al. 2008). Of course, the potential benefits vary with the HRC, the geographic location of use, the way the farmer uses the HRC, and different components of environmental impact. Since nature is not static, the environmental impact will change with time as farmers using HRC technology adjust their methods to deal with changing weed and other problems. Whether HRCs have increased or decreased herbicide use in terms of the amount of material used per hectare of crop can be argued either way, depending on many factors. However, in terms of environmental impact this factor is not useful, as the

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relative toxicity and environmental fate of glyphosate or glufosinate compared with the herbicides that they replace in HRCs can be large. Glyphosate and glufosinate have relatively low mammalian toxicity (Ebert et al. 1990; Giesy et al. 2000; Williams et al. 2000), compared to many of the herbicides that they can replace when used with transgenic crops. Glyphosate, in particular, has lower acute toxicity than aspirin or many other commonly ingested compounds. In a study, using acute mammalian toxicity data, Gardner and Nelson (2008) compared the number of LD50 doses per unit area that were decreased by GR crops in the United States. Depending on the crop and the location, they calculated that conventional weed management with other synthetic herbicides could result in as much as 3,000 more LC50 doses per hectare with maize, more than 375 with cotton, and more than 90 with soybean. An example of some of their data with cotton is provided in Fig. 3.4. They concluded that GR technology has a positive environmental effect. In a European study, Devos et al. (2008) found that use of GR maize and glufosinate-resistant maize would lower the pesticide occupational and environmental risk or weed management. The main benefits were due to the lower potential of these chemicals to contaminate groundwater and their lower acute toxicity. Glyphosate gave slightly greater reductions than glufosinate. Their calculations were done with the assumption that other herbicides would not be used with glyphosate nor glufosinate. However, glyphosate is increasingly used with other

Fig. 3.4 Increase in LD50 doses per hectare of herbicides needed if GR cotton were not available in the US. From Gardner and Nelson (2008)

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herbicides in GR crops because of evolved glyphosate resistance and shifts to naturally resistant weed species. Neither glyphosate nor glufosinate are problematic pesticides in the context of surface and groundwater contamination of the herbicides that they replace (reviewed by Duke and Cerdeira 2005; Cerdeira and Duke 2006; Borggaard and Gimsing 2008). Glyphosate does not move well in soil because of its strong sorption to soil minerals (e.g., Mamy and Barriuso 2005). Furthermore, in most soils, it degrades more rapidly than the herbicides that it replaces (e.g., Mamy et al. 2005). In addition to microbial degradation, both glyphosate and AMPA can undergo nonbiological degradation in soil (Barrett and McBride 2005), although the relative contribution of the two types of processes is unknown. Concern over AMPA contamination of groundwater has recently been expressed (Mamy et al. 2005, 2008). AMPA is more persistent than glyphosate in the environment, and it more readily leaches in soil (Mamy et al. 2008; Simonsen et al. 2008). Both glyphosate and AMPA are leached from soil containing high phosphate levels than soil with low levels (Simonsen et al. 2008). The relative toxicities of AMPA versus contaminants from conventional herbicide use have not been adequately analyzed. Fuel utilization associated with weed management has been reduced by GR crops because of reduced tillage and fewer trips across the field to spray herbicides (reviewed by Cerdeira and Duke 2006). Bennett et al. (2004) estimated that there would be a 50% fossil fuel savings in growing sugar beet in Europe by switching to GR crops. Brookes and Barfoot (2006) estimated that GR crop use in 2005 worldwide reduced carbon emissions approximately the same as the removal of four million family automobiles from the road. Effects of the herbicides used with HRCs on nontarget organisms are a concern. Obviously, since glyphosate is a herbicide, glyphosate drift to nontarget plants can be harmful (e.g., Bellaloui et al. 2006). This is not a new problem, as there have been problems with herbicide drift since the advent of herbicides that are sprayed. Concentrations reaching nontarget plants are generally only a small fraction of the recommended dose for weed control. At very low doses that one might expect with drift, herbicides can often stimulate growth, activate host defense systems against pathogens, or enhance nitrogen utilization, depending on the herbicide (reviewed by Duke et al. 2006). The phenomenon of a stimulatory effect at subtoxic concentration of a toxin is termed hormesis. Glyphosate clearly enhances growth of plants at very subtoxic application rates (e.g., Schabenberger et al. 1999; Wagner et al. 2003; Cedergreen et al. 2007; Velini et al. 2008). But, at least in barley, the effect is not sustained over time (Cedergreen 2008). The mechanism(s) of herbicide-caused hormesis is poorly understood and may well differ between herbicides with different molecular target sites. Perhaps, the greatest damage done by conventional agriculture, other than taking land out of its natural state, has been caused by tillage. The primary reason for tillage has been weed management. Glyphosate use in GR crops has resulted in significantly reduced tillage, especially in soybean and cotton (Fig. 3.5) (American Soybean Association 2001; Penna and Lema 2003; Dill et al. 2008; Locke et al. 2008;

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Powles 2008b). Even in some nonGR crops, reductions in the price of glyphosate and the increases in the cost of diesel fuel have favored conservation tillage rather than traditional tillage (Nail et al. 2006). Reduced tillage sometimes reduces soil compaction (Koch et al. 2003; Shukla et al. 2003). To our knowledge, this aspect of the influence of HRCs has not been studied. However, as both evolved and naturally resistant GR weeds increase, some farmers of GRCs are returning to occasional tillage for more complete weed management. There is concern about the potential of HRCs to create new weed problems, either themselves becoming a weed or the HR transgene escaping to relatives, either feral crops or related species, to create new weed problems. Gene flow to native populations of species with which the HRC can crossbreed could result in unwanted agricultural and/or environmental effects. The HR transgene confers no advantage where the matching herbicide is not sprayed, so the HR crop is no more likely to invade a natural habitat than the nonHR crop. None of the crops that have been made herbicide resistant with transgenes are crops that become weeds outside of agricultural fields. However, HRCs are sometimes problems in agricultural fields in which the same herbicide is used in

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subsequent years with a different HRC. This has already been a problem with GR crops (e.g., York et al. 2004; Soltani et al. 2006; Beckie and Owen 2007), requiring the use of herbicides other than glyphosate to control the “volunteer” GR crop. Gene flow to nontransgenic crops of the same species has been a commercial and political problem, but not an environmental threat. Organic farmers cannot retain the organic status of their crops if transgene presence is above a set limit, nor can crops be sold to markets that require the product to be nontransgenic if transgenic occurrence is above the level set the regulatory jurisdiction. For crops like soybean, outcrossing is not a problem, but considerable outcrossing can occur with maize, rice, sugar beet, and canola. Substantial gene flow can occur between HR and nonHR canola (e.g., Hall et al. 2000; Reiger et al. 2002; Mallory-Smith and Zapiola 2008). Outcrossing may account for the contamination of nontransgenic rice with the glufosinate-resistant gene (Vermij 2006), even though glufosinate-resistant rice has only been grown experimentally. To our knowledge, no herbicide-resistant transgenes have been found to be a problem in nonherbicide-resistant maize, although there is significant potential for this to happen (Allnutt et al. 2008). There has been considerable controversy about whether Mexican maize landraces are contaminated with transgenes (reviewed by Cerdeira and Duke 2006). No gene flow from transgenic to nontransgenic cotton has been reported in cultivated fields, but it should occur because of insect pollination. Gene flow from GR alfalfa to organic alfalfa was the ostensible reason for its re-regulation. Flow of a GR transgene from bentgras being evaluated for commercial use to nontrangenic bent grass occurred rapidly, and mitigation efforts have not been effective (MallorySmith and Zapiola 2008; Zapiola et al. 2008). A much bigger concern is the potential effect of gene flow from HRCs to weedy relatives. The only environmental aspects of transgenic crops that are not also associated with nontransgenic crops are those associated with the transgenes. HR transgenes offer no advantage in natural ecosystems where the herbicide is not used, but when coupled with transgenes imparting traits that would improve fitness in a natural ecosystem (e.g., insect or drought resistance), the HR trait would improve the likelihood of introgression of the gene into the unintended recipient species (see below). Once a transgene escapes to another species, it is unlikely that it could be eliminated in the population by human efforts. Indeed, removal of a GR transgene from a nontransgenic population of bentgrass has not been accomplished with the mitigation methods used (Zapiola et al. 2008). Gene flow or introgression involves the movement of a gene or genes into a sexually compatible species. This type of gene flow is also termed vertical gene flow. The first generation is generally not very fit (e.g., Scheffler and Dale 1994), but subsequent backcrosses to the noncrop species will eventually fully introgress the transgene into this species. Yearly spraying of the herbicide to which the transgene confers resistance should facilitate the process by selecting only crosses with the transgene and removing competing weeds to give the unfit F1 and usually the F2 generations a significant survival advantage. Transmission of HR transgenes could make weedy relatives of the HRC much more problematic for the farmer. This has not happened with soybean, cotton, and

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maize, presumably because there are few or no weedy species with which they are sexually compatible in the places in which they are grown. Canola outcrosses with several weedy related Brassica species, including related Brassica crops (e.g., cabbage, cauliflower, broccoli, etc.). These crosses are generally quite unfit, but introgression of a GR transgene from canola to its weedy relative, bird rape (Brassia rapa) has occurred in field situation, and the introgressed gene appears to be stable in the population, even in the absence of spraying glyphosate (Warwick et al. 2008). A potential exists for a transgene to move from through compatible relative (a bridge species) to a third Brassica species to increase the means by which a GR gene could spread (Brown and Brown 1996). Work by Darmency et al. (2007) indicates that gene flow from sugar beets to weedy beets is sufficient to cause problems for the farmers growing HR beets within a few years if herbicides are not rotated. However, the degree of outcrossing that they measured varied considerably between lines of transgenic beets, years, and field locations. Rice outcrosses readily with feral rice (e.g., red rice), as well as weedy related species. Nontransgenic, imidazolinone (IMI) herbicide-resistant rice created by selection in cell culture (Tan et al. 2005) was commercialized in the USA in 2002. Outcrossing to produce IMI-resistant weeds occurs (e.g., Shivrain et al. 2007). The incidence of IMI-resistant weeds in rice is growing in locations where IMI-resistant rice is grown (Valverde 2007), although whether this is due to evolved resistance or gene flow is unclear in most cases, because IMI resistance evolves quickly in some species when exposed to this herbicide class (Heap 2008). We can expect that gene flow from rice to feral rices and to sexually compatible species will occur with any transgene, including those imparting herbicide resistance. Keeping HRCs out of areas in which sexually compatible species exist is a daunting task. For example, transgenic, GR and glufosinate-resistant canola are not approved as crops in Japan, but the seed can be imported for processing. Saji et al. (2005) found these HRCs growing along routes to processing plants in Japan. There is considerable literature on strategies and technologies for mitigating or eliminating vertical gene flow (reviewed by Gressel 2002; Cerdeira and Duke 2006). Much of this literature deals with simply reducing movement of the genes to plants other than the transgenic crop (e.g., Devos et al. 2005). However, if the gene would pose a potential problem in a natural environment, a fail-safe approach would be preferable. Strategies coupling one or more technologies such as genetic use restriction technology genes to make transmission of a functional transgene impossible (Oliver et al. 1998) or linking the HR transgene to one that would confer unfitness to a wild plant (e.g., genes that prevent shattering or dormancy; Al-Ahmad et al. 2004; Gressel and Al-Ahmad 2004) have the potential for fulfilling this need. In the latter approach, the HR gene(s) should be in the same construct as the unfitness gene(s) to keep the genes from segregating. Lastly, some have voiced concern that there could be gene flow from HRCs and other transgenic crops to totally unrelated organisms (horizontal gene transfer), especially soil or gut microbes (e.g., Bertolla and Simonet 1999; Giovannetti 2003), from degraded HRCs in the field or through ingestion of food composed of GRCs.

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Almost all of the transgenes currently used for HRCs are from soil microbes. These genes are much more likely to be transferred to unrelated microbes from the natural, microbial sources than from HRCs (Kim et al. 2005). Levy-Booth et al. (2008) found that degradation of the cp4-epsps transgene from GR soybean leaf material in the soil was rapid, but could still be detected in soil after 30 days. Degradation of the transgene and a natural soybean gene in soil were similar. Dale et al. (2002) concluded that there is no compelling argument that there would be any more likelihood of such gene transfer transgenic crops than from nontransgenic crops. There has so far been no credible evidence of gene transfer from transgenic crops to microbes (Dunfield and Germida 2004), although some have criticized detection methods (e.g., Nielsen and Townsend 2004). Nevertheless, after 14 years of growing these crops on huge areas, there have been no reports of transgenes being transferred to microbes of any type.

3.2.6

Herbicide-Resistant Crops and Crop Disease

There are some untended benefits of GR crops and perhaps glufosinate-resistant crops. Both glyphosate and glufosinate are fungitoxic (reviewed by Duke et al. 2007). Thus, when used at full application rates, these herbicides may, under some circumstances, be providing sufficient protection from plant pathogens to prevent crop damage or to preclude spraying with a fungicide. Perhaps, the most carefully studied example is that of glyphosate effects of Asian rust in GR soybeans (Feng et al. 2005, 2008), in which glyphosate applications to GR soybeans reduced rust infection and damage, both as a preventative and a curative (Fig. 3.6) treatment. However, in many field situations, the optimal timing of glyphosate application for effective weed management is unlikely to also be most effective for rust control (Bradley and Sweets 2008). The effects of glyphosate on reducing this disease will % ASR incidence (N=15)

a

b

Water 1DAI Fungicide A 1DAI Glyp1x 1DAI Glyp1x 3DAI Glyp1x 6DAI

60 40

Surf 1DAI Water 1DAI Glyp.5x 3DAI Glyp1x 3DAI Glyp2x 3DAI

60 40

20

20

0 0

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40

0

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10 20 30 Days After Inoculation

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Fig. 3.6 (a) Curative activity for Asian rust in glyphosate-resistant soybeans sprayed with glyphosate (Glyp, 1 at 0.84 kg AE ha
1) at 1, 3 or 6 days after inoculation (DAI), water and fungicide A (carbendazim + flusilazole) at 1 DAI. (b) Curative activity as a function of glyphosate dose (0.5, 1 or 2) from spray application at 3 DAI, surfactant (1 g L
1 MON 0818) or water alone at 1 DAI. From Feng et al. (2008)

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Table 3.4 Reports of glyphosate interactions and lack of interactions with plant disease in glyphosate-resistant crops Crop Disease Effect References Soybean Phakopsora pachyrhizi Reduces Feng et al. (2005, 2008) Fusarium spp Increases Kremer et al. (2005) S. sclerotiorum No effect Lee et al. (2003) Increases Nelson et al. (2002) F. solani Increases Sanogo et al. (2001), Nijiti et al. (2003) Cotton Rhizoctonia solani Reduces Pankey et al. (2005) Wheat Puccinia triticina Reduces Feng et al. (2008), Anderson and Kolmer (2005) Sugarbeet Rhizoctonia solani Increases Larson et al. (2005) Fusarium oxysporum Increases Larson et al. (2005)

probably increase as glyphosate use increases, both in application rates and number of applications, because of the evolution of GR weeds and weed species shifts to more naturally GR weeds. On the other hand, glyphosate treatment is known to predispose nontransgenic plants to plant disease by more than one mechanism associated with the mechanism of action of glyphosate as a herbicide (reviewed by Duke et al. 2007), although these mechanisms should not exist in GR crops. There are many reported cases of exacerbation of plant disease symptoms with glyphosate, but these studies have generally been done with glyphosate-susceptible plants (reviewed by Duke et al. 2007). Nevertheless, there are a few reports of this phenomenon in GR crops (Table 3.4). Likewise, there are reports of reduced crop disease with glyphosate (Table 3.4). Thus, the interaction of glyphosate, fungal plant diseases, and GR crops is variable, depending on the crop, the disease, and perhaps the timing of herbicide application and infection. Also, differences in cultural practices between HRCs and nontransgenic crops can influence plant disease (e.g., Lee et al. 2005). Glyphosate effects on root exudates and soil moisture can influence root-borne soybean diseases (Kremer et al. 2005; Means and Kremer 2007; Means et al. 2007). Larson et al. (2005) found the that several factors, including the disease isolate, whether or not the crop was sprayed with glyphosate, and the variety of GR sugar beet determined whether there was increased disease with the GR crop. In a recent review, Powell and Swanton (2008) concluded that there was insufficient information from realistic field studies to determine whether glyphosate influences Fusarium spp. diseases in GR crops.

3.3 3.3.1

Coming Herbicide-Resistant Crops Transgenes for Herbicide Resistance

Many transgenes have been reported and/or patented for the production of HRCs. Only a few of them are listed in Table 3.5. Technology is at a stage at which resistance to any herbicide can be imparted by transgene technology. Yet, only

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Table 3.5 Some of the transgenes that have been used for making crops resistant to herbicides or classes of herbicides Herbicide or herbicide class Gene source and gene product References 2,4-D Microbial degradation enzyme Llewellyn and Last (1996), Bisht et al. (2004) Asulam Resistant microbial dihydropteroate Surov et al. (1998) synthase Dalapon Microbial degradation enzyme Buchanan-Wollaston et al. (1992) Dicamba Microbial degradation enzyme Behrens et al. (2007), Herman et al. (2005) Hydroxyphenylpyruvate Microbial, herbicide-resistant HPPD Matringe et al. (2005) dioxidase (HPPD) inhibitors Paraquat Chloroplast superoxide dismutase Sen Gupta et al. (1993) Phenmedipham Microbial degradation enzyme Streber et al. (1994) Phytoene desaturase (PDS) Genes from microbes and the aquatic Sandmann et al. inhibitors weed Hydrilla encoding resistant PDS (1996), Arias et al. (2005) Protoporphyrinogen oxidase Resistant microbial PPO and resistant Li and Nicholl (2005) (PPO) inhibitors Arabidopsis thaliana PPO

three types of HRCs have been marketed; crops resistant to bromoxynil, glufosinate, and glyphosate, utilizing only five transgenes. Considering the huge economic impact of the GR crops, one would expect that other HRCs would have been marketed. Considerable effort and expense was put into development of HRCs that resist both hydroxyphenylpyruvate dioxygenase- and protoporphyrinogen oxidase-inhibiting herbicides (Li and Nicholl 2005; Matringe et al. 2005), yet the decision was apparently made to postpone or terminate plans to commercialize these HRCs. Devine (2005) attributed the few HRCs making it to the market place to the high cost of developing and getting regulatory approval of HRCs. Further complications are international trade issues and the impact on the existing registration of the herbicide to which they are made resistant. Realistically, none of the herbicides on the market to which crops can be made resistant offer the advantages of glyphosate (Duke and Powles 2008b) or perhaps even glufosinate, both relatively safe, broad-spectrum herbicides. In addition to the few number of herbicides used (only two), the number of crops represented among all the currently available HRCs is few (Table 3.1). This is partly because the cost of obtaining approval of HRCs is considered too great for minor crops (Devine 2005). However, this does not explain the absence of GR wheat and rice and glufosinate-resistant wheat, where the market could be very large, worldwide. GR wheat was almost marketed in North America, but concern over acceptance of wheat products from this source in Europe prevented it being marketed. A similar concern slowed the acceptance of GR sugar beets until 2008. The horrendous problems with outcrossing of rice with weedy feral and other species of may be related to GR rice being unavailable.

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Table 3.6 Recently deregulated HRCs (http://www.aphis.usda.gov/brs/not_reg.html) that are not yet on the market and HRCs that have recent approval for field-testing in the USA (http://www.isb. vt.edu/cfdocs/fieldtests1.cfm) Crop Herbicide(s) Company Resistance gene(s) Petition for deregulation Glyphosate acetyltransferase and a modified Soybeana Glyphosate and ALS Pioneer inhibitors soybean ALS Cotton Glyphosate and Bayer Crop Maize EPSPS modified by site-directed glufosinate Sci. mutagenesis and bar gene Maize Glyphosate and ALS Pioneer Glyphosate acetyltransferase and a modified inhibitors maize ALS Approval for field-testing Alfalfa Glyphosate/ sulfonylureas Soybean Glyphosate/dicamba Glufosinate/dicamba Glyphosate Glyphosate/isoxazole Not available Glufosinate Not avaiable Maize Glyphosate Bentgrass Glufosinate

Pioneer Monsanto Monsanto Pioneer MS

Glyphosate acetyltransferase and a modified plant ALS CP4 EPSPS and a demethylase CP4 EPSPS and a demethylase Glyphosate acetyltransferase Technologies

MS Pioneer HybriGene, LLC

Technologies Glyphosate acetyltransferase Phosphinothrichin acetyl transferase

a

Deregulation approved

Many types of HRCs have gotten to the point of being field-tested, but few have proceeded to be deregulated (approved for commercial use). Table 3.6 lists products for which deregulation has been applied, as well as recently approved applications for field-testing in the USA. This information on the USDA/APHIS website provides the best indication of what new products are in the offing. Considering that many of these crops have glyphosate resistance, we consider these products separately.

3.3.2

Glyphosate-Resistant Crops

Considering the enormous success of GR crops and the fact that glyphosate is now a generic herbicide, other companies have discovered or created new glyphosateresistant transgenes and, in at least one case, are going forward with the commercialization process. This crop uses an artificially evolved glyphosate-resistant gene (Castle et al. 2004; Siehl et al. 2005). A gene from the soil bacterium Bacillus licheniformis, which encoded a weak glyphosate N-acetyltransferase (GAT) was put through 11 iterations of gene shuffling to increase its activity by almost four orders of magnitude. Properties of the resultant GAT are described by Siehl et al. (2005, 2007). Plants made resistant to glyphosate with this transgene were

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ca. 100-fold more resistant to glyphosate than to nontransgenic lines (Green et al. 2008). Other glyphosate-inactivating enzymes are apparently encoded by genes of soil microbes, because there are other routes of degradation or inactivation. For example, a C-P lyase that converts glyphosate to inorganic phosphate and sarcosine is found in several bacteria, including Arthrobacter spp., Rhizobium spp., and Pseudomonas spp. (Kishore and Jacob 1987; Liu et al. 1991; Dick and Quinn 1995). Not all bacterial C-P lyases will degrade glyphosate (White and Metcalf 2004). A good C-P lyase transgene for use in GR crops has apparently not yet been developed. A microbial transgene-encoded EPSPS with some properties that might be superior to that used in commercialized GR crops is available (Vande Berg et al. 2008). Thus, transgenes for glyphosate resistance are available for companies that do not have GR crops.

3.3.3

Other Herbicide-Resistant Crops

Crops with both the GAT gene and a gene for resistance to acetolactate synthase inhibitors are in the final stage of development (Green et al. 2008), including testing for food safety (Table 3.6) (McNaughton et al. 2007). Monsanto is developing a dicamba (3,6-dichloro-2-methoxybenzoic acid) resistance trait (Table 3.6) that detoxifies this herbicide by demethylating it (Herman et al. 2005; Behrens et al. 2007). Dow Agroscience has developed HRCs with resistance to broadleaf-killing auxinic herbicides like 2,4-D [(2,4-dichlorophenoxy)acetic acid] and grass-killing aryloxyphenoxypropionate herbicides such as diclofop (()-2-[4-(2,4-dichlorophenoxy) phenoxy]propanoic acid). These crops are apparently being field-tested, but there is no specific information on the APHIS website as to what genes are being used. Thus, we have left them out of Table 3.6. A recent abstract mentions that a transgene encoding an a-ketoglutarate-dependent dioxygenase is used to confer resistance to these two herbicide classes (Simpson et al. 2008). A patent had been filed for such a unique bacterial (Ralstonia eutropha)-derived transgene by Dow Agroscience in 2005 (Wright et al. 2005).

3.4

The Future of HRCs

The future of HRCs will be influenced by the development of other approaches to weed management. Availability of acceptable new technologies that are economically competitive with HRCs may eventually stem their rapidly increasing adoption. There are other strategies for using transgenes for weed management (Gressel 2002; Duke 2003, 2006) including enhancing crop competitiveness and allelopathy, and enhancement of weed biocontrol agents. There is little likelihood of these approaches having a significant effect in the next decade, but ultimately they could have substantive impacts.

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To some extent, regulatory requirements regarding environmental impacts of weed management methods will affect this scenario. The increasing costs of meeting the requirements for deregulation of transgenic crops and the conflicting regulatory processes in different parts of the world have hampered the adoption of HRCs. Eventual harmonization of this process could benefit this technology. There are many other factors that could have an impact on the future of HRCs, such as the amount of biofuel crops that will be grown (Baylis 2008). Since these crops are not meant for human consumption, and, in some cases, could not be used for human or animal consumption, the regulatory requirements for deregulation of HRC biofuel crops may be simplified and less costly to fulfill. Indeed, GR sugarcane is being developed in Brazil (http://monsanto.mediaroom.com/index. php?s = 43&item = 656). Still, the issue of herbicide resistance management and gene flow will be as important for these crops as for those meant for human and livestock consumption. Agriculture has clearly found HRCs to be of great value, but their utility is being eroded by the evolution of GR weeds and weed species shifts in GRCs. Both of these processes are the result of over reliance on this highly effective technology. Some of the new HRCs that are to be introduced will help cope with this situation, but we see overreliance on glyphosate continuing to undermine its effectiveness. The unique properties of glyphosate as a herbicide (Duke et al. 2003a; Duke and Powles 2008b), almost necessitate that its utility be preserved for future harvests (Duke and Powles 2008a; Powles 2008a). It will be shame to see the evolution of GR weeds continue unabated, as the strategies for preventing it are well known. Finally, the future of HRCs has been and may continue to be affected by public opinion. For example, transgenic crops have made meager inroads in Europe for this reason. Even though Europeans grow almost no transgenic crops, they buy substantial amounts of transgenic crops grown elsewhere for animal feed. At the time of this writing, this situation does not appear to be changing significantly. In countries such as Australia and those of South America, resistance to transgenic crops has waned considerably and adoption is increasing rapidly. We expect available HRCs to be almost universal outside of Europe and perhaps a few small areas of resistance within a few more years.

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modified corn line MON 810 resistant to European corn borer and line GA 21 resistant to glyphosate: a chemical study. Voprosy Pitaniia 70:25–27 Valverde BE (2007) Status and management of grass-weed herbicide resistance in Latin America. Weed Technol 21:310–323 Vande Berg BJ, Hammer PE, Chun BL, Schouten LC, Carr B, Guo R, Peters C, Hinson TK, Beilinson V, Shekita A, Deter R, Chen Z, Samoylov V, Bryant CT, Stauffer ME, Eberle T, Moellenbeck DJ, Carozzi NB, Koziel MG, Duck NB (2008) Characterization and plant expression of glyphosate-tolerant enolpyruvylshikimate phosphate synthase. Pest Manag Sci 64:340–345 Vasil IK (1996) Phosphinothricin-resistant crops. In: Duke SO (ed) Herbicide-resistant crops. CRC Press, Boca Raton, FL, pp 85–91 Velini ED, Alves E, Godoy MC, Meschede DK, Souza RT, Duke SO (2008) Glyphosate applied at low doses can stimulate plant growth. Pest Manag Sci 64:489–496 Vencill WK (ed) (2002) Herbicide Handbook, 8th edn. Weed Science Society of America, USA, p 493 Vermij P (2006) Liberty Link rice raises specter of tightened regulations. Nat Biotechnol 24:1301–1302 Vila-Aiub MM, Vidal RA, Balbi MC, Gundel PE, Trucco F, Ghersa CM (2008) Glyphosateresistant weeds of South American cropping systems. Pest Manag Sci 64:366–371 Wagner R, Kogan M, Parada AM (2003) Phytotoxic activity of root absorbed glyphosate in corn seedlings (Zea mays L.). Weed Biol Manag 3:228–232 Wakelin AM, Preston C (2006) A target-site mutation is present in a glyphosate-resistant Lolium rigidum population. Weed Res 46:703–705 Warwick SI, Legere A, Simard M-J, James T (2008) Do escaped transgenes persist in nature? the case of an herbicide resistant transgene in a weedy Brassica rapa population. Mol Ecol 17:1387–1395 Wauchope RD, Estes TL, Allen R, Baker JL, Hornsby AG, Jones RL, Richards RP, Gustosfson DI (2002) Predicted impact of transgenic, herbicide tolerant corn on drinking water quality in vulnerable watersheds of the mid-western USA. Pest Manag Sci 58:146–160 Werth JA, Preston C, Taylor IN, Charles GW, Robets GN, Baker J (2008) Managing the risk of glyphosate ressistance in Australian glyphosate-resistant cotton production systems. Pest Manag Sci 64:417–421 White AK, Metcalf WW (2004) Two C-P lyase operons in Pseudomonas stutzeri and their roles in the oxidation of phosphonates, phosphite, and hypophosphite. J Bacteriol 186:4730–4739 Williams GM, Kroes R, Munro IC (2000) Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regul Toxicol Pharmacol 31:117–165 Wright TR, Lira JM, Merlo DJ, Hopkins N (2005) A bacterial gene for an aryloxyalkanoate dioxygenase conferring resistance to phenoxy auxin and aryloxyphenoxypropionate herbicides. Patent Application WO2005US1437 20050502 Yasuor H, Abu-Abied M, Belausov E, Madmony A, Sadot E, Riov J, Rubin B (2006) Glyphosateinduced anther indehiscence in cotton is partially temperature dependent and involves cytoskeleton and secondary wall modifications and auxin accumulation. Plant Physiol 141:1306–1315 York AC, Steward AM, Vidrine PR, Culpepper AS (2004) Control of volunteer glyphosateresistant cotton in glyphosate-resistant soybean. Weed Technol 18:532–539 Zablotowicz RM, Reddy KN (2004) Impact of glyphosate on the Bradyrhizobium japonicum symbiosis with glyphosate-resistant transgenic soybean: a mini review. J Envron Qual 33:825–831 Zablotowicz RM, Reddy KN (2007) Nitrogenase activity, nitrogen content, and yield responses to glyphosate in glyphosate-resistant soybean. Crop Prot 26:370–376 Zapiola ML, Campbell CK, Butler MD, Mallory-Smith C (2008) Escape and establishment of transgenic glyphosate-resistant creeping bentgrass Agrostis stolonifera in Oregon, USA: a 4-year study. J Appl Ecol 45:486–494 Zhu Y, Li D, Wang F, Yin J, Jin H (2004) Nutritional assessment and fate of DNA of soybean meal from Roundup Ready or conventional soybeans using rats. Arch Anim Nutr 58:295–310

Chapter 4

Understanding and Manipulation of the Flowering Network and the Perfection of Seed Quality Stephen L. Goldman, Sairam Rudrabhatla, Michael G. Muszynski, Paul Scott, Diaa Al-Abed, and Shobha D Potlakayala

4.1

Introduction

In spite of an impressive 2,263 metric tons reported for cereal production in 2004, sustainable output has not kept pace with global population increases. By 2020, the world’s population is expected to exceed 8 billion and the projected minimum annual 1.3% output increase needed to feed the population is not likely to be met. Complicating the problem further is that the projected expansions are not expected to be distributed evenly. Among the Asian Countries, India could become the most densely populated country in the world. Although India is second to the US in arable acreage, food is already being imported. As reported by Saritha Rai in the New York Times (2006), India has imported 2.2 million tons of wheat. This has been attributed to global climate change resulting in decreases in rainfall in the arid/subhumid zones of the country. Temperature increases and significant alterations in rainfall pattern are expected to acerbate in the absence of global enforceable environmental laws designed to mitigate sustainability while doing no harm to the biosphere. Similar problems are expected to plague the Philippines and Indonesia. The world’s governments can no longer look at agricultural production statistics on a country-by-country basis as though the resolution of hunger is confined within national borders. Although the largest population increases are expected in Asia, the highest percent rise falls in Africa. The number of people is expected to multiply at an annual rate of three percent accompanied by significant demographic changes as people move from rural areas to the city, thus decreasing farm output. By 2020, Africa alone is projected to require 60 million additional metric tons from import suppliers.

S. Rudrabhatla (*) Environmental Engineering, College of Science, Engineering and Technology, Pennsylvania State University, Middletown, PA 17057, USA e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_4, # Springer-Verlag Berlin Heidelberg 2010

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Where are these food sources to be found? India is already importing cereal and its demand will increase over the next two decades. Moreover, China, the world’s most populous country cannot sustain its growing population, since it has only 7% of the globe’s arable land. Already America is experiencing the tension resulting from an insufficient amount of corn where the interests of energy production compete with the interests of the cereal’s use as food, forage, and commercial additive. This tension is predictable as energy demand has resulted in historic per bushel prices exceeding US$7.00, with significant increases in food cost. Placing more land in production has its drawbacks, especially if increased cultivation results in global loss with respect to biodiversity. In this connection, threats to deceases in biodiversity with respect to land-use change have already resulted in successful legal challenges. Given the aforementioned facts, it is likely that the most immediate relief to projected global food shortages will come not only as a function of increased seed quality (either through standard breeding or transgenic production), but also as a function of being able to manipulate flower development. Indeed, new plants will have to be developed whose time-to-flower cycle is altered, thus mitigating challenges to both pollen production and fertilization associated with increased temperature and diminished water supplies. The ability to alter development by changing flowering time, life cycle length or, for that matter, where a plant is cultivated should result not only in significant increases in the availability of land usage per se, but also in production. If these alterations are likewise accompanied by improvements in seed quality as measured by protein or fat content, for example, positive movement is made towards the requisite goal of feeding people globally. All aspects of flowering must be dissected and understood if its mechanism(s) is(are) to be manipulated toward this end, and it is to this understanding that this review is dedicated.

4.2 4.2.1

The Regulation of Flowering The Floral Transition

The production of grain crops depends on the successful union of gametes at an optimal time to ensure plentiful generation of seed for harvest. The production and union of fertile gametes occur at flowering, which is a critical event in the life cycle of all higher plants. In order to flower, plants must first transition from vegetative to reproductive growth. During vegetative development, the shoot apical meristem (SAM), a population of totipotent cells at the growing point of the plant, gives rise to leaves and other above-ground organs. To switch to reproductive growth, the SAM ceases leaf production and becomes committed to the production of reproductive structures, such as an inflorescence bearing flowers (Fig. 4.1). The period when the SAM is reprogrammed is called the floral

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Fig. 4.1 The floral transition in maize, when the vegetative shoot apical meristem (left) ceases leaf production and adopts a reproductive fate (right). Scale bar is 200 mM

transition and the timing of the transition largely determines when a plant flowers. Flowering time is often measured by counting the number of leaves produced prior to the transition of the SAM and, thus, is a direct measure of the timing of the transition. Accordingly, late flowering varieties produce more leaves than early flowering varieties, as the transition is delayed and the SAM remains in the vegetative stage of growth for a longer period of time. The timing of the floral transition has been manipulated by plant breeders for centuries. Elite varieties have been selected to balance the extent of vegetative growth with the duration of grain fill to maximize yield in their adapted geographic locations. Identifying key regulators of the floral transition and understanding their functions will allow for the development of varieties improved for productivity and yield within a rapidly changing environment. Higher plants have developed sophisticated genetic mechanisms to ensure that flowering coincides with an optimal time for reproductive success (Blazquez and Weigel 2000; Hayama and Coupland 2003; Izawa et al. 2003; Komeda 2004; Putterill et al. 2004; Simpson et al. 2004). Previous physiological studies revealed that a combination of various environmental inputs and endogenous cues affect the timing of the transition. For many species, day length is the predominant input, while other species are day-neutral and rely almost entirely on endogenous signals. These early studies also established several relevant points regarding the floral transition (Bernier and Perilleux 2005). First, the inductive flowering signal originates in leaves, whether the transition is triggered by external or endogenous cues. Second, the inductive signal, often referred to as florigen (F), is mobile and is transmitted from the leaves through the phloem to the shoot apex. Lastly, the SAM, the target of the inductive signal in the shoot apex, must be competent to perceive the signal in order to transition. Thus, we know that leaves and the shoot apex are key tissues involved in the initiation, propagation and perception of floral signaling.

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The Floral Transition Network

At present, the molecular mechanisms that regulate the floral transition have been primarily elaborated in the model plant Arabidopsis thaliana using the wealth of flowering time mutants available in that species. Proper timing of the transition is controlled by signaling through a complex network of floral inductive and repressive activities. These activities function through four main regulatory pathways: autonomous, photoperiod, gibberellin and vernalization (Koornneef et al. 1998; Mouradov et al. 2002; Simpson and Dean 2002). The four regulatory pathways converge on the following key floral integrators; Leafy (LFY), Suppressor of Overexpression of Constans (SOC1), Flowering Locus T (FT) and Flowering Locus D (FD) (Lee et al. 2000; Samach et al. 2000; Abe et al. 2005; Michaels et al. 2005; Parcy 2005; Corbesier et al. 2007). From these integrators, the flowering signals are transmitted to early-acting floral meristem identity MADS-box genes Apetala1 (AP1), Cauliflower (CAL) and Fruitful (FUL) that promote formation of inflorescence meristems which ultimately produce flowers (Fig. 4.2; Yanofsky 1995; Ferra´ndiz et al. 2000). Less is known about what controls the floral transition in the cereals, but a growing body of evidence suggests some of the floral regulators and their signaling modules are conserved in Oryza sativa (rice), Triticum aestivum (wheat) and Zea mays (maize) (Table 4.1; Danyluk et al. 2003; Hayama et al. 2003; Izawa et al. 2003; Izawa 2007; Yan et al. 2003, 2006; Muszynski et al. 2006; Adam et al. 2007; Chengxia and Dubcovsky 2008; Danilevskaya et al. 2008). The genes known or presumed to regulate flowering time in the major cereal crops are listed in Table 4.1. The core photoperiod regulatory module in Arabidopsis – GIGANTEA (GI), CONSTANS (CO) and FT – has been shown to be conserved in rice; although, under long-day photoperiods, rice FT expression is suppressed, leading to suppression of flowering, which is the opposite regulation of FT in Arabidopsis (Fig. 4.2; Yano et al. 2000; Hayama et al. 2002; Kojima et al. 2002). Signaling downstream of FT to the floral meristem identity genes also appears conserved among Arabidopsis, rice, wheat and maize (Lim et al. 2000; Lee et al. 2003). Although a number of studies show that diverse plant species share parts of the regulatory pathways defined in Arabidopsis, whether all the components of these regulatory pathways exist in other plants is not apparent. Furthermore, for those components that are conserved, how they integrate within the larger network is unknown. Further analysis of floral regulatory mechanisms in several species is required to define common pathways as well as uncover unique regulatory elements that may have evolved to accommodate particular environmental conditions and species-specific physiologies. For example, although both Arabidopsis and rice predominantly rely on photoperiod to regulate the timing of the transition flowering is promoted in Arabidopsis by long days and in rice by short days. Unlike rice, many temperate cereals, such as wheat, are long-day plants that also require vernalization, a period of prolonged cold temperature, to promote flowering. Conversely, although domesticated from teosinte, an obligate short-day species, temperate maize is largely photoperiod insensitive and relies primarily on endogenous

4

Understanding and Manipulation of the Flowering Network Autonomous

Photoperiod Response Arabidopsis (LD) circadian clock LEAF

Rice (SD)

Maize (DN) RID1

id1

GI

OsGI

CO

Hd1

(F)

FT

Hd3a

ZCN8(?)

FD + FT

OsDlf1(?) + Hd3a

LFY APEX

171

dlf1 + ZCN8(?) x

AP1/CAL/FUL

OsMADS14/18(?)

zfl1, zfl2(?) ZMM4, ZMM15

Fig. 4.2 The floral transition networks in Arabidopsis (left), rice (center) and maize (right). Under inductive photoperiods (long day for Arabidopsis and short day for rice), the circadian clock drives expression of key leaf-expressed flowering time genes (GI and CO or OsGI and Hd1). These activate the downstream mobile floral stimulus FT or Hd3a which moves from the leaves through the phloem to the shoot apex. In the shoot apex, the mobile floral stimulus interacts with the floral inductive gene FD or OsDlf1(?) to activate the floral meristem identity MADS-box genes AP1/ CAL/FUL or OsMADS14/18(?). In Arabidopsis, LFY integrates inductive signals from the leaf to the floral meristem identity genes in a parallel pathway. In rice, the LFY ortholog does not appear to participate in floral inductive signaling; but RID1, an id1 ortholog, has been shown to control initiation of floral induction. In temperate maize, which is relatively day-neutral, an autonomous signal induces expression of id1 in leaves that regulates a florigenic factor (F). Signals downstream of F (perhaps ZCN8, a possible maize FT ortholog) are transmitted to the shoot apex and regulate dlf1 expression or DLF1 activity. Interaction of DLF1 and ZCN8 presumably activates downstream targets x and the ZMM4 and ZMM15. Redundant inductive signaling through an id1independent alternate pathway converges downstream of dlf1. The position of genes marked by (?) is speculative, hypothetical interactions are marked by dotted lines and the alternate pathway in maize is colored blue

cues to flower. The molecular nature of the endogenous signaling pathways is not known but some parts of the maize flowering network are shared with other cereals and Arabidopsis (Table 4.1). The succeeding section describes what is known about the floral transition regulatory network in maize and how the network components relate to components from other species.

4.2.3

Flowering in Maize

Growth of maize (Zea mays) is largely determined by the activity of the SAM (Fig. 4.1). During the first 3–4 weeks after germination, the SAM produces vegetative organs, such as leaves and stem tissue. After about 4–5 weeks of growth, the

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Table 4.1 Genes regulating flowering time in the major cereal crops; maize, rice and wheat Similarity to Reference Gene/locus Activitya Protein homology Arabidopsisb Maize Constans of Zea mays1 UNK B-box zinc finger CONSTANS (CO) Miller et al. (conz1) (2008) Delayed flowering1 P Basic-leucine FLORAL LOCUS D Muszynski et al. (dlf1) zipper (bZIP) (FD) (2006) Gigantea of Zea mays1 UNK Nuclear protein GIGANTEA (GI) Miller et al. (gigz1A and B) (2008) Indeterminate1 (id1) P C2H2 zinc finger None Colasanti and Sundaresan (2000) Phytochrome B (phyb) R Phytochrome PHYTOCHROME B Sheehan et al. (2007) Zea FLORICAULA/ P DNA-binding LEAFY (LFY) Bomblies et al. LEAFY (zfl1 and protein (2003) zfl2) Zea mays UNK PhosphatidylFLORAL LOCUS T Danilevskaya CENTRORADIALIS ethanolamine (FT) and et al. (2008) (ZCN) binding TERMINAL protein FLOWER (TFL) Zea mays MADS-box4 P MIKC-type APETALA1 (AP1), Danilevskaya (ZMM4) MADS-box CAULIFLOWER et al. (2008) protein (CAL) and FRUITFUL (FUL) ZmRap2.7 R AP2/ethyleneTARGET OF EAT1 Salvi et al. (2007) responsive (TOE1) elementbinding protein Rice Early heading date1 (Ehd1) GRAIN number, plant height and heading date7 (Ghd7) Heading date1 (Hd1)

P R

P

Heading date3a (Hd3a) P

Heading date6 (Hd6)

R

OsGI

P

OsMADS14

P

OsMADS51

P

B-type response None regulator Zinc-finger CCT None domain protein B-box zinc finger CO PhosphatidylFT ethanolamine binding protein a-subunit protein CK2 kinase Nuclear protein GI MIKC-type MADS-box protein Type I MADSbox protein

AP1/CAL/FUL

Doi et al. (2004)

Yano et al. (2000) Kojima et al. (2002)

Takahashi et al. (2001) Hayama et al. (2002) Jeon et al. (2000)

None (continued )

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Table 4.1 (continued) Gene/locus OsSOC1 (OsMADS50)

Rice FLO-LFY (RFL) Rice Id1 (RID1) Photoperiodic sensitivity5 (se5)

Activitya Protein homology P MIKC-type MADS-box protein P DNA-binding protein P C2H2 zinc finger

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Similarity to Arabidopsisb SOC1

Lee et al. (2003)

LFY

Rao et al. (2008)

none

Wu et al. (2008a,b) Izawa et al. (2000)

R

Heme oxygenase HY1

Wheat TaFDL2

P

Basic-leucine FD zipper (bZIP)

TaFT (VRN3)

P

Reference

Chengxia and Dubcovsky (2008) Yan et al. (2006)

PhosphatidylFT ethanolamine binding protein TaVRN1 P MIKC-type AP1/CAL/FUL Yan et al. (2003) MADS-box protein TaVRN2 R Zinc-finger CCT None Yan et al. (2004) domain protein TaVRT2 R MIKC-type SHORT Kane et al. MADS-box VEGETATIVE (2007) protein STAGE3 (SVP3) a Gene activity has been shown to either promote (P) or repress (R) flowering or has not yet been determined (UNK) b Similarity to Arabidopsis flowering time genes

SAM switches from vegetative over to reproductive growth during the period of the floral transition. This period is distinguished in the SAM by the cessation of leaf initiation and its rapid increase in size (Shaver 1983; Irish and Nelson 1991). The SAM continues to increase in size as it progresses through the floral transition, which terminates with the SAM acquiring inflorescence identity to become the tassel primordium (Fig. 4.1). In maize, the tassel is the apical inflorescence, which bears the male flowers that produce pollen. A similar transition occurs later in time, leading to the conversion of several axillary meristems into ear primordia. In maize, the ear is the axillary inflorescence, which bears the female flowers that exsert silks (elongated pistils). Flowering in temperate maize is largely controlled by endogenous signals, possibly involving plant size or leaf number (Irish and Nelson 1991). Much of our understanding regarding the molecular regulation of flowering in maize comes from the analysis of genetic variants with altered flowering time. Three single gene late-flowering mutants are described in maize; the recessive indeterminate1 (id1) and delayed flowering1 (dlf1) mutations, and the dominant Leafy (Lfy) mutation.

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All three mutations delay the floral transition and therefore we expect functional id1 and dlf1 to promote flowering, while normal Lfy function is difficult to deduce (Shaver 1983; Neuffer et al. 1997; McSteen et al. 2000). The id1 mutants flower extremely late, producing many more leaves and flowering many weeks later than normal. Additionally, id1 mutants often have aberrant morphology, such as the absence of ears (Galinat and Naylor 1951). The id1 gene has been cloned and encodes a unique type of Zn-finger transcription factor, defined by the ID domain (IDD). id1 is specifically expressed in immature leaf tissue and has been suggested to control a florigenic signal (Colasanti et al. 1998; Colasanti and Sundaresan 2000). Subsequent analysis showed that the ID1 protein localizes to nuclei and binds DNA suggesting it regulates the transcription of downstream flowering-time genes (Kozaki et al. 2004). Furthermore, ID1 protein accumulates soon after germination and high protein levels are detected in discrete regions of immature leaves up to the time of the floral transition, after which time levels decline (Wong and Colasanti 2007). id1 is unique to grasses and a putative homolog was not found in Arabidopsis suggesting id1 participates in a monocot-specific pathway (Colasanti et al. 2006). Recently, a rice ortholog of id1, RID1, was identified that was proposed to control the initiation of floral induction and appears to regulate multiple inductive pathways (Wu et al. 2008a,b). Plants lacking RID1 activity never flower and remain in a perpetual vegetative state of growth. The other recessive late-flowering mutation, dlf1, has a more moderate late-flowering phenotype, with mutant plants producing a modest number of additional leaves and flowering 10–14 days later than normal (Neuffer et al. 1997; Muszynski et al. 2006). The dlf1 gene was cloned recently and encodes a basic leucine-zipper (bZIP) homologous protein that resembles the Arabidopsis floral integrator FD (Muszynski et al. 2006). Similar to FD, dlf1 is expressed in shoot apices, peaking in expression near the time of the floral transition. dlf1 was shown to function downstream of id1, thereby defining an id1-dlf1 floral inductive module (Fig. 4.2). To date, an id1-dlf1 homologous flowering module has not been described in rice or other grass species, although an apparent dlf1 ortholog was identified in wheat (Chengxia and Dubcovsky 2008). Leafy (unrelated to the Arabidopsis LEAFY gene or similar homologous genes) is a unique, dominant, late-flowering mutation, which increases the number of leaves specifically between the uppermost ear and tassel (Shaver 1983). Additionally, the Lfy mutation disrupts the coordination of inflorescence maturation, such that the ear matures before the tassel, resulting in mutant plants exserting silks prior to shedding pollen (M.G. Muszynski, unpub.). A mutation with a similar phenotype has not been identified in other grasses. Little is known about the morphological and developmental aspects of this dominant mutation and it has yet to be molecularly isolated. Although relatively uncharacterized from a genetic perspective, it has been used to a modest degree in maize breeding programs in Canada to increase leaf biomass as a means to improve yield (Costa et al. 2002; Subedi and Ma 2005; Subedi et al. 2006). Additional information regarding the molecular control of the floral transition in maize comes from the study of flowering time variants which display a quantitative mode of inheritance. Two quantitative trait loci (QTL) mediating early flowering

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were identified on chromosome 8 and named vegetative to generative transition1 (Vgt1) and Vgt2 (Vladutu et al. 1999; Salvi et al. 2002). Positional cloning and association mapping recently pinpointed the molecular position of Vgt1 to an intergenic region ~70 kb upstream of an APETALA2 (AP2)-like transcription factor, designated ZmRap2.7 (Salvi et al. 2007). Variation in flowering time in various inbred lines was associated with sequence changes in this putative cis-element that presumably controls the expression of ZmRap2.7. Transgenic analysis showed that overexpression of ZmRap2.7 cDNA caused late flowering, whereas downregulation in antisense maize plants caused early flowering, demonstrating ZmRAP2.7 is a repressor of the floral transition. The similarity of ZmRAP2.7 to members of a family of Arabidopsis AP2-like genes that also have a negative effect on flowering suggests a potential orthologous role for ZmRap2.7 in the regulation of the floral transition (Aukerman and Sakai 2003). How ZmRAP2.7 functions within the maize flowering network is currently unknown. Homology-based investigations have also informed our understanding of flowering in maize. A recent study has identified maize homologs of GI (gigantea of Zea mays1, gigz1A and B) and CO (constans of Zea mays1, conz1) that display different diurnal expression patterns in response to varied day lengths, suggesting temperate maize does sense and respond to photoperiod (Miller et al. 2008). Whether these homologs affect the floral transition was not determined. Associations with flowering time and inflorescence architecture were correlated with differences in gene copy number of duplicate maize genes homologous to FLORICAULA of Antirrhinum majus and LEAFY of Arabidopsis (Bomblies and Doebley 2006). It was also shown that mutation of both paralogous Zea FLO/LFY1 (zfl1) and zfl2 genes leads to very mild late flowering and altered inflorescence patterning (Bomblies et al. 2003). Additional maize floral regulatory candidates have been identified based on their sequence homology to FT, which was recently shown to be part of the mobile floral stimulus in several species (Jaeger and Wigge 2007; Lin et al. 2007; Mathieu et al. 2007; Tamaki et al. 2007). Maize has at least 25 FTlike and related paralogos TERMINAL FLOWER (TFL)-like genes, designated ZCN (for Zea mays CENTRORADIALIS) that could encode candidates for a conserved florigenic protein (Danilevskaya et al. 2008). Determining which family member(s) participate in florigenic signaling will require functional analysis of each gene but ZCN8 stands out as a likely candidate based on its expression pattern and ability to interact with the DLF1 protein in yeast two-hybrid assays (Danilevskaya et al. 2008). Comprehensive phylogenetic analyses have determined that for the meristem identity AP1/CAL/FUL MADS-box gene clade, only FULlike homologs exist within maize and other noneudicot species (Litt and Irish 2003; Malcomber et al. 2006). The grass FUL-like genes fall into three subgroups; FUL1 and FUL2 are sister clades that are both sister to the FUL3 clade. A recent study has identified two maize FUL1-like genes, ZMM4 and ZMM5, which are initially expressed near the time of the floral transition (Danilevskaya et al. 2008). Overexpression of ZMM4 promotes early flowering and is able to suppress the late-flowering phenotype of id1 and dlf1 mutants, suggesting ZMM4 has floral inductive activity.

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Collectively, these studies have enabled the configuration of a maize floral transition regulatory gene network (Fig. 4.2). Unlike Arabidopsis and rice, where signaling is photoperiod-responsive and linked to the circadian clock, signaling in maize is principally autonomous and not connected to day length. In the maize network, id1 sits at the top of an inductive signaling hierarchy initiated in leaves by unknown autonomous inputs. id1 is proposed to regulate the production or transmission of a florigenic signal (F) that moves from the leaves to the shoot apex (Fig. 4.2). The relationship between F and ZCN8, a possible maize FT ortholog, is not known. Signals downstream of F are transmitted to the shoot apex and regulate dlf1 expression or DLF1 protein activity. The model network predicts interaction of DLF1 and ZCN8, which presumably activates several targets downstream of dlf1, including an early target (x) which feedback regulates dlf1 expression and one or more ZMM MADS-box floral identity genes (Muszynski et al. 2006). Redundant inductive signaling through an id1-independent alternate pathway converges downstream of dlf1 to activate both x and the ZMM MADS genes. The positions of ZmRAP2.7 and the zfl genes are not known, although the zfl genes make attractive candidates for participating in the alternate inductive pathway. Modern maize inbreds are exquisitely adapted to a wide range of geographical and environmental conditions, with selection for specific maturities to flower at particular latitudes. In fact, most inbreds are selected to flower as late as possible, but early enough to assure that yield is maximized. Inbreds developed for one area of adaptation are rarely used in another because of limitations of flowering, grain fill or kernel maturation. Thus, breeding between inbreds with different maturities is uncommon, leading to a reduction in germplasm diversity and impeding the transfer of superior alleles to new lines. A comprehensive understanding of the genetic determinants regulating flowering would enable breeders to manipulate maturity through molecular breeding or transgenic methods and in this way increase the diversity of germplasm utilized in a selection program. This is especially significant at a time when climate change may dramatically alter an environmental component of a particular geography, thus rendering elite lines maladapted to these new conditions.

4.3

In Vitro Flowering: An Alternative Route to Rapid Flower Production

Plants differ with respect to the length of their life cycle and the timing of the floral transition, to say nothing of robustness with respect to gamete and seed production. Seeds likewise differ with respect to ease of germination. When these limitations are factored against challenges arising from both biotic and abiotic stressors, the accessibility of year round flower production has broad economic implications. This is especially true for secondary plant products whose biosynthesis is restricted either to the inflorescence or to the flower. Recently concentrated efforts have been directed towards producing fertile flowers in vitro whose seed may be harvested

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directly from the culture dish. The ideal in vitro flowering protocol couples a reduction in flowering and seed maturation, in effect shortening the entire life cycle, thus increasing the number of segregating populations that can be produced annually. Hence, the ability to manipulate in vitro flower production will have an incalculable impact on the introgression of transgenes into robust breeding lines to say nothing of plant breeding per se. In vitro flowers have already been produced in culture using a number of different hormone regimes being sensitive to particular cytokinins in a concentration-dependent fashion. In vitro flowers in plant species as diverse as legumes, pearl millet, ornamentals and bamboo (Abou-Alaiwi 2007) result following sequentially the formation of in vitro shoots that lead to flowers. While flowers have been produced, the technical problems associated with the development of a medium are complex. Specifically, from a population of explants, flowers may be produced through an intervening shoot or through the induction of vegetative shoots or infertile shoots where the inflorescence develops aberrantly leading either to the absence or the production of defective gametes. Most elusive of all to date has been the expression of direct flowering where the vegetative development is completely inhibited (Lin et al. 2005; Rudrabhatla and Goldman 2009, US Patent # 7, 547, 548). For the purpose of this discussion, a true in vitro flower is one that is fertile and therefore capable of developing seeds that are indistinguishable from those harvested from traditional crosses.

4.3.1

In vitro Flowering in Grasses

4.3.1.1

Pearl Millet

Devi et al. (2000) reported the production of in vitro flowers from cultured apical meristems of Pennisetum glaucum (L.) R. Br. that is hormone- and concentrationdependent. If the apices were cultured with low levels of 2,4-dichlorophenoxyacetic acid (2,4-D) and the concentration of benzyladenine (BA) varied, the shoot apical meristem enlarged and adventitious shoots developed. At higher concentration of BA, somatic embryos were also observed. In the absence of 2,4-D, shoot tips produced many leaves and in vitro flowers but only upon the inclusion of BA in the medium. Notably, the plants developed from in vitro shoots are fertile. The varied developmental profiles reported are indistinguishable among the four genotypes tested and determined solely as a function of media composition with inclusion of BA being an obligate requirement for in vitro flower development. 4.3.1.2

Bamboos

As a general rule, in vitro flowers have been derived from cultured shoots following a number of manipulations that require the addition of specific concentrations of cytokinin. Working with Dendroclamus strictus Nees, a bamboo, Singh et al. (2000)

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produced flowers from cultured shoots on a modified Murashige and Skoog medium containing thidiazuron (TDZ). While shoots were robustly produced over a wide concentration of cytokinin (0.01–1.0 mg/l), in vitro flower production was restricted only to medium containing TDZ between 0.5 and 1.0 mg/l. The formation of anthers did not necessarily specify the development of normal flowers. Not only were there problems with pollen dehiscence but also with pollen development. In fact, only 20% of the grains were normal, with the remaining 80% being empty. Lin et al. (2003) described in vitro flowering in another bamboo species, Bambusa edulis, albeit with significant shoot formation. Unlike Singh et al. (2000) the shoots were initiated from in vitro derived spikelets. Specifically, these were cultured on Murashige and Skoog (MS) medium containing 9.3 mM kinetin and kinetin þ 13.57 mM 2,4-D. The callus-derived somatic embryos were then germinated on media supplemented with 0.5 uM TDZ plus 3% sucrose. After several subcultures, shoots were recovered. The shoots then were transferred to media containing different cytokinins and the effects determined by testing with a number of different concentrations. Coincident with this transfer, cytokinin is not only sufficient but necessary for the induction of in vitro flowers. Following the production of proliferating intervening shoots, culture on different cytokinins altered the developmental fate of the cells of the axillary meristems. When BA is substituted for TDZ, the number of flowering shoots is profoundly reduced; an effect further complicated as a function of exogenous hormone concentration. Hence, the sensory apparatus of cytokinin signaling can discriminate not only differences among cytokinin molecules but also in concentration leading to profound alterations in morphogenesis. This is in keeping with the earlier observation of Singh et al. (2001) who demonstrated that differences among endogenous concentrations can have profound and antagonistic effects on root and shoot meristem development. Finally, auxins can also effect in vitro development. Although not sufficient in themselves to specify flower formation, the addition of auxin in the media modifies morphogenetic fate in bamboo. If NAA is included in the media during shoot proliferation at high auxin to cytokinin ratios, the number as well as the percentages of reproductive shoots was significantly reduced and, accordingly, in vitro flowering decreased.

4.3.1.3

Maize

Maize in vitro flower induction originating from split-seed was observed after 3–4 weeks in medium containing BA and kinetin (Fig. 4.3 a, b; Al-Abed 2007;

<

Fig. 4.3 (a) In vitro flowers originating from maize split seed explants. Close images of in vitro induced ears (a) and (b) and tassels (b) and (c). (b) Comparisons of maize hybrids and inbred lines for the induction of in vitro flowers on various concentrations of BAP and Kinetin. R 23, Hi-II, B73 and LH 198  LH 227 are different maize hybrid and inbred lines. Numbers 1–6 correspond to different concentration combinations of BAP and kinetin. All percentage data were transformed using arc-sin transformation before statistical analysis

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Al-Abed et al. 2006). Although both ears and tassels were formed, only the ears were fertile. The induction frequency rose as a function of cytokinin concentration with an optimum of 17.6 mM 6-benzylaminopurine (BAP) and 22 mM kinetin. Four genotypes were tested to determine genotype independence, and no significant differences were observed in the frequency of in vitro flowering among the varieties. Interestingly, although the incidence of inflorescence production among the varieties B73, Hi-II and LH198  LH227 proved to be cytokinin concentration dependent, there were no significant differences for in vitro flower induction frequency among the four genotypes when the concentration of BAP was stabilized at 17.6 mM and kinetin varied between 4.6 and 18.4 mM. Similarly, when the concentration of BAP was stabilized at 22 mM and kinetin concentration varied as before no differences in induction were observed. Of equal interest, TDZ could not successfully substitute for either BA or kinetin; an incorporation of TDZ in the culture media resulted in no in vitro inflorescences formed. When the media contained BAP alone there was a significant difference in flower induction frequency among the four micromolar concentrations tested (8.8, 13.2, 17.6 and 22 mM). The number of flowers increased as BAP concentrations increased up to 22 mM. Shoots from line R23 were induced to flower, although the number of ears induced was higher than the number of tassels. On the other hand, there was no significant difference between the number of ears and tassels induced from B73, Hi-II and LH198  LH227. With further increases in BAP concentration (26.4 mM and higher), the shoots were stunted and additional rounds of multiple shoot production began. From these results, it may be concluded that flower induction frequency is correlated with incremental changes of BAP and kinetin concentration. Moreover, prolonged culture in cytokinin-supplemented media affects the development of the ear and subsequent silk formation. If shoot-derived ears were kept on 17.6 mM BAP þ 9.2 mM kinetin for more than 4 weeks, none of the ears formed silks. In contrast, if the shoots were cultured on 17.6 mM BAP þ 9.2 mM kinetin and then transferred to MS lacking hormones, the silks developed with the ears growing larger and more robust.

4.3.2

In Vitro Flowering in Dicots

4.3.2.1

Tomato

Dielen et al. (2001) investigated the floral transition in the tomato mutant uniflora (uf). Under winter growth conditions, homozygous plants exhibit a prolonged vegetative phase and flower production is suppressed. Floral transition can be induced on a medium containing sucrose and a variety of cytokinins, in addition to nitrogenous nutrients. The inclusion of gibberellic acid (GA3) was found to inhibit transition.

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Sheeja and Mandal (2003) produced in vitro tomato flowers through an intervening callus on medium containing 2 mg/l BAP. Flowers, however, required continuous light (2.2 mM2/s) in order to open. The number of flowers produced was low, averaging approximately 11 per explant. The flowers were fertile and tomatoes set 160 days after pollination. Flower production capacity was limited with respect to variety. Of the seven varieties tested on MS þ 2 mg/l BAP, only one variety, KS118, was able to produce fully formed flowers from callus developed from leaf explants.

4.3.2.2

Roses

In vitro flowering was induced in six vegetatively produced rose cultivars in MSmodified media containing different phytohormone combinations. All contained the auxin a-naphthaleneacetic acid (NAA) at 0.1 mg/l and a cytokinin which was varied. Media containing 0.5 mg/l TDZ or 0.5 mg/l zeatin induced in vitro floral bud production at frequencies exceeding 40%. The phytohormone response of each rose cultivar was not uniform.

4.3.2.3

Ginseng

Lin et al. (2005) reported the direct formation of Panax gensing inflorescences. In this connection, plantlets derived from somatic embryos produced clusters of dormant buds when cultured on B5 medium containing BA (1 mg/l) and GA3(1 mg/l). The buds were subcultured again on the same medium and approximately 15% of the buds cultured produced inflorescences directly without a vegetative phase. In addition to direct production of flowers, a mixture of plant organs developed on the explant including vegetative shoots and indirect flowers in addition to dormant buds. If TDZ was substituted for BA, the number of flowers produced without an intervening shoot increased as did the number of dormant buds. These observations confirm the importance of cytokinin as a prerequisite for flower development (Chang and Hsing 1980; Lim et al. 1997). While no gross structural or morphological differences could be detected among flowers produced either by direct or indirect flowering, Lin et al. (2005) reported neither the production of functional gametes nor seed. Hence, the utility of a season-independent, direct in vitro flowering system remains a goal.

4.3.2.4

Soybean

To date, in vitro flowers have also been obtained from dicots through culturing an intervening shoot as shown by Franklin et al. (2000) using the legume Pisum sativum. Following germination on hormone-free MS media, cotyledonary node and shoot tip explants were transferred to an MS medium containing 2mg/l BAP.

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The resulting elongated shoots were removed 15 days post-implantation and incubated on medium containing either auxin alone (indole-3-butyric acid, IAA; or NAA) or in combination with (GA3). Following culture on this medium roots and mature pods were formed within 25 days. The number of shoots per cotyledonary node or shoot tip is BAP concentration dependent. The inclusion of BAP, either below 0.5 mg/l or above 5.0 mg/l, results in the complete absence of shoots derived from cotyledonary node explants. Indeed, shoot formation is maximized between 1 and 2.0 mg/l. Consistent with this observation is the fact that the number of shoots per shoot tip explant is also BAP concentration dependent, albeit over a much narrower range. While BAP results in shoots regardless of the explant, a component of shoot development is fine-tuned based on the tissue that is the donor cell source. The in vitro flowers formed on these shoots, however, are dependent on the subsequent inclusion of specific auxins at highly defined concentrations. While indole-3-butyric acid (IBA) can induce flowering over a wide range of cytokinin concentrations (0.1–2.0 mg/l with or without GA3), substitution with NAA cannot. Not only is GA3 required but the range over which it is effective is restricted to 0.1–0.5 mg/l. In contrast, Rudrabhatla and Goldman (2009), (patent pending) report that culture of soybean cotyledons or radicles on MSB5 medium supplemented with specific cytokinin combinations produced in vitro flowers either directly or indirectly through an intervening shoot. For example, after 7–15 days in MSB5 medium containing TDZ and BAP, the bulging of cotyledons is prominent and small greenish protuberances appear at the proximal end of the cotyledon (Fig. 4.4a). The cotyledons are transferred to a modified MS basal medium containing B5 vitamins but lacking hormones. The small greenish protuberances morph into bud-like structures (Fig. 4.4b). Both shoot buds and/or flower buds form at the proximal end of the cotyledon. The formation of soybean in vitro shoots and/or flowers is not only defined by the cytokinin molecule per se but is also concentration dependent. As will become apparent, soybean tissues have the capacity to discriminate differences among classes of cytokinins on the same explant and in doing so alter the developmental fate of that fixed cell population. If soybean cotyledons are challenged with BAP alone, only multiple shoots were observed (Barwale et al. 1986), while a low frequency of flower buds were observed with TDZ alone. The highest frequency of flower buds was observed in the combination treatment of TDZ (2.0 mg/l) and BAP (1.0 mg/l) (Fig. 4.4c). A decrease in the frequency of flower buds (8.75%) was observed when the concentration of TDZ was kept constant and the concentration of BAP increased to 2.0 mg/l (Fig. 4.4d). Further increase in BAP (3.0 mg/l) resulted in the complete suppression of flower buds with a concomitant increase in the production of multiple shoot buds. Hence, using the same cell population and manipulating the growth regulators, the meristem identity of the tissue was changed from flower buds to shoots. All the flower buds were clumped or grouped together, even in those cultures producing multiple shoots along with flowers suggesting the flower buds arise from a specific group of cells. Among all the combinations tested, 2.0 mg/l TDZ along

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Fig. 4.4 Effect of hormones on in vitro flowering and viable seed set in soybean. Cotyledons producing direct flowers (a) and multiple shoots (b) on MSB5. Direct pod formation from cotyledons without vegetative phase (c and d)

with 1.0 mg/l BAP induced 70–75 flower buds. As soybean is a self-pollinated species, most of the in vitro flowers produced pods and set seed. The in vitro pods fully matured within 35 days after anthesis, and had well-developed seeds. Those in vitro developed seeds, when cultured on MSB5 basal medium, readily germinated and formed fully fertile plants that were easily hardened and grown in the greenhouse. This developmental course can be predictably varied by changing the concentration of BAP and TDZ in the media. In this connection, cotyledons incubated on MSB5 containing 3 mg/l BAP and 1 mg/l TDZ suppressed direct flower formation leading directly to the development of in vitro soybean shoots. From these shoots, fertile flowers developed and seeds indistinguishable from those grown in the field or greenhouse were recovered. The elimination of TDZ from this media likewise produced shoots, which eventually gave rise to fertile flowers at a low frequency (Rudrabhatla and Goldman 2009, patent pending). Similar results have also been obtained using radicle explants as summarized in Fig. 4.5a–c. Three-day-old germinated seeds were collected, the seed coats

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Fig. 4.5 In vitro flowering from radical explants in soybean. Soybean in vitro flowering from radical explants through in vitro developed shoots (a), flower (b) and pod (c)

removed and segments of the radicle and plumule excised. These were placed on MSB5 supplemented with TDZ (2 mg/l) and BAP (1 mg/l) and directly formed clustered fertile in vitro flowers (Fig. 4.5a). If the concentration of BAP was raised to 3 mg/l and the concentration of TDZ remained either constant at 1 mg/l or was raised incrementally to 2 mg/l, direct flower formation was suppressed and shoots formed. Notably, these shoots too developed normal fertile flowers. Taken as a whole, the phenomenon of in vitro flowering supports the hypothesis that cytokinins are essential not only for meristem maintenance and integrity but also as a determinant of cell fate. This conclusion is based on the fact that attempts to induce in vitro flowering through manipulation of cytokinin has resulted in the formation of shoots lacking flowers, shoots producing sterile flowers, shoots producing fertile flowers, direct sterile in vitro flowers where the intervening shoot is absent and direct fertile in vitro flowers. Each of these outcomes may be attributed to tension that results when cytokinin is supplied exogenously thus challenging the plant’s ability to maintain phytohormone homeostasis in the meristem. The role of cytokinin and its obligate requirement for meristem stability, integrity, and function is now well established (Werner et al. 2001, 2003; Riefler et al. 2006;

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Kurokawa et al. 2007). Transgenic tobacco plants overexpressing any one of the cytokinin oxidase genes, AtCKX1, AtCKX2, AtCKX3 or AtCKX4, produce plants with significantly reduced cytokinin concentration. Such plants are not only stunted but show significant size reduction in the shoot apical meristem (Werner et al. 2001). In subsequent experiments using Arabidopsis, Werner et al. (2003) demonstrated that overexpression of any of the six members of the AtCKX oxidase/ hydrogenase gene family has profound effects on the aerial portions of the plant impacting both shoot and floral development. The effect on development is likely a function of perturbations in differential cytokinin activity that is normally associated with growth zones, which are reduced because of the expression of the AtCKX genes that shrink the size of the SAM. Key to the production of in vitro flowers and the route by which they are produced is likely to be, in part, a function of challenges to cytokinin homeostasis in the meristem. T-DNA insertion mutants into the cytokinin receptor genes Arabidopsis histidine kinase genes (AHK2, AHK3) and cytokinin response (CRE/ AHK4) result in profound developmental changes (Riefler et al. 2006). The effect of these mutated genes is to reduce signaling and lead not only to increases in endogenous cytokinin concentration but to do so in a defined manner leading to the accumulation of specific metabolites. The ahk2 and ahk3 double mutants affect leaf development reducing cell number and total chlorophyll content while showing reduced sensitivity to cytokinin-dependent inhibition of dark-induced chlorophyll loss. The effect of the triple mutants on flowering remains a matter for discussion. While Higuchi et al. (2004) and Nishimura et al. (2004) claimed complete plant sterility, Riefler et al. (2006) demonstrated that some plants may be rescued. The said effect of disturbing homeostasis on flower development is unmistakable and consistent with the observation that mutants express anywhere from 16- to 19-fold increases in zeatin metabolites. While reduced signaling increases cytokinin accumulation, it remains unclear as to whether the changes seen in the development result from increased biosynthesis or decreased degradation. A decreased signaling results in increased endogenous cytokinin activity diminishing homeostasis and resulting in disturbances in the development. Indeed, the source of the change in endogenous cytokinin concentration is unimportant. Recently, Kurokawa et al. (2007) have detailed the expression of lonely guy (LOG) gene in rice. Plants overexpressing LOG produce plants with diminished panicles, reductions in the number of floral organs and a flattened meristem resulting in anomalies in organ development. The wild-type gene is definitive for the final step of cytokinin biosynthesis in the shoot tip. The differential response to cytokinin on in vitro flowering described here substantiates the fact that the uptake of exogenous cytokinin disturbs the delicate internal homeostatic balance needed if plant development is to proceed normally. The parameters regulating in vitro flowering are complex and result by signaling appropriate developmental pathways and repressing others. While cytokinin signaling is initiated through a number of histidine kinase receptors followed by phosphor relay, it remains unknown how increases in hormone concentration effect

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differential gene expression as a result of perturbations in homeostasis. This said, while the precise genes that are either expressed or repressed are unknown at this time, their definition is made possible. Our results clearly indicate that the unique hormonal combination applied at appropriate stage of in vitro culture seems to be a key factor that defines whether floral meristems are formed or not. Hence, it should be possible to determine if any, of the sentinel cloned genes at which all floral pathways converge are expressed, as well as those that are unique. Indeed, the collapsing of the vegetative phase and the appearance of flower buds beginning the 3rd or 4th week after transfer to flower induction medium raises questions as to whether the four flower induction pathways previously described for Arabidopsis finds a parallel in soybean or, alternatively, whether the in vitro flowering system expresses a regulatory pathway previously unknown. Identification of the genes regulating in vitro production of soybean flowers may offer the possibility of transferring genes that are absent to complete the pathway or identifying those genes that are uniquely expressed in response to the specific combination of the floral induction medium.

4.4 4.4.1

Genes for Improving Grain Quality in Corn and Soybeans Key Issues

Grain crops form the basis of the global food supply, either through direct consumption or in animal feed to produce meat. A number of trends lead to an increasing global demand for grain. First, the global population is increasing. The current population is 6.5 billion and this is expected to increase to 9 billion by 2050. Second, global meat consumption is increasing in developing countries. In the past 25 years, meat consumption in developing countries has doubled (Speedy 2003). Meat production by nonruminant animals often depends on grain, and conversion of grain to meat is relatively inefficient. Thus, an increase in meat consumption creates a larger demand for grain. Third, increasing amounts of grains are used to produce renewable biofuels such as ethanol and biodiesel. This increasing demand is compounded by environmental factors such as reductions in fresh water and arable land. It is clear that crop yields will need to be increased substantially to keep pace with the demand for grain. In addition to increasing crop yields, it is important to ensure that our grain supplies are used efficiently. It may be possible to use less grain for a given purpose if that grain is particularly well suited to that purpose. For example, oil crops that contain more oil require consumption of less grain to meet the demand for oil. Thus, part of the demand for grain can be met by producing better grain. The definition of grain quality depends on the end use of the grain. This chapter will examine the major uses of grain and the genes that have been important in improving grain for these purposes.

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4.4.2

Protein

4.4.2.1

Amino Acid Balance

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Meeting protein nutrition needs is one of the greatest challenges because plants tend to be low in protein and this protein has poor nutritional quality. Animal protein sources have much higher nutritional value. The nutritional quality of a protein source is defined, in part, by its amino acid balance. Of the 20 common natural amino acids, nonruminant animals can produce 10 amino acids from other sources and the remaining 10 are required in the diet. While the exact amino acid requirement depends on the animal species, developmental stage and other factors, in general, monocot grains tend to be deficient in lysine and tryptophan while dicot grains tend to be deficient in the sulfur-containing amino acids, cysteine and methionine. These deficiencies are largely a consequence of the amino acid balance of the seed storage proteins that accumulate to high levels in the seed and support the seedling until it is capable of photoautotrophic growth. The major seed storage proteins of cereals tend to be prolamins, which are characterized by low levels of lysine and tryptophan, while the main seed storage proteins in legumes tend to be globulins, which have low levels of sulfur-containing amino acids. These deficiencies lead to poor utilization of plant protein in diets, so improving the balance of amino acids is an important grain quality objective. Several approaches have been used to improve the amino acid balance of grain. Because the seed storage proteins have such a large impact on determining the amino acid balance, one approach to solving this problem is to alter seed protein deposition. In cereals, several genes involved in deposition of the prolamin family of seed storage proteins called zeins have been found to impact amino acid balance. Recessive mutant alleles of the opaque 2 (o2) gene of maize cause an increase in the lysine and tryptophan content of the grain (Mertz et al. 1964). The o2 gene encodes a basic leucine zipper (bZIP) transcription factor (Hartings et al. 1989) that binds to promoter elements of some zeins (Schmidt et al. 1990). Unfortunately, the soft, lowdensity kernels produced on these mutant plants are not well suited to agronomic production. Development of nutritionally improved maize with acceptable agronomic properties has required an extensive breeding effort to overcome the unfavorable effects of the mutation. The resulting varieties are called Quality Protein Maize (QPM) and carry the o2 gene as well as a number of unidentified “modifier loci” that condition desirable agronomic properties (Prasanna et al. 2001). Mutation in the floury-2 (fl2) gene also improves the lysine and methionine content of maize (Mertz et al. 1965). This mutation encodes a zein with an altered signal sequence that interferes with zein deposition (Coleman et al. 1995). The delta zein regulator (dzr1) gene conditions elevated methionine levels in maize and was originally identified in a screening program aimed at identifying high lysine genotypes (Phillips et al. 1981). This gene regulates the deposition of a methionine-rich seed storage protein, the 10 kDa delta zein by altering the mRNA stability of the gene (Cruz-Alvarez et al. 1991) and has been used to produce a series of high methionine inbred lines (Olsen et al. 2003).

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Transgenic approaches to improving the amino acid balance have taken advantage of information gained from the naturally occurring mutants to achieve effects similar to those of the natural mutations. Downregulation of zein transcription results in improved amino acid balance (Segal et al. 2003; Huang et al. 2004) as would be predicted from studies of o2, and enhancing the stability of the methioninerich delta zein message improves methionine content (Lai and Messings 2002) as would be predicted from studies of dzr1. A different transgenic approach involves modifying genes encoding enzymes involved in metabolism of amino acids of interest. Introduction of feedbackinsensitive versions of an enzyme in the lysine biosynthetic pathway results in increased levels of total and free lysine when used in combination with a transgene designed to downregulate the zein storage proteins (Huang et al. 2005). Downregulation of degradative enzymes is also effective is increasing free lysine levels (Houmard et al. 2007). An approach that involved both expression of a feedbackinsensitive biosynthetic enzyme and downregulation of a degradative enzyme with a single transgene was effective in increasing the free lysine levels 40-fold over nontransgenic controls. Approaches to improving the amino acid balance of dicot grain have been recently reviewed (Krishnan 2005). Expression of methionine-rich proteins from other species has been widely used to address this problem. For example, the 2S albumin from Brazil nut is a methionine-rich seed storage protein that has been shown to increase methionine content when introduced into the seeds of transgenic plants (Altenbach et al. 1989). Analysis of transgenic soybeans containing this protein indicated that the allergenic properties of this protein were present in the transgenic grain (Nordlee et al. 1996) and this approach has not been pursued further. Maize methionine-rich zein genes have also been expressed in transgenic soybeans (Dinkins et al. 2001; Kim and Krishnan 2004). While the transgeneencoded proteins accumulated in soybean seeds, the change to methionine content was modest. 4.4.2.2

Antigenicity

Allergenicity limits the utility of plant proteins in diets. Genetic approaches have been used to produce varieties with lacking certain antigens. In soybean, for example, the P34 protein is a major allergen. A transgene was introduced that uses gene silencing to prevent accumulation of this protein (Herman et al. 2003). In addition, several null alleles that fail to accumulate this protein were identified through screening of a germplasm of the Glycine genus (Joseph et al. 2006). These resources may allow the development of varieties with reduced antigenicity. 4.4.2.3

Digestibility

The digestibility of soybeans is reduced by the presence of trypsin inhibitors in the seeds. A null allele for the Kunitz trypsin inhibitor allele has been identified and

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designated ti (Orf and Hymowitz 1979). A line containing this mutation was shown to have improved protein efficiency ratio when compared to a nonmutant isoline. This difference was attributed to improved digestibility due to reduced trypsin inhibitor activity.

4.4.2.4

Oil

The fatty acid composition of vegetable oil has a large impact on its functional properties and its dietary impact on health. The specific composition with the highest value depends on the end use of the oil. Given the wide range of uses of vegetable oils, many types of modifications have added value. The fatty acid biosynthetic pathway has been manipulated by mutagenesis or genetic engineering to produce oils with many potentially valuable compositions (reviewed in Fehr 2007). Manipulation of fatty acid levels is complicated by the fact that most steps in the biosynthetic pathway of these compounds are encoded by families of genes.

4.4.2.5

Oxidative Stability

Unsaturated fatty acids, such as linolenic acid, are susceptible to oxidation at the high temperatures used in frying. This oxidation results in undesirable flavors and odors, and therefore low-linolenic acid oils are desirable. Breeders have used EMS (ethyl methyl sulfonate) mutagenesis to produce lines with low linolenic acid (Hammond and Fehr 1983; Wilcox et al. 1984). Reduced linolenic acid levels in these lines have been attributed to lesions in the members of the omega-3 fatty acid desturase (FAD3) gene family (Byrum et al. 1997).

4.4.2.6

Health Benefits

High levels of saturated fatty acids are correlated with coronary heart disease. In soybean oil, palmitic acid is the most abundant saturated fatty acid. Alleles of several genetic loci have been used by breeders to reduce palmitic acid levels in soybean, but most have not been molecularly characterized. The exception is FATB, which encodes a 16:0 thioesterase.

4.4.2.7

Flavor

Lipoxygenases are enzymes that catalyze the oxidation of polyunsaturated fatty acids (reviewed in Feussner and Wasternack 2002). These reactions produce a number of aromatic compounds that contribute to undesirable beany flavors in bean products. Three major isoforms of soybean lipoxygenase are designated LOX1, LOX2 and LOX3. Soybeans lines containing null alleles of these isoforms

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have been identified (Kitamura et al. 1983; Davies and Nielsen 1986). Characterization of soybeans containing LOX null alleles has been shown to accumulate reduced levels of compounds responsible for undesirable flavors (Davies and Nielsen 1987; Hildebrand et al. 1990; Moreira et al. 1993; Nishiba et al. 1995) and that tofu and soymilk made from LOX null soybeans had a less beany flavor than similar products made from normal beans (Davies and Nielsen 1987; Torres-Penaranda et al. 1998).

4.4.3

Starch

Starch accumulates in maize kernels as insoluble granules. When heated in the presence of water, starch gelatinizes. This behavior makes starch useful for many purposes in food as an adhesive and thickening agent and industrial applications such as making paper, paint and cosmetics. With such a broad range of applications, nearly any modification to the physical properties of starch will make it better suited to one of its end use.

4.4.3.1

Industrial Applications

The physical properties of starch are determined by its chemical structure. Starch is a polymer of glucose, but it can be fractionated into two polymers with different degrees of branching. Amylose is a largely linear polymer, while amylopectin contains a higher degree of branches. The ratio of amylose to amylopectin has a large impact on the physical properties of starch. Several genes control this ratio, and mutations in these genes are used in varieties that produce different industrial starches. It was shown that the waxy (wx1) gene lacks a glucosyl transferase activity (Nelson and Rines 1962), later determined to be due to the granule-bound starch synthase. Mutants of wx1 do not accumulate amylose (Sprague et al. 1943) and are particularly useful as adhesives. The amylase extender (ae1) gene of maize accumulates high levels of amylose, and encodes starch branching enzyme IIb (Stinard et al. 1993). The high amylose starches derived from this mutant forms strong gels well suited to confections and thickening agents.

4.4.3.2

Food Quality

In addition to mutants that change starch structure, mutants that change the amount of starch produced are valuable. Sweet corn that is used directly for human consumption is based on mutations that reduce the level of starch in the kernel. This reduction in starch is accompanied by a concomitant increase in the sugar content of the developing kernels, resulting in the sweetness and flavor desired by

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consumers. The sugary-1 (su1) gene encodes a starch debranching enzyme that is essential for the production of normal starch granules (James et al. 1995). Recently, the shrunken-2 (sh2) mutation has been employed to develop “supersweet” types of sweet corn (Tracy 1997). The sh2 gene encodes an ADP-glucose pyrophosphorylase (Bhave et al. 1990).

4.4.4

Other Components

Plants store phosphorus in the form of a phosphorylated sugar called phytic acid. Nonruminant animals including humans cannot efficiently digest phytate, leading to a deficiency in phosphorous in grain-based diets. In addition, phytate is an efficient chelator of several minerals, such as iron and zinc that are limiting in some diets, reducing their biological availability. In addition, undigested phosphate in animal manure contaminates ground water and contributes to eutrophication of streams and lakes. Thus, reduction of phytic acid is desirable for both nutritional and environmental reasons. Mutants of maize (Raboy et al. 2000) and soybean (Wilcox et al. 2000; Hitz et al. 2002) with reduced content of phytic acid identified, however, pleiotropic effects on seed weight in maize (Raboy et al. 2000) and seedling emergence in soybean (Oltmans et al. 2005) are a barrier to the development of commercial varieties with reduced phytate. A transgenic approach involving embryo-specific downregulation of the maize lysophosphatidic acid receptor (lpa1) gene may successfully overcome these problems (Shi et al. 2007).

4.5

Concluding Remarks and Future Prospects

Boosting global production of quality grain to feed our planet’s increasing population demands advanced understanding of plant biology and creative strategies to manipulate the inherent development and physiology of our food crops. One strategy is to couple altering the timing of the floral transition with in vitro flowering to change the length of the crop plant’s life cycle, thereby, allowing faster breeding, quicker adaptation to new environments and increased production of higher-quality grain. To be successful, a more complete understanding of the mechanistic interactions that regulate flowering in diverse crop plants is required. For example, the nature of the primary endogenous flowering stimulus in maize is unknown and represents a serious gap in our understanding of how the maize floral transition network functions. Identifying the primary floral stimulus is essential to predictably manipulate flowering in this grain crop. Further, how treatment with different hormone regimes impacts floral network function is not understood. Dissecting how hormone and floral network signaling intersect is an important area for future research. Increased investment in both basic and applied plant

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biology research will enhance our understanding of these key developmental processes and provide the components necessary to enable the modulation of a crop’s life cycle. In conjunction with improvements to grain quality, progress can be made towards the goal of feeding people globally.

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Chapter 5

Biotechnological Interventions to Improve Plant Developmental Traits Avtar K. Handa, Alka Srivastava, Zhiping Deng, Joel Gaffe, Ajay Arora, Martı´n-Ernesto Tiznado-Herna´ndez, Ravinder K. Goyal, Anish Malladi, Pradeep S. Negi, and Autar K. Mattoo

5.1

Introduction

Unprecedented progress during the last three decades in our understanding of the principles of a living cell, particularly the identification of genes and signaling pathways involved in cell differentiation and organ development, has brought us a broader insight into plant biological processes. Technological advancements are revealing new and fundamental knowledge at the molecular and cellular levels, knowledge that is critical towards achieving the goal of precision-based crop improvement. Modernday genetic engineering has emerged as a promising precision-based technology for boosting up food production in the world and introducing desirable traits such as nutritional enhancement and disease and pest resistance, both important components of agricultural sustainability (Chrispeels et al. 2002; Fatima et al. 2008; Negi and Handa 2008). Achieving results that benefit the world will depend on the success of applying new knowledge to real-world field scenarios. The challenge, therefore, is also to simultaneously obtain knowledge on agroecosystem structure and function to understand how manipulation and control of specific gene expression will translate into directing processes at the ecological scale (Mattoo and Teasdale 2009). Developmental traits are coordinated at various levels in a plant and involve organ-to-organ communications via long-distance signaling processes that integrate transcription, hormonal action and environmental cues. Thus, plant architecture, root–soil–microbe interactions, flowering, fruit (and seed) development, and fruit ripening (and seed germination) are highly regulated genetic programs that are also impacted by processes such as organ abscission, organ senescence (and ripening), and programmed cell death (PCD). We note that belowground processes provide the anchor for a healthy and robust plant (Mattoo and Teasdale 2009) but in this

A.K. Handa (*) Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA e-mail: [email protected]

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chapter we have focused on the aboveground plant processes, bringing together information on the genes and biotechnological applications that can potentially affect production and quality of crop plants.

5.2

Shoot Branching

Shoot branching (tillering in monocots) involves the initiation and development of axillary buds of a leaf, which form new branches or flowers. It is one of the most important traits of plant architecture, affecting plants’ ability to intercept light energy for photosynthesis and adaptation to grow in different environmental conditions. Both branching number and branch angle are traits that control crop yield. Branching is regulated by endogenous plant hormones and environmental signals. Plant hormones such as auxin, cytokinin, and strigolactones or their precursors, have major roles in controling branching. Auxin is actively transported basipetally down the shoot to inhibit shoot branching (Ongaro and Leyser 2008), while cytokinin and strigolactones are transported acropetally to promote and inhibit bud growth, respectively. Modification of genes involved in the biosynthesis, transport, or perception of these three hormones is expected to affect branching. For example, three rice mutants, d3 (Ishikawa et al. 2005), htd1 (Zou et al. 2006) and dwarf10 (Arite et al. 2007), which are either deficient in strigolactone perception or biosynthesis, display increased branch numbers. D3, a leucine-rich repeat F-box protein, is required for strigolactone perception, while HTD1 and DWARF10, which encode different members of carotenoid cleavage dioxygenases, are required for strigolactone biosynthesis. Domestication of major cereal crops involved modification of shoot branching, allowing for denser planting and less shelf-shading. Cultivated varieties usually have fewer branches compared to their wild ancestors (Doust 2007). Some of the genes affecting branching number or angle have been cloned from cereal crops (Table 5.1). For example, teosinte, wild ancestor of maize, displays many tillers with slender ears, while maize shows strong apical dominance and typically develops a few tillers with thicker ears (Fig. 5.1). The apical dominance in maize is mainly controlled by teosinte branched1 (tb1) (Doebley et al. 1997). This gene was cloned by transposon tagging and shown to repress growth of axillary buds and promote female inflorescences. Overexpression of a rice TB1 gene under the constitutive rice actin promoter reduced lateral branching, whereas a rice line with nonfunctional TB1 exhibited enhanced lateral branching (Takeda et al. 2003). Similarly, overexpression of a maize TB1 gene in wheat suppressed tiller development (Lewis et al. 2008), while a loss of function of the Arabidopsis TB1 homolog, Branched1, in the T-DNA knockout lines or RNAi lines (Chap. 1-6), resulted in increased branching (Aguilar-Martı´nez et al. 2007). These results indicate that TB1mediated control of branching is conserved between monocots and dicots. Since branching is also affected by environmental and growth conditions, it remains to be seen whether fine-tuning of expression levels of branching-related genes via bioengineering (Chap. 1-7) will improve crop yield.

Tiller Angle Control 1 (TAC1) LAZY1 (LA1)

PROSTRATE GROWTH1 (PROG1) MONOCULM1 (MOC1) Promotes bud initiation and development Increases tiller angle

Promotes erect growth Loss of function in rice promotes prostrate growth (wider angle)

GRAS family protein, putative transcription factor Unknown function

Unknown function

Overexpression in rice increases tiller number, and loss of function in rice reduces tillering capacity Overexpression in rice causes wider tiller angle

Increases branching and tiller angles

Li et al. (2007a), Yoshihara and Iino (2007)

Yu et al. (2007)

Li et al. (2003)

Phenotypes Reference Overexpression in rice and maize Lewis et al. (2008), Takeda et al. decreases branching, loss of function (2003), Aguilar-Martı´nez et al. in Arabidopsis increases lateral (2007) branching Overexpression in rice causes prostrate Jin et al. (2008), Tan et al. (2008) growth

Zinc finger transcription factor

Table 5.1 Genes with potential to modify plant branching Gene Function Effect TCP transcription Inhibits shoot TEOSINTE factor branching BRANCHED1 (TB1)

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Fig. 5.1 Mature plants and inflorescence of teosinte branched 1 mutant and wildtype maize. a, b, the mutant; c, d, an inbred maize line. Modified from Doebley et al. (1997)

5.3

Male Sterility

Male sterility in higher plants is characterized by a failure to produce functional anthers, pollen or male gametes. It is a useful trait, both for studying the male developmental pathway and for exploiting heterosis to obtain high-yielding crop plants. Kaul (1988) classified male sterile types as structural, sporogenous

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or functional. Structural male sterile plants may show absence of stamens with anthers or presence of anthers that are devoid of sporogenous tissues. Sporogenous male sterile plants show normal anther development but the development of pollen is impaired because of irregularities in the division of sporogenous cells. Plants that show anthers with viable pollen development but are still incapable of affecting fertilization because of anther dehiscence failure, abnormal exine morphology or failure of pollen tube germination, are classified as functional male sterile types. Additionally, Taylor and Jorgensen (1992) have proposed the term “conditional male fertility” for the type of male sterility in which viable pollen is unable to germinate and shows pollen tube growth in self-crosses of the chalcone synthase-deficient progeny. At the genetic level, three types of plant male sterilities have been identified, namely, genic male sterility (GMS), cytoplasmic male sterility (CMS) and gene-cytoplasmic male sterility.

5.3.1

Genic Male Sterility

GMS is caused by spontaneous or induced mutations in nuclear genes. It is ordinarily governed by ms, a single recessive gene. GMS is inherited in Mendelian fashion and has been reported in approximately 175 species of angiosperms, including crop plants such as barley, maize, pea, tomato, rice and pepper. These plants can be propagated when pollinated by wild type, male fertile plants. Several male sterile plants exhibit structurally malformed male sexual organs. For example, genes such as apetalla-3 (ap-3), pistillata (pi) and antherless (at) in Arabidopsis affect the development of male sexual organs resulting in sterility (Chaudhury 1993). Mutations also affect genes participating in the differentiation and function of such anther cell types as the stomium, tapetum, endothecium and the archesporial and sporogenous layers of the anther primordium. Arabidopsis mutant ms2 shows defective tapetal layer development resulting in pollen abortion shortly after release from the tetrads (Aarts et al. 1993). Defective meiosis in Arabidopsis (msW and msY; Dawson et al. 1993) and tomato mutants (ms3, ms15 and ms29; Rick 1948) also resulted in male sterile plants. Furthermore, mutations in genes controlling metabolic pathways may also result in sterile/nonviable pollens. Induced petunia white anther (wha) mutant is characterized by the inability of the pollen grains to germinate in vitro. This phenotype is caused by a lesion in chs (chalcone synthase) gene that encodes the first enzyme in the flavonoid biosynthesis pathway (Napoli et al. 1999). Such examples of spontaneous or induced GMS are limited in their use for plant breeding purposes by the availability of a fertility restoration gene. Genetic engineering approaches to introduce a dominant male sterile gene into plant cells (Chap. 1–3) have overcome such barriers and resulted in male sterile transgenic lines in many crop plants. These approaches have been discussed in greater detail under biotechnological advances in Sect. 5.3.3.

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Cytoplasmic and Gene-Cytoplasmic Male Sterility

CMS, in most cases, is determined by mutation in mitochondrial genes (Hanson and Bentolila 2004; Pelletier and Budar 2007). CMS has been reported in approximately 150 species, comprising both wild and cultivated plants (Raghavan 1997). It is maternally inherited, easier to maintain in crop plants and therefore of much value to plant breeders. When the vegetative plant part is the harvested product, CMS alone can be used to obtain higher-yielding hybrid lines. However, when seed or fruit is the desired product, the heterotic hybrid lines should be self-pollinating and able to set seeds. A fertility restorer (Fr) gene must be introduced in such hybrids. Restorer genes, also known as Restorer of fertility (Rf) or Fr, are nuclear genes that mask the male sterility effect of CMS, such that the plant is rendered male fertile in their presence (Schnable and Wise 1998). Rf and CMS together form a binary system (genic-cytoplasmic or nucleo-cytoplasmic) that regulates the mode of reproduction of individuals in populations (Pelletier and Budar 2007). It is possible to remove the effect of Rf genes through interspecific crosses or somatic hybridization such that the nuclear genome of one plant species is present in the male sterility-inducing cytoplasmic background of another plant species (Chase 2007). Male sterility arising as a result of interspecific nuclear-cytoplasmic combination is known as alloplasmic male sterility (Hanson and Bentolila 2004). The absence of Rf gene from the alloplasmic nuclear-cytoplasmic combination unmasks the male sterility-inducing effect of the mitochondrial genes. However, in some cases, hybridization itself has proven mutagenic causing novel cytoplasmic genome configuration that arises because of disturbances in cytoplasmic genome replication and organization (Hanson and Conde 1985). CMS systems have been extensively used to generate high-yielding hybrid varieties in crop plants. In maize, several types of male sterility-inducing cytoplasms have been identified of which CMS-T, -S and -C are the most widespread and well characterized. These CMS systems are differentiated by their reaction to restorers; CMS-T is restored by Rf1 and Rf2, CMS-S is restored by Rf3, and CMS-C is restored by Rf4 (Sofi et al. 2007). Many CMS systems have been found in Brassica crops including the well-studied ogura (ogu), polima (pol) and napus (nap) CMS systems. The pol and nap CMS systems are restored by Rfp and Rfn genes, respectively. Both Rfp and Rfn map to the same chromosomal position and they are allelic (Li et al. 1998). In rice, CMS-BT (Boro type), CMS-WA (wild abortive) and CMS-HL (Honglian) are well established: Rf1a and Rf1b that confer fertility to CMS-BT and Rf5 and Rf6(t) that confer fertility to CMS-HL are all located on chromosome 10 (Fuji et al. 2008). The restorer genes, Rf3 and Rf4, which confer fertility in CMS-WA are located on chromosomes 1 and 10, respectively. CMS systems and their restorer genes in other crop plants are listed in Table 5.2. Many male sterility-conferring cytoplasmic genes have now been identified as open reading frames (ORFs, Table 5.2). For example, the male sterility-inducing CMS systems T-urf13 in maize, pvs-orf239 in bean and orf138, orf224 in Brassica are ORFs formed as a result of complex mitochondrial DNA (mtDNA)

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Table 5.2 Plant cytoplasmic male sterility systems Restorer locus Plant CMS system Mt ORFa/gene Radish Ogura orf 138 and orfB Rfo Kosena orf125 Rf K Rice CMS-BT Orf 79/B-atp6 Rf1a, Rf1b CMS-WA Unknown Rf3, Rf4 CMS-HL orf H79/atp6 Rf5, Rf6 (t) Petunia Pcf Rf1 Maize CMS –T T-urf13 Rf2, Rf1, Rf8, Rf* orf355-orf77,atp6, CMS-S Cob Rf3 CMS-C atp6-C Rf4, Rf5, Rf-I Brassica pol orf224/atp6 Rfp nap orf222 Rfn(Mmt) Sunflower PET1 orfB,orf552 Rf1 Sorghum A1 Rf1 A3 Orf107 Rf3, Rf4 a Mitochondrial open reading frames associated with CMS

205

Reference Brown et al. (2003) Koizuka et al. (2003) Wang et al. (2006a, b) Li et al. (2007a) Zhang et al. (2007a) Bentolila et al. (2002) Wise et al. (1999) Wen and Chase (1999) Dewey et al. (1991) Singh et al. (1996) Homme et al. (1997) Horn et al. (2003) Klein et al. (2005) Kuhlman et al. (2006)

rearrangement (Hanson and Bentolila 2004). These ORFs often comprise a mix of conventional mitochondrial genes and sequences of unknown origins. They are co-transcribed with essential mitochondrial genes and encode proteins with at least two shared features: lower molecular size (<30 kDa) and at least one hydrophobic domain. Studies comparing the respiratory activities of CMS and fertile lines have shown differences in the activity level of the respiratory complexes (Conley and Hanson 1995; Sabar et al. 2000). ATP synthase as well as cytochrome oxidase are associated with the inner membrane of the mitochondria (Hanson and Bentolila 2004). The presence of ATP synthase subunits in the CMS-associated chimeric genes raises the possibility of an impaired ATP synthase activity being the cause of disrupted pollen development that results in male sterility. Premature PCD, in which mitochondria play a central role, has also been suggested to be a possible cause of CMS. According to this suggestion, PCD destroys the tapetal cells that provide nourishment to the developing pollen inside the anthers and thereby causing male sterility (Balk and Leaver 2001; Ku et al. 2003; Rogers 2006). Fr genes encode members of pentatricopeptide repeat protein (PPR) family. PPR genes have been found in the restorer loci of Petunia, Brassica, Raphanus and rice (Hanson and Bentolila 2004). These proteins, targeted to chloroplast and mitochondria, are involved in organelle biogenesis in plants (Lurin et al. 2004). Studies involving nuclear gene mutants with compromized plastid functions have revealed PPR proteins to be involved in RNA processing, intron splicing and translation (Stern et al. 2004; Shikanai 2006). These studies indicate probable functions for mitochondria-targeted PPR proteins including those encoded by restorer genes. In most fertility-restored CMS lines, the protein product for the CMS determining locus does not accumulate. This loss of protein accumulation is accompanied by lower accumulation of CMS-associated transcripts or truncated transcripts. Whether the decreased transcript level or transcript truncation results in the lack

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of protein accumulation or is a by-product of failed translation or imperfect editing of CMS-associated transcript in the presence of a restorer gene is not known (Hanson and Bentolila 2004; Newton et al. 2004; Linke and Borner 2005; Chase 2007).

5.3.3

Bioengineering Male Sterility for Hybrid Plants

Mariani et al. (1990) successfully engineered the first GMS plant, thereby paving the way for the development of more transgenic male sterile crop plants. The ablation of tapetal cells inside anthers results in failure of pollen development. The barnase gene product (barnase) is an extracellular RNase found in Bacillus amyloliquifaciens that protects the bacterium from microbial predators (Hartley 1989). The bacterium is protected by the cytotoxic effect of barnase by intracellular barstar, a barnase-specific RNase inhibitor. A chimeric gene comprising tapetumspecific gene promoter TA29 and barnase gene coding sequences was used to develop transgenic male sterile tobacco and oilseed rape plants. These plants showed ablation of tapetal cells and impaired pollen development. However, these transgenic plants did not show any adverse effect on tapetal cells. When male sterile plants containing TA29/barnase genes were crossed with male fertile plants containing TA29/barstar gene, the resultant progeny was male fertile (Mariani et al. 1992). The barnase/barstar system has been used extensively to obtain male sterile lines in oilseed rape, tomato, cotton, corn and wheat (De Block et al. 1997). Following the pioneering efforts of Mariani et al. (1990), several biotechnological strategies to generate genic male sterile plants have been tested (Perez-Prat and van Lookeren Campagne 2002), which include: 1. Tissue-specific expression of a gene encoding a protein that can disrupt the cell function and thereby development of tissues that are vital for pollen development – barnase/barstar is one such system. Chemical induction of male sterility by selective expression of genes encoding a protein that converts a pro-herbicide into a herbicide only in male reproductive tissues (O’Keefe et al. 1994; Dotson et al. 1996; Kriete et al. 1996). Alternatively, a male sterility gene engineered such that it is induced by the application of a chemical (Goff et al. 1990). Fertility restorer gene or a repressor of male sterility under the control of a chemically inducible promoter employed for fertility restoration (Ward et al. 1993). 2. Manipulation of metabolite levels essential for normal pollen development. Genetic alteration of the levels of amino acids, sugars (Goetz et al. 2001), flavonols (Derksen et al. 1999), jasmonic acids (McConn and Browse 1996; Browse 1997; Sanders et al. 2000), biotin (Albertsen and Howard 1999) and auxins (Spena et al. 1992) to develop sterile lines. In such systems, fertility is inducible by applying the altered metabolite (McConn and Browse 1996; Browse 1997; Albertsen and Howard 1999; Sanders et al. 2000). 3. Crossing two transgenic parental lines, each carrying a gene encoding for an inactive part of a toxin in the male reproductive tissue. When brought together in

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the progeny, these inactive parts combine together to form the active toxin, which results in male sterility (Fabijanski and Arinson 1995; Gutterson and Ralston 1998). 4. Use of transgenic maintainer lines for natural or induced GMS lines. These lines allow the propagation of natural or induced mutations in such a way that male sterile plants may be obtained or male fertile plants may be visually identified and separated. Such maintainer lines are isogenic to the genic male sterile lines except at the transgenic locus comprised two components: (a) A restorer gene that renders the maintainer lines male fertile; and (b) genes or set of genes, which upon crossing a maintainer line with an isogenic male sterile line, either prevent the maintainer locus from passing into the progeny (such plants are called pollen lethality maintainers) or reveal the maintainer locus by simultaneously expressing a gene that results in pigment accumulation (color maintainers). In the latter crosses, 50% plants are male fertile; however, these seeds can be identified and separated according to pigment accumulation (Perez-Prat and van Lookeren Campagne 2002). GMS is limited in its use for hybrid seed production as it is inherited in Mendelian fashion. The plants segregate for male fertility and sterility. This presents a practical problem of separating the male fertile and male sterile plants. CMS, on the other hand, is inherited maternally. Breeders have widely exploited CMS systems to develop male sterile lines. Engineering male sterility through somatic hybridization has resulted in improved CMS systems as well as expansion of the CMS systems to a larger group of crop plants (Pelletier and Budar 2007). Somatic hybridization involves isolation, culture and fusion of protoplasts of two distantly or closely related or even unrelated plants at interspecific, intergeneric, intraspecific or interfamily levels. The heterokaryons resulting from fused protoplasts are selected based on a marker gene and then cultured to develop into complete plants. In some of these heterokaryons, nucleo-cytoplasmic interactions result in formation of cytoplasmic hybrids (cybrids), which carry the nuclear genome of only one parent but the cytoplasmic genomes from both parents. The nuclear genome of the mitochondrial donor should carry a restorer gene (Pelletier and Budar 2007). Male sterile cybrids have been developed in Brassica species (Budar et al. 2004), rice (Brar and Khush 2006) and tomato (Melchers et al. 1992). Natural CMS system is not found in all crop plants and its use for breeding purposes is limited if an appropriate restorer system is not present. Although transgenic dominant genic male sterile plants can be developed, this approach is restricted by the Mendelian segregation of the progeny. Stable, maternally inherited male sterility can be induced in crop plants by organelle transformation (Chap. 1-8). Male sterility induction has been reported by plastid transformation using b-ketothiolase gene (phaA) in tobacco plants (Ruiz and Daniell 2005). The phaA gene, under the control of light-inducible psbA promoter, alters the course of fatty acid synthesis, which in turn leads to pollen grain collapse and male sterility. Male sterility was reversed by growing these plants under continuous illumination. Plastid transformation holds great promise for genetic transformation of crop

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plants. It will be possible to take better advantage of this technology for developing male sterile crop plants and their restorer systems as more plant species become amenable to plastid transformation (Kuchuk et al. 2006). Studies on GMS and CMS systems have not only revealed the fundamental aspects of male developmental pathways, but also the molecular players in male sterility and fertility restoration in plants. Also, genetic engineering was employed to inhibit flower development and introduce reproductive sterility. Expression of a ribosome-inactivating protein (DTA) gene under the control of PTD gene – homologous to the MADS box genes DEFICIENS and APETALA3 – resulted in sterile flowers and consequently impaired dissemination of the transgene and the confinement of specific trait (Skinner et al. 2003; Wei et al. 2006). Biotechnological advances have allowed the expansion of these male sterility systems in a wider range of crop plants. Identification of mitochondrial genes controlling male sterility and those that restore fertility and understanding their mode of function will allow a better control over these systems. Advances in organellar transformation will provide a better process for inducing male sterility/fertility restoration in plants. Transformation of mitochondria remains the next hurdle to overcome and will prove a breakthrough advance towards understanding the role of this organelle in male development and sterility aspects.

5.4

Flowering Induction

Flower induction is under the control of several interconnected pathways involving light irradiation, temperature, plant growth regulators and the physiological state of the plant (Jaeger et al. 2006; Kobayashi and Weigel 2007; Wilkie et al. 2008). The genetic basis of these pathways are the subject of intense research in Arabidopsis (Ba¨urle and Dean 2006), as well as in maize, rice (Chap. 4.2) (Izawa 2007), soybean (Wong et al. 2009) and other plants of economic interest. The control of flower induction is a major agronomical event in crop productivity, e.g., cereal (Cockram et al. 2007), fruit (Carmona et al. 2008), plant fibers (Abdurakhmonov et al. 2007) or wood industry (Yuceer et al. 2003; Bo¨hlenius et al. 2006). In potato, more or less the same genetic components regulate flowering and tuberization (Rodrı´guez-Falco´n et al. 2006). In cereal crops, regulation of flower induction is critical for seed set and development. The day length and temperature were found to be the key elements for the growth attributes of wheat and maize when initially grown in the Fertile Crescent and Central America, respectively. In recent times, however, the area for growing cereals has widely expanded during centuries of selection and improvement (Cockram et al. 2007). In Arabidopsis, promotion of flowering involves long-day photoperiod, gibberellins, an autonomous route, and vernalization (Jack 2004; Putterill et al. 2004). The photoperiod pathway perceives light quantity and circadian clock – long day promotes flowering in Arabidopsis. In order to induce flowering at an appropriate time, the day length has to be in sync with an internal time keeper (Ba¨urle and

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Biotechnological Interventions to Improve Plant Developmental Traits

Table 5.3 Genes regulating flowering time in plants Gene Function Phenotype CO

Transcription factor

Biotechnology Reference intervention Constitutive Martı´nezoverexpression Garcı´a et al. (2002) Overexpression Kardailsky et al. (1999)

Altered flowering time. Impaired potato tuberization FT Kinase inhibitor Early flowering Arabidopsis Forms CO/ FT regulatory module pTFT1 Kinase inhibitor Early flowering. Timing of Overexpression Populus FT ortholog flowering and seasonal growth cessation in trees Ehd 2 (rice) Zinc finger Promotes flowering Mutation protein ortholog of maize INDETER MINATE1 OsCO3 (rice) Transcription Inhibition of flowering factor Reduced juvenile phase Overexpression Leafy (LFY) Apetala1 (AP1) Arabidopsis

209

Bo¨hlenius et al. (2006) Matsubara et al. (2008)

Pen˜a et al. (2001)

Dean 2006). Gibberellins promote flowering, especially under short-day photoperiod. The autonomous pathway is independent of photoperiod control, and causes flowering in Arabidopsis under short days. Vernalization refers to promotion of flowering by extended cold exposure, as naturally happens in plants during winter. Different pathways of flower-regulation genes converge at a few defined floral pathway integrators that include Flowering Locus T (FT), Suppressor of Overexpression of Constans 1 (SOC1) and Leafy (LFY), which activate floral meristem identity genes and trigger the transition from vegetative to reproductive phase (Table 5.3) Details of the transition from vegetative meristem to floral transition are described in Chap. 4.2. Two genes, Constans (CO) and Flowering Transition (FT), whose expression is tuned to the change in day length and involved in flowering, were initially isolated from Arabidopsis (Kobayashi and Weigel 2007). CO is a transcription activator that belongs to the B Box Zinc finger protein family. FT is a 23-kDa protein from the RAF kinase inhibitor family involved in regulatory cascade and signal transduction (Turck et al. 2008). FT protein is synthesized in the companion cell of the leaf phloem and mobilized to the shoot apical meristem to initiate the flowering (Cockram et al. 2007; Turck et al. 2008). In Arabidopsis, FT protein accumulation is directly under the CO control.

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CO protein does not bind DNA by itself but interacts with transcriptional complex (Kobayashi and Weigel 2007). The biochemical mode of action of CO is still unclear. Orthologs of CO have been identified in rice (Izawa 2007), potato (Martı´nez-Garcı´a et al. 2002) and aspen tree (Bo¨hlenius et al. 2006). CO transcription is under the control of circadian regulation through CDF1 (cycling DOF Factor). Its transcripts accumulate from the afternoon to the middle of the night either in long day (LD) or short day (SD) but with a slight shoulder at the end of the afternoon during LD (Fig. 5.2; Imaizumi et al. 2005). CO transcripts are degraded in LD plants at the end of the day and during the night but in SD this takes place only during the night (Valverde et al. 2004). Proteosome-mediated degradation of CO protein occurs in the dark as well as under red light. During LD conditions, CO protein accumulates at the end of the light cycle and activates FT transcription but under SD it is rapidly degraded and therefore unable to activate FT transcription (Fig. 5.2). In rice, a CO ortholog Hd1, activates flowering only slightly under the SD condition but inhibits the accumulation of FT under LD. Patterns of CO and Hd1 transcript accumulation are the same (Izawa 2007). Transgenic potato expressing Arabidopsis CO exhibited reduced growth and delayed tuberization, indicating an inhibitory effect of AtCO on tuberization (Martı´nez-Garcı´a et al. 2002). CO-FT regulatory module also controls timing of flowering and seasonal growth cessation in aspen trees (Bo¨hlenius et al. 2006). Overexpression of FT ortholog, PtFT1 in aspen, reduced the flowering delay in transgenic tree from several years to 6 months. Unexpectedly, it also regulated the SD-induced growth cessation and bud setting in the fall. This may explain growth cessation displayed by aspen trees a

c CO CO CO CO CO CO CO CO

b

CO CO CO CO CO CO CO CO CO

d CO CO CO CO CO CO CO

CO

CO CO

DegradedCO

CO CO CO CO CO CO CO CO

FLOWERING

ActiveCO

Fig. 5.2 Diurnal regulation of CO transcripts (—) CO protein (▪ ▪ ▪) and FT protein (- - -) accumulation of in plants under long day and short day growth conditions. (a) Accumulation of CO transcripts is regulated by circadian clock but not by the light status, (b) whereas accumulation the CO protein is light dependent (adapted from Bau¨rle and Dean 2006). (c) Under the short day conditions CO protein is rapidly degraded and inactivated by the proteasome during the dark period, (d) but when plants are grown under long day conditions it accumulates at the end of the afternoon before the beginning of dark period. Active CO proteins induce the flowering (adapted form Valverde et al 2004)

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sampled across a latitudinal gradient spanning northern Europe. LFY or APETALA1 (AP1) are other genes that promote flower initiation in Arabidopsis. When these genes were overexpressed under CaMV 35S promoter in hybrid citrus (Citrus sinensis L. Osbeck x Poncirus trifoliata L. Raf.), juvenile phase was reduced from 7 years to about 15–20 months (Pen˜a et al. 2001). Expression of AP1 was as efficient as LFY in the initiation of flowers, and no severe developmental abnormality was observed. Modifying transition phase from juvenile to mature plants should greatly improve the possibilities for breeding of trees for agronomical and industrial use.

5.4.1

Flower Senescence

Senescence results in significant losses of cut flowers and offers challenge for postharvest researchers trying to delay this process. The process of flower senescence has been shown to be a genetically programmed event (Stead et al. 2006; Arora 2008). Flower petals are ideal tissues for cell death studies as they are relatively homogeneous, short-lived tissue that can be manipulated by exogenous chemical treatment without substantial wounding. Plant hormones, membrane stability, water availability, cellular proteolysis and carbohydrate metabolism act in concert to determine the differential rate of senescence for different floral organs. However, the death of floral tissues is affected by several factors including the developmental stage, pro-senescence signals (e.g., pollination-induced petal senescence), and stress-related metabolism in response to temperature, wounding, and nutrient starvation (Stead et al. 2006; Arora 2008). Floral senescence is a developmental continuum in the flower that is preceded by tissue differentiation, growth and maturation of the petal, growth and development of seeds. Genome-wide searches have resulted in isolation and identification of a number of genes associated with flower senescence from several species, including Alstroemeria (Breeze et al. 2004), carnations (Verlinden et al. 2002), Chrysanthemum (Narumi et al. 2005), daffodil (Hunter et al. 2002), daylily (Panavas et al. 1999), rose (Channeliere et al. 2002), iris (van Doorn et al. 2003), Sandersonia aurantiaca (Eason et al. 2002), Petunia (Jones et al. 2005) and Gladiolus (Arora and Singh 2004; Arora et al. 2006). Characterization of their function has provided insights into the roles played by ethylene signaling, proteolysis, nucleic acid and chlorophyll breakdown, and lipid and nitrogen remobilization in the progression of flower senescence (Gan and Amasino 1997; Stead et al. 2006). The future genetic analysis of floral senescence will likely identify molecular mechanisms that help maintain a non-senescent “juvenile” state and provide novel interventions to extend post-harvest life of not only cut flowers but also fruit and vegetable crops (Mattoo and Handa 2008). Ethylene is one of the plant hormones with profound effects on plant growth and development including senescence and ripening processes (Fluhr and Mattoo 1996; Mattoo and Handa 2004). Key genes regulating biosynthesis of ethylene

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include S-adenosylmethionine (SAM) synthase, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase (Fluhr and Mattoo 1996). Different plant organs show responses to ethylene to different degrees. Even cultivars within flower species exhibit a significant variability in sensitivity to ethylene. Most modern carnation cultivars exhibit much reduced sensitivity to ethylene indicating possibilities of genetically engineering flower vase life by reducing ethylene biosynthesis or perception. Impairing ethylene biosynthesis by expressing an antisense ACC oxidase gene resulted in dramatic reduction of ethylene production with a concomitant increase in the longevity of carnation and Torenia fournieri flowers (Savin et al. 1995; Aida et al. 1998). However, similar attempts in Begonia failed and transgenic flowers did not show prolonged flower life (Einset and Kopperud 1995). Increasing longevity of carnation cut flowers was also achieved by reducing expression of ACC synthase by co-suppression (Michael et al. 1993). Another avenue to control floral and leaf senescence is to manipulate perception of ethylene. Characterization of an ethylene receptor gene from Arabidopsis, AtETR1 (Chang et al. 1993), and tomato (Zhou et al. 1996a,b) led to the identification of a family of ethylene receptor genes in plants. Tomato genome contains six members, LeERT1–LeETR6, with each of the receptor genes having a distinct expression pattern during plant development and in response to external stimuli (Klee and Tieman 2002). Ethylene-insensitive 3 (EIN3) encodes a transcription factor that functions downstream from the ethylene receptors in the ethylene signal transduction pathway (Wang et al. 2002). EIN3 possesses two mitogen-activated protein kinase (MAPK) phosphorylation sites that have opposing effects on EIN3 stability. Homologs of the Arabidopsis EIN3 gene are present in tomato: LeEIL, LeEIL1, LeEIL2 and LeEIL3. EIN3-BINDING F-BOX1 and 2 (EBF1/2) coordinately control 26S proteasome degradation of the transcription factors, EIN3 and EIL1. ETHYLENE-INSENSITIVE5 (EIN5), which encodes the exoribonuclease XRN4, represses expression of EBF1/2. However, functional significance of each member of the ethylene receptor family is not fully understood (Kevany et al. 2007, 2008). A mutated ERS gene (ethylene response sensor, an Arabidopsis gene uncovered by cross-hybridization with the Arabidopsis ETR1 gene) conferred dominant ethylene insensitivity to wild-type Arabidopsis (Hua et al. 1995). Overexpression of a mutated dominant Arabidopsis ethylene resistance gene etr1-1 under the CaMV 35S promoter conferred ethylene insensitivity to Petunia (Wilkinson et al. 1997; Clark et al. 1999; Gubrium et al. 2000; Celvenger et al. 2004). Among the effects produced by the introduction of ethylene-related transgenes in plants were: enhanced ethylene production in pollinated flowers; slightly faster flowering; poor root development of cuttings; lower seed weight; less-efficient seed germination and rooting; delayed seedling growth; and reduced flower senescence, abscission and fruit ripening. The delayed flower senescence was seen both in the presence and absence of ethylene in pollinated and non-pollinated flowers. Most of these effects can be explained by the roles played by ethylene in different developmental processes. The enhanced ethylene production in flowers is probably due to the uncoupling of the feedback regulation from the plant’s response to pollination.

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213

However, the poor rooting ability of cuttings limits the use of this methodology. Overexpression of a mutated ers homolog from Brassica oleracea (Boers) also imparted ethylene insensitive to Petunia and induced prolonged flower longevity (Shaw et al. 2002). An unintended effect of this transformation, however, was increased susceptibility of the transgenic plant to fungal diseases, most likely because of an interference with the ethylene-induced defense activated by ethylene. The use of a flower-specific promoter from Petunia (fbp1) to express etr1-1 in carnation resulted in stronger insensitivity to ethylene without the undesired side effects as observed in earlier experiments (Bovy et al. 1999). Transformation of an ornamental Nemesia strumosa with a mutated etr1 melon homolog under a constitutive promoter also exhibited enhanced flower longevity by altering ethylene perception (Cui et al. 2004). Overexpression of isopentenyl phosphotransferase (ipt gene) from Agrobacterium tumefaciens under a senescence-regulated promoter Psag12 increased cytokinin levels with concomitant delay in ethylene production and enhanced ethylene tolerance of the flowers of the transgenic plants (Chang et al. 2003). The flower longevity was prolonged by 100% in non-pollinated flowers and approximately 450% in pollinated flowers. Interactions between ethylene, cytokinins, sugars and various hydrolytic enzymes are known to differentially mediate the progression of flower senescence. However, individual signals appear to be species-specific and vary among the plant variety and floral organs. The challenge for post-harvest scientists is to identify a hierarchy of regulators or specific patterns underlying the progression of flower senescence. The use of tissue-specific promoters (Chap. 1-5) is recommended since these may reduce undesired effects of the introduced gene, particularly in modifying plant hormone levels (Clark et al. 1999; Shaw et al. 2002). Conventional breeding has made significant advances in increasing the number of flowering buds, extending the longevity of inflorescence and improving post-harvest performance, as demonstrated in Lilium (Van der Meulen-Muisers et al. 1999). Coupling transgenic approach with molecular breeding might pave a way to greatly improve flowers, fruits and vegetables with desirable traits including shelf life.

5.5 5.5.1

Fruit Fruit Shape, Size and Mass

Domestication of almost all fruit species has invariably led to the selection for increased fruit size. In addition to size, fruit crops have been bred for shape, texture, flavor, shelf life and nutrient content. Genetic evaluation of various species, especially tomato, has provided a wealth of information about loci that control fruit size, weight and shape (Frary et al. 2000; Tanksley 2004; Cong et al. 2008). However, their quantitative nature has impeded characterization of individual genes regulating these fruit attributes (Grandillo et al. 1999). Table 5.4 lists some of the genetic

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Table 5.4 Transgenic genetics of fruit developmental attributes Enzyme/protein Transgene Phenotype Fruit shape, size and mass fw 2.2 Negative Overexpression Reduced fruit size, regulator of cell fruit weight division; possibly involved in cell-tocell communication SUN, IQ67 domain– Overexpression More elongated fruit containing family shape Ethylene regulated ripening ACC oxidase Underexpression Prolonged fruit shelf life ACC synthase Antisense Impaired ethylene production and fruit ripening. Extended shelf life Ethylene receptor LeETR1 Delayed abscission, antisense shorter internode length & reduced auxin transport. No effect on pigmentation and fruit softening SAM decarboxylase Overexpression Spermidine and spermine accumulation. High lycopene, improved juice quality and longer vine life Ripening mutations Ripening-inhibitor (rin) Overexpression Complements mutation MADS-box Regulates fruit transcription factor ripening Colorless non-ripening VIGS SPB-box transcription (Cnr) Factor Inhibits fruit ripening high-pigmentOverexpression Elevated carotenoid and 2 DETIOLATED1 flavonoid accumulation Fruit Texture Polygalacturonase (PG) Overexpression Increased pectin solubilization Antisense Slightly firmer fruit. Increased juice viscosity. Reduced pectin solubilization PG-b-subunit

Antisense

Reference Fray et al. (2000)

Xiao et al. (2008)

Xiong et al. (2005) Oeller et al. (1991)

Whitelaw et al. (2002)

Mehta et al. (2002)

Vrebalov et al. (2002)

Manning et al. (2006)

Davuluri et al. (2005)

Giovannoni et al. (1989)

Langley etal. (1994), Schuch et al. (1991), Kramer et al. (1992), Brummell and Labavitch (1997) Increased fruit softening, Watson et al. (1994), decreased middle Chun and Huber lamella cohesion. (1998) Reduced tissue integrity (continued)

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Biotechnological Interventions to Improve Plant Developmental Traits

Table 5.4 (continued) Enzyme/protein Pectin methylesterase

Pectate lyase b-galactosidase1

b-galactosidase3

b-galactosidase4

Expansin Expansin and PG

PG and PME Cellulase

endo-1,4-b –glucanase

Transgene Antisense

Phenotype No effect on softening but reduced tissue integrity in overripe fruit. Increased degree of methylesterification. Reduced pectin degradation. Increased soluble solids. Increased juice and paste viscosity. Increased serum viscosity Antisense Increased fruit firmness in strawberry. Antisense No effect on cell wall galactose. No effect on fruit softening. Antisense Reduction in TBG1 and TBG4 transcripts. Higher GalA in cell wall. Reduced fruit deterioration. Antisense Reduced tomato fruit softening. Higher GalA in cell wall. Antisense Increased firmness; reduced polymer. Both antisense Increased fruit firmness Modified juice rheology. Both antisense Modified juice rheology. Antisense No reduction in xyloglucan. depolymerization, pepper. Overexpression No increase in xyloglucan. depolymerization, pepper

215

Reference Tieman et al. (1992), Hall et al. (1993), Tieman and Handa (1994), Thakur et al. (1996a, b)

Jime´nez-Bermu´dez et al. (2002) Carey et al. (2001)

de Silva and Verhoeyen (1998)

Smith et al. (2002)

Brummell et al. (1999b) Kalamaki et al. (2003)

Errington et al. (1998) Harpster et al. (2002a)

Harpster et al. (2002b)

loci and genes that play a role in determining fruit shape, size and weight. Although so far these genes have not been used to develop cultivars with altered fruit physical attributes, they provide a resource to genetically engineer fruit crops for different phenotypic attributes. Fruit size and shape seem closely related as more extreme shapes were more confined to larger fruit phenotype than smaller fruits (van der Knaap and Tanksley 2003). Mutation in three genes (ovate, sun and fs8.1) affected fruit shape through

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modulation of early stages of carpel development (Gonzalo and van der Knaap 2008). About 30 quantitative trait loci (QTLs) have been identified regulating tomato fruit size and shape but less than 10 loci could account for majority of the changes associated with tomato domestication (Grandillo et al. 1999; Tanksley 2004). The effect of these loci appears to be largely confined to fruit mass except fw3.1 (van der Knaap et al. 2002; van der Knaap and Tanksley 2003). The fw2.2 has the strongest effect on fruit size and is associated with a hyper mitotic index during cell division stage just after anthesis. It encodes a negative repressor of cell division particularly during fruit development and has a sequence similarity to the human oncogene c-H-ras p21A (Frary et al. 2000; Cong et al. 2002). Introduction of fw2.2 into a large-fruited cultivar causes expected reduction in fruit size. fasciated locus on chromosome 11 and the locule number locus on chromosome 2 are reported to increase fruit size by increasing the number of carpels in the flower, which after fertilization develops into locules resulting in larger and wide fruits (Lippman and Tanksley 2001; Barrero et al. 2006). All the large-fruited, multilocular tomatoes carry mutations in either one or both of these loci while plants carrying mutated alleles of fasciated can produce more than 15 locules that affect the shape of the fruit. Although the exact nature of the genes at these loci is yet to be characterized, putative tomato homologs for fascinated locus have been identified in Arabidopsis. Thus far, a complete separation between the loci that control fruit size and those that control fruit shape has not been achieved. The organ-determining genes fasciated and locule number affect both the final size and shape of the fruit. However, the fruit size loci, fw1.1, fw2.2, fw3.1 and fw4.1 exert their effects largely on fruit growth resulting in changes in size with no or little change in shape. Three major loci, ovate (chromosome 2), sun (chromosome 8), and fs8.1 (chromosome 8), that modulate fruit shape have minimal effect on size. Ovate locus could account for both pear and elongated shape of tomato and the sequence comparisons of OVATE alleles indicated that all tested pear-shaped varieties of tomato share the same nonsense mutation that causes truncation of the predicted protein and could account for the loss-of-function (recessive) nature of these alleles (Ku et al. 1999; Liu et al. 2002). A single major locus, fs8.1, is responsible for both blocky and slightly elongated appearance of processing tomatoes (Grandillo et al. 1996). Changes in fruit shape caused by fs8.1 are initiated very early in floral/carpel development (Ku et al. 2000).

5.5.2

Ripening Mutant Genes

A large number of genes that regulate fruit development and ripening have been cloned and functionally characterized (Alba et al. 2005; Giovannoni 2007; Seymour et al. 2008). Genes responsible for attenuated ripening in ripeningimpaired tomato mutants have provided an attractive model to modify fruit ripening. Table 5.4 lists some of the genes that have been used to modify fruit ripening

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217

process. Several single gene tomato-ripening mutants that exhibit attenuated ripening have been known for quite some time. Identification and molecular characterization of these mutant genes have opened new avenues for modifying ripening process in fruit crops by transgenic approaches. RIPENING-INHIBITOR (Vrebalov et al. 2002) and COLOURLESS NON-RIPENING (CNR) mutants encode a MADS-box and SPB-box (Manning et al. 2006) genes, respectively. These genes are required for ripening and their study has begun to provide an insight into mechanisms that control ripening upstream of the plant ripening hormone, ethylene. Delving into the Never-ripe mutant led to the cloning of the first ethylene receptor gene from tomato (Wilkinson et al. 1995). Studies on Green-ripe identified a gene that encodes a fruit-specific, novel component that likely affects ethylene receptor-copper homeostasis during ethylene signaling (Barry and Giovannoni 2006). Gene mutations responsible for the high-pigment 1 (Damaged DNA Binding Protein 1 (DDB1)) and high-pigment 2 (Detiolated1 (DET1)) phenotypes should lead to dissection of regulatory pathways that impact metabolic content and associated fruit quality (Mustilli et al. 1999; Liu et al. 2004).

5.5.3

Ethylene-Regulated Fruit Ripening

Fruit ripening is a genetically regulated transitional period during which many dynamic processes occur which are manifested into perceivable and altered changes including firmness, pigmentation, and weight loss. Although these changes are desirable for human consumption, overripeness leads to wastage and discarding large portions of harvested fruit. Once initiated, ripening is a relatively irreversible process. Treatment with inhibitors of ethylene biosynthesis (e.g., aminoethoxyvinylglycine) or ethylene perception [e.g., silver salts or 1-methylcyclopropene (1-MCP)] results in delaying fruit ripening (Mattoo and Suttle 1991; Lurie and Paliyath 2008). However, the recognition of involvement of ethylene-dependent as well as ethylene-independent processes in ripening and fruit quality is impacted by preventing the ethylene responses in most fruits. Nevertheless, since ethylene plays a critical role in fruit ripening (Oeller et al. 1991), especially in climacteric fruits, approaches altering ethylene biosynthesis or its perception have been largely used to engineer ethylene insensitivity in several plant species. Fruit ripening control has been achieved by downregulating the expression of ACC synthase (Oeller et al. 1991) or ACC oxidase (Hamilton et al. 1990). Overexpression of a bacterial ACC deaminase (Klee et al. 1991) or SAM hydrolase (Good et al. 1994) also resulted in attenuated fruit ripening in tomato by reducing the levels of ethylene biosynthesis substrates ACC and SAM, respectively. Engineered suppression of a member of ethylene receptor family, LeETR4, in a fruit-specific manner was found to result in early fruit ripening without large effects on fruit size, flavor or yield (Kevany et al. 2008). These authors suggested that ethylene receptors might work as biological clocks to regulate the onset of fruit

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ripening. A progeny from a cross between two transgenic tomato lines impaired in the expression of LeEtr1 or LeERT2 using antisense RNA technology had a phenotype similar to that observed for LeERT1 antisense plants indicating its role in ethylene signaling during tomato growth and development (Wang et al. 2006a, b). The functional significance of members of ethylene-receptor family and downstream signaling is complex (Kendrick and Chang 2008). Degradation of ethylene receptor was found correlated with enhanced sensitivity to ethylene (Kevany et al. 2007).

5.5.4

Transgenic Interventions to Alter Fruit Texture

The economic value of post-harvest fruit softening has generated considerable interest in understanding the biochemical changes associated with fruit textural modifications (Brummell 2006; Vicente et al. 2007; Negi and Handa 2008). Ripening-associated fruit textural changes usually involve structural modification of the polysaccharide components of the primary cell wall and middle lamella. Structural proteins, such as expansins, also play an important role in this process (Brummell 2006; Vicente et al. 2007). The overall interplay between primary cell wall and middle lamella components is a very complex process. Loosening the structure of and cross-links between the cell wall and middle lamella have been implicated in the transition of a firm unripe fruit to a softer/crispy and juicy fruit (Brummell 2006). Genetic engineering approaches have led to the identification of the complexity of the cell wall chemistry and the role various cell wall modifying enzymes play in fruit softening and texture. Table 5.4 lists the phenotypes of transgenic plants obtained by decreasing the expression of genes encoding some of these enzymes. Antisense inhibition of polygalacturonase (PG) had only a slight effect on fruit softening but significantly impacted depolymerization of pectins, resulting in increased juice viscosity due to higher molecular size of the constituent pectins (Thakur et al. 1997). Similarly, transgenic plants expressing an antisense gene of pectin methylesterase (PME) showed >95% reduction in fruit PME activity concomitant with markedly improved juice viscosity and increased total soluble solids (Tieman et al. 1992; Gaffe et al. 1994; Thakur et al. 1996a,b). Although softening of transgenic fruits with reduced PME activity occurred to similar levels as the nontransgenic fruits, the transgenic fruits exhibited loss of tissue integrity after extended storage (Tieman and Handa 1994). Homology-dependent silencing of TBG1 resulted in about 90% reduction in its transcript accumulation but it did not affect the total exogalactanase activity, cell wall galactose content or fruit softening, indicating little, if any, role of this gene in fruit texture (Carey et al. 2001). Suppression of TBG3 by antisense RNA caused reduction in TBG1 and TBG4 transcript levels and ~75% loss in extractable exo-galactanase activity with retention of the cell wall galactose content but with little effect on fruit softening (de Silva and Verhoeyen 1998). However, reduction in TGB4 transcripts by antisense

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219

RNA caused about 90% reduction in extractable exogalactanase activity, reduced galactose levels in mature green fruits, and produced firmer fruits than the control (Smith et al. 2002). Specific members of the ß-galactosidase, expansin and pectate lyase (PL) gene families partially regulate softening in tomatoes and strawberry (Brummell et al. 1999b; Jime´nez-Bermu´dez et al. 2002; Smith et al. 2002). However, functional analyses of specific PGs, PMEs, xyloglucan endotransglucosylase/hydrolases (XTHs) and 1,4-endoglucanases (EGases) in tomatoes, strawberries and peppers have failed to establish their roles in softening (Lashbrook et al. 1998; Brummell et al. 1999a; Woolley et al. 2001; Harpster et al. 2002a,b). Fruit firmness was not significantly altered when PG or expansin was suppressed using transgenic tomatoes, but when genes encoding both proteins were simultaneously downregulated fruit firmness was retained for longer duration (Powell et al. 2003).

5.6

Cuticle Modification

Cuticle is the outermost layer of plants (Fig. 5.3), which plays important roles in preventing plant dehydration by limiting non-stomatal water loss, improving resistance of plants to biotic and abiotic stresses (See Chaps. 2-1 and 2-2), controlling plant morphology, and regulating organ fusion (Pollard et al. 2008; Samuels et al. 2008). As a barrier to pathogen attack, cuticle negates germination of pathogen spores (Gniwotta et al. 2005). Further, cuticle plays a role in osmotic adjustment as well as in protecting plants against the negative effects of light, temperature and pollution (Shepherd and Griffiths 2006). By regulating gas exchange, this layer likely plays a significant role in extending post-harvest shelf life of fruits and vegetables (Saladie et al. 2007). The cuticle layer is composed of two main lipophilic components: cutin and cuticular wax. Cutin is lipid-derived polyester composed mainly of a- and o-hydroxyl and epoxy C16 and C18 fatty acids and glycerol (Nawrath 2006). The wax component of cuticle is made up of aliphatic C24–C34 compounds made

Fig. 5.3 Section of a leaf showing the cuticle with respect to other tissues. Adapted from Purves et al. (1997)

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entirely of carbon and hydrogen or including functional groups like alcohol and ketone (Kunst and Samuels 2003). The pathways responsible for the formation of this component including its transport to the outside of a plant organ as well as the intercellular and extracellular assembly remain to be elucidated (Pollard et al. 2008). Microarray analysis of transcripts (Chaps. 2-10) from the top epidermis of Arabidopsis led to the isolation of 85 upregulated genes playing a role in lipid metabolism (Chung et al. 2005), whereas the same analysis in the wax deposition zone in barley leaf led to the isolation of five upregulated wax-deposition genes (Richardson et al. 2007). Although more experimental evidence is needed, these data suggest that the number of genes involved in cuticle assembly is rather few compared to the number of genes participating in the biosynthetic pathway of the cuticle components. Double knockouts of glycerol-3-phosphate acyltransferase (GPAT), gpat4/gpat8, caused significant reduction in cutin. The mutant plants were less resistant to desiccation and infection by the fungus Alternaria brassicicola. Also, overexpression of GPAT4 or GPAT8 led to about 80% increase in the levels of C16 and C18 cutin monomers in leaves and stems (Li et al. 2007b). In order to modify cutin composition, these authors overexpressed the acyltransferase GPAT5 and the cytochrome P450-dependent fatty acyl oxidase CYP86A1, two enzymes associated with suberin biosynthesis. The simultaneous overexpression of both enzymes caused accumulation of new C20 and C22 o-hydroxyacids and a,odiacids typical of suberin with altered fine structure and water-barrier function of the cuticle (Li et al. 2007b). Substantial experimental evidence has accumulated on the roles that cuticle plays in various developmental and physiological processes. Modification of cuticle has been shown to result in a number of attributes such as: (a) Altered leaf and petal epidermal cell structure, trichome number and branching as well as density of stomata (Aharoni et al. 2004) (b) Reduced leaf size and plant growth, seed production and seed germination (Schnurr et al. 2004) (c) Abnormal bending of embryos, ectopic adhesion between cotyledons, a permeable epidermal structure and an altered distribution pattern of stomata in several tissues (Tsuwamoto et al. 2008) (d) Dwarf appearance due to amorphous and smaller cells and enhanced tendency to dehydrate (Cominelli et al. 2008) (e) Altered morphology of trichomes and pavement cells (Panikashvili et al. 2007) (f) Post-genital organ fusions and stunted growth (Sieber et al. 2000; Bird et al. 2007; Luo et al. 2007; Ukitsu et al. 2007) (g) Hypersensitivity to drought (Chen et al. 2004; Ukitsu et al. 2007) (h) Disruption of microspore normal development along with reduced pollen development (Jung et al. 2006) (i) Irregular shape of pollen (Ukitsu et al. 2007). In tomato fruits, the manipulation of cuticle by expressing the gene cwp1 (cuticular water permeability 1), which imparts the microfissure/dehydration phenotype in the wild species Solanum habrochaites, caused a microfissured fruit

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Table 5.5 Genes with potential to modify cuticles Gene Function Physiological effect CaCAF1 DQ672569

Transcription and mRNA decay

WXP1 TC107019

Ethylene response transcription factor Ethylene response transcription factor

WXP2 TC94548

GPAT5 Glycerol acyl At3g11430 transferase CUTE Cutinase protein M29759 SHN1 AP2/EREBP At1g15360 transcription SHN2 factors At5g11190 regulating lipid SHN3 biosynthesis At5g25390

Biotechnology Reference intervention Growth enhancement, Overexpression Sarowar thicker cell walls and pCaMV35S et al. cuticle layers. Increased (2007) resistance against Phytophtora infestans Enhanced drought and Overexpression Zhang freezing tolerance pCaMV35S et al. (2007b) Enhanced drought Overexpression tolerance. Increased pCaMV35S low-temperature sensitivity. Reduced growth. Increased drought tolerance Overexpression Li et al. pCaMV35S (2007b) Total resistance to Botrytis Overexpression Chassot cinerea pCaMV35S et al. (2007) Increased cuticular wax, Overexpresion Aharoni drought tolerance and pCaMV35S et al. recovery. (2004)

cuticle leading to a dehydrated fruit (Hovav et al. 2007). Table 5.5 lists some of the genes that have been manipulated to modify cuticle and their effects on plant growth and development. Transgenic tomato plants overexpressing CaCAF1, a CCR4-associated factor 1 protein belonging to the CCR4-NOT complex that plays an important role in the control of transcription and mRNA decay in yeast and mammals, exhibited significant growth enhancement, with thicker leaves with twofold enlarged cell size, thicker cell walls and cuticle layers, and enhanced resistance against Phytophthora infestans compared to the control plants (Sarowar et al. 2007). In the same study, virus-induced silencing (Chap. 1-2) of CaCAF1 in pepper resulted in significant growth retardation and enhanced susceptibility to bacterial pathogen Xanthomonas axonopodis pv. vesicatoria. Introduction of putative ethylene-responsive transcription factor (ERF) genes, WXP1 and WXP2, from Medicago truncatula into Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response to freezing tolerance (Zhang et al. 2007b). Both WXP1 and WXP2 transgenic plants showed increase in n-alkanes, the major wax component in Arabidopsis, but only the WXP1 transgenic plants exhibited increase in the amount of primary

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alcohols. The WXP1 transgenic plants showed no change, but the WXP2 plants exhibited increased chlorophyll bleaching. Both WXP1 and WXP2 transgenic plants retained more water than the control and exhibited significantly enhanced plant drought tolerance. On the basis of electrolyte leakage from detached leaves, the WXP1 plants showed increased freezing tolerance while the WXP2 plants were more sensitive to low-temperature stress than the control plants. WXP1 overexpressing plants showed no obvious effects on plant growth and development, but the expression of WXP2 resulted in slower plant growth. It is becoming increasing clear that the manipulation of cuticle will help designing plants with higher resistance to pathogens and abiotic stresses. Stress due to fungi and water loss are significant factors causing post-harvest losses of fruit and vegetable crops (Troncoso-Rojas and Tiznado-Herna´ndez 2007). The cuticle engineering has a high potential to reduce these losses.

5.7

Abscission

Abscission is a process by which plant organs (leaves, flowers and fruits) detach (are shed) from the parent plant. This process is under the cue of developmental signals as well as environmental stimuli. Thinning or reduction of crop load, a common practice in the production of fruit crops, involves manual or chemicalinduced detachment of excess fruits to optimize fruit size. Harvesting of fruit crops is one of the most demanding aspects of fruit production both in terms of labor as well as economic inputs. There is currently renewed interest in mechanical harvesting to circumvent these issues. Efficiency of mechanical harvesting systems can be greatly improved by facilitating abscission-related processes. However, untimely detachment of immature fruit can lead to crop loss and low profitability, and therefore needs to be avoided. In seed crops, pod shattering and seed loss can be prevented through better understanding of dehiscence, a process that is somewhat similar to abscission. Hence, knowledge of abscission and its manipulation can greatly benefit crop production. Abscission occurs at specialized regions, termed abscission zones (AZ), which are located proximal to the organ that will subsequently detach (Fig. 5.4). Cells within the AZs are often small and rounded, and usually undergo extensive cell division prior to organ separation (Sexton and Roberts 1982; Goren 1993). Cell separation in the AZ occurs within a group of cells called the separation layer. Activity of cell wall hydrolytic enzymes, in response to the abscission stimulus, leads to dissolution of the primary cell wall and the middle lamella within cells of the separation layer (Taylor and Whitelaw 2001; Roberts et al. 2002). Subsequent loss of adhesion or collapse of cells in the separation layer leads to organ detachment due to the weight of the subtending organ. Either during the process of separation or immediately following it, cells within the layer proximal to the separation layer expand to form a protective layer (Patterson 2001).

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Biotechnological Interventions to Improve Plant Developmental Traits

Abscission Zone Differentiation

JOINTLESS; BOP1; BOP2 (AZ)

SHATTERPROOF SHATTERPROOF2 (DZ)

223

FRUITFULL

AZ (Separation Layer) ETHYLENE (ACC SYNTHASE; ETR1; EIN2)

Abscission Signaling

AUXIN (ARF2) ETHYLENE INDEPENDENT (DAB; IDA)

Cell Separation

Cell Wall hydrolases IDA

Fig. 5.4 Progression of abscission is represented here in three phases involving: abscission zone (AZ) differentiation; abscission signaling; and cell separation. AZ differentiation is regulated by genes such as the JOINTLESS gene in tomato and the BLADE ON PETIOLE (BOP1; BOP2) genes in Arabidopsis. The dehiscence zone (DZ) differentiation is regulated by expression of the SHATTERPROOF genes which are in turn inhibited by the FRUITFULL gene in Arabidopsis siliques. Abscission signaling is antagonistically regulated by ethylene and auxin. Additionally, ethylene-independent mechanisms involved in Arabidopsis flower abscission have been reported in the delayed abscission (dab) and inflorescence deficient in abscission (ida) mutants. Cell separation is facilitated by the degradation of the middle lamella and the cell wall, activities that are largely facilitated by cell wall hydrolases. Additionally, the IDA gene may also be involved in regulating final stages of cell separation

5.7.1

Development of the Abscission Zone

Development and differentiation of AZs are under tight genetic control and usually occur at specific sites within the plant (Fig. 5.4). Several genes associated with the development of AZ and those that have an impact on the abscission process are

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Table 5.6 Genes with potential to modify abscission Gene Function Role in Biotechnology abscission intervention JOINTLESS MADS-box AZ Mutation; over Development expression; silencing SHATTERPROOF, MADS-box Dehiscence zone Mutation SHATTERPROOF2 development FRUITFULL MADS-box Dehiscence zone Mutation; development overexpression

AGL15

MADS-box

Progression of abscission

Mutation

CEL1, CEL2

Cell wall modification

Promote cell separation

Silencing

Polygalacturonase

Cell wall modification

Promote cell separation

Silencing

ACC synthase

Ethylene biosynthesis

Promotes floral organ abscission

Overexpression; silencing

ETR1; EIN2

Ethylene Ethyleneperception mediated and signaling abscission signaling

Mutation; silencing

DAB; IDA

Unknown

Delayed floral organ abscission

Mutation

ARF2

Auxin response Delayed floral factor organ (transcription abscission factor)

Mutation

Reference Mao et al. (2000) Liljegren et al. (2000) Ferrandiz et al. (2000), Østergaard et al. (2006) Fernandez et al. (2000) Lashbrook et al. (1998), Brummel et al. (1999a) GonzalezCarranza et al. (2007), Jiang et al. (2008) Lanahan et al. (1994), Ecker and Theologis (1994) Bleecker and Patterson (1997), Whitelaw et al. (2002) Patterson and Bleecker (2004), Butenko et al. (2006) Ellis et al. (2005)

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listed in Table 5.6. One of the key genes regulating AZ development was isolated using a spontaneous mutation in tomato, jointless. The jointless mutants do not possess the AZ that typically forms midway along the flower/fruit pedicel. The JOINTLESS gene was isolated using map-based cloning and found to encode a MADS-box transcription factor that is essential for the development of the pedicel AZ (Mao et al. 2000). Overexpression of the JOINTLESS gene in the mutant background restored AZ development albeit not in the same location as in the wild type, while antisense suppression of this gene resulted in loss of the flower pedicel AZ in tomato (Mao et al. 2000). Several other MADS-box genes have been implicated in dehiscence. Two such genes are SHATTERPROOF (SHP1) and SHATTERPROOF2 (SHP2) both of which redundantly control cell separation by affecting dehiscence zone formation and its lignification in Arabidopsis siliques (Liljegren et al. 2000; Lewis et al. 2006). In contrast, FRUITFULL, another MADSbox gene, negatively regulates dehiscence zone development by inhibiting expression of the SHATTERPROOF genes (Ferra´ndiz et al. 2000). Another MADS-box gene, AGL15, is also implicated in regulating abscission through delaying progression of the process (Fernandez et al. 2000). Recently, two redundant BLADE ON PETIOLE genes, BOP1 and BOP2, have been identified as essential regulators of abscission zone development in Arabidopsis. These genes belong to the nonexpressor of PR1 protein (NPR1) family and appear to be involved in the establishment of the floral AZ as well as the vestigial cauline leaf AZ in Arabidopsis (McKim et al. 2008). Such genes affecting AZ zone development have immense potential for genetic manipulation of organ abscission in crops. In fact, the jointless mutant background has been widely used for developing “stemless” processing tomatoes that are not physically damaged during storage (Mao et al. 2000). Also, ectopic expression of FRUITFULL has been used to prevent unwanted pod dehiscence in Brassica (Østergaard et al. 2006).

5.7.2

Hormonal Control of Abscission

Ethylene has long been known to be a primary natural regulator of abscission (Jackson and Osborne 1970). Increase in ethylene evolution is often associated with the abscission process (Goren 1993; Taylor and Whitelaw 2001). Experiments involving genetic manipulation of ethylene biosynthesis, perception and signaling support a role for ethylene in promoting abscission of various plant organs. Constitutive expression of ACC synthase resulted in enhanced ethylene production and earlier flower abscission in tomato (Lanahan et al. 1994), whereas antisense suppression of ACC synthase delayed abscission in Arabidopsis (Ecker and Theologis 1994). The ethylene receptor mutant, etr1, and the ethylene insensitive mutant, ein2, exhibit delayed abscission of floral organs in Arabidopsis and tomato (Bleecker and Patterson 1997; Whitelaw et al. 2002; Patterson and Bleecker 2004). One of the main mechanisms through which ethylene aids cell separation is by enhancing expression and activity of cell wall hydrolyzing enzymes in the AZ.

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However, recent work with the delayed abscission (dab) and inflorescence deficient in abscission (ida) mutants, which exhibit altered floral abscission responses in an ethylene-independent manner, have led to the suggestion that ethylene may not be absolutely required for abscission in Arabidopsis but may act as an accelerator of the abscission process (Patterson 2001; Patterson and Bleecker 2004; Butenko et al. 2006). Nonetheless, alteration of ethylene biosynthesis and/or perception continues to be a promising approach for genetically altering abscission in plants. Auxin plays a dual role in regulating abscission. While auxin generally inhibits progression of abscission during early stages of the process, application of auxin at later stages accelerates abscission by increasing ethylene biosynthesis (Brown 1997). In fact, the ratio of ethylene and auxin levels at the AZ is considered a key factor determining progression of abscission (Sexton and Roberts 1982; Brown 1997). A flux of auxin across the AZ is thought to be essential to prevent abscission (Sexton and Roberts 1982). Auxin inhibits cell separation by decreasing the activity of cell wall hydrolyzing enzymes (Taylor and Whitelaw 2001). While rapid progress has been made in understanding auxin biosynthesis, transport and signaling, its role in regulating abscission has not been adequately addressed at the molecular and genetic level. Evidence is accumulating for the involvement of auxin signaling genes in regulating abscission. Expression of several AUX/IAA genes is altered in an auxin-dependent manner at the leaf and stem AZs in Mirabilis (Meir et al. 2006). Auxin response factors (ARFs) are transcription factors that, in conjunction with AUX/IAA genes, either activate or inhibit downstream auxin responsive genes. Loss of ARF2 function in Arabidopsis delayed floral organ abscission supporting a role for auxin signaling in the progression of abscission (Ellis et al. 2005). Further analysis of the molecular machinery involved in auxin transport and signaling during abscission should lead to the identification of potential candidates that can be utilized to alter abscission in plants.

5.7.3

Cell Wall Degradation during Cell Separation in the Abscission Zone

Enzymes facilitating dissolution of the middle lamella and the cell wall, and degradation of the cell membrane are obvious candidates for manipulating abscission. These include cellulases (Cels), PGs, pectin methylesterases (PME), expansins and phospholipases (Cho and Cosgrove 2000; Roberts et al. 2002; Malladi and Burns 2008). AZ-specific Cels and PGs have been identified and isolated from many plants (Tucker et al. 1988; Kalaitzis et al. 1995; del Campillo and Bennett 1996; Kazokas and Burns 1998). Antisense inhibition of the cellulase gene, Cel1, reduced floral abscission in tomato (Lashbrook et al. 1998). Similarly, antisense inhibition of the Cel2 gene increased the force required for detachment at the pedicel AZ in tomato by almost 50% (Brummell et al. 1999a). Loss of a PG gene in Arabidopsis resulted in delayed floral organ abscission in air or in the presence

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of the abscission-promoting hormone, ethylene (Gonzalez-Carranza et al. 2007). Virus-induced gene silencing (Chap. 1–2) of a tomato abscission-related PG gene, TAPG1, delayed ethylene-regulated petiole abscission and increased petiole break strength (Jiang et al. 2008). An analysis of the ida mutant in Arabidopsis revealed a function for this gene in regulating the final steps of cell separation, possibly through the secretion of arabinogalactan proteins. Constitutive expression of IDA results in enhanced floral organ abscission as well as ectopic abscission at several locations in Arabidopsis (Stenvik et al. 2006). These studies demonstrate practical applicability of altering cell wall/membrane modifying enzymes to manipulate organ abscission.

5.8

Programmed Cell Death

PCD is a highly coordinated and complex process (Fig. 5.5) that involves dismantling of the cellular apparatus. It serves an important function in the development and defense of the organism. Plants use a well-defined and self-regulated program of cell death during vascular bundle formation, defense management and senescence of leaf, flower or fruit. Whether senescence in plants is a form of PCD or precedes PCD is being debated in the literature; one school of thought considers senescence as a complete overlap with PCD (van Doorn and Woltering 2005), while others view the latter as culmination of senescence events (Mattoo and Handa 2004; Reape et al. 2008). The molecular events during senescence and hypersensitive response (HR)-induced cell death share some common features but vary in the time frame of their completion. PCD is believed to be more spontaneous and has a shorter span than the senescence. Both plants and animals employ PCD to complete their developmental process and manage defense against pathogenic (biotic) or abiotic stresses. Advances in our understanding of PCD have provided opportunities for biotechnological improvements in the plants ranging from a markedly improved tolerance against stress to delayed senescence for higher biomass (Table 5.7) Cell death is fundamental to the life cycle of an organism. In nature, it forms a link in the evolutionary process that facilitates every form of life to adapt to the natural vagaries. In multicellular organisms, the cell death serves the purpose of removing damaged or redundant cells. In animals, PCD occurs in two morphologically distinct ways – apoptosis and autophagy. Another mode of cell death is necrosis whose initial events bear footprints of PCD. Autophagy is a highly conserved mechanism involving degradation of cytoplasmic contents by the lysosomal vacuoles. It helps in the mobilization of cell constituents before the cell death. An autophagic type of PCD is often observed in plants during growth and development of growing cells (tissues), e.g., in tracheary elements or root growth and expansion. Animal cell apoptosis is characterized by cell shrinkage, nuclear fragmentation, formation of apoptotic bodies and their engulfment by the lysosomic action of another cell (Adrain and Martin 2001). In plants, the engulfment of apoptotic bodies by another cell is unknown. Characteristics such as retraction of protoplast, DNA

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Fig. 5.5 A model of programmed cell death in plants. JA jasmonic acid; MAPK mitogen-activated protein kinase; NO nitric oxide; PAMPs pathogen-associated molecular patterns; PK protein kinase; PM plasma membrane; PRI pathogen R-gene protein interaction; RLK receptor-likekinase(s); ROS reactive oxygen species; SOD superoxide dismutase

fragmentation and involvement of caspase-like proteins are shared by plant apoptosis. However, owing to yet-to-be-found other animal apoptotic characteristics, some investigators have preferred to identify the phenomenon in plants as apoptotic-like PCD (AL-PCD) (van Doorn and Woltering 2005; Reape and McCabe 2008). In plants, HR-induced PCD is a key defense mechanism to restrict the spread of pathogens during a compatible host–pathogen interaction. Also, induction of HR takes place during abiotic stresses due to cold, heat, drought, salinity, high light intensity, or ultraviolet radiation to mitigate the stress effect. The core components

Table 5.7 Genes related to programmed cell death and their potential to modify plant development or agronomic traits Gene Product/Function Effect Biotechnology intervention Fungal or bacterial resistance CaPO2 Extracellular peroxidase Resistance to P. syringae pv. Overexpression of pepper gene in Arabidopsis tomato OsAOS2 Allene oxide synthase Resistance to Magnaporthe Overexpression of the gene in rice grisea (rice blast) RCT1 TIR-NBS-LRR - R gene protein Resistance to anthracnose Expression of M. truncatula gene in alfalfa disease Pto Serine/threonine protein kinase – Resistance to P. syringae Overexpression of tomato gene R-gene protein Rpi-blb1 CC-NBS-LRR-R gene protein Resistance to Phytophthora Expression of Solanum bulbocastanum in potato infestans (late blight) (Expression of wild species potato gene in cultivated potato) Rxo1 NBS-LRR-R gene protein Resistance to Xanthomonas Expression of maize gene in rice oryzae N1141-flaA Flagellin Resistance to M. grisea (rice Expression of bacterial gene in rice blast) AtNPR1 NPR1 protein Resistance to Fusarium Expression of Arabidopsis gene in wheat head blight Cf9 & Avr9 R-gene proteins Resistance to Leptosphaeria Expression of tomato genes in Brassica napus maculans Viral protection Bcl-xL Or Animal cell death suppressor protein Protection from virusExpression of animal gene(s) in tomato Ced-9 induced necrosis Insect resistance ProsysteA signal protein for JA synthesis Resistance to herbivores Overexpression in tomato min gene Tolerance to abiotic stress Antioxidant Removal of superoxide radical Enhanced salt, drought Overexpression genes tolerance See Ashraf (2008) (continued)

Li et al. (2002)

Xu et al. (2004)

Zhao et al. (2005) Takakura et al. (2008) Makandar et al. (2006) Hennin et al. (2001)

Choi et al. (2007) Mei et al. (2006) Yang et al. (2008) Tang et al. (1999) van der Vossen et al. (2003)

Reference

5 Biotechnological Interventions to Improve Plant Developmental Traits 229

Transcription factor

Poly (ADP-ribose) polymerase

DREB1

PARP

Plastid NDH gene

Delayed leaf senescence

Delayed leaf senescence

OsDOS

Nuclear-localized CCCH-type zinc protein – negative regulator of JA A component of chloroplast NDH complex

Delayed leaf senescence

Bcl-xL or Animal cell death suppressor protein Ced-9 Delayed senescence AtNAP Transcription factor

Improved tolerance to drought, cold, and salinity Tolerance to high light, heat, and drought Salinity, cold, and wound tolerance

Enhanced drought tolerance Expression of tobacco gene in maize

Protein kinase

NPK1

Suppression of the gene in tobacco

Overexpression in rice

Suppression of the gene

Overexpression of animal gene(s) in tobacco

Suppression of the gene

Overexpression of the gene

Expression of tobacco gene in maize

Freezing tolerance

Effect Biotechnology intervention Enhanced drought tolerance Expression of IPT gene

Table 5.7 (continued) Gene Product/Function IPT Isopentenyl transferase – in cytokinin biosythesis MAPKKK Phosphorylation of regulatory proteins

Guo and Gan (2006) Kong et al. (2006) Zapata et al. (2005)

De Block et al. (2005) Qiao et al. (2002)

Reference Rivero et al. (2007) Shou et al. (2004a) Shou et al. (2004b) Ito et al. (2006)

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of PCD include perception of stimulus (extrinsic or intrinsic), signaling cascade and execution of cell death. The mechanism of perception of biotic stimulus is relatively better understood than that of any abiotic stimulus. The pathogens are recognized by receptors at the cell surface through pathogen-associated molecular patterns (PAMPs), which are highly conserved structural components required to support the lifestyle of a pathogen. The host receptors are termed pattern-recognition receptors, which resemble in function with toll-like receptors in animals (Nurnberger et al. 2004; Zipfel et al. 2004). A receptor–pathogen interaction triggers HR that helps to contain the proliferation and spread of pathogens to neighboring cells. An endeavor to survive – a basic principle of evolution – allows certain pathogens to evade host resistance by blocking the interaction-driven HR through secretion of “effectors.” Interestingly, recognition of these effectors by “R” (resistance) gene products enables plants to become resistant (Chisholm et al. 2006). Characterization of R genes has become a potential tool in marker-assisted breeding and genetic engineering to develop disease-resistant plants. Once pathogen is recognized, a defense response is initiated and decision on the fate of cell is made. The available information suggests the involvement of a multicomponent mechanism, which is conserved in animals and plants in its basic form (Hoeberichts and Woltering 2003; Franklin-Tong and Gourlay 2008; Williams and Dickman 2008). Some regions of the R-gene product show significant similarity with apoptotic proteins from humans (APAF) and Caenorhabditis elegans (CED4). The apoptotic proteins recruit caspases, which are specific type of cysteine proteases capable of acting on large number of substrates to effect cell death. In plants, though true caspases are yet to be identified, caspase-like proteases called metacaspases have been detected (see Mattoo and Handa 2004). The animal cell apoptosis is regulated by pro-apoptotic (e.g., CED4, APAF, Bax) and anti-apoptotic (e.g., CED9, Bcl-2, Bcl-xL) proteins. Bioinformatics has allowed predicting the presence of antiapoptotic proteins in plants despite the lack of sequence similarity with the animal counterparts. An ectopic expression of animal anti-apoptotic proteins suppressed apoptosis and imparted tolerance against certain type of stresses in plants (Table 5.7). In HR-induced PCD, reactive oxygen species (ROS) and nitric oxide (NO) have emerged as early messengers in the signaling cascade leading to defense response. The most notable species are superoxide radical (O2-1) and hydrogen peroxide (H2O2). A superoxide radical is highly reactive and dismutates to H2O2 by superoxide dismutase (SOD). The perception of a stimulus due to a pathogen or an abiotic stress is marked by an oxidative burst resulting in the synthesis and accumulation of ROS in large amounts. A similar increase in NO has been observed in plants (Zeidler et al. 2004). It is thought that a fine-tuned NO/ROS balance is responsible for the induction of genes during HR (Grun et al. 2006). ROS act in concert with MAPK to activate a host of biochemical responses, such as cell wall cross-linking, lignification, synthesis of pathogenesis-related (PR) proteins and phytoalexins. They are also known to regulate the synthesis of hormones, such as jasmonic acid, which in turn may initiate a defense response. NO interacts with salicylic acid and jasmonic acid, particularly during wounding, which indicates its role in stress signaling (see Grun et al. 2006). Miller et al. (2008) have reviewed the

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mechanisms of ROS signaling. Besides mediating a stress signal, ROS can deleteriously affect pathogen or host cell through oxidative damage of its biochemical constituents. Further, recent studies suggest that both ROS and NO act as signaling molecules in the development of plants (Gapper and Dolan 2006; Grun et al. 2006). Because ROS play a crucial role in the cell function, their intracellular level is tightly controlled. Different mechanisms are known to operate in the production and removal of ROS. Following pathogen recognition, the accumulation of ROS in apoplast is attributed to the action of more than just one enzyme. Respiratory burst oxidase homologs (Rboh) with similarity in function to mammalian neutrophil oxidase are recognized to play a dominant role (see Apel and Hirt 2004). More recently, extracellular peroxidases have also been shown to participate in the synthesis process (Bindschedler et al. 2006; Choi et al. 2007). During PCD in mammalian cells, mitochondria serve as a source of ROS when there is a transient change in their permeability because of stress and cytochrome-c is released to the cytoplasm. In plants, chloroplasts seem to significantly contribute to ROS generation. Accordingly, the degeneration of chloroplast is observed prior to leaf senescence and necrosis. Several antioxidant enzymes – dismutases, catalases and peroxidases – are active during HR-induced PCD, and their suppression in transgenic plants has been observed to severely compromise the ability of these plants to withstand stress. On the other hand, the increased antioxidant activity through overexpression of the genes results in enhanced tolerance against a variety of environmental stress (Table 5.7). Even though progress has been made in our understanding of ROS role in plants, their signaling pathways and metabolic crosstalk with other components are yet to be fully elucidated. Similarly, as roles of NO in plant development and stress responses are revealed (Parani et al. 2004; Lindermayr et al. 2005; Abat et al. 2008), the manipulation of its intracellular levels can serve yet another tool for biotechnological improvements in crop plants.

5.9

Future Perspectives

Molecular and genetic dissection of plant developmental processes has provided a map of complex interactions that regulate desirable plant traits. This knowledge is enabling scientists to search for regulatory genes that can be manipulated to enhance desirable developmental traits in agronomical and horticultural crops (Rothstein 2007). Details on the interactions among hormones, environment and physiology are being unraveled and have begun to reveal how differentially mediated progression of various processes regulate the life span of plants. The challenge is to identify a hierarchy of regulators or a specific pattern of events that control desirable attributes and then genetically modify critical and beneficial processes without negatively impacting other beneficial attributes in crop plants. Thus far, studies on transgenic plants and effectiveness of a gene construct have reinforced the advantage of using tissue-specific and regulatable promoters to tightly control the expression of introduced transgene and avoid affecting non-targeted developmental

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processes. Thus, the need is for identifying and characterizing tissue-specific promoters and promoter elements that control both tissue specificity and the level of expression of the introduced transgene. We also need to develop transgenic plants particularly suited to grow well in ecofriendly, sustainable agricultural systems that have minimal reliance on chemical input and synergistically (positively) influence plant metabolism (Neelam et al. 2008; Mattoo and Teasdale 2009).

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Chapter 6

Transgenics for Biofuel Crops Anjanabha Bhattacharya, Pawan Kumar, and Rippy Singh

6.1

Introduction

Fossil fuels, like petroleum and coal, are fast-depleting, nonrenewable sources that were produced over million years ago, when the higher- and lower-order plants were buried under the surface of the earth by volcanic activities and sedimentation. These were further acted upon by microorganisms leading to the formation of nonrenewable fuels. There is an urgent need to find alternative sources of energy in order to cope with the energy demands of the human race and the much-debated global climate change in the coming years. So intense is the problem that the world has been divided into energy-independent producers and energy-dependent consumer economies, triggering trade war and political unrest, leading to monopoly over production and pricing issues. In such a scenario, there is a greater urge for the fossil-fuel-dependent economies to look for alternative sources of energy to become more self-reliant when it comes to their own energy needs (Hill et al. 2006). Several alternative energy sources, which exist and are being exploited today, include wind, solar, hydroelectric, nuclear energy and possibly methane gas reserves entrapped in the underground seabed. These may provide a part of the solution. There are practical problems associated with harnessing, storage and transport of these new renewable resources compared with other popular nonrenewable sources. Biofuels or agrofuels are basically carbon-derived fuels (solid, liquid or gaseous state) where the source of carbon is either plants or animals, and are therefore indirectly solar energy sources. They can be derived from animal fats, vegetable oils (biodiesel), or from agro-residues (bioethanol, biomethanol, or biobutanol), or can be derived from solid forms (like refuse fuels, pellets, wood, sewage, and

A. Bhattacharya (*) National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_6, # Springer-Verlag Berlin Heidelberg 2010

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History 200 Liquids

150

Coal

100

Natural Gas

Renewables

50 Nuclear 0 1990

2000

2005

2010

2020

2030

Fig. 6.1 Present and projected global energy demand. Adapted from Energy Information Administration (EIA), Official Energy Statistics from US government site available at http://www.eia. doe.gov/oiaf/ieo/world.html. Original source: International energy annual report 2005 and EIA, World energy projection plus (2008).

briquettes) (Petrou and Pappis 2009), which can be used to produce energy. They are generally less efficient than fossil fuels but their usage results in less emission of greenhouse gasses (Vogt et al. 2009). Besides, biofuels are termed green fuels because they emit less carbon dioxide compared to the fossil fuels, contain no sulfur and thus decrease the global warming effect (Vogt et al. 2009). They are biodegradable and contribute to sustainability (Puppan 2002). The concept of biofuel usage in automobiles is in fact not new, but dates back to the early nineteenth century, when Rudolf Diesel used peanut oil to drive engines used in agriculture machinery (ijalwan et al. 2006; Murugesan et al. 2009). However, the availability of low-cost fossil fuels in practical abundance resulted in the neglect of biofuel research. Rapid industrialization and fast economic growth of several developing nations, including China and India, in the twentieth century resulted in greater consumption of once abundant resources, which led to investigations of the feasibility of alternative fuels, and biofuels in particular. The annual global fossil fuel consumption and biodiesel production is estimated to be about 4.018 and 0.107 billion tons, respectively (Demirbas 2008), and is increasing every year (Fig. 6.1). Initially, biofuel production came from food crops like sugarcane, sunflower, soybean, sugarbeet, rapeseed, and corn, which have been blamed for triggering a food crisis in recent years. This raises the following question: Which should be given a priority when it comes to making a choice between energy and food resources? The answer to this paradox lies in the recently identified potential biofuel crops, for example, poplar, hemp, members of the grass family (switch grass, burmuda grass, miscanthus, prairie and wheat) and oil-rich crops like Jatropha and Pongamia sp.

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Biofuels

Gaseous

Liquid

Solid

Briquettes Pellets Wood

Bioethanol Biomethanol Biobutanol

Biodiesel

Biogas

Municipal Agriculture & Forestry residue

Sugar and Starch rich crop plants

Oil rich crop plants

Decomposing Biomass

Fig. 6.2 Classification of biofuels

A prerequisite to using crops in efficient biofuel production is to design the most effective way to break down complex carbohydrates into simple sugars. One of the ways to circumvent this issue is to biotechnologically engineer transgenic crops with modified lignin, tannin and cellulosic biosynthetic pathway, which will make their decomposition into biofuels quicker and more efficient when acted upon by microbes. Plants provide us with food, feed and fiber. We can genetically engineer parts of plants often thrashed away, like the straw in wheat and paddy, stalks of cotton, corn and several other crop plants, to degrade more quickly than they do naturally. This will not only enhance the rate of production of biofuels but also improve efficiency of the whole production process. At the same time, we can also genetically engineer the microbial strains used in this process to increase their rate of action. Some biofuel-related terminology has been briefly explained in this section and illustrated in Fig. 6.2.

6.1.1

Bioalcohol

The simple and complex polysaccharide plant food reserves are broken down into simple sugars, and fermented to produce bioalcohol. They are specifically designated as Bioethanol and Biobutanol.

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1:6CO2 þ 6H2 O þ Solar energy

!

C6 H12 O6 þ 6O2

Glucose=simplesugar ðFermentationÞ

2: C6 H12 O6 ! 2C2 H5 OH þ 2CO2 þ energy Glucose

6.1.2

ðBioÞethanol

Biogas and Biohydrogen

Biogas is produced from decomposed organic substances by pyrolysis in the presence of anaerobic bacteria, releasing gaseous substances (generally a mixture of hydrogen, methane and carbon dioxide). Recently, scientists have developed hydrogen and nitrogen-fixing bacteria capable of producing biogas.

6.1.3

Biodiesel

Plant lipids are composed of long-chain fatty acids which are broken down into short-chain fatty acids by esterification to produce biodiesels. Typical source material for biofuels include: (a) Starch and sugars. The first generation of biofuel crops included food crops like sugarcane, corn, barley, wheat and cassava. (b) Lignocellulasic material. Unlocking the cellulose from plant cell walls and efficiently converting it to ethanol is the key to make ethanol a universal, inexpensive form of fuel for the future. Aquatic plants like lotus, lily, etc. are naturally low in lignin content, which is less than 10% (Gunnarsson and Petersen 2007), and they grow at a rapid pace (Ripley et al. 2006). (c) Plant lipids. Crop plants like soybean, corn, sunflower, oil palm, castor seed, rapeseed, and mustard, along with primitive photosynthetic microrganisms like algae (including microalgae, kelps and fucoids), cynobacteria and phytoplanktons, are naturally rich in oil or starch, which can be converted into biofuel in mutistage processing. Biodiesel is better than conventional diesel fuel with respect to sulfur content, flash point, aromatic content, and biodegradability (Hanna et al. 2005).

6.2

Transgenics Approach to Biofuels

Conventional breeding has been largely responsible for the introduction of novel traits and has played an important role in the development of new cultivars. This includes domestication of wild relatives and selection of novel sports from popular cultivated species. However, complex polygenic traits are difficult to manipulate using this method and can take considerable time (Zuker et al., 1995; Mishra and

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Srivastava 2004). Crop plants in the past were never selected on the basis of their amenability for biofuel production, and traits such as low lignin content or high cellulose were lost systematically from the gene pool. Thus, limited gene pool and failure of distant crosses in conventional breeding have lead to the exploitation of genetic transformation in generating plants of relevance for the rapidly growing biofuel industry. Growth and crop productivity are polygenic traits making their breeding difficult under natural conditions. Successful genetic manipulation depends upon the integration of transgene(s) into the host genome, the regeneration capacity of the transformed cells often via tissue culture, and subsequent expression and integration over several generations with exceptions in vegetatively propagated plants (Robinson 1998). However, a transgenic crop production approach of modifying plant architecture by making available the gene of interest (manipulating biofuel productivity traits or pathways) in a cloned form and robust plant regeneration and transformation system paves the way for integration of transgene(s) in a highly directed manner. This will ensure the development of new transgenic varieties previously difficult to obtain through the conventional breeding. With the advent of high-throughput, low-cost sequencing technology, genomes of many plant species have been sequenced and annotated. An example of the possible pathway, which could be adopted to genetically modify crop plants to yield biofuel is described in Fig. 6.3. Many genes of importance in relation to polysaccharide biosynthesis pathways have been identified using database information, and a number of expression studies have been conducted to verify such results (Ragauskas et al. 2006). For example, overexpression of expansin genes, which bring about the loosening of the cell walls of plants, may be considered for modification in biofuel crops (Cosgrove 2005). Also, emphasis must be given to identification of quantitative trait loci (QTLs) controlling lignification in plants, and breeding programs for biofuel production must emphasize selection of cultivars with low lignin content. RNA interference (RNAi) and antisense approaches can also be adopted to selectively silence the genes involved in complex carbohydrate synthesis.

6.2.1

Manipulating Genes and Pathways Involved in Starch and Sugar Metabolism

Starch is produced by all green plants for energy storage (Fig. 6.4a). It is a large polysaccharide made up of glucose linked via a-1,4 and a-1,6 glycosidic linkages (amylose and amylopectin). These are stored in plant in a form of dense semicrystalline structure. Gelatinization of starch is required for enzymatic digestion and fermentation, which can be achieved by heating starch at 60 C. Hydrolysis by glucoamylase enzyme converts the starch molecule into D-glucose. Sucrose, on the other hand, is a disaccharide of glucose and fructose. Enzymatic hydrolysis of sucrose is catalyzed by invertase, converting sucrose into glucose and fructose (both C6H12O6).

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Identify major genes and / QTL involved in complex polysaccharide biosynthesis by bioinformatics and subsequently find analogs or orthologs in other plant species.

Generate ectopic expressor /knockout (T-DNA insertion or by TILLING)/antisense/ RNAi induced silenced lines to hasten tissue degradation

Genetically engineer complex metabolic pathway like cellulose, lignin, tannin biosynthesis. Harvest and subsequent storage of the biomass

Pretreatment with genetically modified microorganism and other enhancer chemicals

Genetically manipulate microorganism / enzymes

Recovery of product and residue mix

Liquid Biofuel

Residue can be used as sillage or biofertilzer or solid / biohydrogen biofuels

Fig. 6.3 Possible pathway to genetically engineer crops to yield biofuel

C12 H22 O11 ! C6 H12 O6 þ C6 H12 O6 ðSucroseÞ

ðGlucoseÞ

ðFructoseÞ

Enzymatic hydrolysis is followed by a fermentation process for the production of bioethanol. Commercial yeast such as Saccharomyces cerevisiae converts glucose molecule into ethanol under anaerobic conditions. ðFermentationÞ

C6 H12 O6 ! ðGlucoseÞ

2 C2 H5 OH þ2CO2 ðEthanolÞ

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a Sucrose

Cytosol

Uridine diphosphate Glucose

Glucose 1phosphate

Adenosine glucose pyrophosphorylase

Adenosine diphosphate glucose Debranching enzymes

Plastid Amylose, Amylopectin

(Starch Granule)

b

Fig. 6.4 (Continued)

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Fig. 6.4 Strategies for manipulating (a) Starch (b) Cellulose (c) Lignin (d) Lipids

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Therefore, cloning genes responsible for rapid conversion to ethanol and then expression of such genes in plant system will enhance the production of ethanol. Myers et al. (2000) reported that transgenic plants with suppressed debranching enzyme produce soluble phytoglycogen, thus no gelatinization by pretreatment is required in this condition. Aside from genetic engineering of plants for reducing the production costs, specific genetic manipulation may be required depending on the source categories of biofuel. For example, in sugarcane, sugar content was increased by targeting sugar accumulated in the vacuoles and subsequently converted to isomaltulose by the enzymatic rearrangement of the glycosidic linkage from a (1–2)-fructoside in sucrose to a (1–6)-fructoside (Wu and Birch 2007). This new arrangement resulted in doubling the total sugar concentration in these transgenic lines (Gressel 2008). The increase in sugars in transgenic lines directly translates into higher yield of biofuel. Smith (2008) reported that manipulation of ADPglucose pyrophosphorylase (which plays a role in the sugar signaling pathway) to increase the yields of starch have been met with limited success. They also advocated partitioning of photosynthate in storage organs and increasing the flux of photoassimilate from source to sink. Earlier studies concentrated on manipulating carbohydrate composition of major food crops by altering the enzymes which act on photosynthesis.

6.2.2

Manipulating Genes and Pathways Involved in Cellulose and Lignin Biosynthesis

With the recent advent of increasing prices of food products and their shortage, the first generation of biofuel crops were discouraged. Instead, research was vastly concentrated on non-food crops and waste (nonutilizable biomass) from food crops, which could be converted into biofuels without compromising the food needs of mankind. This strategy could lead to potential sources of employment, and at the same time help in land reclamation with little or no subsequent maintenance. Here, we needed more advanced technologies to extract biofuels. Cellulose (long chains of glucose molecules bound by b-glycosidic bonds) is a component of plant cell walls, making up between a quarter and half of them in terms of mass. However, it is not easy to extract, as the cellulose in a plant is embedded in a cross-linked matrix with other components such as hemicellulose (branched polymers of xylose, arabinose, galactose, mannose, and glucose that bind bundles of cellulose strands together), lignin (a complex polymer into which the above-described bundles are matrixed and thus unavailable for conversion to fermentable sugar), pectins, xylene, and proline-rich proteins. Further, lignin limits the access of cellulase enzymes needed for fermentation of sugars. The area of ethanol production showing the most promise, both in terms of potential energy savings and in terms of national security interests, is ethanol derived from cellulose. Current processes for producing cellulosic ethanol are time-consuming, low-yielding, and, above all, expensive. The process of cellulose

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biochemistry is still poorly understood. Cellulose degradation pathways require multiple steps of varying temporal, environmental, and efficiency penalties. The major hurdle to producing biofuel is the high cost of conversion of cellulose and hemicellulose into ethanol. Therefore, the price of cellulosic ethanol is two- to threefold higher than bioethanol produced from corn grains. These production costs could be cut down by bioengineering crop plants in such a way that the necessary cell wall degrading enzymes, such as cellulase and hemicellulase, are produced within the plant system, thereby reducing the dependency on microbial bioreactors for enzymes. The concept of self-destructive plants could be coined to such transgenic varieties. Ziegler et al. (2000) advocated the restriction of the hydrolytic enzymes produced by the transgenic plants in the apoplast region of the cell to avoid reaction with other symplast elements. Further, Schillberg et al. (2003) advocated the potential of selectively degrading protein bodies in subcellular compartments of the endoplasmic reticulum (ER), thereby preventing interference with other cellular activities. Afterwards, the same enzyme will help in increasing the degradation process upon extraction from the transgenic plants. It is very important to ensure that enzymes produced for cell wall degradation should not interfere with other cellular activities. The other strategy could be directing the expression of these enzymes in the chloroplast of the plant cells. Chloroplast transformation with transgenes has been shown to be consistently predictable and less prone to silencing. The other strategy could involve genetically engineering plants in a tissue-specific manner, so that the transgene does not interfere with parts used for human consumption (Dai et al. 2000). Further cost reduction could also be achieved by genetically engineering the cellulose biosynthetic pathway in plants so that there is a minimal need for expensive pretreatment. Another strategy is to upregulate the cellulose biosynthesis pathway enzymes for increased polysaccharide production, which in turn will increase the yield of cellulosic biofuel. Good et al. (2005) showed that ectopic expression of glutamate dehydrogenate from bacteria resulted in increase in biomass of tobacco. Oxenboll Sorensen et al. (2000) reported that pectin arrangement in rice and potato plants was drastically changed (confirmed by Fourier transform infrared microspectroscopy, immune-gold labeling, and fermentable sugar accumulation studies) by the ectopic expression of endo-galactanase gene from fungus under the influence of a tissue-specific promoter without altering the plant architecture. Skjot et al. (2002) also reported decrease in arabinose content in the cell wall of potato plants. Sticklen (2008) reviewed the production of Acidothermus cellulolyticus E1 endo-1,4-b-glucanase in different crop plants for efficient conversion to biofuels, which is a stepping stone in the production of biofuel from cellulosic material. Attempts have been made to increase cellulose-binding molecules in plants, which results in increase in cellulose production without subsequent increase in lignin production. This may provide an opportunity to increase cellulose content in plants and hence increase proportion of fermentable sugars (Fig. 6.4b). Another strategy could be manipulating C3 mechanism of photosynthesis in favor of C4 mechanism, which is present in the grass family and can only be grown in tropical regions.

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Van Camp (2005) overexpressed two enzymes from cyanobacteria in tobacco, resulting in enhanced rate of photosynthesis that translated into increase in biomass. This could ensure equal distribution and usage of biomass over the globe. Jing et al. (2004) reported increase in tree height and hence biomass by ectopically expressing glutamine synthase gene (GS1).Also, manipulating genes for enhanced solar energy capture and conversion of photosynthate to sugars, and genes which provide endurance to plants growing under saline conditions, or land unutilizable for cultivation, should be emphasized (Antizar-Ladislao and Turrion-Gomez 2008). In plants, lignin (composed of phenylpropanoid groups) acts as a polymer around the hemicellulose microfibrils, binding the cellulose molecules together and protecting them against chemical degradation. Lignin cannot be converted into sugars. Thus, it is not practical in biofuel production. Their degradation is a high-energy process. A simplified illustration of genetic manipulation of lignin has been described in Fig. 6.4c. Vanden Wymelenberg et al. (2006) reported a large number of genes involved in breakdown of lignin from the fungus Phanerochaete chrysosporium genome. Such genes could be expressed in plants to induce cell wall breakdown in the latter stages of plant maturity. Ralph et al. (2006) reported genetic manipulation of monolignins in such a way that they continue to provide crosslinkage to cellulose and hemicellulose microfibrils required for the firm framework of the plant and at the same time increase biomass conversion efficiency to biofuels. Studies with the model plant Arabidopsis have shown that secondary wall thickening is lost when knockout lines (nst1, nst2) are produced, without affecting vascular bundle architecture (Mitsuda et al. 2007). This provides documented evidence that lignin biosynthesis can be selectively altered without rendering the plants prone to lodging by vascular collapse or increasing their susceptibility to insects, pests, and disease. The major share of the cost in biofuel production is the expense incurred in the production of cellulase enzymes in microbial bioreactors. Secondly, the cost of pretreating lignocellulosic matter for its breakdown to the intermediate components, and subsequent removal of the lignin, is very high. Three families of enzymes responsible for digesting lignin, laccases, manganese dependent peroxidases and lignin peroxidases (Kirk and Farrell 1987) could be used in transgenic plant production to produce plants with low lignin content amenable for biofuel production. Li et al. (2003) showed that by downregulating a single lignin biosynthetic gene, 4-hydroxycinnamoyl CoA ligase (4CL), a prominent reduction in lignin composition was observed along with an increase in biomass. Ralph et al. (2006) found that decreasing expression of 4-coumarate 3-hydroxylase (C3H) in alfalfa (Medicago sativa) drastically altered the lignin profile and structure. This could enhance the fermentation process in the production of biofuels. Gressel (2008) reported the isolation of brown midrib (bmr) mutations in sweet-stemmed sorghum and maize with low lignin content that were highly digestible by industrial carbohydrases. Besides the grain component, a large portion of biomass is left unutilized as lignocellulosic material. Lignin is a polyphenolic polymer (composed of monolignin units) and is highly resistant to degradation. In a typical study, downregulation of a lignin biosynthesis gene, cinnamyl alcohol dehydrogenase (CAD) in

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alfalfa resulted in altered lignin biosynthesis, thus increasing the processibility (Baucher et al. 1999). Chabannes et al. (2001) found that reduction in expression of cinnamoyl CoA reductase (CCR) in transgenic tobacco resulted in reduced lignin content with a corresponding increase in glucose in plant cells. Blaschke et al. (2004) reported that downregulation of O-methyl transferase (OMT) enzyme in Nicotiana tabacum enhanced biomass content, though proportion of lignin content was decreased. Ragauskas et al. (2006) suggested that often downregulation of a lignin biosynthesis gene results in altered lignin architecture in transgenic plants. Similarly, Chen and Dixon (2007) reported that downregulation of six different lignin biosynthetic pathway enzymes in alfalfa may eliminate the pretreatment process associated with extraction of fermentable sugars. Hu et al. (1999) reported alteration in lignin content and subsequent increase in cellulose content in quaking aspen (Populus tremuloides) by decreasing the enzyme hydroxycinnamate- CoA/5-hydroxyferuloyl-Co-A ligase. Bate et al. (1994) similarly advocated that suppression of phenylalanine ammonia lyase (PAL) may decrease lignin content in poplar plants. Ralph et al. (2006) found that flexible polymerization is possible in plants, whereby monoligins can be actively substituted by polyphenols. Similarly, Riboulet et al. (2009) identified lignin biosynthesis genes from microarray analysis in maize, which are differentially expressed during plant development. Such information will be vital in developing new transgenic crop programs and plant breeding. This in turn may not affect the development process of plants, but will facilitate the extraction of saccharides needed for biofuel production from the plants. Among several other renewable sources biomass (includes residual non-food parts of cultivated crop plants, garden and kitchen waste) is probably the most promising alternative to fossil fuel resources. It has several advantages, such as it will be sustainably available in the future and it is considered to be environmentally friendly, since it has low net release of carbon dioxide and sulfur content. This will also help us to mitigate the problem of landfilling in industrial and urban generated waste. Biomass is a complex material primarily made up of cellulose (30–50%), hemicellulose (20–40%) and lignins (15–30%). Complex carbohydrates like cellulose and hemicellulose are first converted into their component sugars through a hydrolysis process and then the sugars are anerobically fermented into biofuel such as bioethanol. At present, only the cellulose and hemicellulose components of plant biomass can be converted into biofuels by the action of anaerobic microbes. The major obstacle in increased biofuel production is to find an efficient way to break down complex plant polymers into simpler derivatives.

6.2.3

Manipulating Genes and Pathways Involved in Lipid Metabolism

Oils from crops like palm, castor, soybean and oilseed rape are presently being used to make biodiesel. The biosynthesis of oils in plants starts de novo in the plastids

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where photosynthesis provides the carbon source, which are assembled into short chains to produce palmatic (C16), stearic (C18), oleic and linoleic acids (C20). In general, a greater proportion of short-chain fatty acids is more desirable in biodiesel production (Fig. 6.4c). The higher proportion of short-chain fatty acids like oleic acid will render oxidative stability to the oils even at high temperature (Metzger and Bornscheuer 2006). The fatty acids are then transported to the endoplasmic reticulum (ER) by an energy-dependent pathway, and there long-chain fatty acids are assembled from the short-chain ones. The final products (oil or fatty acids) are transported out of the ER to be stored in vacuoles as oil bodies, which act as a source of reserve energy for the plants. Therefore, the pathway to synthesize fatty acids and triacylglycerols will need to be modified to facilitate the production of biodiesel from these crops (Durrett et al. 2008). All the genes acting on the lipid biosynthesis have been cloned by Zhang et al. (2005). They further suggested that improving the composition of the fatty acid components of plant oils will enhance the acceptability of biodiesel in temperate regions of the world. For example, Lardizabal et al. (2008) achieved increase in biodiesel production from soybean by ectopically expressing diacylglycerol acyltransferase 2A (DGAT2A) from Umbelopsis ramanniana fungus during the seed development stage. Similar results have been reported in rapeseed by overexpressing a laurate-specific acylACP thioesterase gene from California bay tree and a laurate-specific LPAAT gene from coconut (Knutzon et al. 1999; Wiberg et al. 2000). Wiberg et al. (2000) also described that an increase in the content of caprylic acid (C8) and capric acid (C10) in rapeseed had resulted in a 30% increase of short-chain fatty acid in the seed. Also, the production of short chain fatty acids or oils should be enhanced from the non-oil-producing tissues of the plant. Recent studies have shown that plant storage tissue can store high quantities of oil bodies without any drastic effect of cell membrane polarity and hence on plant growth (Napier 2007). Increasing the percentage of short-chain fatty acids by a transgenic approach will reduce the cost associated with the production of biodiesel. Most vegetable oils are large, branched molecules; therefore, could not be directly used in diesel engines. These large and branched molecules of bio-oils are converted into smaller and straight-chain molecules through transesterification, making them suitable for regular diesel combustion engines. Production of high-quality short-chain fatty acids by using advanced biotechnological techniques (TILLING, RNAi, antisense approach, T-DNA insertion lines) may come to our rescue. Targeted induced local lesion in genomes (TILLING) is a very popular technique used today to identify mutant lines for any gene of interest. For example, a mutation in fatty acid gene (FAD1 in peanut) will cause the plant to accumulate short-chain fatty acids, as its capability to convert the short-chain fatty acids into long-chain fatty acids will decrease. Again, RNAi and antisense expression of the fatty acid gene will reduce translation of the gene, and thus the amount of long fatty acids will be decreased. Random T-DNA insertion is a technique introduced to prevent the production of a gene of interest. Random T-DNA may get integrated in the exonic region of the gene and thus translation may come to a premature stop.

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Conclusion

The era of cheap fossil fuel is rapidly coming to an end. Therefore, the need of the hour is to look at the alternative forms of energy, a strategy that was hardly emphasized in the past. There are also concerns about global climate change and severe food shortage. Biomass is the least expensive and most globally available resource. Therefore, priority should be shifted towards utilizing biomass, leaving aside food for human consumption. Plant architecture have been naturally modified, in fact, ever since the evolution of land plants commenced several million years ago and through the selection of desirable phenotypes by crop improvement (around 10000 BC, when man started cultivating plants for food and shelter) to the more sophisticated methods of using genetic manipulation and plant biotechnology to produce modern-day transgenic biofuel crops. Modified carbohydrate and lignin biosynthetic pathway has been a major target for chemical or genetic intervention in recent years. The use of transgene genes could also underpin conventional breeding to modify biofuel production, in view of the growing public concern over the potential environment damage and health hazards by the use of fossil fuel, and the limited availability of suitable genes to modify synthesis of sugars, lignin and lipids in plants, by adopting classical breeding approaches. Tissue-specific antisense expression of transgenes involved in biofuel production in vegetative parts will prevent unwanted assimilate partitioning and direct photosynthate towards storage organs (seeds or underground storage like bulbs, corms and rhizomes), increasing productivity besides contributing to biofuel processing and production. One of the aspects for biofuel production is synchronous maturity of the crops used for biofuel production. Many chemicals that promote synchronous maturity like NAA (napthalene acetic acid) could be useful. Also, application of gibberelic acid (GA) may result in high yield. Anti-GA substances like pacrobutrazol can be used to control plant height, or ectopic expression of GA2-oxidase will result in dwarf plants (Dijkstra et al. 2008). This will allow easy harvest of the produce with low cost on resources and saving time. Compact plants are also less prone to damage by biotic and abiotic stress. Genetic manipulation strategies should lay emphasis on downregulating lignin biosynthesis genes without drastically affecting plant architecture and making plants prone to infestation of disease, insects and pests. Therefore, production of transgenic plants with the above mentioned traits would help in developing a more robust plant phenotype that was not always possible through conventional or molecular breeding. This was because traits amenable for biofuel production did not mostly go hand in hand with increased food crop production. Thus, suitable traits for biofuels were lost in rounds of selection of superior genotypes responsible for increased crop production practiced over several thousand years. Now, crop breeders have been combining tissue culture approaches with traditional breeding in order to broaden the gene pool available for improvement of productivity and to introduce new, desirable traits. With the development of DNA microarray chips, it is now possible to identify novel genes and to generate plants with novel traits

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(in this case, traits amenable for biofuel production), and not just the development of varieties restricted to insect pests (Wang et al. 1996), disease (Marchant et al. 1998) and herbicide tolerance (Slater et al. 2003). Manipulating monolignins, and perhaps modifying three-dimensional lignin architecture, could also be an additional possibility. Therefore, modification of traits related to biofuel production may require manipulation of the multiple hormones, complex carbohydrate synthesis pathways, which may involve the introduction of multiple genes into plants. Such studies may also lead to a better understanding of the roles of the different hormone classes in biofuel crop development. Future research must be directed towards identifying tissue-specific or species-specific inducible promoters that could, for example, drive gene expression in the vegetative tissues, such as stem, internodes, and leaf petioles, thus ensuring that flowering and seed set, both qualitatively and quantitatively, are not affected. Furthermore, the possibility of using constructs carrying genes associated with enhanced biofuel production, but lacking the nptII selectable maker gene (Holn et al. 2001), should be explored in order to facilitate the acceptance of transformation technology to regulate plant biomass. This can be achieved by selecting plants directly, based on phenotype, such as with dwarf growth. It is also possible to use T-DNA related sequences, which have originated from plant genomes. These have been referred to as cisgenic plants, as their transformation vectors, including promoters and terminators, are derived from the same plant species, which is transformed with the gene of interest (Chandler and Tanaka 2007). Also, vectors from plant-originated selectable marker, recombinase recognition sequences (Cre-lox system) can also be beneficial (Zuo et al. 2001). The use of co-transformation (Komari et al. 1996) or the use of chimeric plants (De Vetten et al. 2003) may also be considered. Further, introduction of transgenes in male-sterile plants may be another viable option, or the use of terminator gene technology may be considered. The exact role of each lignin biosynthetic gene on plant development must be accessed to pinpoint genes (in turn enzymes) bringing about the greatest effect. The use of a specific promoter may be pivotal in driving gene expression under study. Low transformation efficiency can be correlated with a constitutive promoter, such as CaMV 35S (Zhu et al. 2008), which may result in poor regeneration and low recovery of transgenic plants (Petty et al. 2003; Zhu et al. 2004; Busov et al. 2006). The use of a viral promoter, like 35S, may result in gene silencing in plants as observed (Bhattacharya et al. unpub.). Furthermore, an ubiquitin-like promoter (Christensen and Quail 1996) may result in high efficiency of transformation compared to 35S promoter, and future genetic manipulation studies should include such ubiquitin-like promoters. Genetic modification paves the way for the development of new cultivars by gradually incorporating one or more genes (Miflin 2000). However, expression of genes across plant species is not always entirely predictable, leading to a large number of trial and error methods to reach a generalized conclusion. Another area of emphasis will be manipulating oilsynthesizing genes in lower primitive plants like algae, cynobacteria and phytoplankton. Exploiting everyday-generated crop waste will also help to lay the foundation of a carbon-balanced world.

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Antagonism to this technology has also restricted the development of the technology (Azevedo and Araujo 2003; Phillips 2004). There is no uniform system for international regulation on genetically modified crop plants with significant difference between countries (Halsberger 2006). Since transgenic biofuel crop plants are not anyway intended for human consumption, they should be theoretically more acceptable to the public. The Cartagena protocol (http://bch.biodiv.org) is the first attempt to bring uniformity in laws governing genetic modification, and many countries are adding these laws to their existing law in order to come to a unified attempt to regulate genetically regulated products. In conclusion, biofuel may not replace the use of fossil fuel in the foreseeable future; however, it can become a major component of alternative fuels, and may add towards the carbon economy in the age of rapid industrialization.

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Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M (2007) NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19:270 Murugesan A, Umarani C, Subramanian R, Nedunchezhian N (2009) Bio-diesel as an alternative fuel for diesel engines-A review. Renew Sustain Energ Rev 13:653–662 Myers AM, Morell MK, James MG, Ball SG (2000) Recent progress toward understanding the biosynthesis of the amylopectin crystal. Plant Physiol 122:989–998 Napier J (2007) The production of unusual fatty acids in transgenic plants. Annu Rev Plant Biol 58:295–319 Oxenboll Sorensen S, Pauly M, Bush M, Skjot M, McCann M, Borkhardt B, Ulvskov P (2000) Pectin engineering: modification of potato pectin by in vivo expression of an endo-1, 4-b-Dgalactanase. Proc Natl Acad Sci USA 97:7639–7644 Petrou EC, Pappis CP (2009) Biofuels: A Survey on Pros and Cons. Energ Fuel 23:1055–1066 Petty LM, Harberd NP, Carre IA, Thomas B, Jackson SD (2003) Expression of the Arabidopsis gai gene under its own promoter causes a reduction in plant height in chrysanthemum by attenuation of the gibberellin response. Plant Sci 164:175–182 Phillips AL (2004) Genetic and transgenic approaches to improving crop performance. In: Davies PJ (ed) Plant hormones: biosynthesis, signal transduction, action! Kluwer, Dordrecht, The Netherlands, pp 582–609 Puppan D (2002) Environmental evaluation of biofuels. Periodica Polytechnica Soc Manag Sci 10:95–116 Ragauskas A, Williams C, Davison B, Britovsek G, Cairney J, Eckert C, Frederick W, Hallett J, Leak D, Liotta C (2006) The path forward for biofuels and biomaterials. Science 311:484–489 Ralph J et al (2006) Effects of coumarate 3-hydroxylase down-regulation on lignin structure. J Biol Chem 281:8843–8853 Riboulet C, Guillaumie S, Mechin V, Bosio M, Pichon M, Goffner D, Lapierre C, Pollet B, Lefevre B, Martinant JP, Barriere Y (2009) Kinetics of Phenylpropanoid gene expression in maize growing internodes: relationships with cell wall deposition. Crop Sci 49:211–223 Ripley BS, Muller E, Behenna M, Whittington-Jones GM, Hill MP (2006) Biomass and photosynthetic productivity of water hyacinth (Eichhornia crassipes) as affected by nutrient supply and mirid (Eccritotarus catarinensis) biocontrol. Biol Con 39:392–400 Robinson A (1998) Developing sustainable global agriculture for the 21st century. In: Rice Reports, John Innes Centre, Norwich, UK, pp 1–8 Schillberg S, Fischer R, Emans N (2003) Molecular farming of recombinant antibodies in plants. Cell Mol Life Sci 60:433–445 Skjot M, Pauly M, Bush M, Borkhardt B, McCann M, Ulvskov P (2002) Direct interference with rhamnogalacturonan I biosynthesis in Golgi vesicles. Plant Physiol 129:95–102 Slater A, Scott N, Fowler M (2003) Plant biotechnology – the genetic manipulation of plants. Oxford University Press, Oxford, UK, pp 7–18 Smith AM (2008) Prospects for increasing starch and sucrose yields for bioethanol production. Plant J 54:546–558 Sticklen MB (2008) Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat Rev Genet 9:433–443 Van Camp W (2005) Yield enhancement genes: seeds for growth. Curr Opin Biotechnol 16:147–153 Vanden Wymelenberg A, Sabat G, Mozuch M, Kersten PJ, Cullen D, Blanchette RA (2006) Structure, organization, and transcriptional regulation of a family of copper radical oxidase genes in the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl Environ Microbiol 72:4871–4877 Vogt KA, Vogt DJ, Patel-Weynand T, Upadhye R, Edlund D, Edmonds RL, Gordon JC, Suntana AS, Sigurdardottir R, Miller M, Roads PA, Andreu MG (2009) Bio-methanol: how energy choices in the western United States can help mitigate global climate change. Renew Energ 34:233–241

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Chapter 7

Plant-Produced Biopharmaceuticals Jared Q. Gerlach, Michelle Kilcoyne, Peter McKeown, Charles Spillane, and Lokesh Joshi

7.1

Introduction

The term “biopharmaceutical” is commonly used to denote a therapeutic protein produced by recombinant (genetic) engineering. In this process, genes encoding proteins or peptides of interest from humans or other organisms are identified, cloned, inserted into an expression vector and the protein or enzyme produced within a prokaryotic or eukaryotic expression host “production platform” organism. Such production platforms may typically be bacteria, yeasts, insects, mammals or plants. Batch cultures of cell suspensions are also commonly used for large-scale production of recombinant therapeutic proteins, a method often referred to as “fermentation culture.” Bacteria and yeast can be grown in closed-loop bioreactors, as can insect, mammal and plant cell-lines, which are generated from whole organisms. This ensures several generations of stable cell line propagation, allowing production of the protein or enzyme of interest. The use of animal cell lines and bacterial or yeast strains to produce therapeutic proteins has resulted in several successful products of medical interest (Walsh 2006). Such pharmaceutical products include glycosylated human erythropoietin (rhEPO), which acts as a red blood cell stimulating hormone, recombinant human clotting factor VIII (rhCFVIII), used for treating hemophilia, and recombinant insulin, used to treat diabetes mellitus. Sales of rhEPO were estimated at US $10.7 billion for 2005, while those of rhCFVIII reached an estimated US$1 billion by the same year (Walsh 2006). The total market for biopharmaceuticals is expected to reach approximately US$70 billion by 2010 (Walsh 2006), with insulin estimated to generate US$14 billion from 16,000 kg of protein (Moloney et al. 2003) and monoclonal antibodies contributing approximately US$20 billion (Hiatt and Pauly 2006). L. Joshi (*) Glycoscience and Glycotechnology Group, Martin Ryan Institute and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland e-mail: [email protected]

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Non-human mammalian cell lines, such as Chinese hamster ovary (CHO) cells, are currently considered to be the most desirable expression systems as they produce proteins similar to those from human cells, particularly with regard to glycosylation or other post translational modifications (Hamilton and Gerngross 2007). However, one of the major challenges associated with mammalian cell culture technology of particular public concern is that contaminating pathogens, which are capable of infecting other mammalian species (i.e., xenozoonosis), may be transferred to humans during product recovery. These include viruses, endogenous retroviruses and internalized bacteria and mycoplasmas, as well as prions. This inherent biological limitation poses a particular challenge to the development of pathogen-free Good Manufacturing Practice (GMP) procedures for mammalian cell culture systems. Furthermore, as complex growth media is required for mammalian cell bioreactors, minute variations in media components and fermentation conditions can lead to undesirable changes in the end product(s). Even under the most stringent conditions, maintaining consistency from batch to batch is challenging as the interactions of cells with each other and their environment may also create variations in end products (Butler 2006). Also, endogenously produced immunogenic agents, such as proteins or species-specific glycosylation, harvested as a co-extracted or incorporated component of biopharmaceuticals produced in CHO cells, may create unwanted effects in patients even at extremely low levels (Hamilton and Gerngross 2007). In addition to the above, there are a number of other key drivers for the development of improved production platforms for biopharmaceutical production. The costs associated with growing and maintaining mammalian cells for pharmaceutical production remain relatively high compared to yeast and bacteria (Fischer et al. 1999). Costs for establishing a pharmaceutical-grade mammalian cell culture facility may exceed €250 million (Sardana et al. 2007), and each requires extensive testing and certification with the result that only a relatively small number of such facilities worldwide can produce biopharmaceuticals on a large (kilograms per year) production scale. Hence, any approach that leads to a manufacturing cost-saving will gain competitive advantages over time. Increasing numbers of new protein therapeutics coming on stream are generating a production capacity crisis within the available mammalian cell culture systems. This situation is likely to worsen – as of 2006, there were 2,500 biotech drugs in discovery phase, 900 in preclinical trials and 1,600 in clinical trials (Walsh 2006). Moreover, the advent of high-volume protein therapeutics targeted at much larger numbers of people (e.g., monoclonal antibody-based therapeutics such as Herceptin, Enbrel, Mylotarg, Remicade, etc.) and the need to produce generics or biosimilars are placing strains on current biopharmaceutical production platforms to deliver at the scale and cost levels needed. To produce 300 kg of secretory IgA antibodies per year, plant-based production systems have the lowest capital costs (maize or tobacco; €0.5–2.0 million/300 kg per year) when compared to platforms based on transgenic goats or mammalian cell culture (~€6–7 million/300 kg per year) (Richard McClosky,

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Centocor). Finally, with escalating elderly populations in developed countries increasing product requirement, and patients and governments demanding affordability, there is a definite need to innovate and develop more cost-effective platforms for large-scale production of biopharmaceuticals.

7.2

Transgenic Plants as Production Systems for Biopharmaceuticals

Transgenic plants (or derived plant suspension cell lines) are considered very attractive alternatives to mammalian cell systems for the production of recombinant mammalian glycoproteins. Plants as multicellular eukaryotes have many advantages as production platforms for recombinant proteins or enzymes, including similar eukaryotic biosynthetic pathways, high protein yields, freedom from animal pathogens and bacterial endotoxin contamination and low production cost per unit protein overhead (Gomord et al. 2005). Indeed, cost savings associated with plant technologies and end-product purification may lead to a 10- to 100-fold advantage over currently used mammalian fermentation systems such as CHO cells (Vidi et al. 2007). Additionally, with the use of organ-specific promoters to drive protein expression, the seeds, stems, roots, fruits, and leaves of whole plants suitable for growth in fields or greenhouses can be targeted for transgenic protein production (Fischer et al. 2004). To date, the highest yields have generally been achieved by targeting recombinant proteins to chloroplasts, oil bodies and seed protein bodies of whole plants (see below). It has been estimated that 250 acres of greenhouse production of transgenic plants producing hepatitis B vaccine could meet Southeast Asia’s demand. Indeed, the scalability of plant platforms for biopharmaceutical production is one compelling advantage of this approach, e.g., in response to rapid demand for therapeutics for any future pandemics. The possibility of plant systems for facilitating freedom from refrigeration would be a tremendous advantage for economically challenged populations, where maintenance of a “cold chain” for handling and storage may simply not be possible (Rademacher et al. 2008). The capacity of plant storage organs (seeds or tubers) to act as protein storage systems is truly remarkable (Fischer et al. 1999) – antibodies in plant seeds stored at room temperature have been found to be stable and fully active after several years. The market potential of plant-based recombinant technology was first demonstrated in the production of pure, research-grade proteins. By 2005, three major recombinant proteins had been produced in corn (Zea mays) – the biotin-receptor, avidin, and the enzymes, trypsin and b-glucuronidase – and were commercially available (Ma et al. 2005). Seed storage protein bodies (PBs) are of particular interest as the recombinantly expressed proteins can be accumulated in a comparatively high

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proportion into a relatively stable, compact mass, greatly contributing to relative ease of harvest, storage and purification of products (Stoger et al. 2005). Despite the clear value of seed storage targeting, for many purposes it is more appropriate to make use of plant cells propagated as suspension cultures. Such techniques also aid containment (see below). Plant suspension cells are derived from calli, clusters of undifferentiated cells. Calli are first stimulated from mature plant tissues through direct contact with hormone-enriched solid growth media. After reaching a suitable size, the frangible calli are transferred to a liquid growth medium, agitated to shake the cells apart and cells continue to divide within the liquid growth medium (Hellwig et al. 2004). Plant suspension cells are grown using media that is much simpler in formulation than that used for insect and mammalian systems, typically containing just a few salts and minerals, sucrose and a plant hormone (Kolewe et al. 2008). A wide range of plant species are used commercially for molecular pharming purposes, including well-known biological models (e.g., Arabidopsis thaliana, Medicago truncatula), food crops, and non-food crop species specialized for particular purposes (Twyman et al. 2003; Fischer et al. 2004; Stoger et al. 2005). Amongst the seed crops, molecular pharming is underway in cereals (maize, wheat, rice, barley), legumes (peas, soybean) and oilseeds (safflower, rapeseed). The root and tuber crops, carrot and potato, are used for pharming, while lettuce, spinach, alfalfa, banana and tomato are examples of fruit and green leaf crops being used for pharming. Non-food crops in use for pharming include tobacco and falseflax, while non-cultivated species include duckweed (Lemna spp.) and moss (Physcomitrella patens), and also unicellular photosynthetic organisms, such as microalgae (Chlamydomonas reinhardtii). Importantly, all these eukaryotes share the common characteristics of eukaryotic protein machinery and being amenable to genetic transformation. Transformation of plants and plant cells is typically done using one of two species of Agrobacteria which are able to transfer transgenes (encoding recombinant biopharmaceuticals) into the host plant genome. Agrobacterium tumefaciens is competent to transform foliar and floral tissues and cultured cells (Broothaerts et al. 2005), while A. rhizogenes is competent to transform root tissue or cultures (Shi and Lindemann 2006). First, the Agrobacteria are transformed with DNA plasmids containing the gene of biopharmaceutical interest, under the control of a suitable plant promoter. In response to the patent landscape (Dunwell 2005) and the need to develop complementary approaches for transferring desirable genes and associated traits to plants, a range of other techniques for plant transformation have been developed (Broothaerts et al. 2005; Vain 2007). These include plant transformation by particle bombardment, otherwise known as the gene gun method (Brereton et al. 2007). Plasmid vectors adhered to minute particles of a carrier agent, often gold particles, are accelerated by compressed air to penetrate and transform the nuclear genome of the target cells. Vectors for transformation can be rapidly assembled using high-throughput cloning techniques, such as Invitrogen’s Gateway system, which allows combination of specific features (Curtis and Grossniklaus 2003). Plasmid vectors may be constructed to allow

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for selection of successfully transformed cells by transformation with genes conferring antibiotic or herbicide resistance and subsequent growth in media containing these agents or encoding reporter enzymes or fluorescent molecules (Sala et al. 2003). Promoters may be chosen for constitutive high expression throughout the plant, or tailored for expression in cell culture (i.e., inducible systems) or a particular organ, such as the seed or tuber. These transformation techniques can also be targeted for chloroplast expression, a relatively new alternative (Hou et al. 2003). Directing genes for recombinant proteins to chloroplasts in whole plants may allow yields of up to approximately 45% of total leaf protein (Arlen et al. 2008). The chloroplast is an endosymbiotic organelle of prokaryotic origins with its own genome. Hence, chloroplast genes and translational machinery differ from those of the nucleus, although there is regulatory interaction and protein trafficking between these two cellular compartments (Pogson et al. 2008; Woodson and Chory 2008). An important attribute of transgenic chloroplast systems is that chloroplasts are maternally inherited and therefore rarely transmitted via pollen (there are some species exceptions) and therefore transgenes located in chloroplast genomes are typically biologically contained (Ruf et al. 2007). Lipoprotein particles found within chloroplasts (plastoglobules) may be specifically targeted as depositories for recombinant proteins. Vidi and colleagues fused recombinant yellow fluorescent protein (YFP) to a naturally occurring plastoglobulin protein to study the viability of the process (Vidi et al. 2007) and found that plants expressing the YFP fusion protein did not have a significantly altered phenotype. Moreover, such plants had similar growth characteristics to those expressing recombinant proteins targeted to oil bodies. One disadvantage of chloroplast expression systems is a lack of access to the post translational machinery of the secretory pathway (see below). Hence, proteins produced by chloroplasts cannot be endogenously glycosylated, deacylated or phosphorylated (Anisimov et al. 2007). Some recombinant proteins may be improperly folded or even rendered biologically inactive without these modifications (Gomord and Faye 2004; Byrne et al. 2006; Eichler et al. 2006; Mitra et al. 2006). High-level production of recombinant proteins within cytoplasmic compartments of plant cells depends on promoters to drive the expression of the gene of interest. Many promoters have been used in both monocot and dicot species to date, and the sophistication of such promoters is increasing as understanding of plant gene regulation grows (Venter 2007, see also Vol. 1, Chap. 5). Low-level expression using dicot promoters in cereals has highlighted the necessity of developing monocot-specific promoters (Stoger et al. 2005). Additionally, the use of an engineered plant virus-based transient expression systems (e.g., magnICON from Icon Genetics; Gleba et al. 2005) can allow a significantly faster turnaround time and a much higher protein yield than other stable plant transformation techniques (Mallory et al. 2002; Gleba et al. 2005), although the higher mutation rate of RNA-based viral expression systems is a consideration for the generation of homogeneous protein product (Domingo et al. 2006).

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Challenges Regulatory Challenges

Despite the distinct advantages transgenic plants offer, some specific challenges remain. As transgenic plants will be producing protein compounds with known pharmaceutical effects, it is imperative for the success and acceptability of the molecular pharming field that such production operates in a biologically contained system and is also compliant with current GMP (cGMP) procedures for the production of therapeutic grade proteins. Given the issues with controlling the abiotic and biotic environment, open-field production of food crops or other cultivated species producing high-potency pharmaceutical compounds may not be desirable for molecular pharming industry development. Closed-loop production systems which ensure that pharma-producing seeds or plant materials are not used for any other purposes are essential. This would include ensuring that any biotic predators or pests are not adversely impacted by the pharmaceutical-producing plants. However, pharmaceutical-producing plants have been deliberately cultivated since the first pharmacopeias were compiled for apothecaries in the Middle Ages. Physic gardens, which trained the earliest physicians in the use of medicinal plants, were the forerunners of today’s botanic gardens, many of which were originally associated with the faculties and departments of pharmacy in universities. Today, there are multiple examples of highly potent (non-transgenic) medicinal or toxic plants (e.g., opium poppy, high erucic acid rapeseed), which are cultivated for the production of pharmaceuticals without any adverse effects on humans or environment. Nonetheless, to further bolster public trust and to avoid any unforeseen secondary consequences of plant-based recombinant technologies, it will be prudent to apply efficient measures of biological containment so that pharma-producing seeds or transgenes are not used outside of the closed-loop production systems for which they were developed. Hence, methods for the containment of reproductive material (e.g., male sterility, maternal inheritance systems such as chloroplasts or autonomous apomixis) in conjunction with the use of genetically-linked, visible phenotypic markers for any transgenic seeds/plants expressing pharma proteins will be advisable. Other approaches worth considering are inducible expression systems. In general, the most prudent containment will be to use the best greenhouse-controlled environment conditions (for whole transgenic plant systems) or photobioreactors (for cell culture suspensions) for commercial production of the first generation of plant-derived biopharmaceuticals. It is certain that plant-made pharmaceutical (PMP) production will be like any other pharmaceutical production system in terms of its strict regulatory control. The regulations for pharmaceutical production in plants are not currently harmonized internationally with different governing bodies operating in the USA and European Union (EU). In the USA, regulatory oversight for PMPs is under the aegis of the following bodies: the US Department of Agriculture

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(USDA: http://www.usda.gov/wps/portal/usdahome) regulates growth of plant and defines the required safeguards for the production and transport of the product; the Food and Drug Administration (FDA) Centre for Biologics Evaluation and Research (CBER: http://www.fda.gov/%20BiologicsBloodVaccines/default.htm) regulates biologic products, their manufacture and distribution; the FDA Centre for Food Safety and Applied Nutrition (CFSAN: http://www.foodsafety.gov/list. html) and Centre for Veterinary Medicine (CVM: http://www.fda.gov/cvm/default. html) are involved in consultations on food and feed safety, and finally the PMP developer is responsible for safety assessments, drug master files, chemistry manufacturing controls and biologics license application for the client’s drug registration (Streatfield 2005). In the European Union, regulatory procedures for PMPs are in flux due to a need to tailor the existing regulations to deal with PMPs. Any field-grown plant pharming would require notification under EU Directive 2001/18/EC (http:// eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32001L0018:EN:HTML), which covers both food and non-food transgenic crops. Containment-grown plant pharming would fall under EU Directives 90/219/EEC (http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=CELEX:31990L0219:EN:HTML) and 98/81/EC (http://ec.europa.eu/environment/biotechnology/pdf/dir98_81.pdf). For any commercial release, the European Food Standards Agency (EFSA) would be involved in the review process under 1829/2003/EU Food and Feed Guidelines (http:// eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:268:0001:0023:EN: PDF). All pharma products produced from plants within the EU must adhere to the 2309/93/EU regulations (http://www.biosafety.be/EMEA/2309_93en.pdf). All decisions at the clinical trial stage for the PMP would be dealt with by the relevant national authorities in each EU Member State. At the commercial evaluation stage, the PMP would be regulated by the European Agency for Evaluation of Medicinal Products (EMEA). See also Vol. 2, Chap. 11 for discussion.

7.3.2

Biological Challenges

The potential allergenicity of any co-extracted endogenous protein is a further consideration. For example, targeting expression in plant seeds, which naturally produce high levels of storage proteins, is a means of generating high yields of recombinant products (Reggi et al. 2005; Nykiforuk et al. 2006). However, species such as certain legumes contain endogenous proteins which can induce allergic reactions. Several potent protein allergens have been identified in peanut (Arachis hypogeae) including storage proteins Arah1, Arah2 and Arah3 (Barre et al. 2005), and soybean (Glycine max) such as the vicilin Bd 28k (Tsuji et al. 2001). In plant species and organs where there are known allergens, isolation of recombinant protein products from the endogenous components requires stringent and exhaustive purification, generally requiring multiple steps, to minimize risk of allergic reaction.

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Glycosylation and the Production of Clinical Grade Biopharmaceuticals

One of the major roadblocks in the use of plants as recombinant protein production platforms is the absence of human-type glycosylation (Wilson 2002; Gomord and Faye 2004; Paccalet et al. 2007; Bakker et al. 2008). Protein glycosylation is an issue that has to be taken into account for all protein-production platforms for biopharmaceuticals that are glycosylated for parenteral administration by injection or diffusion, whether mammalian, yeast, plant, insect or E. coli (Fig. 7.1). Typically, the objective will be to replicate the glycosylation profile of the protein generated when the protein is expressed in the cells of the species in which the particular protein or gene variant is found. However, in some instances differences in glycosylation profiles may have no functional consequences in terms of therapeutic efficacy (Ko et al. 2003). Plants are capable of assembling oligosaccharides with linkages not found in humans and these moieties are immunogenic in humans (Fo¨tisch and Vieths 2001; Sourrouille et al. 2008). These motifs include b(1!2)-linked xylose (Xyl), a(1!3)-linked fucose (Fuc) and b(1!3)-linked galactose (Gal). The latter contrasts with the b(1!4)-Gal extension typically found at the distal ends of mammalian N-linked oligosaccharides which allows the correct attachment of terminal sialic acid structures (Sourrouille et al. 2008). There are a number of strategies

Bacteria

Yeast

xylose galactose mannose

Insect

Plant

N-acetylneuraminic acid N-acetylglucosamine fucose polypeptide

Fig. 7.1 Typical N-linked oligosaccharides from bacterial to mammalian

Mammalian

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being developed to “humanize” the plant glycosylation machinery (Lerouge et al. 2000; Bardor et al. 2006) to better refine the efficacy of their products, boost useable production and ultimately to lower costs (Castilho et al. 2008). These strategies are based on (a) inhibition of plant-specific glycosylation enzymes and pathways (Strasser et al. 2004) and/or (b) introduction of human-specific glycosylation enzymes and pathways (Bakker et al. 2001). On the other hand, it should be noted that topical or orally administered recombinant therapeutics may often contain plant glyco-epitopes with no negative consequences to test subjects. While plants do have many advantages, there are also competing (and complementary) technology platforms such as yeast where there are parallel efforts to develop humanized glycosylation pathways (Chiba and Jigami 2007; Hamilton and Gerngross 2007). Transformation of plants with a gene for human b(1!4) galactosyltransferase (b1,4GalT) is one way to combat the formation of natural plant glyco-epitopes on recombinant proteins (Sourrouille et al. 2008). Human b1,4GalT in its native state and fused with the Golgi targeting domain of a natural plant GlcNAc transferase were found to be effective at a1!3Fuc and b(1!2)-Xyl linkage-containing structures on plant proteins when cloned into alfalfa. The same study also found that siRNA-mediated silencing of sequences encoding alfalfa XylTs and FucTs was effective at decreasing the occurrence of plant-specific glyco-motifs on recombinant protein products (Sourrouille et al. 2008). The moss P. patens has been engineered to reduce endogenous synthesis of a(1!3)-Fuc and b(1!2)-Xyl linkages within N-linked oligosaccharides (Weise et al. 2007). P. patens plants were transformed with the gene encoding human erythropoietin (hEPO) and produced a highly glycosylated protein which still contained the essential N-linked oligosaccharides, but lacked the immunogenic a(1!3)-Fuc and b(1!2)-Xyl motifs. In small scale bioreactors (maximum 10 L), the moss plants secreted a maximum accumulation of rhEPO (250 mg g–1 dry plant weight) after 6 days of growth. In humans, a predominant terminal sugar structure is the nine-carbon sialic acid, 5-N-acetyl-D-neuraminic acid (Neu5Ac), which is most often found in either an a(2!3) or a(2!6) linkage to Gal or N-acetylgalactosamine (GalNAc), although polysialic acid termination with a(2!8) linkages also occurs. The efficacy of recombinant proteins can be greatly enhanced by the inclusion of complete human-type oligosaccharides which allow them to mimic naturally occurring blood-borne proteins and evade rapid removal by the body, thereby increasing the half-life (Goochee et al. 1991; Paccalet et al. 2007). hEPO is reported to have only a 2 min half-life without sialylation, but this is extended to 3 h when sialylated (Ngantung et al. 2006). Clotting Factor VIII has an unsialylated half-life of just 5 min while that of the fully sialylated version is 4 h (Ngantung et al. 2006). The presence of sialic acids has been reported in A. thaliana suspension cells, buckwheat and other plant species (Mayer et al. 1964; Onodera et al. 1966; Bourbouze et al. 1982; Shah et al. 2003). These findings have been subject to much debate in the literature, partly based on the identification methods used and partly due to the lack of plant homologs corresponding to mammalian or bacterial enzymes in their complete sialic acid biosynthetic pathways (Fig. 7.2; Lerouge et al. 2000;

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CYTOSOL GOLGI

UDP-GlcNAc

Sialylated glycoconjugate

ATP GNE

ManNAc-6-P

STr

PEP NANS

glycoconjugate

Neu5Ac-9-P

ST

NANP CTP Neu5Ac

CMP-Neu5Ac CMAS NUCLEUS

Fig. 7.2 Mammlian biosynthetic pathway of sialic acid after Castilho et al. (2008). Enzyme abbreviations are GNE, UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase; NANS, N-acetylneuraminic acid phosphate synthase; NANP, Neu5Ac-9-phosphate phosphatase; CMAS, CMP-Neu5Ac synthetase; ST, CMP-Neu5Ac transporter; STr, sialyltransferase

Faye et al. 2005; Zeleny et al. 2006). Recently, it has been reported that plants may be able to transport sialic acid (Bakker et al. 2008) and some species may have transferases capable of attaching sialic acid structures (Takashima et al. 2006). However, present or not, based on the quantities of sialic acid reported, plants would not produce sufficient sialylation to be of use in recombinant technologies regardless of the endogenous mechanism and, thus, molecular engineering to augment any endogenous pathways would be required (Paccalet et al. 2007). It would be useful to discover and use plant genes whose products perform the same function as those from mammals (Lerouge et al. 2000; Bakker et al. 2001; Bakker et al. 2008). However, this goal comes with the caveat that genes for enzymes of similar function in plants and mammals may not display high sequence homology. For example, the discovery of a typically mammalian Gal-b-(1!3) GalNAc-a-O-Ser/Thr structure on proteins from rice seeds (Kishimoto et al. 1999) implies the presence of a GalNAc transferase (GalNAcT) gene encoding the enzyme responsible for mucin-type (short oligosaccharide chains with GalNAc-aSer/Thr linkage to the protein backbone) O-glycosylation initiation. Despite the functional homology, no gene with significant sequence homology could be found

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in the rice genome via an in silico search (Kilcoyne et al., 2009). This could imply that certain enzymes of homologous function can differ widely from plant to mammals, or that the same function is fulfilled by other enzymes in planta. While the basic mechanisms of asparagine-linked core glycosylation in plants are now considered to be well understood (Lerouge et al. 2000; Wilson 2002; Faye et al. 2005), many questions still remain concerning exactly how plants perform fully extended N- and O-linked glycosylation of proteins.

7.4

Plant-Produced Biopharmaceuticals

Biopharmaceuticals have already had an enormous global impact from both health and economic perspectives. As demand is expected to exceed current supply capabilities, less expensive alternative production platforms are under intense investigation with plants firmly at the forefront in terms of biological and costefficiency (Giddings et al. 2000; Daniell et al. 2001; Goldstein and Thomas 2004; Yano and Takekoshi 2004). To date, many experimental products produced using plant recombinant technologies have been explored and validated. These will be discussed as three different groups of biopharmaceuticals: vaccines, antibodies and protein therapeutics (Table 7.1).

7.4.1

Vaccines

Vaccines comprise the largest class of recombinant biopharmaceuticals (Carter 2006). Vaccinations, either therapeutic or prophylactic, are a cost-effective method of reducing the occurrence of infectious disease in animals and humans. In general, vaccines are composed of an antigenic agent from the bacterium or virus containing an epitope known to induce an immunogenic response in the host, or else are based on an inactive or attenuated pathogen (e.g., Sabin polio vaccine). Effective vaccines have been developed and produced for a wide range of diseases, such as hepatitis B (Keating and Noble 2003; Sojikul et al. 2003; Greco et al. 2007) and rabies (see below), and are in development for many other diseases, such as HIV (see below). However, there are large populations vulnerable to major disease burdens, especially in developing nations, who remain unprotected due to the prohibitive costs of accessing even traditional or existing vaccines. Oral delivery of vaccines could address some of the issues that increase vaccine cost. Elimination of needles and reduced levels of training, expertise and equipment for delivery of oral vaccines would be beneficial for reducing the cost of vaccine delivery. Oral delivery can target the antigen to the mucosal surface of the intestinal lining, which can prime other mucosal surfaces, and this is especially relevant for gastrointestinal or sexually transmitted diseases. However, this delivery method also typically leads to degradation of the vaccination agent in the gastrointestinal

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Table 7.1 Three groups of biopharmaceuticals: vaccines, antibodies and protein therapeutics with examples and references Product Expression platform References Vaccines NV-VLP Tobacco, potato Mason et al. (1996), Zhang et al. (2006) HBsAg Tobacco, potato, tomatillo Mason et al. (1992), Richter et al. (2000), Kong et al. (2001), Gao et al. (2003), Huang et al. (2005), Youm et al. (2007), Sojikul et al. (2003) HIV-1/HBV fusion Arabidopsis, tobacco Greco et al. (2007), Guetard et al. (2008) protein FI-V fusion protein Tobacco, tomato Jones et al. (2003), Williamson et al. (2005), Mett et al. (2007), Alvarez et al. (2006), Santi et al. (2006), Arlen et al. (2008) LTB Tobacco, corn, potato, Wagner et al. (2004), Lamphear et al. soybean, carrot (2002), Moravec et al. (2007), Mason et al. (1998) PyMSP4/5 Tobacco, tomato Wang et al. (2008), Chowdhury and Bagasra (2007) Antibodies Anti-CD4/28 receptor scFv Anti-HBsAg Mab Anti-ErbB-2 scFv 2G12 Mab

Wheat

Brereton et al. (2007)

Tobacco Tobacco Corn

hsv81sc IgA Anti-HBsAg scFv

Micro algae Tobacco

Yano and Takekoshi (2004) Galeffi et al. (2006) Rademacher et al. (2008), Ramessar et al. (2008) Mayfield and Franklin (2005) Pujol et al. (2007)

Therapeutics MuIL-12 hGM-CSF rh-insulin

Tobacco Tobacco, rice, sugarcane Safflower, Arabidopsis

IFN-a Gcase Novokinin

Rice Tobacco Soybeans

Liu et al. (2008) Wang et al. (2005) Moloney et al (2003), Markley et al. (2006), Nykiforuk et al. (2006) Shirono et al. (2006) Reggi et al. (2005) Yamada et al. (2008)

tract, and to overcome this, large quantities of antigen have to be administered to ensure that enough will survive to induce immunogenicity. The amount of vaccine required for delivery of oral vaccines will place strains on the ability of current mammalian systems to cost-effectively deliver the quantities required. Ideally, a plant-produced vaccine would require minimal downstream processing. In this case, an edible (or orally ingested) plant vaccine would deliver the antigen to the intestinal mucosa in sufficient quantities and could protect the antigen from degradation in the harsh stomach environment as it is encapsulated within plant cells. A current drawback for making an edible vaccine a viable option is the

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low levels of accumulation of the recombinant protein in plant tissues, but advances in gene expression systems for plants will allow such issues to be overcome. Biosafety concerns about transgenic foods (i.e., ensuring production and delivery is within a strictly controlled closed-loop production and regulatory system) and consistency (cGMP-related) concerns regarding batch-to-batch consistency are also barriers to the implementation of edible vaccines. Biosafety issues for the production of medicinal plants (e.g., opium poppies) or toxic organs of food plants (e.g., poisonous leaves of Solanaceous species, such as potatoes) have been dealt with for decades and, based on these previous experiences, should be possible to address. In terms of the consistency issues, low-cost downstream processing approaches involving concentration or partial purification step(s) could make plant-produced vaccines feasible for commercial development. Plant-produced vaccines for livestock have made more progress in getting to market than those for humans, as they face fewer regulatory issues. Dow Agro Sciences LLC received the first regulatory approval in January 2006 for a plantmade injectable poultry vaccine against Newcastle Disease virus (NDV) from the USDA Center for Veterinary Biologics. The antigen is a haemagglutinin neuraminidase protein from NDV that was produced in a transgenic tobacco cell culture system. Diseases targeted for animal vaccine trials include foot and mouth disease virus, canine parvovirus, rabies virus, porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus (Ma et al. 2005). Diseases and antigens currently targeted for human vaccinations are discussed below. Noroviruses (NoV) belong to the Caliciviridae family and consist of one species, Norwalk virus (NV; Xi et al. 1990). The species is genetically diverse and genotypes are distributed over five genogroups (GGI–GGV; Ramirez et al. 2008). NoVs are a group of non-enveloped, single-stranded RNA viruses and are the leading cause of viral gastroenteritis in humans worldwide (Xi et al. 1990; Vinje et al. 1997; Fankhauser et al. 1998). They are transmitted directly or indirectly by the fecal-oral route but may also become airborne and are highly contagious. There is currently no commercially available vaccine for norovirus but the economic and public health impact of gastroenteritis epidemics make NoV an ideal candidate for vaccine development. Previously, when the capsid protein was expressed in insect cells by recombinant baculovirus, it self-assembled into empty 38-nm virus-like particles (VLPs), which were similar to the native virus in morphology and antigenicity (Jiang et al. 1992; Prasad et al. 1994). These VLPs were immunogenic in CD1 and BALB/c mice when orally administered (Ball et al. 1998) and the intranasal route induced higher serum IgG and fecal IgA responses (Guerrero et al. 2001). Additionally, recombinant Norwalk (rNV) VLPs orally administered without an adjuvant to humans in a phase I study were found to be safe and produce a dose-dependent serum IgG response (Ball et al. 1999). The potential of rNV VLPs for use as an oral immunogen for a mucosal vaccine has made it a popular target for production in plants. Partially purified rNV VLPs from transgenic tobacco plants elicited humoral and mucosal antibody responses specific for rNV in mice, as did feeding with

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transformed potato tubers (Mason et al. 1996). In another study by this group, the plant optimized gene for rNV capsid protein was expressed in tomato and potato plants and the product successfully assembled VLPs. Lyophilized tomato fruit induced dose-dependent NV-specific serum IgG and mucosal IgA production in mice. However, larger quantities of freeze-dried potato tuber (1 g) were required to elicit the same response. It was found that rehydrated potato tuber was less immunogenic due to VLP instability caused by phenolic compound oxidation and that air-dried tomato fruit was more immunogenic than lyophilized tomato fruit and dried potato tubers (Zhang et al. 2006). Furthermore, when raw transgenic potato expressing rNV capsid protein was fed to human volunteers, 19 out of 20 volunteers developed an immune response, although the increase in serum antibody level was of limited magnitude (Tacket et al. 2000). These VLPs have also been produced in Nicotiana benthamiana leaves using an engineered plant virus-based transient expression system (magnICON). Oral immunization with partially purified rNV VLPs without adjuvant induced serum IgG and fecal and vaginal IgA response in mice (Santi et al. 2008). Coupled with a low-cost concentration and partial purification step, as demonstrated in this study (Santi et al. 2008), the goal of manufacturing oral vaccines in plant tissue for humans is becoming more achievable. Over two billion people worldwide are infected with hepatitis B virus (HBV), which is associated with chronic liver disease and liver cancer (WHO 1998). Hence, there is a need for an inexpensive vaccine for widespread immunization, especially in the developing world. Engerix-B1 (GlaxoSmithKline) and Recombivax HB1 (Merck and Co.) are commercial vaccines that confer seroprotection and consist of recombinant hepatitis B surface antigen (HBsAg) produced in the yeast Saccharomyces cerevisiae (Adkins and Wagstaff 1998; Keating and Noble 2003). The surface envelope glycoprotein exists in three isoforms produced by alternative splicing and initiation – large (L), middle (M) and small (S) – of which the commercial vaccine is the recombinantly produced S form (Fig. 7.3). This envelope protein is reported to have an important role in attachment to the host cell surface and subsequent infection (Paran et al. 2003). HBsAgs assemble VLPs and, when expressed in tobacco leaves, the subviral particles formed were similar to those produced in yeast (Mason et al. 1992). SHBsAg MHBsAg

12

pre-

108 109

pre-

163 164

S

389

LHBsAg

Fig. 7.3 The three isoforms of hepatitis B virus surface envelope glycoprotein (HBsAg) produced by alternative splicing and initiation – large (L), middle (M) and small (S)

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The partially purified antigen from tobacco elicited B- and T-cell responses when injected in a mouse model and the T-cell antigen epitope was found to be a partial sequence of the S region of HBsAg (Thanavala et al. 1995). HBsAg VLPs were then produced in potato tubers with some improvements to increase the yield, such as targeting the gene product for retention in the plant cell endoplasmic reticulum (ER; Richter et al. 2000). Mice fed HBsAg-transgenic potatoes with a cholera toxin adjuvant generated HBsAg-specific serum antibodies. A long-lasting secondary antibody response was obtained on parenteral boosting (Kong et al. 2001) and, conversely, by parenteral prime followed by an oral boost (Richter et al. 2000). HBsAg has also been produced in transgenic cherry tomatillo (Physalis ixocarpa) that was orally immunogenic in mice (Gao et al. 2003). Additionally, the serum anti-HBsAg titre increased in over half the group of immunized humans who were fed uncooked transgenic potatoes without a mucosal adjuvant or buffering for stomach pH (Thanavala et al. 2005). MHBsAg from transformed tobacco resulted in an enhanced antibody titre in mice after intraperitoneal (i.p.) injection (Huang et al. 2005). This same result was also observed when mice were fed potato-derived MHBsAg (Youm et al. 2007). In an attempt to overcome low accumulation of antigenic proteins in plant tissues, a fusion protein of a soybean signal protein with HBsAg was introduced into suspension-cultured tobacco cells which resulted in greater accumulation. The fusion protein was more immunogenic in mice than the unmodified HBsAg and this may have been due to greater antigen stability, improved presentation of the antigenic determinant in the S domain and increased oligomerization (Sojikul et al. 2003). The highly immunogenic hepatitis B core antigen (HBcAg) has also been recombinantly produced in transgenic tobacco and was found to assemble into spherical particles 25 to 30 nm in diameter. In the hemagglutination-inhibition test, partially purified VLPs demonstrated serologic properties comparable to those produced in E. coli (Tsuda et al. 1998). High levels of production of HBcAg (up to 7.14% of total soluble protein (TSP)) were reported 7 days post-infection using the magnICON system (Icon Genetics). The product also self-assembled into VLPs and the partially purified product evoked strong serum antibody response in mice when injected i.p. Moreover, mucosal immunization (oral and nasal) with no adjuvant gave HBcAg-specific serum IgG and intestinal IgA response (Huang et al. 2006). This rapid, high-level antigen production of strong immunogenicity makes a potential oral vaccine from plants more feasible. Enterotoxin produced by pathogenic strains of E. coli is the major cause of death in developing countries and claims 1.6 million lives per annum (Tacket 2007). The heat-labile toxin is similar to cholera toxin and is composed of the toxic A 27 kDa subunit and the non-toxic B subunit (LTB) which is a 55 kDa homopentamer of 11.6 kDa subunits. LTB is a strong immunogen that binds to the GM1 ganglioside on enterocytes and is a popular choice for a vaccine candidate. It has been expressed in potato (Mason et al. 1998), maize (Lamphear et al. 2002), tobacco (Wagner et al. 2004) and soybean (Moravec et al. 2007); volunteers who consumed transgenic potato developed serum and/or mucosal immune response (Tacket et al. 1998).

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Transgenic defatted corn meal was generally well tolerated and was immunogenic in volunteers (Tacket et al. 2004). However, as raw potato and uncooked corn meal can be unpalatable to many, LBT was expressed in transgenic carrot taproots (Rosales-Mendoza et al. 2008), which can be eaten raw. The product was found to be immunogenic and also protected against cholera toxin challenge in mice (Rosales-Mendoza et al. 2008). A bivalent vaccine of recombinant HIV-1/HBV fusion protein VLPs has been produced in A. thaliana and tobacco (Greco et al. 2007). The HIV-1 portion consisted of a polyepitope comprised of eight epitopes from five major HIV-1 proteins. These particular epitopes were expressed together as HIV-1 and HBV have similar transmission pathways and co-infection with HBV occurs in more than 30% of HIV-1 patients. Oral administration to mice elicited anti-HIV-1 specific CD8+ T-cell activation (Guetard et al. 2008). Malaria is another major effective vaccine target. Its prevalence in areas of extreme poverty that lack infrastructure also makes it an ideal candidate for an oral or edible vaccine. Malaria is caused by the parasite protozoan genus Plasmodium transmitted to humans through the bites of infected female Anopheles mosquitoes. The species Plasmodium vivax and P. falciparum cause most infections in humans resulting in death and morbidity and are thus the main targets of vaccine development (Chowdhury and Bagasra 2007). However, the parasite’s complexity, its ability to change through its life cycle in humans and mosquitoes, and its ability to evade the immune system make this a challenge (Chowdhury and Bagasra 2007). An edible vaccine using transgenic tomatoes of different sizes, shapes and colors to deliver multiple antigens for the various stages of malarial infection has been proposed (Chowdhury and Bagasra 2007). In an attempt to immunize against one life stage, surface protein 4/5 (PyMSP4/5) of the murine P. yoelii merozoite, the homolog of P. falciparum merozoite surface proteins 4 and 5, has been selected. The merozoite life stage takes place in hepatocytes and PyMSP4/5 has been shown to provide protection for mice against lethal challenge (Kedzierski et al. 2001). PyMSP4/5 from tobacco parenterally delivered to mice induced antigen-specific antibodies but antibody levels were not high enough to provide protection against lethal challenge (Wang et al. 2008). The virulent Gram-negative bacterium, Yersinia pestis, is the cause of plague. The most common form, bubonic plague, is spread to humans by bites from fleas that have previously fed on infected animals and is then distributed systematically in the body. The disease results in swollen, tender lymph nodes (buboes), which can hemorrhage and become necrotic. The pneumonic form of the disease is almost always fatal and can also be transmitted by inhalation of infected aerosolized droplets, making plague a potential bioterrorism agent. Recently, there have been plague outbreaks in India, Algeria, Congo, Zambia and Malawi (WHO 2007) and over 2,000 cases are reported annually. Vaccines that are currently available include killed whole cells, which are not protective against pneumonic plague and have many undesirable side effects, and a live attenuated vaccine, which also suffers from unacceptable side effects and has not been approved for use in the United States (Anisimov and Amoako 2006). However, vaccines based on the

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antigenic subunits of F1 (fraction 1 anti-phagocytic capsular envelope glycoprotein), V (a secreted immunomodulatory protein) and an F1–V fusion protein have been intensely investigated and were successful immunogens in animal and human trials (Jones et al. 2003; Williamson et al. 2005; Mett et al. 2007). The F1–V fusion protein was expressed in tomato in an attempt to make a useable oral vaccine. The transgenic tomatoes were immunogenic