Plant Abiotic Stress: Molecular Biology and Biotechnological Advances 9781119463689, 9781119463672, 9781119463696, 1119463688

Table of contents :
Cover......Page 1
Title Page......Page 5
Copyright......Page 6
Contents......Page 7
List of Contributors......Page 17
1.1 Introduction......Page 23
1.2 Drought Tolerance......Page 25
1.3 Cold Tolerance......Page 32
1.4 Salinity Tolerance......Page 34
1.5 Need for More Translational Research......Page 38
References......Page 39
2.1 Introduction......Page 51
2.2 Drought‐induced Adaptations......Page 54
2.3 Cold‐induced Adaptations......Page 55
2.4 High Temperature‐induced Adaptations......Page 56
2.6 Heavy Metal‐induced Adaptations......Page 57
2.7 Roles of Auxin, Ethylene, and ROS......Page 58
2.8 Conclusion......Page 59
References......Page 60
3.1 Introduction......Page 67
3.2 Stomatal Morphology......Page 68
3.3 Stomatal Movement Mechanism......Page 69
3.5 Drought Stress Signaling Pathways......Page 70
3.5.2.1 Plant Hormones......Page 71
3.5.3.1 Role of CO2 Molecule in Response to Drought Stress......Page 74
3.5.3.4 Phospholipid Role in Signal Transduction in Response to Drought Stress......Page 75
3.6 Mechanisms of Plant Response to Stress......Page 76
3.8 Conclusion......Page 78
References......Page 79
4.1 Introduction......Page 87
4.2 Reactive Oxygen Species......Page 88
4.2.1.1 Superoxide Radical (O2·−)......Page 89
4.2.1.2 Singlet Oxygen (1O2)......Page 90
4.2.2 Sites of ROS Generation......Page 91
4.2.2.3 Mitochondria......Page 92
4.2.3 ROS and Oxidative Damage to Biomolecules......Page 93
4.2.4 Role of ROS as Messengers......Page 95
4.3.1.1 Ascorbate......Page 96
4.3.1.3 Tocopherols......Page 97
4.3.2 Enzymatic Components......Page 98
4.3.2.3 Peroxidases......Page 99
4.3.2.4 Enzymes of the Ascorbate–Glutathione Cycle......Page 100
4.3.2.7 Glutathione Reductase......Page 101
4.4 Redox Homeostasis in Plants......Page 102
References......Page 103
5.1 Introduction......Page 113
5.2 Osmolyte Accumulation is a Universally Conserved Quick Response During Abiotic Stress......Page 114
5.3 Osmolytes Minimize Toxic Effects of Abiotic Stresses in Plants......Page 115
5.4 Stress Signaling Pathways Regulate Osmolyte Accumulation Under Abiotic Stress Conditions......Page 116
5.5 Metabolic Pathway Engineering of Osmolyte Biosynthesis Can Generate Improved Abiotic Stress Tolerance in Transgenic Crop Plants......Page 117
References......Page 119
6.1 Introduction......Page 127
6.2 Plant Hormones and Other Elicitor‐mediated Abiotic Stress Tolerance in Plants......Page 128
6.4 Signaling Role of Nitric Oxide in Abiotic Stresses......Page 131
6.6 Conclusion......Page 136
References......Page 137
7.1 Introduction......Page 145
7.2 Se Bioaccumulation and Metabolism in Plants......Page 146
7.3.2 The Antioxidant Properties of Se......Page 147
7.4.1 Se and Salt Stress......Page 148
7.4.2 Se and Drought Stress......Page 149
7.4.4 Se Ameliorating the Effects of UV‐B Irradiation......Page 150
7.5 Conclusion......Page 151
References......Page 152
8.1 Introduction......Page 157
8.2.1 PAs Scavenge Reactive Oxygen Species......Page 158
8.3.2 Ethylene and PAs......Page 159
8.3.4 Abscisic Acid and PAs......Page 160
8.4 Conclusion and Future Perspectives......Page 161
References......Page 162
9.1 Introduction......Page 167
9.2 Deep Evolutionary Roots......Page 168
9.3 ABA Chemical Structure, Biosynthesis, and Metabolism......Page 173
9.4 ABA Perception and Signaling......Page 175
9.5 ABA Regulation of Gene Expression......Page 176
9.5.1 Cis‐regulatory Elements......Page 177
9.5.2 Transcription Factors Involved in the ABA‐Mediated Abiotic Stress Response......Page 178
9.5.2.2 MYC and MYB......Page 179
9.5.2.3 NAC Family......Page 181
9.5.2.4 AP2/ERF Family......Page 182
9.5.2.5 Zinc Finger Family......Page 184
9.6 Post‐transcriptional and Post‐translational Control in Regulating ABA Response......Page 186
9.7 Epigenetic Regulation of ABA Response......Page 189
9.8 Conclusion......Page 190
References......Page 191
10.1 Introduction......Page 207
10.2 Ethylene: Abundance, Biosynthesis, Signaling, and Functions......Page 208
10.3 Abiotic Stress and Ethylene Biosynthesis......Page 209
10.4 Role of Ethylene in Photosynthesis Under Abiotic Stress......Page 210
10.5 Role of Ethylene on ROS and Antioxidative System Under Abiotic Stress......Page 216
References......Page 218
11.1 Introduction......Page 231
11.3.1 Crosstalk Between ABA and GA......Page 232
11.3.3 Crosstalk Between ABA and ET......Page 233
11.3.4 Crosstalk Between ABA and Auxins......Page 234
11.4 Conclusion and Future Directions......Page 235
References......Page 237
12.1 Introduction......Page 243
12.2.1 Structure and Functions of sHSP Family......Page 245
12.2.2 Structure and Functions of HSP60 Family......Page 246
12.2.3 Structure and Functions of the HSP70 Family......Page 247
12.2.3.1 DnaJ/HSP40......Page 249
12.2.4 Structure and Functions of HSP90 Family......Page 250
12.2.5 Structure and Functions of HSP100 Family......Page 251
12.3 Regulation of HSP Expression in Plants......Page 252
12.4 Crosstalk Between HSP Networks to Provide Tolerance Against Abiotic Stress......Page 253
12.5 Genetic Engineering of HSPs for Abiotic Stress Tolerance in Plants......Page 254
References......Page 256
13.1 Introduction......Page 263
13.2 Sources of Cl− Ion Contamination......Page 264
13.3 Role of Cl− in Plant Growth and Development......Page 265
13.4 Cl− Toxicity......Page 266
13.5.2 Mechanism of Cl− Efflux at the Membrane Level......Page 267
13.5.3 Differential Accumulation of Cl− in Plants and Compartmentalization......Page 268
13.6 Electrophysiological Study of Cl− Anion Channels in Plants......Page 269
13.7 Channels and Transporters Participating in Cl− Homeostasis......Page 270
13.7.1 Slow Anion Channel and Associated Homologs......Page 271
13.7.2 QUAC1 and Aluminum‐activated Malate Transporters......Page 273
13.7.3 Plant Chloride Channel Family Members......Page 275
13.7.4 Phylogenetic Tree and Tissue Localization of CLC Family Members......Page 277
13.7.5 Cation, Chloride Co‐transporters......Page 279
13.7.6 ATP‐binding Cassette Transporters and Chloride Conductance Regulatory Protein......Page 280
13.7.8 Chloride Channel‐mediated Anion Transport......Page 281
13.8 Conclusion and Future Perspectives......Page 282
References......Page 283
14.2 P. indica: An Extraordinary Tool for Salinity Stress Tolerance Improvement......Page 291
14.4 P. indica‐induced Biomodulation in Host Plant under Salinity Stress......Page 292
14.6 Role of Calcium Signaling and MAP Kinase Signaling Combating Salt Stress......Page 294
14.7 Effect of P. indica on Osmolyte Synthesis and Accumulation......Page 295
14.8 Salinity Stress Tolerance Mechanism in Axenically Cultivated and Root Colonized P. indica......Page 296
14.9 Conclusion......Page 299
References......Page 300
15.1 Introduction......Page 305
15.2 Bacterial Symbionts‐mediated Abiotic Stress Tolerance Priming of Host Plants......Page 306
15.3 AM Fungi‐mediated Alleviation of Abiotic Stress Tolerance of Vascular Plants......Page 308
15.4 Other Beneficial Fungi and their Importance in Abiotic Stress Tolerance Priming of Plants......Page 309
15.4.1 Piriformospora indica: A Model System for Bio‐priming of Host Plants Against Abiotic Stresses......Page 310
15.5 Implication of Transgenes from Symbiotic Microorganisms in the Era of Genetic Engineering and Omics......Page 311
15.6 Conclusion and Future Perspectives......Page 312
References......Page 313
16.1.1 Why is Legume–Rhizobium Interaction Under the Scientific Scanner?......Page 323
16.2.1 Nodule Structure and Formation: The Sequential Events......Page 324
16.2.2 Nod Factor Signaling: From Perception to Nodule Inception......Page 326
16.2.3 Reactive Oxygen Species: The Crucial Role of the Mobile Signal in Nodulation......Page 327
16.2.5 Autoregulation of Nodulation: The Self Control from Within......Page 328
16.3.1 How Do Abiotic Stress Factors Alter Rhizobial Behavior During Symbiotic Association?......Page 329
16.3.2 Abiotic Agents Modulate Symbiotic Signals of Host Legumes......Page 330
16.4 Conclusion: The Lessons Unlearnt......Page 331
References......Page 332
17.1 Introduction......Page 337
17.2 Engineered Nanoparticles in the Environment......Page 339
17.3 Nanoparticle Transformations......Page 340
17.4 Plant Response to Nanoparticle Stress......Page 342
17.5 Generation of Reactive Oxygen Species (ROS)......Page 345
17.6 Nanoparticle Induced Oxidative Stress......Page 346
17.7 Antioxidant Defense System in Plants......Page 348
17.8 Conclusion......Page 349
References......Page 350
18.1 Introduction......Page 357
18.2 Reaction of Plants to Abiotic Stress......Page 358
18.3 Basic Concept of Abiotic Stress Tolerance in Plants......Page 359
18.4 Genetics of Abiotic Stress Tolerance......Page 360
18.5.1 Molecular Markers......Page 361
18.6 Marker‐assisted Selection for Abiotic Stress Tolerance in Crop Plants......Page 363
18.6.1.1 Wheat (Triticum aestivum)......Page 364
18.6.1.4 Grape (Vitis species)......Page 365
18.7 Marker‐assisted Selection for Drought Tolerance......Page 366
18.7.2.1 Rice (Oryza sativa)......Page 369
18.7.2.2 Mungbean (Vigna radiata)......Page 370
18.7.2.3 Oilseed Brassica......Page 371
18.7.2.4 Tomato (Solanum lycopersicum)......Page 372
18.7.3.1 Barley (Hordeum vulgare)......Page 373
18.7.3.2 Pea (Pisum sativum)......Page 375
18.7.3.3 Oilseed Brassica......Page 376
18.7.3.4 Potato (Solanum tuberosum)......Page 377
References......Page 378
19.1 Introduction......Page 391
19.3 Molecular Analysis of Drought Stress Response......Page 392
19.4.1 Transcriptomics......Page 393
19.4.2 Metabolomics......Page 394
19.4.3 Epigenomics......Page 395
19.6 Marker‐assisted Selection......Page 396
19.7 Transgenic Approach: Present Status and Future Prospects......Page 397
19.9 Salinity Stress in Rice......Page 398
19.10 Candidate Genes for Salt Tolerance in Rice......Page 400
19.11 QTL Associated with Rice Tolerance to Salinity Stress......Page 401
19.12 The Saltol QTL......Page 402
References......Page 403
20.1 Introduction......Page 411
20.2.1.1 Roche 454......Page 412
20.2.1.2 ABI SoLid......Page 413
20.2.1.3 ION Torrent......Page 414
20.2.1.4 Illumina......Page 415
20.2.2 Applications of NGS......Page 416
20.2.2.1 Genomics......Page 417
20.2.2.3 Epigenomics......Page 418
20.2.2.4 Transcriptomics......Page 419
20.3.1 Small Interfering RNAs......Page 420
20.4 Criteria and Tools for Computational Classification of Small RNAs......Page 424
20.4.2 Identification and Prediction of miRNAs and siRNAs......Page 425
20.5 Role of NGS in Identification of Stress‐regulated miRNA and their Targets......Page 429
20.5.2 miR159......Page 430
20.5.6 miR167......Page 431
20.5.10 miR393......Page 432
20.6 Conclusion......Page 433
References......Page 434
21.1 Introduction......Page 449
21.3 Combined Drought–Biotic Stresses in Plants......Page 450
21.3.1 Plant Responses Against Biotic Stress during Drought Stress......Page 451
21.4 Varietal Failure Against Multiple Stresses......Page 452
21.5 Transcriptome Studies of Multiple Stress Responses......Page 453
21.6.1 Reactive Oxygen Species......Page 454
21.6.2 Mitogen‐activated Protein Kinase Cascades......Page 455
21.6.3 Transcription Factors......Page 456
21.6.4 Heat Shock Proteins and Heat Shock Factors......Page 458
21.6.5 Role of ABA Signaling during Crosstalk......Page 459
21.7 Conclusion......Page 460
References......Page 461
Index......Page 469
EULA......Page 477

Citation preview

Molecular Plant Abiotic Stress

Molecular Plant Abiotic Stress Biology and Biotechnology

Edited by Dr Aryadeep Roychoudhury Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata-700016, West Bengal INDIA

Dr Durgesh Kumar Tripathi Amity Institute of Organic Agriculture Amity University Uttar Pradesh I 2 Block, 5th Floor, AUUP Campus Sector-125 Noida-201313, Uttar Pradesh INDIA

This edition first published 2019 © 2019 John Wiley & Sons Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Aryadeep Roychoudhury and Durgesh Kumar Tripathi to be identified as the authors of the editorial material in this work has been asserted in accordance with law. Registered Offices John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial Office The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print-on-demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Library of Congress Cataloging-in-Publication Data Names: Roychoudhury, Aryadeep, editor. Tripathi, Durgesh Kumar, editor. Title: Molecular plant abiotic stress : biology and biotechnology / edited by Dr. Aryadeep Roychoudhury, Department of Biotechnology, St. Xavier’s College, Bengal, India, Dr. Durgesh Kumar Tripathi, Amity Institute of Organic Agriculture (AIOA), Amity University, Noida, India. Description: First edition. | Hoboken, NJ : Wiley, 2019. | Includes bibliographical references and index. | Identifiers: LCCN 2019011920 (print) | LCCN 2019012932 (ebook) | ISBN 9781119463689 (Adobe PDF) | ISBN 9781119463672 (ePub) | ISBN 9781119463696 (hardback) Subjects: LCSH: Plants–Effect of stress on–Molecular aspects. | Plant molecular biology. | Plant physiology. | Plants–Adaptation. Classification: LCC QK754 (ebook) | LCC QK754 .M65 2019 (print) | DDC 572.8/2928–dc23 LC record available at https://lccn.loc.gov/2019011920 Cover Design: Wiley Cover Image: © Jose A. Bernat Bacete/Getty Images Set in 10/12pt WarnockPro by SPi Global, Chennai, India

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Contents List of Contributors xv 1

Plant Tolerance to Environmental Stress: Translating Research from Lab to Land 1 P. Suprasanna and S. B. Ghag

1.1 1.2 1.3 1.4 1.5 1.6

Introduction 1 Drought Tolerance 3 Cold Tolerance 10 Salinity Tolerance 12 Need for More Translational Research Conclusion 17 References 17

2

Morphological and Anatomical Modifications of Plants for Environmental Stresses 29 Chanda Bano, Nimisha Amist, and N. B. Singh

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

Introduction 29 Drought-induced Adaptations 32 Cold-induced Adaptations 33 High Temperature-induced Adaptations 34 UV-B-induced Morphogenic Responses 35 Heavy Metal-induced Adaptations 35 Roles of Auxin, Ethylene, and ROS 36 Conclusion 37 References 38

3

Stomatal Regulation as a Drought-tolerance Mechanism 45 Shokoofeh Hajihashemi

3.1 3.2 3.3 3.4 3.5 3.5.1 3.5.2

Introduction 45 Stomatal Morphology 46 Stomatal Movement Mechanism 47 Drought Stress Sensing 48 Drought Stress Signaling Pathways 48 Hydraulic Signaling 49 Chemical Signaling 49

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3.5.2.1 3.5.3 3.5.3.1 3.5.3.2 3.5.3.3 3.5.3.4 3.6 3.7 3.8

Plant Hormones 49 Nonhormonal Molecules 52 Role of CO2 Molecule in Response to Drought Stress 52 Role of Ca2+ Molecules in Response to Drought Stress 53 Protein Kinase Involved in Osmotic Stress Signaling Pathway 53 Phospholipid Role in Signal Transduction in Response to Drought Stress 53 Mechanisms of Plant Response to Stress 54 Stomatal Density Variation in Response to Stress 56 Conclusion 56 References 57

4

Antioxidative Machinery for Redox Homeostasis During Abiotic Stress 65 Nimisha Amist, Chanda Bano, and N. B. Singh

4.1 4.2 4.2.1 4.2.1.1 4.2.1.2 4.2.1.3 4.2.1.4 4.2.2 4.2.2.1 4.2.2.2 4.2.2.3 4.2.3 4.2.4 4.3 4.3.1 4.3.1.1 4.3.1.2 4.3.1.3 4.3.1.4 4.3.1.5 4.3.2 4.3.2.1 4.3.2.2 4.3.2.3 4.3.2.4 4.3.2.5 4.3.2.6 4.3.2.7 4.4 4.5

Introduction 65 Reactive Oxygen Species 66 Types of Reactive Oxygen Species 67 Superoxide Radical (O2 ⋅− ) 67 Singlet Oxygen (1 O2 ) 68 Hydrogen Peroxide (H2 O2 ) 69 Hydroxyl Radicals (OH⋅ ) 69 Sites of ROS Generation 69 Chloroplasts 70 Peroxisomes 70 Mitochondria 70 ROS and Oxidative Damage to Biomolecules 71 Role of ROS as Messengers 73 Antioxidative Defense System in Plants 74 Nonenzymatic Components of the Antioxidative Defense System 74 Ascorbate 74 Glutathione 75 Tocopherols 75 Carotenoids 76 Phenolics 76 Enzymatic Components 76 Superoxide Dismutases 77 Catalases 77 Peroxidases 77 Enzymes of the Ascorbate–Glutathione Cycle 78 Monodehydroascorbate Reductase 79 Dehydroascorbate Reductase 79 Glutathione Reductase 79 Redox Homeostasis in Plants 80 Conclusion 81 References 81

Contents

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Osmolytes and their Role in Abiotic Stress Tolerance in Plants 91 Abhimanyu Jogawat

5.1 5.2

Introduction 91 Osmolyte Accumulation is a Universally Conserved Quick Response During Abiotic Stress 92 Osmolytes Minimize Toxic Effects of Abiotic Stresses in Plants 93 Stress Signaling Pathways Regulate Osmolyte Accumulation Under Abiotic Stress Conditions 94 Metabolic Pathway Engineering of Osmolyte Biosynthesis Can Generate Improved Abiotic Stress Tolerance in Transgenic Crop Plants 95 Conclusion and Future Perspectives 97 Acknowledgements 97 References 97

5.3 5.4 5.5 5.6

6

Elicitor-mediated Amelioration of Abiotic Stress in Plants 105 Nilanjan Chakraborty, Anik Sarkar, and Krishnendu Acharya

6.1 6.2

Introduction 105 Plant Hormones and Other Elicitor-mediated Abiotic Stress Tolerance in Plants 106 PGPR-mediated Abiotic Stress Tolerance in Plants 109 Signaling Role of Nitric Oxide in Abiotic Stresses 109 Future Goals 114 Conclusion 114 References 115

6.3 6.4 6.5 6.6

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Role of Selenium in Plants Against Abiotic Stresses: Phenological and Molecular Aspects 123 Aditya Banerjee and Aryadeep Roychoudhury

7.1 7.2 7.3 7.3.1 7.3.2 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.4.5 7.5 7.6

Introduction 123 Se Bioaccumulation and Metabolism in Plants 124 Physiological Roles of Se 125 Se as Plant Growth Promoters 125 The Antioxidant Properties of Se 125 Se Ameliorating Abiotic Stresses in Plants 126 Se and Salt Stress 126 Se and Drought Stress 127 Se Counteracting Low-temperature Stress 128 Se Ameliorating the Effects of UV-B Irradiation 128 Se and Heavy Metal Stress 129 Conclusion 129 Future Perspectives 130 References 130

8

Polyamines Ameliorate Oxidative Stress by Regulating Antioxidant Systems and Interacting with Plant Growth Regulators 135 Prabal Das, Aditya Banerjee, and Aryadeep Roychoudhury

8.1

Introduction 135

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8.2 8.2.1 8.2.2 8.3 8.3.1 8.3.2 8.3.3 8.3.4 8.4

PAs as Cellular Antioxidants 136 PAs Scavenge Reactive Oxygen Species 136 The Co-operative Biosynthesis of PAs and Proline 137 The Relationship Between PAs and Growth Regulators 137 Brassinosteroids and PAs 137 Ethylene and PAs 137 Salicylic Acid and PAs 138 Abscisic Acid and PAs 138 Conclusion and Future Perspectives 139 Acknowledgments 140 References 140

9

Abscisic Acid in Abiotic Stress-responsive Gene Expression 145 Liliane Souza Conceição Tavares, Sávio Pinho dos Reis, Deyvid Novaes Marques, Eraldo José Madureira Tavares, Solange da Cunha Ferreira, Francinilson Meireles Coelho, and Cláudia Regina Batista de Souza

9.1 9.2 9.3 9.4 9.5 9.5.1 9.5.2

Introduction 145 Deep Evolutionary Roots 146 ABA Chemical Structure, Biosynthesis, and Metabolism 151 ABA Perception and Signaling 153 ABA Regulation of Gene Expression 154 Cis-regulatory Elements 155 Transcription Factors Involved in the ABA-Mediated Abiotic Stress Response 156 bZIP Family 157 MYC and MYB 157 NAC Family 159 AP2/ERF Family 160 Zinc Finger Family 162 Post-transcriptional and Post-translational Control in Regulating ABA Response 164 Epigenetic Regulation of ABA Response 167 Conclusion 168 References 169

9.5.2.1 9.5.2.2 9.5.2.3 9.5.2.4 9.5.2.5 9.6 9.7 9.8

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Abiotic Stress Management in Plants: Role of Ethylene 185 Anket Sharma, Vinod Kumar, Gagan Preet Singh Sidhu, Rakesh Kumar, Sukhmeen Kaur Kohli, Poonam Yadav, Dhriti Kapoor, Aditi Shreeya Bali, Babar Shahzad, Kanika Khanna, Sandeep Kumar, Ashwani Kumar Thukral, and Renu Bhardwaj

10.1 10.2 10.3 10.4 10.5 10.6

Introduction 185 Ethylene: Abundance, Biosynthesis, Signaling, and Functions 186 Abiotic Stress and Ethylene Biosynthesis 187 Role of Ethylene in Photosynthesis Under Abiotic Stress 188 Role of Ethylene on ROS and Antioxidative System Under Abiotic Stress 194 Conclusion 196 References 196

Contents

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Crosstalk Among Phytohormone Signaling Pathways During Abiotic Stress 209 Abhimanyu Jogawat

11.1 11.2 11.3

Introduction 209 Phytohormone Crosstalk Phenomenon and its Necessity 210 Various Phytohormonal Crosstalk Under Abiotic Stresses for Improving Stress Tolerance 210 Crosstalk Between ABA and GA 210 Crosstalk Between GA and ET 211 Crosstalk Between ABA and ET 211 Crosstalk Between ABA and Auxins 212 Crosstalk Between ET and Auxins 213 Crosstalk Between ABA and CTs 213 Conclusion and Future Directions 213 Acknowledgements 215 References 215

11.3.1 11.3.2 11.3.3 11.3.4 11.3.5 11.3.6 11.4

12

Plant Molecular Chaperones: Structural Organization and their Roles in Abiotic Stress Tolerance 221 Roshan Kumar Singh, Varsha Gupta, and Manoj Prasad

12.1 12.2 12.2.1 12.2.2 12.2.3 12.2.3.1 12.2.4 12.2.5 12.3 12.4

Introduction 221 Classification of Plant HSPs 223 Structure and Functions of sHSP Family 223 Structure and Functions of HSP60 Family 224 Structure and Functions of the HSP70 Family 225 DnaJ/HSP40 227 Structure and Functions of HSP90 Family 228 Structure and Functions of HSP100 Family 229 Regulation of HSP Expression in Plants 230 Crosstalk Between HSP Networks to Provide Tolerance Against Abiotic Stress 231 Genetic Engineering of HSPs for Abiotic Stress Tolerance in Plants 232 Conclusion 234 Acknowledgements 234 References 234

12.5 12.6

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Chloride (Cl− ) Uptake, Transport, and Regulation in Plant Salt Tolerance 241 DB Shelke, GC Nikalje, TD Nikam, P Maheshwari, DL Punita, KRSS Rao, PB Kavi Kishor, and P. Suprasanna

13.1 13.2 13.3 13.4 13.5 13.5.1 13.5.2

Introduction 241 Sources of Cl− Ion Contamination 242 Role of Cl− in Plant Growth and Development 243 Cl− Toxicity 244 Interaction of Soil Cl− with Plant Tissues 245 Cl− Influx from Soil to Root 245 Mechanism of Cl− Efflux at the Membrane Level 245

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13.5.3 13.6 13.7 13.7.1 13.7.2 13.7.3 13.7.4 13.7.5 13.7.6 13.7.7 13.7.8 13.7.9 13.8

Differential Accumulation of Cl− in Plants and Compartmentalization 246 Electrophysiological Study of Cl− Anion Channels in Plants 247 Channels and Transporters Participating in Cl− Homeostasis 248 Slow Anion Channel and Associated Homologs 249 QUAC1 and Aluminum-activated Malate Transporters 251 Plant Chloride Channel Family Members 253 Phylogenetic Tree and Tissue Localization of CLC Family Members 255 Cation, Chloride Co-transporters 257 ATP-binding Cassette Transporters and Chloride Conductance Regulatory Protein 258 Nitrate Transporter1/Peptide Transporter Proteins 259 Chloride Channel-mediated Anion Transport 259 Possible Mechanisms of Cl− Influx, Efflux, Reduced Net Xylem Loading, and its Compartmentalization 260 Conclusion and Future Perspectives 260 References 261

14

The Root Endomutualist Piriformospora indica: A Promising Bio-tool for Improving Crops under Salinity Stress 269 Abhimanyu Jogawat, Deepa Bisht, Nidhi Verma, Meenakshi Dua, and Atul Kumar Johri

14.1 14.2

Introduction 269 P. indica: An Extraordinary Tool for Salinity Stress Tolerance Improvement 269 Utilization of P. indica for Improving and Understanding the Salinity Stress Tolerance of Host Plants 270 P. indica-induced Biomodulation in Host Plant under Salinity Stress 270 Activity of Antioxidant Enzymes and ROS in Host Plant During Interaction with P. indica 272 Role of Calcium Signaling and MAP Kinase Signaling Combating Salt Stress 272 Effect of P. indica on Osmolyte Synthesis and Accumulation 273 Salinity Stress Tolerance Mechanism in Axenically Cultivated and Root Colonized P. indica 274 Conclusion 277 Acknowledgments 278 Conflict of Interest 278 References 278

14.3 14.4 14.5 14.6 14.7 14.8 14.9

15

Root Endosymbiont-mediated Priming of Host Plants for Abiotic Stress Tolerance 283 Abhimanyu Jogawat, Deepa Bisht, and Atul Kumar Johri

15.1 15.2

Introduction 283 Bacterial Symbionts-mediated Abiotic Stress Tolerance Priming of Host Plants 284 AM Fungi-mediated Alleviation of Abiotic Stress Tolerance of Vascular Plants 286

15.3

Contents

15.4 15.4.1 15.4.2 15.5 15.6

Other Beneficial Fungi and their Importance in Abiotic Stress Tolerance Priming of Plants 287 Piriformospora indica: A Model System for Bio-priming of Host Plants Against Abiotic Stresses 288 Fungal Endophytes, AM-like Fungi, and Other DSE-mediated Bio-priming of Host Plants for Abiotic Stress Tolerance 289 Implication of Transgenes from Symbiotic Microorganisms in the Era of Genetic Engineering and Omics 289 Conclusion and Future Perspectives 290 Acknowledgements 291 References 291

16

Insight into the Molecular Interaction Between Leguminous Plants and Rhizobia Under Abiotic Stress 301 Sumanti Gupta and Sampa Das

16.1 16.1.1 16.2 16.2.1 16.2.2 16.2.3

Introduction 301 Why is Legume–Rhizobium Interaction Under the Scientific Scanner? 301 Legume–Rhizobium Interaction Chemistry: A Brief Overview 302 Nodule Structure and Formation: The Sequential Events 302 Nod Factor Signaling: From Perception to Nodule Inception 304 Reactive Oxygen Species: The Crucial Role of the Mobile Signal in Nodulation 305 Phytohormones: Key Players on All Occasions 306 Autoregulation of Nodulation: The Self Control from Within 306 Role of Abiotic Stress Factors in Influencing Symbiotic Relations of Legumes 307 How Do Abiotic Stress Factors Alter Rhizobial Behavior During Symbiotic Association? 307 Abiotic Agents Modulate Symbiotic Signals of Host Legumes 308 Abiotic Stress Agents as Regulators of Defense Signals of Symbiotic Hosts During Interaction with Other Pathogens 309 Conclusion: The Lessons Unlearnt 309 References 310

16.2.4 16.2.5 16.3 16.3.1 16.3.2 16.3.3 16.4

17

Effect of Nanoparticles on Oxidative Damage and Antioxidant Defense System in Plants 315 Savita Sharma, Vivek K. Singh, Anil Kumar, and Sharada Mallubhotla

17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8

Introduction 315 Engineered Nanoparticles in the Environment 317 Nanoparticle Transformations 318 Plant Response to Nanoparticle Stress 320 Generation of Reactive Oxygen Species (ROS) 323 Nanoparticle Induced Oxidative Stress 324 Antioxidant Defense System in Plants 326 Conclusion 327 References 328

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Marker-assisted Selection for Abiotic Stress Tolerance in Crop Plants 335 Saikat Gantait, Sutanu Sarkar, and Sandeep Kumar Verma

18.1 18.2 18.3 18.4 18.5 18.5.1 18.5.2 18.6 18.6.1 18.6.1.1 18.6.1.2 18.6.1.3 18.6.1.4 18.7 18.7.1.1 18.7.1.2 18.7.1.3 18.7.1.4 18.7.2 18.7.2.1 18.7.2.2 18.7.2.3 18.7.2.4 18.7.3 18.7.3.1 18.7.3.2 18.7.3.3 18.7.3.4 18.8

Introduction 335 Reaction of Plants to Abiotic Stress 336 Basic Concept of Abiotic Stress Tolerance in Plants 337 Genetics of Abiotic Stress Tolerance 338 Fundamentals of Molecular Markers and Marker-assisted Selection 339 Molecular Markers 339 Marker-assisted Selection 341 Marker-assisted Selection for Abiotic Stress Tolerance in Crop Plants 341 Marker-assisted Selection for Heat Tolerance 342 Wheat (Triticum aestivum) 342 Cowpea (Vigna unguiculata) 343 Oilseed Brassica 343 Grape (Vitis species) 343 Marker-assisted Selection for Drought Tolerance 344 Maize (Zea mays) 344 Chickpea (Cicer arietinum) 345 Oilseed Brassica 346 Coriander (Coriandrum sativum) 346 Marker-assisted Selection for Salinity Tolerance 347 Rice (Oryza sativa) 347 Mungbean (Vigna radiata) 348 Oilseed Brassica 349 Tomato (Solanum lycopersicum) 350 Marker-assisted Selection for Low Temperature Tolerance 351 Barley (Hordeum vulgare) 351 Pea (Pisum sativum) 353 Oilseed Brassica 354 Potato (Solanum tuberosum) 355 Outlook 356 References 356

19

Transgenes: The Key to Understanding Abiotic Stress Tolerance in Rice 369 Supratim Basu, Lymperopoulos Panagiotis, Joseph Msanne, and Roel Rabara

19.1 19.2 19.3 19.4 19.4.1 19.4.2 19.4.3 19.5 19.6 19.7

Introduction 369 Drought Effects in Rice Leaves 370 Molecular Analysis of Drought Stress Response 370 Omics Approach to Analysis of Drought Response 371 Transcriptomics 371 Metabolomics 372 Epigenomics 373 Plant Breeding Techniques to Improve Rice Tolerance 374 Marker-assisted Selection 374 Transgenic Approach: Present Status and Future Prospects 375

Contents

19.8 19.9 19.10 19.11 19.12 19.13

20

20.1 20.2 20.2.1 20.2.1.1 20.2.1.2 20.2.1.3 20.2.1.4 20.2.2 20.2.2.1 20.2.2.2 20.2.2.3 20.2.2.4 20.3 20.3.1 20.3.2 20.4 20.4.1 20.4.2 20.5

Looking into the Future for Developing Drought-tolerant Transgenic Rice Plants 376 Salinity Stress in Rice 376 Candidate Genes for Salt Tolerance in Rice 378 QTL Associated with Rice Tolerance to Salinity Stress 379 The Saltol QTL 380 Conclusion 381 References 381 Impact of Next-generation Sequencing in Elucidating the Role of microRNA Related to Multiple Abiotic Stresses 389 Kavita Goswami, Anita Tripathi, Budhayash Gautam, and Neeti Sanan-Mishra

Introduction 389 NGS Platforms and their Applications 390 NGS Platforms 390 Roche 454 390 ABI SoLid 391 ION Torrent 392 Illumina 393 Applications of NGS 394 Genomics 395 Metagenomics 396 Epigenomics 396 Transcriptomics 397 Understanding the Small RNA Family 398 Small Interfering RNAs 398 microRNA 402 Criteria and Tools for Computational Classification of Small RNAs 402 Pre-processing (Quality Filtering and Sequence Alignment) 403 Identification and Prediction of miRNAs and siRNAs 403 Role of NGS in Identification of Stress-regulated miRNA and their Targets 407 20.5.1 miR156 408 20.5.2 miR159 408 20.5.3 miR160 409 20.5.4 miR164 409 20.5.5 miR166 409 20.5.6 miR167 409 20.5.7 miR168 410 20.5.8 miR169 410 20.5.9 miR172 410 20.5.10 miR393 410 20.5.11 miR396 411 20.5.12 miR398 411 20.6 Conclusion 411 Acknowledgments 412 References 412

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21

Understanding the Interaction of Molecular Factors During the Crosstalk Between Drought and Biotic Stresses in Plants 427 Arnab Purohit, Shreeparna Ganguly, Rituparna Kundu Chaudhuri, and Dipankar Chakraborti

21.1 21.2 21.3 21.3.1 21.3.2 21.4 21.5 21.6 21.6.1 21.6.2 21.6.3 21.6.4 21.6.5 21.7

Introduction 427 Combined Stress Responses in Plants 428 Combined Drought–Biotic Stresses in Plants 428 Plant Responses Against Biotic Stress during Drought Stress 429 Plant Responses Against Drought Stress during Biotic Stress 430 Varietal Failure Against Multiple Stresses 430 Transcriptome Studies of Multiple Stress Responses 431 Signaling Pathways Induced by Drought–Biotic Stress Responses 432 Reactive Oxygen Species 432 Mitogen-activated Protein Kinase Cascades 433 Transcription Factors 434 Heat Shock Proteins and Heat Shock Factors 436 Role of ABA Signaling during Crosstalk 437 Conclusion 438 Acknowledgments 439 Conflict of Interest 439 References 439 Index 447

xv

List of Contributors Krishnendu Acharya

Supratim Basu

Department of Botany University of Calcutta Kolkata, West Bengal India

NMC Biolab New Mexico Consortium Los Alamos, New Mexico USA

Nimisha Amist

Renu Bhardwaj

Plant Physiology Laboratory Department of Botany University of Allahabad Allahabad, 211002 India

Plant Stress Physiology Lab Department of Botanical & Environmental Sciences Guru Nanak Dev University Amritsar, 143005 India

Aditi Shreeya Bali

Department of Botany M.C.M. DAV College for Women Chandigarh, 160036 India

Deepa Bisht

School of Life Sciences Jawaharlal Nehru University New Delhi India

Aditya Banerjee

Department of Biotechnology St. Xavier’s College (Autonomous) Kolkata, West Bengal India

Dipankar Chakraborti

Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata, 700016, West Bengal India

Chanda Bano

Plant Physiology Laboratory Department of Botany University of Allahabad Allahabad, 211002 India

Nilanjan Chakraborty

Department of Botany Scottish Church College Kolkata, West Bengal India

xvi

List of Contributors

Rituparna Kundu Chaudhuri

Budhayash Gautam

Department of Botany Krishnagar Govt. College Krishnagar, 741101, West Bengal India

Sam Higginbottom University of Agriculture, Technology and Sciences Allahabad, Uttar Pradesh India

Francinilson Meireles Coelho

S. B. Ghag

Universidade Federal do Pará Belém, PA Brazil

School of Biological Sciences UM-DAE Centre for Excellence in Basic Sciences Kalina campus, Santacruz (East) Mumbai, Maharashtra India

Solange da Cunha Ferreira

Universidade Federal do Pará Belém, PA Brazil Prabal Das

Department of Botany University of Calcutta Kolkata, West Bengal India Sampa Das

Division of Plant Biology Bose Institute, P1/12, CIT Scheme, VIIM Kolkata, West Bengal India Meenakshi Dua

School of Environmental Sciences Jawaharlal Nehru University New Delhi, 110067 India

Kavita Goswami

International Centre for Genetic Engineering and Biotechnology New Delhi India Sumanti Gupta

Department of Botany Rabindra Mahavidyalaya Hooghly, West Bengal India Varsha Gupta

National Institute of Plant Genome Research New Delhi India Shokoofeh Hajihashemi

Shreeparna Ganguly

Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata, 700016, West Bengal India

Plant Biology Department Behbahan Khatam Alanbia University of Technology Khuzestan Iran Abhimanyu Jogawat

Saikat Gantait

Department of Genetics and Plant Breeding Faculty of Agriculture Bidhan Chandra Krishi Viswavidyalaya Mohanpur, Nadia, West Bengal, 741252 India

National Institute of Plant Genome Research New Delhi India

List of Contributors

Atul Kumar Johri

Sandeep Kumar

School of Life Sciences Jawaharlal Nehru University New Delhi India

Department of Environmental Sciences DAV University Sarmastpur, Jalandhar, 144012, Punjab India

Dhriti Kapoor

Vinod Kumar

School of Bioengineering & Biosciences Lovely Professional University Punjab, 144411 India Kavi Kishor PB

Center for Biotechnology Acharya Nagarjuna University Guntur, 522510 India

Department of Botany DAV University Sarmastpur, Jalandhar, 144012, Punjab India Maheshwari P

Center for Biotechnology Acharya Nagarjuna University Guntur, 522510 India Sharada Mallubhotla

Kanika Khanna

Plant Stress Physiology Lab Department of Botanical & Environmental Sciences Guru Nanak Dev University Amritsar, 143005 India Sukhmeen Kaur Kohli

Plant Stress Physiology Lab Department of Botanical & Environmental Sciences Guru Nanak Dev University Amritsar, 143005 India Anil Kumar

School of Biotechnology Shri Mata Vaishno Devi University Katra, J&K India

School of Biotechnology Faculty of Sciences Shri Mata Vaishno Devi University Katra, 182320, J&K India Deyvid Novaes Marques

Universidade Federal do Pará Belém, PA Brazil Neeti Sanan-Mishra

International Centre for Genetic Engineering and Biotechnology New Delhi India Joseph Msanne

NMC Biolab New Mexico Consortium Los Alamos, New Mexico USA

Rakesh Kumar

Nikalje GC

Department of Botany DAV University Sarmastpur, Jalandhar, 144012, Punjab India

Department of Botany Savitribai Phule Pune University Pune, 411007 India

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List of Contributors

Nikam TD

Aryadeep Roychoudhury

Department of Botany Savitribai Phule Pune University Pune, 411007 India

Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata, West Bengal India

Lymperopoulos Panagiotis

NMC Biolab New Mexico Consortium Los Alamos, New Mexico USA Manoj Prasad

National Institute of Plant Genome Research New Delhi India Punita DL

Center for Biotechnology Acharya Nagarjuna University Guntur, 522510 India Arnab Purohit

Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata, West Bengal India Roel Rabara

NMC Biolab New Mexico Consortium Los Alamos, New Mexico USA

Anik Sarkar

Department of Botany University of Calcutta Kolkata, West Bengal India Sutanu Sarkar

Department of Genetics and Plant Breeding Faculty of Agriculture Bidhan Chandra Krishi Viswavidyalaya Mohanpur, Nadia, West Bengal, 741252 India Babar Shahzad

School of Land and Food University of Tasmania Hobart, Tasmania Australia Anket Sharma

Plant Stress Physiology Lab Department of Botanical & Environmental Sciences Guru Nanak Dev University Amritsar, 143005 India Savita Sharma

Rao KRSS

Center for Biotechnology Acharya Nagarjuna University Guntur, 522510 India

School of Biotechnology Shri Mata Vaishno Devi University Katra, J&K India Shelke DB

Sávio Pinho dos Reis

Universidade Federal do Pará Belém, PA Brazil

Department of Botany Savitribai Phule Pune University Pune, 411007 India

List of Contributors

Gagan Preet Singh Sidhu

Ashwani Kumar Thukral

Department of Applied Sciences UIET Chandigarh, 160014 India

Plant Stress Physiology Lab Department of Botanical & Environmental Sciences Guru Nanak Dev University Amritsar, 143005 India

N. B. Singh

Department of Botany University of Allahabad Allahabad, Uttar Pradesh India Roshan Kumar Singh

National Institute of Plant Genome Research New Delhi India Vivek K. Singh

School of Physics Shri Mata Vaishno Devi University Katra, J&K India Cláudia Regina Batista de Souza

Universidade Federal do Pará Belém, PA Brazil

Anita Tripathi

International Centre for Genetic Engineering and Biotechnology New Delhi India Durgesh Kumar Tripathi

Amity Institute of Organic Agriculture Amity University, Uttar Pradesh I 2 Block, 5th Floor, AUUP Campus Sector-125 Noida, 201313, UP India Nidhi Verma

School of Life Sciences Jawaharlal Nehru University New Delhi, 110067 India Sandeep Kumar Verma

P. Suprasanna

Nuclear Agriculture and Biotechnology Division Bhabha Atomic Research Centre Trombay, Mumbai, Maharashtra India

Institute of Biological Science SAGE University Kailod Kartal, Indore Madhya Pradesh, 452020 India Poonam Yadav

Eraldo José Madureira Tavares

Empresa Brasileira de Pesquisa Agropecuária Petrolina, PE Brazil Liliane Souza Conceição Tavares

Universidade Federal do Pará Belém, PA Brazil

Plant Stress Physiology Lab Department of Botanical & Environmental Sciences Guru Nanak Dev University Amritsar, 143005 India

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1

1 Plant Tolerance to Environmental Stress: Translating Research from Lab to Land P. Suprasanna 1,3 and S. B. Ghag 2 1

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, 400 085 Mumbai, India Department of Biology, UM-DAE Centre for Excellence in Basic Sciences, Kalina campus, Santacruz (East), Mumbai 400 098, India 3 Homi Bhabha National Institute, Mumbai, 400 095, India 2

1.1 Introduction Food security for a burgeoning human population in a sustainable ecosystem is an important goal. However, the threat from climate change and unpredictable environmental extremes (Abberton et al. 2016) to plant growth and productivity (Lobell and Gourdji 2012; Gray and Brady 2016; Tripathi et al. 2016a) is increasing. Climate change-driven effects, especially from erratic environmental fluctuations, can result in increased prevalence of abiotic stresses and, pests and pathogens in crop plants (Chakraborty and Newton 2011; Batley and Edwards 2016). Various abiotic stresses such as drought, salinity, temperature, and heavy metals have been shown to diminish average yields by more than 50% for major crops (Wang et al. 2003; Pereira 2016; Tripathi et al. 2016c). Over the years, considerable information has become available on the stress-related genetic repertoire of genes, quantitative trait loci and molecular networks governing plant responses to drought, salinity, heat, and other abiotic stresses (Krasensky and Jonak 2012; Liu et al. 2018). This knowhow about genes and their regulation will enable improvements in stress tolerance in crops, in the face of the imminent threat of climate change, impacting crop genetic diversity and the productivity of staple food crops. Global temperature rises of 2–3 ∘ C are predicted to push crops toward extinction and even wild species that have so far been considered valuable genetic resource may also be affected. This will have negative consequences locally as well as globally, because the key traits for adaptiveness to climate change and variability adaptation may be lost forever. It is hence desirable that additional genetic variability should be introduced through mutagenesis or other approaches. Over the past few decades, great success has been achieved through selection, breeding, hybridization, recombination, and mutation to broaden genetic variability for important traits conferring adaptation of many species to changing biotic, climatic, and environmental pressures. Crop plants are susceptible to climate-driven abiotic (elevated CO2 , heat, drought, salinity, flooding) and biotic effects (Chapman et al. 2012). Several reviews have critically discussed the impact of climate change on various crop systems (Ahuja et al. 2010; Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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1 Plant Tolerance to Environmental Stress

Yadav et al. 2011; Tripathi et al. 2016a). Abiotic stresses elicit a plethora of morphological, physiological, biochemical, and molecular alterations (Singh et al. 2015a,b; Tripathi et al. 2016b, 2017; Singh et al. 2017; Suprasanna et al. 2018). The impact of stress has been shown to induce modulated gene function of structural genes, regulatory genes, and other master regulators (Zhu 2016; Patel et al. 2018). Plant defenses are endowed with molecular components of stress signal perception, osmotic and ionic homeostasis, hormone signaling, reactive oxygen species (ROS) scavenging systems, metabolic pathways, etc. (Figure 1.1). There are specific responses that are osmotic, hormonal, ionic, signal transduction, and transcription factor based, and there

ENVIRONMENTAL STRESS FACTORS Salinity, Cold, Drought, Heat, etc.,

Stress Response: Osmotic, Ionic, Oxidative

Damage and/or Disruption of Ionic And Osmotic Homeostasis, Membrane, Proteins PERCEPTION OF SIGNAL AND TRANSMITTAL Osmosensors, phospholipid-cleaving enzymes, second messengers, MAP kinases, Ca2+ sensors, calciumdependent protein kinases

TRANSCRIPTION FACTORS

OSM

OPR OTE C

ION

IFICAT

TION

DETOX STRESS RESPONSIVE MECHANISMS

ES

N RO

PE CHA

WAT E MOV R, ION EME NT

Revival of cellular homeostasis, functional and structural protection of proteins and membranes

TOLERANCE/ADAPTATION

Figure 1.1 Abiotic stress impact and plant responses (Lokhande et al. 2012).

1.2 Drought Tolerance

are also nonspecific responses that are activated by ROS (Mittler and Blumwald 2010, Muchate et al. 2016). Despite tremendous knowledge that has been generated in understanding abiotic stress responses, an integrated information gateway is needed to combine all of the genomics, proteomics, and metabolomics data concerning field conditions to achieve plant tolerance of environmental change (Roychoudhury et al. 2011, Edwards 2016). This has become a challenge that requires concerted effort. Hirayama and Shinozaki (2010) outlined some considerations (see Box 1.1) which should pave the way toward achieving this goal. Box 1.1 • Sensor(s) and signaling pathways – perception and transduction of local stress signals under single and combined stresses. • Molecular basis of interaction among biotic and abiotic stresses. • Key factors in the crosstalk between abiotic stress responses and other plant developmental pathways. • Long-term stress-associated responses under multiple abiotic stress conditions. • Experimental conditions that simulate natural field conditions for testing and functional validation. Modified after Hirayama and Shinozaki (2010).

Research into plant abiotic stress biology has two dimensions: the first, is the need to develop a detailed mechanistic view of plant responses to single and/or combined stresses to create a resource of gene targets and regulatory circuits for the improvement of stress-tolerant crop plants; and the second is the translation of research outcome into environmentally challenging field conditions. Physiological, biochemical, and molecular studies have generated data and great understanding of the mechanisms of how a plant will respond to a given stress or combined stress factors. Transcriptomic studies have demonstrated that the adaptation or responses are controlled by either upor down-regulation of several genetic pathways and processes associated with stress perception and signaling (Munns and Tester 2008; Roychoudhury and Banerjee 2015). Transgenic approaches are available as the existing strategies for crop improvement programs based on biotechnology (Jewell et al. 2010). Genetic engineering for improved stress tolerance has been made possible through the manipulation of a single or a few effector genes or regulatory genes (Wang et al. 2016) or those that encode osmolytes, antioxidants, chaperones, water, and ion transporters (Chen et al. 2014; Paul and Roychoudhury 2018; Suprasanna et al. 2018). Various genes involved in the synthesis of osmoprotectants have been explored for their potential in improving abiotic stress tolerance (Reguera et al. 2012). In this article, we have reviewed the progress made in genetic engineering for abiotic stress tolerance, especially drought, salinity and cold, and highlight the potential areas for translational research in this field.

1.2 Drought Tolerance Paucity of water is the most important environmental stress affecting crop plants, accounting for ∼70% loss of potential yield worldwide (Shiferaw et al. 2014). Daryanto

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et al. (2016) investigated the data published from 1980 to 2015 that reported up to 21% and 40% yield reductions in wheat and maize, respectively, owing to drought worldwide. With changing climatic conditions and limited water supply, it is necessary to develop crop plants that can sustain drought conditions without reduced yield. Moreover, much lands are left barren due to poor water supply. Generating plants that can withstand drought stress will improve the food security for the growing population. Understanding of the physiological and biochemical basis of drought response and the gene regulatory networks relating to drought tolerance in plants is necessary. Remarkable studies have been carried out that identify the key regulators of drought response at different stages. These can be classified as: (i) drought induced transcriptional factors such as dehydration-responsive-element-bindings (DREBs), abscisic acid responsive element binding proteins (AREBs)/abscisic acid responsive element binding factors (ABFs), nuclear factor Y-B subunits (NF-YB), and tryptophan–arginine–lysine–tyrosine (WRKY) (Oh et al. 2005; Nelson et al. 2007; Xiao et al. 2009; Wu et al. 2009; Banerjee and Roychoudhury 2015); (ii) posttranscriptional and/or posttranslational modifications (Wang et al. 2008; Xiang et al. 2007; Kim et al. 2017); and (iii) production of osmoprotectant and molecular chaperones (Xiao et al. 2009; Bhaskara et al. 2015; Liu et al. 2015). Overexpressing or downregulating drought-responsive genes has yielded success in the laboratory. However, field studies demonstrating drought tolerance in plants are required to confirm the results. Drought stress induces the synthesis or transportation of the phytohormone abscisic acid (ABA), which is a key molecule regulating signal events during drought impact (Fang and Xiong 2015). The initial perception of accumulation of ABA is through a complex of PYR (pyrabactin resistance)/PYL (PYR1-like)/RCARb (regulatory component of abscisic acid response), PP2C (protein phosphatase 2C), and SnRK2 (sucrose nonfermenting1-related protein kinase 2), which induces the expression of transcription factors NF-YA, SNAC (stress and abscisic acid-Inducible NAC), and AREBs (Roychoudhury and Paul 2012). These proteins further regulate the opening and closing of stomata to reduce transpirational water loss. Drought stress is also perceived by another regulatory loop through calcium-dependent protein kinase (CDPK) and calcineurin B-like protein-interacting protein kinase (CIPK), which activates AREB and DREBs that bind to the dehydration responsive element and abscisic acid responsive element cis-elements of downstream genes to produce the effector proteins such as late embryogenesis abundant protein (LEA), heat-shock protein (HSP), proline, glycine betaine, sugars, and polyamines (Yang et al. 2010). The overexpression of these transcription factors in drought-sensitive plants has improved tolerance of water-deficit conditions (Table 1.1). Moreover, some plants constitutively expressing drought-responsive transcription factors displayed growth retardation (Suo et al. 2012). To lessen this undesirable effect, researchers have employed stress-inducible promoters such as HVA22P to drive the expression of these transgenes in transgenic plants (Bhatnagar-Mathur et al. 2007; Xiao et al. 2009). However, when the drought stress is extended, it induces continuous expression of these genes in the transgenic plants, resulting in growth anomalies. To circumvent this problem, researchers have used stress-inducible tissue-specific promoters such as Responsive To Dehydration 29A (RD29A) for expressing these transgenes (Ito et al. 2006; Kasuga 2004). RD29A promoter is expressed only in the root tissues of rice plants under abiotic stress conditions. However, a small problem in root development could circumvent its use.

Table 1.1 List of genes used to generate drought-tolerant transgenic plants. Target gene

Source of gene

Target plant

Evaluation

Functional change

References

AtABF3

Arabidopsis thaliana

Oryza sativa cv. Nakdong

Greenhouse

No visible growth abnormality, increased drought tolerance

Oh et al. 2005

SNAC1

Rice IRAT109

Rice (japonica)

Greenhouse, field

No growth anomaly, drought tolerance

Hu et al. 2006

OsNAC6

Rice cv. Nipponbare

Rice cv. Nipponbare

Greenhouse

Growth retardation, poor reproductive yields, increased tolerance to dehydration and enhanced resistance to blast disease

Nakashima et al. 2007

DREB1A

Arabidopsis thaliana

Triticum aestivum

Greenhouse

Delayed drought symptoms

Pellegrineschi et al. 2004

Arabidopsis thaliana

Arachis hypogaea L. cv. JL 24

Greenhouse

40% higher transpiration efficiency than the untransformed controls

Bhatnagar-Mathur et al. 2007

OsDREB1G

Oryza sativa L. ssp. japonica cv. Zhonghua 11

Oryza sativa L. ssp. japonica cv. Zhonghua 11

Greenhouse

Improved tolerance to drought stress

Chen et al. 2008

OsDREB2B

Oryza sativa L. ssp. japonica cv. Zhonghua 11

Oryza sativa L. ssp. japonica cv. Zhonghua 11

Greenhouse

Improved tolerance to water deficit stress

Chen et al. 2008

OsDREB1F

Oryza sativa

Oryza sativa and Arabidopsis

Greenhouse

Enhanced tolerance to salt, drought, and low temperature

Wang et al. 2008

GhDREB

Gossypium hirsutum

Triticum aestivum L.

Greenhouse

Improved tolerance to drought, salt, and freezing stresses, increased accumulation of soluble sugar and chlorophyll in leaves under stress conditions

Gao et al. 2009

HhDREB2

Halimodendronhalodendron

Arabidopsis

Greenhouse

Increased tolerance to salt and drought stresses

Ma et al. 2015

GmDREB2

Glycine max L.

Arabidopsis and tobacco

Greenhouse

Enhanced tolerance to drought and high-salt stresses, high proline levels

AtDREB2A-CA

Arabidopsis thaliana

Gossypium hirsutum L.

Greenhouse

Improved shoot development, improved morphometrics roots traits under water deficit

Lisei-de-Sá et al. 2017

(continued)

Table 1.1 (Continued) Target gene

Source of gene

Target plant

Evaluation

Functional change

References

HARDY

Arabidopsis

O. sativa ssp. Japonica cv. Nipponbare

Greenhouse

Increased leaf biomass and bundle sheath cells, enhanced photosynthesis assimilation

Karaba et al. 2007

Arabidopsis

Trifolium alexandrinum L.

Greenhouse, field

Thicker stems and more xylem rows per vascular bundle, resistant to lodging in the field, drought tolerance

Abogadallah et al. 2011

ZFP252

Oryza sativa L. cv. Zhonghua 11

Oryza sativa L. cv. Zhonghua 11

Greenhouse

Increased amount of free proline and soluble sugars, high-level expression of stress defense genes and enhanced rice tolerance to salt and drought stresses

Xu et al. 2008

ZFP182

Oryza sativa L. subs. Japonica cv. Zhonghua 11

Oryza sativa L. subs. Japonica cv. Zhonghua 11

Greenhouse

Increased accumulation of free proline and soluble sugars

Huang et al. 2012

DST

Oryza sativa L. cv. Zhonghua 11

Oryza sativa L. cv. Zhonghua 11

Greenhouse

Enhanced drought and salt tolerance in rice

Huang et al. 2009

ZAT10

Arabidopsis thaliana

Oryza sativa L. ssp. Japonica

Greenhouse, field

High spikelet fertility and high yield under drought stress

Xiao et al. 2009

NHX1

Arabidopsis thaliana

Oryza sativa L. ssp. Japonica

Greenhouse, field

High spikelet fertility and high yield under drought stress

Xiao et al. 2009

LOS5

Arabidopsis thaliana

Oryza sativa L. ssp. Japonica

Greenhouse, field

High spikelet fertility and high yield under drought stress

Xiao et al. 2009

Arabidopsis thaliana

Nicotiana tabacum

Greenhouse

Higher water content, better cellular membrane integrity, accumulated higher quantities of ABA and proline, and higher levels of antioxidant enzymes

Yue et al. 2011

Arabidopsis thaliana

Maize

Greenhouse

Reductions in stomatal aperture, higher relative water content and leaf water potential, lower leaf wilting, less electrolyte leakage, less malondialdehyde and H2 O2 content, and higher levels of antioxidative enzymes and proline content

Lu et al. 2013

NPK1

Arabidopsis thaliana

Oryza sativa L. ssp. Japonica

Greenhouse, field

High spikelet fertility and high yield under drought stress

Xiao et al. 2009

LeNCED1

Tomato

Petunia

Greenhouse

Elevated leaf ABA concentrations, increased concentrations of proline, and increase in drought resistance.

Estrada-Melo et al. 2015

AtNF-YB1

Arabidopsis thaliana

Arabidopsis thaliana

Greenhouse

Higher water potential and photosynthesis rate

Nelson et al. 2007

ZmNF-YB2

Zea mays

Maize

Greenhouse, field

Increased chlorophyll content, stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis under stress conditions

Nelson et al. 2007

TaNF-YB3

Triticum aestivum

Tobacco cv. Wisconsin 35

Greenhouse

Improved growth under drought, enhanced leaf water retention capacity, and increased antioxidant enzyme activities and osmolyte accumulation.

Yang et al. 2017

GmNFYB1

Glycine max

Arabidopsis

Greenhouse

Higher seed germination rate, longer root lengths, increased proline accumulation in leaves and decreased water loss under drought and salt stress conditions

Li et al. 2016

Cdt-NF-YC1

Bermuda grass (Cynodon dactylon 9 Cynodon transvaalensis)

Oryza sativa L. ssp. japonica cv. Zhonghua 11

Greenhouse

Increased tolerance to drought and salt stress and increased sensitivity to ABA

Chen et al. 2015a,b

OsWRKY11

Oryza sativa L.

Oryza sativa cv. Sasanishiki

Greenhouse

Slower leaf wilting and less impaired survival rate

Wu et al. 2009

PdNF-YB7

Populus nigra × (Populus deltoides × Populus nigra)

Arabidopsis

Greenhouse

Increased seed germination rate and root length and decrease in water loss, and displayed higher photosynthetic rate

Han et al. 2013

(continued)

Table 1.1 (Continued) Target gene

Source of gene

Target plant

Evaluation

Functional change

References

DnWRKY11

Dendrobium nobile

Nicotiana tabacum cv. Huangmiaoyu

Greenhouse

Higher germination rate, longer root length, higher fresh weight, higher activities of antioxidant enzymes, and lower content of malonidialdehyde

Xu et al. 2014

FcWRKY70

Fortunella crassifolia

Nicotiana nudicaulis and Citrus lemon

Greenhouse

Higher expression levels of arginine decarboxylase and accumulated larger amount of putrescine

Gong et al. 2015

TaWRKY33

T. aestivum cv. Xiaobaimai

Arabidopsis

Greenhouse

Increased germination rates, promoted root growth and reduced water loss

He et al. 2016

FtbHLH3

Fagopyrum tataricum

Arabidopsis

Greenhouse

Lower malondialdehyde, ion leakage, and reactive oxygen species, higher proline content, activities of antioxidant enzymes, and increased photosynthetic efficiency

Yao et al. 2017

Musa DHN-1

Musa spp.

Musa spp.

Greenhouse

Improved tolerance to drought and salt-stress, increased accumulation of proline and reduced malondialdehyde levels

Shekhawat et al. 2011

AnnSp2

Solanum pennellii

Solanum lycopersicum

Greenhouse

Induced stomatal closure and reduced water loss, improved scavenging of ROS, higher total chlorophyll content, lower lipid peroxidation levels, increased peroxidase activities and higher levels of proline

Ijaz et al. 2017

SbPIP1

Salicornia bigelovii

Nicotiana tabacum

Greenhouse

Higher relative water content and proline content, but lower levels of malondialdehyde and less ion leakage

Sun et al. 2017a,b

DRIR

Arabidopsis thaliana

Arabidopsis thaliana

Greenhouse

Increased tolerance to drought and salt stress

Qin et al. 2017

Sly-miR169c

Solanum lycopersicum

Solanum lycopersicum

Greenhouse

Reduced stomatal opening and transpiration rate, lowered leaf water loss, and enhanced drought tolerance

Zhang et al. 2011

miR408

Arabidopsis thaliana

Chickpea

Greenhouse

Stunted growth, regulation of DREB genes

Hajyzadeh et al. 2015

1.2 Drought Tolerance

To address this problem, Kudo et al. (2016) stacked two transcription factors in transgenic Arabidopsis plants, namely DREB1A to improve drought tolerance and the rice Phytochrome-Interacting Factor-Like 1 (OsPIL1) to partially enhance plant growth. OsPIL1 augments cell elongation by regulating cell wall-related gene expression, thereby circumventing the negative effects of overexpression of DREB1A gene. All of these individual strategies can be grouped together, wherein the gene-stacking strategy can be employed along with the use of stress-inducible tissue-specific promoters to impart drought tolerance and at the same time remove the growth-retardation effects. The strategy will be more effective and acceptable if the genes and promoters are chosen from a plant and overexpressed in the same plant. Post-translational modification such as phosphorylation, farnesylation, sumoylation, and poly(ADP-ribosyl)ation (PAR) of drought-responsive proteins in the above regulatory network regulates drought stress tolerance (Wang et al. 2009; Xiang et al. 2007; Kim et al. 2017). Conditional and specific downregulation of farnesyl transferase gene in canola using the AtHPR1 promoter resulted in yield protection against drought stress under field conditions (Wang et al. 2009). Overexpression of protein kinases such as CDPKs and CIPKs in transgenic plants improved tolerance to drought stress (Saijo et al. 2000; Xiang et al. 2007; Vivek et al. 2013; Campo et al. 2014; Wei et al. 2014; Tai et al. 2016; Wang et al. 2018). LEAs are hydrophilic proteins that usually accumulate in embryos during seed desiccation and are known to be involved in adaptive responses to dehydration by binding water molecules, stabilizing proteins or membrane structures and acting as molecular chaperones like HSPs (Bray 1997). The expression of LEA genes in transgenic plants displays increased ABA sensitivity and enhances osmotic tolerance (Duan and Cai 2012; Wang et al. 2014; Yu et al. 2016; Banerjee and Roychoudhury 2016). The accumulation of proline, glycine betaine, sugars like trehalose and polyamines like spermine, spermidine, and putrescine prevents water loss and protects the cellular components from osmotic damage (Zhu et al. 1998; Quan et al. 2004; Lv et al. 2007; Xiao et al. 2009; Bhaskara et al. 2015; Liu et al. 2015; Mwenye et al. 2016; Montilla-Bascón et al. 2017; Liu et al. 2017; Juzo´n et al. 2017). The overexpression of plant or bacterial cold shock proteins in major staple crops such as maize, rice, and wheat has conferred drought tolerance and increased yield under field conditions (Castiglioni et al. 2008; Yu et al. 2017). During stress conditions, modulation of cellular energy homeostasis is the key to improving plant performance and yield stability. Poly(ADP-ribosyl)ation is a unique posttranslational protein modification (mediated by the PARP enzyme) induced in plants during environmental stress conditions. PAR is known to be involved in DNA synthesis and repair, transcription, and cell cycle activities (d’Amours et al. 1999). Inhibition of PARP activity alters photosynthesis and improves stress tolerance in plants (Vanderauwera et al. 2007; Schulz et al. 2012). MicroRNAs and long noncoding RNAs have also been acknowledged in response to drought stress (Ferdous et al. 2015; Qin et al. 2017). Several miRNAs are upor downregulated during drought stress (Ferdous et al. 2015; Shriram et al. 2016; Banerjee et al. 2016). These miRNAs can be targeted to generate transgenic plants using particular promoters. Overexpression of a rice Osa-miR319a in transgenic creeping bentgrass (Agrostis stolonifera) displayed enhanced drought and salt tolerance. These plants showed increased leaf wax content and water retention but reduced sodium uptake (Zhou et al. 2013). Expression of miRNA408 in chickpea and miR169 in tomato

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showed enhanced drought tolerance in these plants (Zhang et al. 2011; Hajyzadeh et al. 2015). Advanced sequencing technologies are now available for deriving the genomic and transcriptomic information during drought tolerance which in turn could reveal the regulatory networks activated during drought (Huang et al. 2014; Chung et al. 2016; Muthusamy et al. 2016; Li et al. 2017a,b,c; Bai et al. 2017). Engineering the components of these regulatory networks will further help in developing better drought-tolerant crops. Since many of the studies carried out until now have been restricted to greenhouse or experimental fields, the tolerant plants should be tested for their full capacity under natural field conditions where other environmental factors accompany drought scenario. It is difficult to measure the performance of transgenic plants under drought conditions as drought is variable from mild to extreme and in duration. A drought-tolerant transgenic maize line, DroughtGardTM , developed by Monsanto, harbors the bacterial cspB gene which was commercialized in 2011 (Castiglioni et al. 2008). The credibility of the gene could not be fully validated during this study because of varying drought conditions along with variable ambient temperatures and soil conditions.The transgenic line displayed tolerance to moderate drought but could sustain extreme drought conditions (Gurian-Sherman 2012). Bayer Crop Science also adopted the Performance Plants Inc. Yield Protection Technology for the development of drought-tolerant and high-yielding cotton in 2009. After repeated field trials for three consecutive years under natural field conditions, these cotton plants showed significant yield advantages under water stress and no undesirable effects on growth under optimal conditions (Performance Plants 2019).

1.3 Cold Tolerance Cold stress is another critical abiotic stress agent that limits crop cultivation and restricts growth and development, hampering crop productivity. Cold tolerance is achieved through acclimation of the crop plants to lower temperatures (chilling temperatures, 0–15 ∘ C) or even below-freezing temperatures (80% of the nonenzymatic lipid peroxidation (Triantaphylides et al. 2008). The singlet oxygen production favored in Arabidopsis mutants caused photooxidative stress which led to a dramatic rise in lipid peroxidation, causing cell death (Triantaphylides et al. 2008). The 𝛽-carotene, tocopherol, or plastoquinone quenched 1 O2 efficiently, and if not, 1 O2 activated the upregulation of genes involved in the molecular defense responses against photooxidative stress (Krieger-Liszkay et al. 2008). The effect of 1 O2 (produced by Rose Bengal, a photosensitizer) on adenosine triphosphate (ATP) hydrolysis and ATP-driven proton translocation activity of CF1-CFo was investigated. It was found that 1 minute of exposure dramatically reduced both activities. It was also shown that oxidized thylakoid ATP synthase was more vulnerable to 1 O2 than CF1-CFo in its reduced state (Buchert and Forreiter 2010).

4.2 Reactive Oxygen Species

4.2.1.3

Hydrogen Peroxide (H2 O2 )

Univalent reduction of O2 produces H2 O2 . H2 O2 has relatively long half-life (1 ms) and it is fairly reactive, whereas other ROS such as O2 ⋅− , OH⋅ , and 1 O2 , have much shorter half-lives (2–4 ms) (Bhattacharjee 2005). Various studies have proved that excess H2 O2 in plant cells leads to oxidative stress. H2 O2 oxidizes thiol groups of enzymes, which may inactivate their activity. High H2 O2 levels were observed in the middle portion of trichomes in Cu-deficient leaves of Morus alba cv. Kanva 2 in comparison with the plants grown under Cu excess (Tewari et al. 2006). Various antioxidant enzymes activities such as SOD, CAT, APX, and glutathione reductase (GR) increased in both Cu-deficient and Cu-excess plants. Oxidative stress conditions are aggravated by excessive ROS production. In the young leaves of mulberry plants, excessive ROS production disturbed the redox couple in Cu-deficient conditions, whereas Cu excess damaged the roots, accelerated the rate of senescence in the older leaves, induced antioxidant responses and disturbed the cellular redox environment (Tewari et al. 2006). Cu/Zn SOD downregulation resulted in the overproduction of H2 O2 and O2 ⋅− in Pisum sativum owing to Cd, because Cd induces a decline in Ca which leads to Cu/Zn SOD downregulation (Rodriguez-Serrano et al. 2009). The photoactive nature of chloroplast makes it an important source of ROS production. H2 O2 at low concentrations acts as a signal molecule that is involved in acclimatory signaling, which is responsible for triggering tolerance to different biotic and abiotic stresses. At high concentrations, it causes PCD (Quan et al. 2008). In most physiological processes, like senescence (Peng et al. 2005), stomatal movement (Bright et al. 2006), photorespiration and photosynthesis (Noctor and Foyer 1998a,b), cell cycle (Mittler et al. 2004) and growth and development (Foreman et al. 2003), H2 O2 acts as a major supervising agent. H2 O2 has been successfully accepted as a second messenger for signals generated by means of ROS owing its long life span and elevated permeability across membranes (Quan et al. 2008). In one study, it was reported that H2 O2 and sodium nitroprusside (SNP) increased the activities of leaf SOD, CAT, APX, and GR along with the induction of related isoform(s) under non-NaCl stress conditions (Tanoua et al. 2009). Reduction in the ASH redox state owing to salinity stress was partially prevented by H2 O2 and SNP pre-treatments. On the other hand, the GSH redox state was enhanced by SNP under normal and NaCl stress. Pre-treatments with H2 O2 and SNP totally reversed the NaCl-dependent protein oxidation (Tanoua et al. 2009). 4.2.1.4

Hydroxyl Radicals (OH⋅ )

Among ROS, OH⋅ is highly reactive. In the presence of neutral pH and optimum temperatures, O2 ⋅− and H2 O2 can produce OH⋅ radicals through an iron-catalyzed superoxide-driven Fenton reaction. For this reaction, suitable transitional metals are required and Fe is the most favorable one. In vivo, OH⋅ radicals are thought to be mainly responsible for mediating oxygen toxicity. OH⋅ can potentially react with biological molecules such as DNA, proteins, lipids, and all constituents of cells. Excess production of OH⋅ in the absence of any enzymatic mechanism for the elimination of this highly reactive ROS ultimately leads to cell death (Vranova et al. 2002). 4.2.2

Sites of ROS Generation

Under both unstressed and stressed conditions, ROS are produced in cells at different locations like chloroplasts, mitochondria, plasma membranes, peroxisomes,

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endoplasmic reticulum, cell walls and apoplast. In various metabolic pathways localized in different cellular compartments, ROS are formed as a byproduct by the unavoidable leakage of electrons through O2 from the electron transport activities. 4.2.2.1

Chloroplasts

The chloroplast is an organelle with a well-developed thylakoid membrane and light-harvesting complexes. The chloroplast has various sites for ROS (O2 ⋅− , 1 O2 , and H2 O2 ) generation, but PSI and PSII of ETC are the main contributors. During normal conditions, excited photosystems generate electron flow to NADP+ , eventually reducing it to NADPH, which ultimately reduces the electron acceptor, CO2 , in the Calvin– Benson cycle. During stressed conditions, electrons are transported from ferredoxin to O2 , and this altered flow of electrons leads to O2 ⋅− formation via the Mehler reaction (Elstner 1991). Further, Takahashi et al. (1988) have established the roles of quinone A (QA) and quinone B (QB) in O2 ⋅− production. 1 O2 is a regular byproduct at PSII under low light intensity (Buchert and Forreiter 2010). The dismutation of O2 ⋅− by Cu/Zn-SOD results in the formation of H2 O2 at the stromal membrane (Takahashi et al. 1988). The availability of Fe2+ at Fe–S centers plays a major role in formation of OH⋅ with the help of H2 O2 in the Fenton reaction. 4.2.2.2

Peroxisomes

Peroxisomes are small, sphere-shaped organelles surrounded by a single lipid bilayer. They are concerned with the oxidation of long-chain fatty acids, and are the major sites for ROS generation in plants. The peroxisome matrix and membrane are the two main sites of O2 ⋅− production (Del Rio et al. 2002). Uric acid formation from xanthine and hypoxanthine owing to the activity of the enzyme xanthine oxidase produces O2 ⋅− in the peroxisome matrix (Corpas et al. 2001). Cytochrome b and nicotinamide adenine dinucleotide along with monodehydroascorbate reductase (MDHAR) contributes toward the generation of O2 ⋅− on the peroxisome membrane (Del Rio et al. 2002). Imbalance of O2 ⋅− , glycolate oxidation, the 𝛽-oxidation of fatty acids and reactions involving flavin oxidases result in H2 O2 generation in peroxisomes (Del Rio et al. 2002, 2006). 4.2.2.3

Mitochondria

Mitochondria are the “powerhouses” of cells and also major sites of ROS production. ETC is the main contributor toward ROS production. Various stresses modify the electron carriers in the ETC leading to increased ROS formation (Noctor et al. 2007; Blokhina and Fagerstedt 2010). Complexes I and III are recognized as the chief site for O2 ⋅− production in mitochondrial ETC, and SOD reduces it to H2 O2 (Sweetlove and Foyer 2004; Quan et al. 2008). About 1–5% of O2 consumed by mitochondria is related to H2 O2 generation, which eventually causes the formation of highly toxic OH⋅ through reaction with Fe2+ and Cu+ (Moller 2001); OH⋅ radicals migrate from mitochondria by membrane penetration (Sweetlove and Foyer 2004; Rhoads et al. 2006). Mitochondria play a major role in controlling ROS generation by energy dissipating systems (Gill and Tuteja 2010). There are also various other known sites of ROS formation in plants, like endoplasmic reticulum, plasma membrane, cell wall, and apoplast (see Figure 4.3)

4.2 Reactive Oxygen Species

CHLOROPLAST

MITOCHONDRIA • Complex I: NADH dehydrogenase segment

• PSII: electron transport chain • •

Fd, 2Fe-2S, and 4Fe-4S clusters PSI: electron transport chain QA and QB Chlorophyll pigments

• Complex II: reverse electron flow to complex I

• Complex III: ubiquinonecytochrome region

• Enzymes • Aconitase, 1-galactono-γ lactone dehydrogenase (GAL)

CELL-WALL

PLASMA MEMBRANE

• Cell-wall-associated peroxidase

• Electron transporting

diamine oxidases

oxidoreductases

• NADPH oxidase, quinone oxidase

ROS ENDOPLASMIC RETICULUM

PEROXISOME • Matrix: xanthine oxidase (XOD) • Membrane: electron transport

• NAD(P)H-dependent electron transport involving Cyt P450

APOPLAST

•

• Cell-wall-associated oxalate oxidase Amine oxidases

chain flavoprotein NADH and Cyt b Metabolic processes: glycolate oxidase, fatty acid oxidation, flavin oxidases, .– disproportionation of O2 radicals

Figure 4.3 ROS generation sites in plants.

4.2.3

ROS and Oxidative Damage to Biomolecules

In order to avoid oxidative stress, elimination of ROS is very important. A cell is said to be in a condition of “oxidative stress” when the level of ROS overcomes the defense mechanisms. Nevertheless, the balance between the formation and removal of ROS is disturbed under a number of stressful conditions like salinity, drought, high light levels, metal toxicity, and pathogens. The ROS increment can cause damage to biomolecules such as lipids, proteins, and DNA, consequently changing the intrinsic membrane properties such as fluidity, ion transport, loss of enzyme activity, protein crosslinking, inhibition of protein synthesis, and DNA damage, ultimately resulting in cell death (Anjum et al. 2015) (see Figure 4.4). Membrane lipid peroxidation and oxidation of -SH groups of proteins have been regarded as an oxidative stress indicator (Boominathan and Doran 2002). Under the influence of ROS or oxidative stress, several byproducts cause protein oxidation,

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LIPID • Chain breakage • Increase in membrane fluidity and permeability

ROS

PROTEIN at high concentration

72

Oxidative Damage

• Site-specific amino acid modification • Fragmentation of the peptide chain • Aggregation of crosslinked reaction products • Altered electric charge • Enzyme inactivation • Increased susceptibility of proteins to proteolysis

DNA • • • • •

Deoxyribose oxidation Strand breakage Removal of nucleotides Modification of bases DNA-protein crosslinks

Figure 4.4 Impact of ROS on lipids, proteins, and DNA under oxidative damage.

which is described as covalent modification of a protein. Generally protein oxidations are essentially irreversible. Various stresses lead to protein carbonylation in tissues. Carbonylation of protein is generally used as a marker of protein oxidation (Moller et al. 2007; Basu et al. 2010). It has been found that reactive metabolites such as OH− , O2 ⋅− , and NO− cause DNA damage described as “spontaneous DNA damage.” Increased ROS concentration can cause damage to cell structures, nucleic acids, lipids, and proteins (Tuteja et al. 2009). Lipid peroxidation is the most damaging process known to take place in living organisms. Membrane damage is sometimes taken as the sole parameter to establish the intensity of lipid destruction under various stresses. Polyunsaturated molecules act as precursors for hydrocarbon products such as ketones, malondialdehyde (MDA), and related compounds formed during lipid peroxidation (LP) (Garg and Manchanda 2009). LP, in both cellular and organelle membranes, takes place when threshold ROS levels are reached, thereby not only directly affecting normal cellular functioning, but also aggravating the oxidative stress through generation of lipid-derived radicals (Roychoudhury et al. 2008; Roychoudhury et al. 2012). There are reports that allelopathic interactions and allelochemicals cause increased membrane leakage and enhanced MDA content in target plant tissues similar to that from oxidative stress (Amist et al. 2015; Bano et al. 2017). Aqueous extract of Callicarpa acuminata and Sicyos deppei increased the generation of free radicals and LP, which resulted in oxidative membrane damage in root tissues (Cruz-Ortega et al. 2002). Sunflower leaf extracts in the presence of allelochemicals showed membrane damage and increased MDA and H2 O2 production (Bogatek et al. 2005). Scrivanti et al. (2003) reported reduced root growth of Zea mays and severe LP in response to volatile oils from Tagetes minuta and Schinus areira. Enhanced LP has been recorded in plants under increasing degree of water stress (Lin and Kao 2000). The deleterious effects of ROS can be established in proteins and nucleic acids, chlorophyll and membrane function.

4.2 Reactive Oxygen Species

Generation of ROS-induced oxidative stress results in cellular injury and plant growth inhibition as a result of the actions of allelochemicals (Yu et al. 2003). Allelopathic stress is considered to be one of the most potent causes of oxidative burst and causes oxidative damage in a variety of crop plants, viz. Phaseolus mungo (Singh et al. 2010b), mungbean (Batish et al. 2006), tomato (Zhang et al. 2010), cucumber (Politycka et al. 2004; Ding et al. 2007), and maize (Singh et al. 2010a). Water stress has the potential to cause oxidative burst and oxidative damage in a variety of crop plants, viz. maize (Jiang and Zhang 2002), sunflower (Bailly et al. 2004), and Triticum aestivum L. (Simova-Stoilova et al. 2009). 4.2.4

Role of ROS as Messengers

ROS act as second messengers which participate in intracellular signaling cascades that mediate several responses in plant cells, such as the closing of stomata (Neill et al. 2002; Yan et al. 2007), PCD (Mittler 2002; Bethke and Jones 2001), gravitropism (Joo et al. 2001), and achievement of tolerance toward different stresses (Torres et al. 2002; Miller et al. 2008). ROS signals a suitable cellular response with the help of some redoxsensitive proteins, calcium mobilization, protein phosphorylation, and gene expression in plants. ROS is also sensed directly by key signaling proteins like tyrosine phosphatase through oxidation of conserved cysteine residues (Xiong et al. 2002). Signaling components such as protein phosphatases, protein kinases, and transcription factor activities are modulated by ROS (Cheng and Song 2006). ROS also correspond with other signal molecules and the pathway forming part of the signaling network that controls the response downstream of ROS (Neill et al. 2002). ROS strength, lifetime, and size of the signaling pool depend on balance between oxidant synthesis and elimination by the antioxidant. Miller et al. (2008) identified a signaling pathway that is activated in cells in response to ROS accumulation using mutants deficient in key ROS-scavenging enzymes. Varied zinc finger proteins and WRKY transcription factors also function as central regulators of abiotic stress responses involved in temperature, salinity, and osmotic stresses. ROS are considered secondary messengers in the abscisic acid (ABA) transduction pathway in guard cells (Neill et al. 2002; Yan et al. 2007). H2 O2 induced by ABA is a vital signal molecule in mediating stomatal closure to decrease water loss through the activation of calcium-permeable channels in the plasma membrane (Pel et al. 2000). Elevated cytosolic H2 O2 participates in ABA-induced stomatal closure, while constitutive increment of H2 O2 is not responsible for stomatal closure (Jannat et al. 2011). In root gravitropism, ROS function as second messengers. Joo et al. (2001) predicted that gravity induces asymmetric movement of auxin within 60 minutes, and then the auxin stimulates ROS generation to mediate gravitropism. ROS scavenging by antioxidants (N-acetylcysteine, AA, and Trolox) inhibited root gravitropism (Joo et al. 2001). ROS participated in dormancy alleviation. Gibberellic acid signaling and ROS content are low in dormant barley grains under control conditions, while ABA signaling is high, resulting in dormancy. Exogenous application of H2 O2 does not change ABA biosynthesis and signaling, but has a more evident effect on gibberellic acid signaling, altering hormonal balance, which results in germination (Bahin et al. 2011). To mediate biotic and abiotic stress responses based on Table 4.1 ROS synthesis, scavenging and signaling, plants have evolved a complex regulatory network. Early events of plant–pathogen interactions showed transient production of ROS that plays a key

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Table 4.1 Role of ROS as secondary messengers in several plant hormone responses in intracellular signaling cascades.

Reactive oxygen species

Stress hormones

Hormone-induced responses in plants

Various reactive oxygen species such as 1 O2 , H2 O2 , O2 ⋅− , and OH⋅ , etc., send message through signal to hormones

Abscisic acid Auxin

Closing of stomata Root gravitropism

Gibberellic acid

Seed germination and programmed cell death

Jasmonic acid

Lignin biosynthesis

Salicylic acid

Osmotic stress and hypersensitivity responses

signaling role in pathogenesis signal transduction regulators. In drought-induced ABA synthesis in plants, ROS play significant roles, suggesting that they may be the signals through which the plant can “sense” the drought condition (Zhao et al. 2001). Metals Cd2+ and Cu2+ induce mitogen-activated protein (MAP) kinase activation via different ROS-generating systems, using pharmacological inhibitors (Yeh et al. 2007).

4.3 Antioxidative Defense System in Plants Halliwell and Gutteridge (1995) defined antioxidants as “any substance that delays, prevents or removes oxidative damage to a target molecule.” Khlebnikov et al. (2007) defined antioxidants as “any substance that directly scavenges ROS or indirectly acts to up-regulate antioxidant defenses or inhibit ROS production.” Plants have multifaceted antioxidative defense system comprising nonenzymatic and enzymatic components to scavenge ROS. Chloroplasts, mitochondria, and peroxisomes are organelles that have systems involved in ROS production and scavenging. ROS scavenging pathways from all cellular organelles are synchronized. Under normal conditions, toxic oxygen metabolites are synthesized in small amounts and there is a suitable balance between the production and quenching of ROS. The equilibrium between the production and quenching of ROS is disturbed by a diverse range of adverse environmental factors, causing quick increases in intracellular ROS levels. Various components of antioxidative defense systems involved in ROS scavenging are categorized under two parts, nonenzymatic and enzymatic defense system. 4.3.1

Nonenzymatic Components of the Antioxidative Defense System

A nonenzymatic component of the antioxidative defense system comprises ascorbate (AsA) and glutathione (𝛾-glutamyl-cysteinyl-glycine, GSH), which function as cellular redox buffers along with tocopherol, carotenoids, and phenolic compounds. 4.3.1.1

Ascorbate

AsA is a low-molecular-weight antioxidant which is found in abundance and plays a chief role in resistance against oxidative stress caused by elevated level of ROS. It is

4.3 Antioxidative Defense System in Plants

considered as a powerful antioxidant owing to its ability to contribute electrons in various enzymatic and nonenzymatic reactions. AsA participates in physiological processes of plants such as growth, differentiation, and metabolism. The AsA pool in plants is generated through the Smirnoff–Wheeler pathway involving d-mannose/l-galactose (Wheeler et al. 1998). It is also synthesized by means of uronic acid intermediates, viz., d-galacturonic acid (Isherwood et al. 1954). AsA is assumed to symbolize the first line of protection against damaging external oxidants. AsA protects essential macromolecules from oxidative damage. AsA exists in a reduced state in chloroplast in normal conditions, functioning as a cofactor of violaxanthin de-epoxidase, contributing toward the dissipation of excess excitation energy (Smirnoff 2000). AsA plays an important role in elimination of H2 O2 via the ascorbate–glutathione (AsA–GSH) cycle (Pinto et al. 2003). In the AsA–GSH cycle, APX reduces H2 O2 to water with simultaneous generation of monodehydroascorbate (MDHA) by utilizing two molecules of AsA. The MDHA radical has a short life time and it impulsively dismutates into dehydroascorbate (DHA) and AsA or it is directly reduced to AsA by the NADP(H)-dependent enzyme MDHAR (Miyake and Asada 1994). Various stresses alter the AsA level in plants (Sharma and Dubey 2005; Maheshwari and Dubey 2009; Radyuk et al. 2009). The level of AsA depends on the balance between AsA biosynthesis and turnover, which is controlled by the antioxidant requirement and influenced by environmental stresses (Chaves et al. 2002). Abiotic stress tolerance in plants is conferred by overexpression of enzymes concerned with biosynthesis of AsA. 4.3.1.2

Glutathione

Tripeptide glutathione (GSH) is a low-molecular-weight nonprotein thiol that contributes significantly to intracellular defense against ROS-induced oxidative damage. It has been reported from all cell compartments such as cytosol, chloroplasts, endoplasmic reticulum, vacuoles and mitochondria (Foyer and Noctor 2003). The cellular redox state is maintained by establishing a balance between glutathione and glutathione disulfide (GSSG). GSH with its reducing power plays a significant role in various biological processes, viz., cell growth/division, regulation of sulfate transport, signal transduction, conjugation of metabolites, enzymatic regulation, synthesis of proteins and nucleic acids, phytochelatin synthesis for metal chelation, detoxification of xenobiotics, and the expression of the stress-responsive genes. GSH has a capability of reacting chemically with O2 ⋅− , ⋅ OH, H2 O2 and thereby functions directly as a free radical scavenger. The macromolecules, viz. proteins, lipids, and DNA, are protected by GSH as it forms adducts with reactive electrophiles (glutathiolation) or donates proton in the presence of ROS or organic free radicals, leading to the formation of GSSG (Asada 1994). It also helps in the regeneration of ascorbate via the AsA–GSH cycle. A GSH pool is formed, either by de novo synthesis or through recycling by GR, using NADPH as a cofactor and electron donor. The GSH/GSSG ratio is altered in plants in response to various stresses (Tausz et al. 2004; Maheshwari and Dubey 2009; Radyuk et al. 2009; Sharma and Dubey 2007). 4.3.1.3

Tocopherols

Tocopherols (𝛼, 𝛽, 𝛾, and 𝛿) comprise a set of lipophilic antioxidants concerned with scavenging of oxygen free radicals, lipid peroxy radicals and 1 O2 . The antioxidant activity of the tocopherol depends upon the number of methyl groups attached to

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the main phenolic ring and upon methylation pattern. There are three methyl substituents in 𝛼-tocopherol, giving it the highest antioxidant activity among tocopherols (Kamal-Eldin and Appelqvist 1996). Only photosynthetic organisms have the capability of synthesizing tocopherols and they appear to be present only in the green parts of plants. Homogentisic acid and phytyl diphosphate act as precursors in the tocopherol biosynthetic pathway. Tocopherols physically quench and chemically react with O2 in chloroplasts, thus protect lipids and other membrane components, especially PSII (Ivanov and Khorobrykh 2003). A single molecule of 𝛼-tocopherol can neutralize a large amount of 1 O2 oxygen molecule before degradation. AsA and GSH help in restoring the oxidized tocopherol back to its reduced form (Fryer 1992). Ascorbate and glutathione pools have been affected by the tocopherol biosynthetic pathway. 𝛼-Tocopherol accumulation caused increased tolerance in various plant species in response to chilling, water deficit, and salinity (Yamaguchi-Shinozaki and Shinozaki 1994; Munné-Bosch et al. 1999; Bafeel and Ibrahim 2008). 4.3.1.4

Carotenoids

Carotenoids are accessory pigments belonging to the group of lipophilic antioxidants with the ability to detoxify various forms of ROS. Carotenoids are found in plants as well as microorganisms. In plants, carotenoids absorb light in the region between 400 and 550 nm of the visible spectrum and pass the captured energy to the chlorophyll. Carotenoids establish their antioxidant properties by scavenging 1 O2 to inhibit oxidative damage and quenching triplet sensitizer (3Chl*) and excited chlorophyll (Chl*) molecules to avoid the creation of 1 O2 and prevent singlet oxygen induction in tissue, thus protecting the photosynthetic apparatus. Carotenoids also participate in the signaling pathway as a precursor of signaling molecules, thus influencing plant development in response to biotic/abiotic stress. The numerous conjugated double bonds in the chain of isoprene residues of carotenoids contribute toward easy energy uptake from excited molecules and dissipation of excess energy as heat (Mittler 2002). 4.3.1.5

Phenolics

Phenolics are a group of secondary metabolites and are most abundant in the plant kingdom. The most common phenolics present in plants include flavonoids, phenolic acids, and tannins. Plant phenolics are chiefly derivatives of cinnamic acid and benzoic acid. According to Grace and Logan (2000), phenolics possess antioxidant properties which have the capability to scavenge and suppress ROS formation. Phenolics are free radical acceptors and reduce the oxidation of lipids and other molecules by contributing hydrogen molecules. Under unfavorable environmental conditions, flavonoids are concerned with neutralization of ROS before they prove damaging to plant cells (Lovdal et al. 2010). Polyphenols decrease membrane fluidity by altering the structure of lipids (Arora et al. 2000). Abiotic and biotic stresses cause an increase in induction of phenolics in plants (Michalak 2006; Choudhury et al. 2013; Choudhary and Agrawal 2014). 4.3.2

Enzymatic Components

The enzymatic components of the antioxidative defense system includes several antioxidant enzymes such as SOD, CAT, GPX, and enzymes of AsA–GSH cycle, i.e. APX, MDHAR, dehydroascorbate reductase (DHAR) and GR (Foyer and Noctor 2005). These

4.3 Antioxidative Defense System in Plants

enzymes operate in different subcellular compartments and respond in distress when cells are exposed to oxidative stress. 4.3.2.1

Superoxide Dismutases

SOD are ubiquitous, widely distributed multimeric metallo-proteins which catalyze the dismutation of O2 ⋅− into H2 O2 . Superoxide radical dismutation to H2 O2 by SOD also subsequently causes the formation of hydroxyl radicals by the Fenton reaction. SODs are classified on the basis of metal ions present in active sites, viz. copper and zinc (Cu/Zn SOD), manganese (MnSOD), and iron (FeSOD) containing SODs. They are differentially distributed within the plant cells, with the Cu/Zn SOD localized in the cytosol, chloroplasts, peroxisome, and mitochondria, Mn SOD in the matrix of mitochondria and peroxisomes, and Fe SOD localized in the chloroplasts of some higher plants and in prokaryotes (Scandalios 1993). The SOD constitute the first line of defense against ROS (Alscher et al. 2002) and are proposed to be important for plant stress tolerance (Apel and Hirt 2004). Eukaryotic Cu/Zn-SOD is present as a dimer and is cyanide sensitive, while the other two (Mn SOD and Fe SOD) may be dimers or tetramers and are cyanide insensitive (Scandalios 1993; Del Río et al. 1998). Overproduction of SOD has been reported to result from elevated oxidative stress tolerance in plants (Logan et al. 2006). 4.3.2.2

Catalases

CATs are tetrameric heme-containing enzymes that catalyze the dismutation of H2 O2 into H2 O and oxygen. They are present mainly in the peroxisomes. This enzyme is present in copious amounts in the glyoxysomes of lipid-storing tissues in germinating barley, where it decomposes H2 O2 formed during the 𝛽-oxidation of fatty acids (Fazeli et al. 2007). CAT is also present in the peroxisomes of the leaves of C3 plants, where it removes H2 O2 produced during photorespiration by the conversion of glycolate into glyoxylate (Jayakumar et al. 2008). There are three CAT genes present in angiosperms. CAT has been classified on the basis of the expression profile of the tobacco genes (Willekens et al. 1997). Class I CATs are synchronized by light and expressed in photosynthetic tissues. Class II CATs are present in high amounts in vascular tissues, whereas Class III CATs are exceedingly abundant in seeds and young seedlings. H2 O2 has been implicated in many stress conditions. The cells under stress rapidly generate H2 O2 through catabolic processes, which is degraded by CAT in an energy-efficient manner. An elevation or depletion of CAT activity in response to environmental stresses has been observed, but it depends on the intensity, duration, and type of the stress (Han et al. 2009; Sharma and Dubey 2005). In general, stresses that reduce the rate of protein turnover also reduce CAT activity. 4.3.2.3

Peroxidases

These are a ubiquitous group of oxido-reductases which are present in most plant tissues. They are heme-containing enzymes that use H2 O2 as the electron acceptor to catalyze a number of oxidative reactions. They exist in large numbers of isoenzyme forms and are implicated in a large number of processes (Passardi et al. 2005). APX belongs to the first peroxidase family while GPX belongs to the third peroxidase family. GPX is a heme-containing protein, which is a roughly 40–50 kDa monomer, with the capability of oxidizing certain substrates at the expense of H2 O2 . The excess

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peroxide produced by metabolic processes under both normal and stress conditions is removed by them. GPX participates in the biosynthesis of lignin and decomposition of indole-3-acetic acid and helps plants defend against biotic stresses by consuming H2 O2 released in cytosol, vacuoles, and extracellular spaces. GPX appears to be more valuable as its two forms, i.e. extra- and intra-cellular forms, participate in the breakdown of H2 O2 . GPXs are widely accepted as “stress enzymes.” Induction of GPX activity has been reported in common bean (Phaseolus vulgaris) nodules in response to salinity stress (Jebara et al. 2005). Many isoenzymes of GPX exist in plant tissues localized in vacuoles, the cell wall, and cytosol. GPX is involved in many important biosynthetic processes and is responsible for increased lignification of cell wall, degradation of indole-3-acetic acid, ethylene biosynthesis, and protection against abiotic and biotic stresses in plants (Kobayashi et al. 1996). GPX is an efficient quencher of reactive intermediary forms of O2 and peroxy radicals under stressed conditions (Vangronsveld and Clijsters 1994). Various stressful conditions of the environment have been shown to induce GPX activity. 4.3.2.4

Enzymes of the Ascorbate–Glutathione Cycle

The change in the ratio of AsA to DHA and GSH to GSSG is critical for the cell to detect oxidative stress and react accordingly. The AsA–GSH cycle, also known as the Halliwell–Asada pathway, is the recycling pathway of AsA and GSH restoration, which also detoxifies H2 O2 . The consecutive oxidation and reduction of AsA, GSH, and NADPH in the AsA–GSH cycle are catalyzed by the enzymes APX, MDHAR, DHAR, and GR, respectively. The AsA–GSH cycle functions in at least four different subcellular locations, viz. cytosol, chloroplast, mitochondria, and peroxisomes. The AsA–GSH cycle plays an important role in combating oxidative stress induced by environmental stresses (Sharma and Dubey 2005; Jiménez et al. 1997). APX is one of the most important antioxidant enzymes of plants. APX breaks down H2 O2 to form H2 O and MDHA, utilizing ascorbate as a hydrogen donor (Asada 1999). Isoforms of APX are active in chloroplasts, cytosol, and microsomes. In different plant species, APX activity increases in response to a variety of biotic and abiotic stresses. APX in combination with the effective AsA–GSH cycle functions to prevent the rise in toxic levels of H2 O2 in photosynthetic organisms (Asada 1999). APX activity is increased during drought stress in the alfalfa nodule (Naya et al. 2007). Hossain et al. (2009) noted that, during water logging, APX activity increased significantly in citrus plants. APX has been categorized on the basis of amino acid sequences. There are five chemically and enzymatically distinct isoenzymes of APX distributed in various subcellular locations in higher plants. The isoforms are found in cytosol, stroma, thylakoid, mitochondria, and peroxisome (Jiménez et al. 1997; Nakano and Asada 1987; Sharma and Dubey 2004). APX of cell organelles scavenges H2 O2 generated within the organelles, whereas H2 O2 produced in the cytosol or apoplast or diffused out from organelles is eliminated by cytosolic APX. The activity occurs in the chloroplast and depends upon AsA, which also acts as an electron donor. Reduction of AsA concentration causes a greater decrease in activity of chloroplastic isoenzymes than cytosolic APX isoforms. The cytosolic APX isoforms along with stromal and thylakoid bound enzymes are less sensitive to AsA (Sharma and Dubey 2004). APX is one of the most extensively distributed antioxidant enzymes in plant cells. APXs are efficient scavengers of H2 O2 under stressful conditions because their isoforms

4.3 Antioxidative Defense System in Plants

have superior affinity for H2 O2 in comparison with CAT. Elevated activity of APX in reaction to abiotic stresses such as drought, salinity, chilling, metal toxicity, and UV irradiation has been reported (Boo and Jung 1999; Sharma and Dubey 2005). 4.3.2.5

Monodehydroascorbate Reductase

MDHA radicals have a short life time and impulsively dismutate into DHA and AsA or are directly reduced to AsA by the NADP(H)-dependent enzyme MDHAR (Miyake and Asada 1994). Monodehydroascorbate reductase (1.6.5.4) is a FAD enzyme. It is also the only known enzyme to utilize an organic radical (MDA) as a substrate with the ability to reduce phenoxyl radicals. MDHAR activity is extensive in plants. In several cellular compartments, viz. chloroplasts, cytosol, mitochondria, and peroxisomes, isoenzymes of MDHAR have been reported (Jiménez et al. 1997). In chloroplasts, MDHAR is responsible for the regeneration of AsA from MDHA and the mediation of the photoreduction of dioxygen to O2 ⋅− when the substrate MDHA is not present (Miyake et al. 1998). MDHAR is involved in O2 ⋅− generation. 4.3.2.6

Dehydroascorbate Reductase

Dehydroascorbate reductase (EC 1.8.5.1) catalyzes the reduction of DHA to AsA using GSH as substrate. It plays a vital role in maintaining the reduced form of AsA. When AsA is oxidized in leaves and other tissues, DHA is always produced. DHA has a very short life span and is usually hydrolyzed irreversibly to 2, 3-diketogulonic acid or regenerated to AsA by DHAR. Overexpressions of DHAR have been reported to increase AsA content, signifying that it plays vital roles in maintaining the pool size of AsA (Qin et al. 2011). DHAR is a monomeric thiol enzyme copiously present in dry seeds, roots, and etiolated green shoots. Environmental stresses have caused elevation in the activity of DHAR in plants (Boo and Jung 1999; Sharma and Dubey 2005; Maheshwari and Dubey 2009; Sharma and Dubey 2007). 4.3.2.7

Glutathione Reductase

GSH functions as an antioxidant participating in enzymatic as well as nonenzymatic oxidation–reduction cycles where it is oxidized to GSSG. In the AsA–GSH cycle, GSH oxidization is carried out under the influence of enzyme DHAR. Glutathione reductase (EC 1.6.4.2) is an NAD(P)H-dependent enzyme responsible for reduction of GSSG to GSH, thus playing a major role in maintaining a high cellular GSH/GSSG ratio. GR belongs to a group of flavoenzymes and contains a disulfide group. The GR isoforms are located in the chloroplasts, cytosol, mitochondria, and peroxisomes, but chloroplastic isoforms are the most active form in the photosynthetic tissue. H2 O2 generated by Mehler reactions in chloroplasts is detoxified with help of GSH and GR. Several authors have reported increased activity of GR under environmental stresses (Boo and Jung 1999; Sharma and Dubey 2005; Sharma and Dubey 2007; Maheshwari and Dubey 2009). The activity of APXs is thought to form a second barrier of defense against H2 O2 /ROS produced in the chloroplasts (Chang et al. 2004). The role of GPXs as an H2 O2 scavenger has received reserved consideration (Mullineaux et al. 1998). The antioxidative defense capacity of the cells is determined by the pool size of the antioxidants and protective pigments. The impact of environmental stresses on plant metabolism may be reflected through a change in antioxidant contents (Herbinger et al. 2002). Antioxidant levels and the activities of ROS-scavenging enzymes have been

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correlated with tolerance to several different environmental stresses (Tewari et al. 2002). The activities of antioxidant enzymes in plants under stress are usually considered as an indicator of the tolerance of the genotypes against stress conditions. Prevention of cell death and stress tolerance has been correlated with the high antioxidant activity of an increased level of antioxidants (Van Der Mescht et al. 1998).

4.4 Redox Homeostasis in Plants ROS production generates an oxidative environment by a specific stress or by combination of multiple stresses (Thorpe et al. 2004). In higher plants, almost all cellular compartments such as chloroplasts, mitochondria, peroxisome, and cytoplasm participate in the production of ROS inside the cell. To sense, transduce, and translate ROS signals into appropriate cellular responses, plants have evolved a built-in mechanism (Das and Roychoudhury 2014). The activity of redox-sensitive proteins, which can participate in oxidation and reduction reactions as well as regulating the switching on or off, depends upon the cellular redox state (Shao et al. 2006). ROS directly or indirectly oxidizes redox-sensitive proteins through the ubiquitous redox-sensitive molecules, such as thioredoxins or glutathione (Nakashima and Yamaguchi-Shinozaki 2006). Redox-sensitive metabolic enzymes directly modulate the cellular metabolism in response to oxidative stress, but the redox-sensitive signaling proteins complete their action through downstream signaling apparatus, such as phosphatases, kinases, and transcription factors (Foyer and Noctor 2005; Li and Jin 2007). In plants and other living organisms, molecular mechanisms for redox-sensitive regulation of protein have also been explained (Cvetkovska et al. 2005; Foyer and Noctor 2005). Heterotrimeric G-proteins and MAP kinase-regulated protein phosphorylation and protein tyrphosphatases are involved in ROS-mediated signaling (Pfannschmidt et al. 2003; Foyer and Noctor 2005; Kiffin et al. 2006). Cellular organelles play a significant role in maintaining redox homeostasis in the plant cell (Andreev 2012; Ferrández et al. 2012; Lázaro et al. 2013). In plants, reduced and oxidized forms of electron transporters are required for competent flux through electron transport cascades. This state is known as redox poisoning and occurs in the respiratory and photosynthetic electron transport chains; it involves an uninterrupted chain of electrons to oxygen molecule. The reactive character of ROS means not only that their escalating level should be controlled, but also that they are capable of acting as signaling molecules. The ROS accumulation level is determined by the antioxidative system which enables the cells to preserve the cellular components in an active state for metabolism. Plants preserve most cytoplasmic thiols in a reduced condition, as the low-thiol disulfide redox potential imposed by millimolar concentrations of glutathione is the thiol buffer. Plant cells produce elevated concentrations of ascorbate, a hydrophilic redox buffer against oxidative stress, which gives a strong defense. Large pools of these antioxidants, which maintain the level of reductants and oxidants in a balanced state, direct redox homeostasis. Tocopherols (vitamin E), liposoluble redox buffers known as major singlet oxygen scavengers, can also efficiently scavenge other ROS (Foyer and Noctor 2005). The tocopherol redox couple has an extra positive midpoint potential over the ascorbate pool; it further increases the range of competent scavenging of superoxide. In plant cells, the redox buffer capacity of the glutathione, ascorbate and

References

tocopherol pools is one of the important characteristics. Homeostatic regulation of the ROS signaling pathways is accomplished by antioxidant redox buffering. The duration and the specificity of the signal of ROS are decided by antioxidants. A high level of ROS generation is handled very cautiously by plant cells. Cellular oxidation is evident in all stress reactions. The intensity and physiological effect of oxidative injury are debatable. Plants with low cytosolic APX and CAT activities show minor stress-induced signs compared with plants which use either one of these enzymes (Rizhsky et al. 2002).

4.5 Conclusion The normal metabolic activity of plants generates ROS, which act as signaling molecules for activating the plant metabolic pathway. It is well documented that various abiotic stresses lead to overproduction of ROS in plants, which are highly reactive and toxic and ultimately results in oxidative stress. Oxidative stress in plants causes injury to the cell membranes (lipid peroxidation) and biomolecules like nucleic acid and protein. To overcome the damaging effect of elevated ROS accumulation, plants have developed efficient ROS scavenging mechanisms. Plants have evolved two major scavenging tools, i.e. antioxidant molecules, viz. AA, 𝛼-tocopherols, glutathione, proline, flavonoids and carotenoids, and scavenging enzymes such as SOD, CAT, MDAR, DHAR, and GR. ROS also work as key signaling molecules interacting with each other and all other cellular antioxidant systems to maintain suitable equilibrium between a variety of cellular metabolic pathways, which get disrupted under adverse environments. The concentration of ROS in the cell decides the effects on plant. The information about ROS generation and their effective scavenging has been documented, but there are still gaps in our understanding of complete ROS scavenging and signaling pathways. Future research in this area will be constructive for designing schemes to determine the potential yield under hostile environments. Tolerance in a variety of crop plants against abiotic/biotic stress has been enhanced to some extent via the transgenic technology of ROS-scavenging components. To achieve greater yields of crops in the future under a rapidly changing climate, there is a need for further improvement by genetic engineering.

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5 Osmolytes and their Role in Abiotic Stress Tolerance in Plants Abhimanyu Jogawat National Institute of Plant Genome Research, New Delhi, 110067, India

5.1 Introduction Plants are sessile organisms which encounter variety of stresses at every developmental stage of their lifespan. The phyllosphere as well as the rhizosphere encounter a variety of abiotic stresses such as cold, heat, UV, submergence, wounding, extremes of temperature, drought, salinity, high metal concentrations, water logging, nutrient deficiency stress, and biotic stresses such as phytopathogenic viruses, bacteria, fungi, algae, nematodes, and insects (Atkinson and Urwin 2012; Suzuki et al. 2014; Singh et al. 2015; Zhu 2016; Tripathi et al. 2016a,b, 2017a,b; Jeandroz and Lamotte 2017; Singh et al. 2015). To defend themselves, plants have developed different kinds of mechanisms that counterattack or restore balance according to the stress agents encountered. Salinity, drought and heavy metal stresses cause some overlapping effects on plants, including high reactive oxygen species (ROS) levels, lipid peroxidation, antioxidant system activation, and accumulation of inert solutes (Arif et al. 2016a,b; Singh et al. 2017; Liu et al. 2018). During heat shock, freezing, osmotic shock, drought, water stress and heavy metal stress conditions, plant cells allow the influx, sequestering and synthesis of some solutes to accumulate them for homeostasis maintenance (Kuznetsov et al. 1999; Parvanova et al. 2004; Yancey 2005; Bohnert and Jensen 1996; Sharma and Dietz 2006; Burg and Ferraris 2008). These inert or nonharmful compatible molecules are widely known as osmolytes. Initially, the term osmolyte referred to molecules that are overproduced and accumulate during osmotic stress to maintain homeostasis in a cell or in the surrounding fluid. Later, the term osmolyte included any solute or metabolite produced and accumulated for protecting the cell environment against harmful effects caused by abiotic stress. The osmolytes include various biochemicals such as sugars, polyamines, secondary metabolites, amino acids, methylamines, and polyols, which protect or neutralize the damaging effects of abiotic stresses, and protect cells and make them tolerant of the particular abiotic stress (Roychoudhury and Chakraborty 2013; Roychoudhury and Das 2014; Roychoudhury and Banerjee 2016). Because of their importance in protecting against different kinds of stresses, the osmolytes are also known as cytoprotectants (Yancey 2005; Groppa and Benavides 2008; Khan et al. 2010). Among amino acids, proline is most important and well known for its role in changing abiotic stress environments (Hayat et al. 2012; Roychoudhury et al. 2015). Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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Biosynthesis and accumulation of various osmolytes comprise one of the earliest responses of host plants for combating osmotic and oxidative stress caused by various stressors. Several studies have shown that the expression of the metabolite biosynthesis pathway genes is regulated by phytohormones, calcium signaling and mitogen activated protein (MAP) kinase pathways under abiotic stress conditions. Thus, the accumulation of osmolytes is the result of various stress signaling pathways and is necessary for survival of plants under an abiotic stress environment. Their increased level protects plants, minimizes damage and rescues them from stress-induced oxidative damage, growth diminution and loss of photosynthesis efficiency. There are also different kinds of secondary metabolites that are accumulated in various kinds of abiotic stresses to protect plant cells from various kinds of damage. Some osmolytes and their biosynthesis have been studied in detail, such as proline, glycinebetaine, and mannitol (Yoshiba et al. 1997; Hare et al. 1998; Ashraf and Foolad 2007; Karakas et al. 1997; Giri 2011). For better survival under such conditions, some plants have been engineered metabolically for induced osmolyte biosynthesis. Interestingly, such transgenic plants have shown improved survival as well as tolerance of various abiotic stresses (Tarczynski et al. 1993; Kishor et al. 1995; Bohnert and Jensen 1996; Karakas et al. 1997; Shen et al. 1997; Hare et al. 1998; Sakamoto and Murata 2002; Parvanova et al. 2004; Yang et al. 2009). In this chapter, we analyzed the different osmolytes and their role in improving salinity, oxidative stress, and the heavy metal stress tolerance of plants.

5.2 Osmolyte Accumulation is a Universally Conserved Quick Response During Abiotic Stress From bacteria to plants, the accumulation of osmolytes is one of the earliest responses for combating osmotic stress (Yancey et al. 1982; Burg and Ferraris 2008). Even bacteria accumulate several kinds of osmolytes, such as amino acids and their derivatives (glutamate, proline, and glycinebetaine) and sugars (trehalose) in response to environmental stresses (Csonka 1989). MAP kinase pathways play an important role in sensing environmental stress signals in organisms (Nakagami et al. 2005; Qi and Elion 2005; Zarubin and Han 2005). There is an osmoregulatory pathway that is conserved in eukaryotes from yeast to mammals, but not plants, which is referred as the high osmolarity glycerol response (HOG) pathway (Westfall et al. 2004; Qi and Elion 2005). The activation of the HOG MAP kinase pathway results in glycerol accumulation in yeast and other fungi (Brewster et al. 1993; Bahn et al. 2007). In plants, there is a different signaling pathway which is known as the salt overly sensitive (SOS) pathway (Ji et al. 2013). The genes involved in the SOS pathway are referred to as SOS genes. The overexpression of SOS genes leads to increased salt tolerance of plants by maintaining ion homeostasis, which might also involve the synthesis of compatible solutes (Yang et al. 2009). The abscisic acid (ABA) stress signaling pathway-mediated increased abiotic stress tolerance mechanism also includes increased biosynthesis and accumulation of various osmolytes such as proline, mannitol, and glycinebetaine (Strizhov et al. 1997; Zhu 2001). Thus, it can be concluded that different stress-response signaling pathways such as MAP kinase signaling, calcium signaling, ABA signaling, and ROS signaling synergistically lead to increased biosynthesis and/or accumulation of different osmolytes for tolerance, acclimatization, and detoxification upon encountering abiotic stresses (Figure 5.1).

5.3 Osmolytes Minimize Toxic Effects of Abiotic Stresses in Plants

Figure 5.1 Schematic representation of overall relation between abiotic stress and osmolytes: upon perception of abiotic stress signal, the related signaling pathway is activated and results in the induction of stress-responsive transcription factors which subsequently upregulate stress-responsive genes related to biosynthesis and accumulation of osmolytes such as free amino acids and their derivatives, carbohydrates and soluble sugars, polyols, polyamines, free amines and other secondary metabolites to protect and make plants tolerant of encountered abiotic stresses.

Abiotic stresses

Abiotic stress signaling

SRTFs

SRGs

Biosynthesis and accumulation of osmolytes Free amino acids Secondary and their derivatives metabolites Carbohydrates Polyamines and soluble and free sugars amines Polyols

5.3 Osmolytes Minimize Toxic Effects of Abiotic Stresses in Plants Abiotic stresses such as high temperature, freezing/cold, drought/water, salinity, heavy metals, UV and nutrient stresses have adverse effects on the physiology, biochemistry, molecular mechanisms, and survival of plants (Atkinson and Urwin 2012; Suzuki et al. 2014; Singh et al. 2015; Zhu 2016; Tripathi et al. 2016a,b, 2017a,b; Jeandroz and Lamotte 2017; Singh et al. 2015). For rescue from such damaging effects under abiotic stresses, plant cells usually synthesize and accumulate organic molecules which are compatible in nature. These osmolytes protect the cells of the organism when they encounter abiotic stresses such as water, salt, drought, and cold (Mahajan and Tuteja 2005). Moreover, these compatible osmolytes rapidly replace damaging inorganic salts and protect them from oxidative damage (Burg and Ferraris 2008). Organic osmolytes play a role in minimizing the harmful effects of abiotic stresses, including amino acids and their derivatives, polyols and sugars, methylamines, and methylsulfonium compounds (Yancey 2005). They act in many ways, for example as ROS detoxifiers (antioxidants), photosynthesis protectants, osmoprotectants, macro-biomolecule stabilizers, and protein folding enhancers (Yancey 2005; Chinnusamy et al. 2007; Giri 2011; Table 5.1). In addition to cell size maintenance by osmotic adjustment, osmolytes also have metabolic benefits. For instance, synthesis of proline and glycine betaine buffers cellular redox potential and affects the allocation of photoassimilate between roots and shoot tissues (Hare et al. 1998). Polyamines, amino acids and their derivatives have very important roles in protecting macromolecules such as DNA, protein, and enzymes in plants during abiotic stresses (Rodríguez et al. 2005; Groppa and Benavides 2008; Roychoudhury et al. 2011; Paul et al. 2017). Regulation of biosynthesis and accumulation of free proline is well known in plants during osmostress and the biosynthesis pathway has been studied in detail in plants (Delauney and Verma 1993; Kishor et al. 2005).

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Table 5.1 Osmolytes and their role in abiotic stresses in crop plants. Plant

Osmolytes

Abiotic stress

References

Rice

Proline, polyamines, glycinebetaine, protein content, soluble sugars

Salt, drought, dehydration, water, heavy metals

Lutts et al. 1999; Majerus et al. 2007; Choudhary et al. 2005; Xu et al. 2008; Farooq et al. 2008; Shah and Dubey 1997; Sharma and Dubey 2005

Wheat

Proline, glycinebetaine, sugars (mannitol, glucitol)

Salt, water, heat, oxidative stress, heavy metals, temperature

Nayyar 2003; Sairam et al. 2002; Nayyar and Walia 2004; Heidari 2009; Kumar et al. 2012; Puniran-Hartley et al. 2014; Bassi and Sharma 1993; Gajewska et al. 2006; Öncel et al. 2000

Barley

Sugars and free amino acids, proline, glycinebetaine

Salt, oxidative stress, heavy metal

Pérez-López et al. 2010; Puniran-Hartley et al. 2014; Tamás et al. 2008

Maize

Proline, glycinebetaine, sugars

Salt, water stress, heavy metals

Brunk et al. 1989; Nayyar 2003; Kholová et al. 2009

Sorghum

Proline, amino acids, glycine betaine, sugars

Salt, water, drought

Heidari 2009, Sonobe et al. 2010; Damame et al. 2014; Nxele et al. 2017

5.4 Stress Signaling Pathways Regulate Osmolyte Accumulation Under Abiotic Stress Conditions In fungi, the production and accumulation of glycerol, trehalose, glycerophosphocholine, and polyols (erythritol, ribitol, arabinitol, xylitol, sorbitol, mannitol, and galactitol) is a rapid response for survival under osmotic stress (Hohmann 2002; Kiewietdejonge et al. 2006; Burg and Ferraris 2008). In response to different kinds of abiotic stresses, the production and accumulation of the osmolytes are known to be regulated by a MAP kinase cascade, i.e. the HOG pathway, during osmotic stress in fungi (Bahn et al. 2007; Hersen et al. 2008). Along with the HOG pathway, the cell wall integrity pathway also plays a role in abiotic stress responses in fungi (Fuchs and Mylonakis 2009). In plants, the SOS pathway, ABA signaling, calcium signaling, and the MAP kinase network respond rapidly and synergistically in various abiotic stresses for establishing osmotic homeostasis by regulating the production and accumulation of osmolytes (Figure 5.2; Roychoudhury et al. 2013; Roychoudhury and Banerjee 2017). The accumulation and biosynthesis of osmolytes comprise one of the important responses of the activation of stress signaling pathways in plants under abiotic stresses, and enables plants to tolerate and adapt quickly under abiotic stresses such as salt, high metals, cold, drought, water, and temperature (Figure 5.1; Zhu 2001; Xiong et al. 2002; Kishor et al. 2005; Wingler and Roitsch 2008; Verbruggen and Hermans 2008). The SOS signaling pathway is important for stress signaling in plants (Mahajan et al. 2008). The SOS pathway is reported to be regulated by MAP kinase pathways and is also affected by the level of osmolyte glycinebetaine in plants (Ashraf and Foolad 2007). ABA signaling has been shown to regulate proline accumulation in response to salinity and drought stress (Verslues and Bray 2005). For biosynthesis of proline under such abiotic stresses, 𝛥1 -pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (PDH) are important genes (Peng et al. 1996). The P5CS gene has been

5.5 Metabolic Pathway Engineering of Osmolyte Biosynthesis

Abiotic stress Sensors ABA Biosynthesis

ABAR

Ca2+ Rise ROS MAPKKK(s)

CaMs/CMLs /CDPKs

MAPKK(s) SnRK2

MAPK(s)

SRTFs Nucleus Osmolyte Biosynthesis and Accumulation Related Gene Activation

Cytosol

Osmolyte Biosynthesis and Accumulation Related Functions for Adaptation and Tolerance under Abiotic Stress

Figure 5.2 Regulation of osmolyte production and accumulation upon perceiving abiotic stress signal: osmolyte production and accumulation are triggered and regulated by various signaling pathways such as ABA signaling, calcium signaling and ROS-MAP kinase networks (Golldack et al. 2014; DeFalco et al. 2010; Jalmi and Sinha 2015).

reported to be highly stimulated during abiotic stresses such as water and salinity. In Arabidopsis, P5CS has two genes. Induction of AtP5CS mRNA by ABA, drought and salinity has been reported. ABA signaling has been proven to play an important role in the regulation of AtP5CS2 gene. In ABA biosynthesis mutant plants such as aba1, abi1, and axr2, AtP5CS2-mediated proline accumulation was lacking (Strizhov et al. 1997). Moreover, ABA stress signaling transcription regulator AREB1 overexpression has been demonstrated to increase drought tolerance in plants by increasing proline biosynthesis and accumulation (Fujita et al. 2005; Roychoudhury and Paul 2012). In plants, MAP kinase kinases MKK2 and MKK4 have been shown to improve the abiotic stress tolerance of plants by increasing levels of osmolytes such as proline and sugars (Teige et al. 2004; Wang et al. 2014). In rice, calcium-induced protein kinase 1 (CIPK1) has been demonstrated to enhance abiotic stress tolerance which was mediated by increasing proline production and accumulation (Abdula et al. 2016).

5.5 Metabolic Pathway Engineering of Osmolyte Biosynthesis Can Generate Improved Abiotic Stress Tolerance in Transgenic Crop Plants Osmolyte accumulation can be improved in crops using traditional plant breeding, marker-assisted selection or genetic engineering for better crop yield in salt-, drought-, and dehydration-stressed fields (Serraj and Sinclair 2002). The genes of the biosynthesis

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and metabolic pathways of the osmolytes can be utilized to generate transgenic crop plants with increased ability to accumulate and produce osmolytes (Hare et al. 1998). The source genes may originate from bacteria, algae, fungi, or other plants. Various genetic engineering efforts have been made in this direction with great success in producing abiotic stress-tolerant plants (Zhang et al. 2000; Wang et al. 2003; Vinocur and Altman 2005; Valliyodan and Nguyen 2006; Bhatnagar-Mathur et al. 2008; Ahanger et al. 2017). The metabolic engineering of pathways for proline, glycinebetaine, and sugars can lead to enhanced abiotic stress tolerance owing to the increased production and accumulation of such osmolytes (Rathinasabapathi 2000; Chen and Murata 2002; Rontein et al. 2002). In the case of proline biosynthesis, either the removal of the inhibitory gene of Δ1 -pyrroline-5-carboxylate synthetase or its overexpression has resulted in increased levels of proline as well as osmotolerant plants (Zhu et al. 1998; Hong et al. 2000). The biosynthesis gene of glycinebetaine, choline oxidase, of Table 5.2 Utilization of the osmolyte production and accumulation pathway-related genes for improving abiotic stress tolerance in plants. Transgenic plant

Abiotic stress tolerance

Plants

Rice, potato, Arabidopsis

Salinity, drought, heavy metals

Zhu et al. 1998; Hong et al. 2000; Anoop, and Gupta 2003; Hmida-Sayari et al. 2005

Mannitol 1-phosphate dehydrogenase

Bacteria, fungi

Tobacco, Sorghum, egg plant, wheat

Water, oxidative stress, salinity, heavy metals

Tarczynski et al. 1993; Shen et al. 1997; Prabhavathi et al. 2002; Abebe et al. 2003; Tang et al. 2005; Maheswari et al. 2017

Glucitol

Glucitol-6-phosphate dehydrogenase

Bacteria, fungi

Tobacco

Oxidative stress, salinity, heavy metals

Tang et al. 2005

Glycine betaine

Choline oxidase

Bacteria

Arabidopsis

Temperature, salinity

Sakamoto and Murata 2001

Choline dehydrogenase

Bacteria

Tobacco, Arabidopsis, Carrot

Salinity, cold

Lilius et al. 1996; Hayashi et al. 1997

Beataine

Betaine aldehyde dehydrogenase

Carrot

Carrot

Salinity

Kumar et al. 2004

Fructan

Fructosyl-transferase

Wheat, Lactuca sativa

Perennial ryegrass, tobacco

Freezing

Hisano et al. 2004; Li et al. 2007

Trehalose

Trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

Yeast, E. coli, rice

Tobacco, potato, rice

Drought, salinity, and cold

Romero et al. 1997; Yeo et al. 2000; Garg et al. 2002; Jang et al. 2003; Li et al. 2011

Ononitol

myo-Inositol O-methyltransferase

M. crystallinum

Tobacco, Arabidopsis, soybean

Salinity, drought and cold

Sheveleva et al. 1997; Chiera et al. 2006; Zhu et al. 2012

Osmolyte

Transgene

Source

Proline

Δ1 -Pyrroline-5carboxylate synthetase

Mannitol

Reference

References

bacterial origin has also been reported to improve the abiotic stress tolerance of plants (Sakamoto and Murata 2001). Overexpression of sugar-related genes such as mannitol 1-phosphate dehydrogenase and glucitol-6-phosphate dehydrogenase genes has been shown to improve the abiotic stress tolerance of plants (Tarczynski et al. 1993; Shen et al. 1997; Abebe et al. 2003; Tang et al. 2005; Khare et al. 2010; Maheswari et al. 2017). High-throughput omics technologies are being employed to determine the genes involved in the biosynthesis and regulation of osmolytes (Bohnert et al. 2006; Shinozaki and Yamaguchi-Shinozaki 2007). It was also argued that osmolyte pathway engineering is not that successful, but in the near future, successful measures can be established to utilize the potential of different genes for developing abiotic stress-tolerant crops (Valliyodan and Nguyen 2006). Despite several limitations, various efforts have successfully demonstrated the potential of osmolytes in improving the abiotic stress tolerance of plants. These efforts have been summed up in Table 5.2.

5.6 Conclusion and Future Perspectives Osmolytes play pivotal role in plants for improving abiotic stress tolerance. The overproduction and accumulation of osmoprotectants is universal process for combating abiotic stress. Various abiotic stresses trigger the biosynthesis and accumulation of osmoprotectants such as proline, glycinebetaine, and soluble sugars. In several studies, the biosynthesis and regulatory genes from different sources have been successfully utilized for generating abiotic stress-tolerant transgenic plants which have better capability to rapidly accumulate sufficient amounts of osmolytes. From various reports, it is clear that the different candidates for the osmolyte biosynthesis pathway can be utilized for better survival of crop plants. In the era of exploding populations and shrinking agricultural land, we need to focus on utilizing the benefits of osmolyte-mediated crop improvement. Advancements in gene editing and manipulation will prove valuable for generating super crops using super genes from different sources by improving their ability to accumulate osmolytes for better productivity as well as ensure survival under various abiotic stresses.

Acknowledgements The author acknowledges the financial support from SERB-National Post doctoral scheme, the Government of India.

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6 Elicitor-mediated Amelioration of Abiotic Stress in Plants Nilanjan Chakraborty 1,2 , Anik Sarkar 1 , and Krishnendu Acharya 1 1 Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, Kolkata 700019, India 2 Department of Botany, Scottish Church College, Kolkata 700006, India

6.1 Introduction Plants deal with various types of biotic (viz. fungi, bacteria, and virus) and abiotic (such as heavy metals, UV radiation, salinity, high and low temperatures, ozone, and drought) stresses in their natural habitats, which ultimately limits the total harvest of a crop (Xiong and Zhu 2001; Thakur and Sohal 2013; Khan et al. 2015; Savvides et al. 2016; Singh et al. 2015; Zhu 2016; Tripathi et al. 2016a, 2017a,b; Singh et al. 2017; Liu et al. 2018). Global food security is being gravely hampered by the gradual increase of urbanization, industrial waste generation, and climate change, which intensify the damaging effects of abiotic stresses on crop health and yield (Lobell and Field 2007; Nagajyoti et al. 2010; Reddy 2015; Arif et al. 2016a,b; Savvides et al. 2016; Tripathi et al. 2016b,c; Chakraborty and Acharya 2017). Regularly, more than 50% of the yield of crops is spoiled by the adverse effects of abiotic factors (Khan et al. 2009; Segnou et al. 2013; Thakur and Sohal 2013; Zhani et al. 2013; Poltronieri et al. 2014). Plants are not passive witnesses to the continuous onslaught of hazardous environments with which they interact. Like other organisms, they defend themselves by the activation of various mechanisms (Repka 2001; Chakraborty et al. 2015). Recent investigations imply the existence of a chemical priming process which may act as a stress sensor and inducer of stress signals in plants (Chakraborty and Acharya 2017). This process greatly relies upon the application of elicitors, low-molecular-weight compounds that mimic either an abiotic stress stimulus or other biotic factors (Acharya et al. 2011a; Baenas et al. 2014; Chandra et al. 2014a). On the basis of their origin, there are two distinct groups of elicitors, viz. biotic elicitors and abiotic elicitors (Baenas et al. 2014). Abiotic elicitors include substances of nonbiological origin and are again grouped as physical (such as variable temperature, UV, etc.) and synthetic chemical factors (such as, CaCl2 , CuCl2 , chitosan, isonicotinic acid, plant hormones like ethylene, salicylic acid, jasmonic acid, etc.). Contrary to this, biotic elicitors are substances of biological origin including polysaccharides originating

Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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from the cell walls of plant (e.g. pectin, chitin, and cellulose), heat-killed or attenuated microbes or inactivated products and plant growth-promoting rhizobacteria (PGPR) (Mejía-Teniente et al. 2010; Thakur and Sohal 2013; Chakraborty and Acharya 2016; Chakraborty et al. 2016). Both types of elicitors have several effects on plants and affect mainly the secondary metabolism. However, in comparison with biotic elicitors, application of small amounts of abiotic elicitors can induce vast range of defense mechanisms in host plants (Thakur and Sohal 2013; Chandra et al. 2015). Earlier reports suggested that elicitors derived from pathogens (primary elicitors) and subsequent endogenous signals (secondary elicitors) may turn on plant defense-related genes, antioxidant enzymes like glutathione S-transferases and peroxidases, other hydrolytic enzymes, cell wall-strengthening components like lignin, pathogenesisrelated proteins synthesis, phytoalexin biosynthetic enzymes, etc. (Wang et al. 2010). Induced resistance can also be attained by the use of various elicitors like jasmonic acid and its derivatives including methyl jasmonate (Moreno et al. 2010; Yang et al. 2011); salicylic acid and its derivatives, including 2,6-dichloro-isonicotinic acid, benzo(1,2,3) thiadiazole-7-carbothioic acid S-methyl ester, and dl-3-amino-n-butyric acid (Chandra et al. 2014a,b); chitin and chitosan (Dos Santos et al. 2012; Yan et al. 2012); benzothiadiazole (Lin et al. 2011); copper sulfate (CuSO4 ), calcium chloride (CaCl2 ), cupric chloride (CuCl2 ), arachidonic acid, oxalic acid, and isonicotinic acid (Aziz et al. 2006; Tian et al. 2006; Acharya et al. 2011a; Chakraborty and Acharya 2016; Chakraborty et al. 2016). Application of those chemicals induces overproduction of diverse defense gene products including polyphenol oxidase, peroxidases, 𝛽-1,3-glucanase, and phenylalanine ammonia-lyase (Maxson-Stein et al. 2002; Pal et al. 2011), along with some other antioxidant enzymes and by elevation of total phenol accumulation (Anand et al. 2009; Chandra et al. 2014b).

6.2 Plant Hormones and Other Elicitor-mediated Abiotic Stress Tolerance in Plants Although phytohormones are produced in very minute amounts, they act as chemical messengers, playing key roles and harmonizing diverse signal transduction pathways in abiotic stress responses (Vob et al. 2014; Wani et al. 2016). They have tremendous potential to control both external and internal stimuli (Kazan 2015). Exogenous treatment with various plant growth regulators (PGRs) as foliar spray or for seed soaking led to varied degrees of abiotic stress tolerance (Roychoudhury et al. 2011; Verma et al. 2016; Awan et al. 2017; Banerjee and Roychoudhury 2018). To adopt sustainable agriculture in developing countries and to reduce the use of hazard-free chemicals, sometimes plant extracts are also used as PGRs (Ashraf et al. 2016). Biologically active compounds found in those plant extracts, like brassinolides in Brassica, zeatin in Moringa oleifera, etc., may act as bio-stimulants of plant growth and simultaneously perform a protective role in a cost-effective manner against different abiotic stresses with which plants interact every day (Ashraf et al. 2016). The roles of various phytohormones and other elicitors in abiotic stress alleviation are listed in Table 6.1.

Table 6.1 Role of plant growth-promoting rhizobacteria (PGPR) in various abiotic stresses. Sample no.

Plants

Plant hormones and other elicitors used

Responses

References

1

Zea mays L.

Salicylic acid (SA)

Provide resistance against drought stress

Elgamaal and Maswada 2013

2

Vigna radiata L.

SA (0.5 mM)

Resistance against cadmium chloride stress

Roychoudhury et al. 2016

3

Oryza sativa L.

SA (100 mg l−1 )

Application of chemical on leaves gave enhanced resistance to drought stress compared with soaking the seeds in the same SA solution

Farooq et al. 2009; Kareem et al. 2017

4

Hydroponic growth solution of young Zea mays L.

SA (0.5 mM)

Induced antioxidant enzymes which directly increased chilling tolerance

Janda et al. 1999

5

Triticum aestivum L.

SA (1–3 mM)

Improved plant growth and superoxide dismutase activity and maintained nitrate reductase activity under water stress

Singh and Usha 2003

6

Bluegrass (Poa pratensis L.)

SA as foliar spray along with humic acid and seaweed extract

Alleviated decline of photochemical efficiency and turf quality under UV-B stress during summer

Ervin et al. 2004

7

Arabidopsis thaliana

Methyl jasmonate

Increased thermo-tolerance and protected plants from the damaging effects of heat shock

Clarke et al. 2009

8

Bean

Triazoles

Showed less electrolyte leakage and tolerance against heat stress

Fletcher et al. 2000

9

Oryza sativa L.

Combination of methyl jasmonate, ascorbic acid, 𝛼-tocopherol, and brassinosteroids

Showed resistance against high-temperature stress by improving plant growth and vigor

Fahad et al. 2016

10

Vigna radiata

Homobrassinolides and aliphatic alcohols

Significantly improved photosynthetic activity resulting in enhanced carbohydrate flux to the budding pods under water stress

Sanadhya et al. 2012

11

Triticum aestivum L.

Abscisic acid and 1-aminocyclopropane1-carboxylic acid, ethylene precursor

Improved shoot growth under mild drought conditions

Valluru et al. 2016

(Continued)

Table 6.1 (Continued) Sample no.

Plants

12

Capsicum annuum L.

13

Plant hormones and other elicitors used

Responses

References

SA, hydrogen peroxide (H2 O2 ), and chitosan

Increased endogenous H2 O2 as well as gene expression of cat1, pal, and pr1; improved enzymatic activities related to oxidative stress

Mejía-Teniente et al. 2013

Ocimum basilicum L.

Chitosan

Improved chlorophyll and carotenoid contents, height of plant, leaf area, and root and shoot growth; checked transpiration under water-deficit conditions

Malekpoor et al. 2016

14

Raphanus sativus L.

Plant leaf extracts of mulberry, Brassica, Sorghum, and Moringa

Foliar application of plant leaf extracts as plant growth regulator showed a promising effect on growth improvement, and biochemical and antioxidant activity of radish plants

Ashraf et al. 2016

15

Lycopersicon esculentum Mill.

Cupric chloride (CuCl2 )

Improved catalase and ascorbate peroxidase enzymes and reduced oxidative stress

Chakraborty et al. 2015

16

Lycopersicon esculentum Mill.

Calcium chloride (CaCl2 )

Increased antioxidant enzymes and restricted oxidative stress

Chakraborty et al. 2016

17

Capsicum annuum

Silicon

Regulated physiology, antioxidant metabolism, and protein expression under salt stress

Manivannan et al. 2016

18

Oryza sativa L.

Polyamines (spermidine and spermine)

Resistance against salt stress by regulating antioxidant and osmolyte levels and gene expression of diverse metabolic pathways

Roychoudhury et al. 2011; Paul and Roychoudhury 2016; Paul et al. 2017; Paul and Roychoudhury 2017

6.4 Signaling Role of Nitric Oxide in Abiotic Stresses

6.3 PGPR-mediated Abiotic Stress Tolerance in Plants PGPRs inhabit the rhizosphere of several plant species and give support to the plant by increasing plant growth and making them competent to counteract biotic agents like fungi, viruses, pathogenic bacteria, and nematodes (Kloepper et al. 2004; Yang et al. 2009). PGPRs have been applied as elicitors to induce systemic resistance in various plants (Van Loon 2007; Acharya et al. 2011b, 2013). Earlier reports suggested that application of PGPR has a great role to play in improving abiotic stress tolerance mechanisms in plants (Yang et al. 2009; Vurukonda et al. 2016). PGPR-mediated abiotic stress tolerance in plants is referred to as “induced systemic tolerance” (Sandhya et al. 2010). A brief overview of PGPR-mediated defense induction in plants against abiotic stresses is listed in Table 6.2.

6.4 Signaling Role of Nitric Oxide in Abiotic Stresses Since all protective actions come at a substantial cost of energy, plants have evolved inducible defense responses that are induced only when they encounter stress (Heil and Ton 2008; Notaguchi and Okamoto 2015). In order to increase their endurance, plants have evolved several strategies to recognize environmental signals and make them competent to continue their development in diverse habitats (Dinant and Suarez-Lopez 2012). This includes the perception of biotic and abiotic factors by specific organs and the subsequent broadcasting of the information to the distal parts of the plant (Heil and Ton 2008; Dinant and Suarez-Lopez 2012). Plants have specific receptors on their surfaces to sense different types of biotic and abiotic stresses. Perception of a stress signal induces different signaling components. Small lipophilic, bioactive gaseous nitric oxide (NO) is the most important stress mediator signaling molecule (Lamattina et al. 2003; De Stefano et al. 2005; Chandra et al. 2014a, 2015; Chakraborty et al. 2015, 2016; Chakraborty and Acharya 2016). In various organisms, like cyanobacteria, green algae, lichens, ferns, gymnosperms, monocots, and dicot plants, endogenous NO production has been observed (Salmi et al. 2007; Catala et al. 2010; Foresi et al. 2010; Sturms et al. 2011; Yu et al. 2012; R˝oszer 2014). The signaling role of NO is observed during foliar application of NO donors, nitric oxide synthase inhibitors and NO scavengers in various plants. Previous studies suggest the involvement of NO in nearly all abiotic stress responses in plants (Misra et al. 2011; Chakraborty and Acharya 2017). The involvement of NO as a ubiquitous signal in different physiological processes including biotic and abiotic stress responses in plants is a well-documented fact (Lamattina et al. 2003; Desikan et al. 2004; Wendehenne et al. 2004; Delledonne 2005; Leitner et al. 2009; Dinant and Suarez-Lopez 2012; Simontacchi et al. 2015). The signaling action of NO at the molecular level can be explained by the identification of NO-synthesizing enzymes and the NO-mediated activity of specific proteins (Hanafy et al. 2001; Kone et al. 2003; Stuehr et al. 2004). It has been observed that NO plays an essential role in the regulation of plant metabolism and senescence (Guo and Crawford 2005; Siddiqui et al. 2011), reduction of seed dormancy and induction of seed germination (Bethke et al. 2006; Zheng et al. 2009), regulation of stomatal movement (Guo et al. 2003; Garcia-Mata and Lamattina 2007), induction of cell death (Pedroso and Durzan 2000), mitochondrial functionality (Zottini et al. 2002), regulation of photosynthesis (Takahashi and Yamasaki 2002), floral induction (He et al. 2004), and gravitropism (Hu et al. 2005). NO also plays a significant

109

Table 6.2 Role of PGPRs in various abiotic stresses. Sample no.

Plants

PGPR used

Responses

References

1

Arabidopsis thaliana

Paenibacillus polymyxa

Generated drought tolerance

Timmusk and Wagner 1999; Yang et al. 2009

2

Capsicum annuum L. and Solanum lycopersicum

Achromobacter piechaudii ARV8

Produced 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which degraded the ethylene precursor ACC and restored normal plant growth against drought stress

Mayak et al. 2004; Glick et al. 2007

3

Lactuca sativa L.

Pseudomonas mendocina

Increased the production of catalase under severe drought conditions to release oxidative stress

Kohler et al. 2008

4

Arabidopsis thaliana

Bacillus subtilis GB03

Gave resistance against salt stress and many plant pathogens

Yang et al. 2009

5

Solanum lycopersicum

Achromobacter piechaudii

Ethylene content was reduced against high salt stress conditions

Mayak et al. 2004; Yang et al. 2009; Tank and Saraf 2010

6

Oryza sativa

Bacillus amyloliquefaciens SN13

Upregulation of SOS1, EREBP, SERK1, and NADP-Me2

Nautiyal et al. 2013

7

Glycine max

Pseudomonas simiae AU

Upregulation of storage proteins and RuBisCO; reduced Na+ accumulation in roots and augmentation in proline and chlorophyll content

Vaishnav et al. 2015

8

Triticum aestivum

Zhihengliuella halotolerans; Bacillus gibsonii; Staphylococcus succinus; Oceanobacillus oncorhynchi; Halomonas sp.

Significantly improved growth under 200 mM NaCl stress

Orhan 2016

9

Triticum aestivum

Serratia sp. Sl–12

Improved high salt tolerance and increased shoot growth

Singh and Jha 2016

10

Zea mays

Pseudomonas fluorescens

Promoted root growth under salt stress

Zerrouk et al. 2016; Ilangumaran and Smith 2017

11

Vigna radiata

Pseudomonas fluorescens Pf1

Increased vigor index, fresh and dry weight, improved catalase, peroxidase and proline content under water stress

Saravanakumar et al. 2011

12

Solanum lycopersicum

Leifsonia xyli SE134

Modulated endogenous amino acid content and overcame Cu stress

Kang et al. 2017

13

Zea mays

Azospirillum brasilense

Increased relative and absolute water content compared with noninoculated plants under drought stress

Casanovas et al. 2002; Vurukonda et al. 2016

14

Zea mays

Azospirillum lipoferum

Secreted abscisic acid and gibberellins to overcome drought stress.

Cohen et al. 2009

15

Triticum aestivum

Bacillus amyloliquefaciens 5113 and Azospirillum brasilense NO40

Upregulation of APX1, SAMS1 and HSP17.8 genes and improved plant growth

Kasim et al. 2013; Vurukonda et al. 2016

16

Sugarcane

Gluconacetobacter diazotrophicus PAL5

Inoculation activated the abscisic acid-dependent signaling genes and improved drought resistance

Vargas et al. 2014; Vurukonda et al. 2016

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6 Elicitor-mediated Amelioration of Abiotic Stress in Plants

6 • Fruit ripening • Senescence

5 • Flowering control • Pollen tube growth regulation

1 • Seed dormancy and germination

Nitric oxide 4 Disease resistance • Biotic stress (bacteria, fungi, virus etc.) • Abiotic stress (drought, heavy metal, ozone, wounding, UV radiation etc.) • Defense gene activation

3 • Hormonal responses

2 • Root formation and growth • Vegetative growth • Vascular differentiation • Chlorophyll biosynthesis • Stomatal movement • Fe homeostasis

Figure 6.1 Role of nitric oxide (NO) in plant system. (1) It breaks seed dormancy and promotes germination of diverse orthodox seeds. (2) NO is also involved in the regulation of root formation and growth, chlorophyll biosynthesis, and stomatal movement. (3) It serves various metabolic pathways by interacting with plant hormones. (4) NO has been found to be promising in inproving tolerance to various abiotic and biotic stresses. (5) NO participates in the development of flower and pollen tube growth. (6) NO also delays fruit ripening and senescence. Source: Adapted from Simontacchi et al. (2015).

role in cell protection by interacting and nullifying the toxicity level of reactive oxygen species and different plant hormones (Lamattina et al. 2003; del Río et al. 2004). Moreover, NO acts as a gaseous signal whisperer just like the plant hormone ethylene (Guo et al. 2003; Yamasaki 2005). It also induces transcriptional changes which might further act on transport, reactive oxygen species production, defense and cell death, signal transduction, basic metabolism, and degradation (Palmieri et al. 2008; Siddiqui et al. 2011). However, NO status is significantly altered during both abiotic and biotic stress conditions in plants irrespective of the nature of stimuli encountered (Durzan and Pedroso 2002). Important functions of NO in plant system are demonstrated in Figure 6.1. There are various reports that deal with exogenous application of NO donor sodium nitroprusside along with certain other chemicals as an elicitor to counteract diverse abiotic stresses (Chakraborty and Acharya 2017). Some of them are listed in Table 6.3.

Table 6.3 Role of sodium nitroprusside (SNP) in various abiotic stresses. Elicitors used

Stress type

Name of the plant

Mechanism of action

References

SNP

Drought

Populus przewalskii

Proline accumulation and activation of antioxidant enzymes

Lei et al. 2007

SNP

Drought

Wheat seedling

Enhanced seedling growth and maintained high relative water content

Tian and Lei 2007

SNP

Heavy metal

Lupinus luteus

Increase in superoxide dismutase, catalase, and POX

Kopyra and Gwó´zd´z 2003

SNP + indole acetic acid

Heat stress

Lycopersicon esculentum Mill.

Enhanced the activity of antioxidant enzymes and NO generation, resulting in the prevention of reactive oxygen species and DNA damage and thus improving the tolerance of the plants to heat stress.

Siddiqui et al. 2017b

SNP + spermidine

Salt stress

Lycopersicon esculentum Mill.

Increased the activities of antioxidant enzymes and enhanced photosynthetic pigment (chlorophyll a and b) and Pro accumulation, as well as reducing H2 O2 and O2 •− and malondialdehyde content, under salt stress

Siddiqui et al. 2017a

SNP

Cd toxicity

Wheat

Reduction in lipid peroxidation, H2 O2 content, electrolyte leakage

Singh et al. 2008

SNP + H2 O2

Salt stress

Strawberry

Increased transcript levels of enzymatic antioxidants

Christoua et al. 2014

SNP

Chilling stress

Cicer arietinum L.

Electrolyte leakage was effectively reduced and plant vigor was increased

Chohan et al. 2012

SNP

Arsenic-induced oxidative stress

Wheat

Antioxidant defense and glyoxalase system

Hasanuzzaman and Fujita 2013

SNP

UV-B stress

Lactuca sativa

Enhanced antioxidant enzyme activities, total phenolic concentrations, antioxidant capacity, and PAL gene expression

Esringu et al. 2016

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6 Elicitor-mediated Amelioration of Abiotic Stress in Plants

6.5 Future Goals Abiotic stresses are the foremost issues that reduce crop production worldwide. Upon facing these stresses, there is a huge change in the metabolism and cell signaling pathways in plants that limits crop productivity (Kumar et al. 2017). Exogenous application of specific chemical, physical, or biological agents (elicitors) can improve stress tolerance in a cost-effective way, by regulating the different cell signaling pathways and gene expression. Elicitors also act on metabolic cycles of plants so that they may influence secondary metabolite production. Therefore, restricted short-term elicitation by different elicitors, for the duration of the pre-harvest and post-harvest phases, can be used as a device by which the producer may obtain improved products with high amounts of nutraceuticals. This improvement may also protect those plants or plant products from abiotic and biotic stresses. Simultaneously, functional foods enriched with extractable active compounds will fulfill the increasing demands of consumers who are concerned about a healthy diet and nutrition. Interestingly, most of the phytohormones may act as PGRs and also play an important role in the stress tolerance of plants. Conventional plant breeding methods can also be replaced by the use of different PGR and other biostimulants which may be a cost-effective and easy approach to combat abiotic stresses. Various phytohormones can be used as a specific metabolic target. However, detailed understanding of signaling crosstalks requires further investigation. Transgenic approaches with the modification of different PGR-sensitive genes can also be regarded as an innovative tool to improve abiotic stress tolerance in plants. Seed priming with different elicitor molecules can also increase stress tolerance in plants by increasing the activities of the antioxidant system and regulating the expression of different stress-tolerance genes. Apart from this, future research requires the development of efficient combinations of microbial formulations for boosting plant resistance against various abiotic stresses that substantially decrease the use of hazardous chemical fertilizers and harmful pesticides. Potential indigenous PGPRs should be isolated from contaminated soils that could be used as biostimulants for commercially important crops that are grown under stressed conditions (Vurukonda et al. 2016). Various fundamental mechanisms of plant–microbe interactions in the rhizosphere could be resolved in near future. Detailed understanding of those signaling crosstalks is essential to use soil microbes for stress mitigation of important crops. In this connection, further research on NO’s function in plant systems as a signaling amplifier molecule in abiotic stressed environments could provide further approaches to plant improvement.

6.6 Conclusion Abiotic stresses comprise a severe environmental constraint that alters agricultural productivity. Elicitors in its various forms play an imperative role in conferring future food security issues by protecting crops from the harmful effects of different stresses. Improvement of different stress-responsive and stress-sensitive genes relies on the activation of transcription factors by elicitor treatment. However, elicitor application may involve activation of downstream secondary signaling cascades. Thus, the overall idea implies that multiscale and multifactorial defense systems which include both the vascular and airborne transport system can reduce the adverse effects of abiotic stress on plants.

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7 Role of Selenium in Plants Against Abiotic Stresses: Phenological and Molecular Aspects Aditya Banerjee and Aryadeep Roychoudhury Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata 700016, West Bengal, India

7.1 Introduction Edaphic Se can exist in four different oxidation states: selenate (Se6+ ), selenite (Se4+ ), elemental Se (Se0 ), and selenide (Se2− ). The physico-chemical properties of the soil, like pH, redox potential and clay content, ultimately dictate the bioavailability of Se. Their content in the soil is determined by the composition of the bedrock, which is exposed to geochemical processes (Dhillon and Dhillon 2003). Oxidized and alkaline soils promote absorption of selenate by the plant, whereas strong adsorption of selenite on iron oxide surfaces and soil clays makes this form less available to plant roots (Mikkelsen et al. 1989). Se is a nonmetal trace element participating in antioxidant defense systems in humans and animals (Rotruck et al. 1973). Both the organic and inorganic forms of Se in the form of selenoamino acids and methylated complexes have been detected in plants (Finley et al. 2001). This indicates the essential nature of these micronutrients in plants. Although Se promotes optimal growth in unicellular green alga, its roles in higher plants are debated. At very high concentrations, Se is toxic to plants, causing selenosis. The symptoms are manifested by high accumulation of reactive oxygen species (ROS) like hydrogen peroxide (H2 O2 ), hydroxyl, superoxide radicals, etc., protein sulfur substitution by Se, and inhibited methylation. The plants exhibit necrotic patches, chlorosis, and wilting (Mengel and Kirkby 1987). Plants have been classified into nonaccumulators, accumulators, and indicators based on their different capacities to accumulate Se. Nonaccumulators like garlic, onion, broccoli, rice, wheat, corn, and beans show sensitivity beyond 2 mg kg−1 Se, whereas the accumulators (belonging to genus Astragalus and Stanleya) can tolerate 4000 mg kg−1 Se (Shrift 1969; Aggarwal et al. 2011). Se being a micronutrient exerts diverse beneficial roles at low concentrations (Finley et al. 2001). It promotes plant developmental physiology under harsh environmental conditions. This review thoroughly discusses the growth-promoting roles of Se in plants which are pivotal reasons for the abiotic stress tolerance conferred on plants by these micronutrients at sparing levels. In this regard, exogenous application of Se at optimum concentrations can be standardized to promote neo-domesticated cultivation of plants under sub-optimal conditions. Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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7.2 Se Bioaccumulation and Metabolism in Plants Owing to their close chemical similarity, the metabolism of Se is related to that of sulfur in higher plants. The uptake of Se from soil varies with the plant species and growth stage. Turakainen (2007) reported high Se accumulation in the aerial biomass compared with root tissues in a majority of plant species. However, in potato plants, Se supplementation increased its accumulation in the upper leaves, roots, stolons, and tubers. Interestingly, during the growth phase, Se micronutrients were channeled from the aerial parts, roots, and stolons into the mature and immature tubers (Turakainen 2007). Thus it appears that Se is utilized in synthesizing the seleno-derivatives of amino acids in the storage organs. It has been proposed that, owing to its rapid incorporation, selenite is more toxic than selenate. Higher plant roots prevent such selenite-mediated toxicity by selectively permitting the higher uptake and distribution of selenate (Hasanuzzaman et al. 2010). Such specificity is incurred by the active sulfate transporters, SULTR1;2 and SULTR1, which transport selenate in Arabidopsis thaliana (El Kassis et al. 2007). Mutational studies with SULTR1;2 knockouts showed that this protein acts as the major selenate transporter in Arabidopsis. Interestingly, Se import is also accelerated during Chloroplast SeMet

SehoCys

SehoCys

• Se competes with S and

Leaf

via

sulfate

transporter

SMT DMSe

Me-SeMet

Me-SeCys

SeCys SMT

DMDSe

transported

SL

A,T Se(0)

Protein

and SeMet in protein chain

SeCys CS

induces toxicity

OAS

SeO32–

• Methylated forms of SeCys APR

A,T

APSe

and

SeMet

are

safely

accumulated APS

SeO42–

Cytoplasm

• Misincorporation of SeCys

A,T

• DMSe and DMDSe are volatilized into atmosphere

Xylem • APS, APR, SMT enhanced

Organic Se

Se

Roots

DMSe SeO32–

tolerance

(T)

and

accumulation (A) in plants

SeO42– • Se affects S and N metabo-

ST

lism at the level of OAS (Competes with SO42–) SeO42–

Soil

Figure 7.1 Se metabolism within plant cells. Source: Extracted from Gupta and Gupta (2017). APS, ATP sulfurylase; APR, APS reductase; CS, cysteine synthase; SL, selenocysteine lyase; SMT, selenocysteine methyltransferase; A, accumulation; T, tolerance.

7.3 Physiological Roles of Se

phosphate deficiency in the plant, indicating the participation of phosphate transporters in mediating Se transport (Li et al. 2008). Within plants, Se is converted to selenite via the catalysis of ATP sulfurylase (APS) and APS reductase (APR). Adenosine phosphoselenate formed by APS is reduced to selenite by APR (Sors et al. 2005). Sulfite reductase reduces selenite to selenide. Glutathiones (GSHs) and glutaredoxins can also catalyze this step (Wallenberg et al. 2010). Cysteine synthase couples selenide to o-acetyl serine to form selenocysteine (SeCys). Brassica oleracea var. italica accumulates large amounts of Se and incorporates it in the form of SeCys and selenomethionine (SeMet) (catalyzed by selenocysteine methyltransferase) into amino acids to form selenoamino acids (Lyi et al. 2005). Se toxicity at high concentrations can also be due to misincorporations of SeCys and SeMet in proteins, resulting in abnormal protein folding (Pilon-Smits and Quinn 2010). Excess SeMet is volatilized as nontoxic dimethylselenide in nonaccumulators and as dimethyldiselenide in accumulators (Pilon-Smits and Quinn 2010) (Figure 7.1).

7.3 Physiological Roles of Se 7.3.1

Se as Plant Growth Promoters

Se acts as a beneficial trace element in higher plants like ryegrass (Lolium perenne) and lettuce (Lactuca sativa) at the very low soil concentration of 0.1 mg kg−1 (Xue et al. 2001). Similar observations were reported in the case of Glycine max. Se delayed senescence by promoting the growth of aging seedlings (Djanaguiraman et al. 2005). The growth and dry matter yield in Brassica juncea were stimulated by selenite (Hasanuzzaman et al. 2010). Chen and Sung (2001) reported that the selenite priming of bitter gourd (Momordica charantia) seeds resulted in higher germination percentage even at unfavorable temperatures. This indicates the capacity of Se to induce the antioxidant machinery of the seeds to tolerate the stress conditions. Foliar application of sodium selenate on barley seedlings at 20 g Se ha−1 increased the accumulation of Se in grains and straws. Spraying of Se solutions improved the growth pattern in ryegrass, lettuce, potato, and green tea leaves. Such developments were due to the increased accumulation of starch in chloroplasts and protection of the cellular components (Hasanuzzaman et al. 2010). Soil fertilization with Se imposes diverse effects on the root system in lettuce plants (Simojoki 2003). The basal and lateral roots exhibited reduced length and surface area on interaction with moderate amounts of Se. However, large additions of Se to the soil promoted a specific volume of the roots (Simojoki 2003). Se increased the photoassimilate allocation for potato tuber growth and created a strong sink for carbohydrates in the young upper leaves, stolons, roots, and tubers (Turakainen 2007). The antioxidant properties of Se delayed senescence in the potato plants (Turakainen 2007). The yield of Cucurbita pepo plants also increased significantly upon treatment with Se (Germ et al. 2005). 7.3.2

The Antioxidant Properties of Se

ROS are generated in almost all kinds of abiotic stresses. The plant succumbs to these toxic molecules if they are not promptly eliminated and neutralized by the cellular

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processes (Banerjee and Roychoudhury 2016a). Stress-sensitive plant species do not possess elaborate antioxidant machineries that are efficient enough to ameliorate the toxic effects by scavenging ROS (Banerjee et al. 2017). The uncontrolled cellular accumulation of ROS degrades proteins and triggers lipid peroxidation in crucial membrane systems (Banerjee and Roychoudhury 2017a). Application of selenate at low concentrations promoted increased activities of antioxidant enzymes like ascorbate peroxidase (APX) and glutathione peroxidase (GPX) along with elevated accumulation of the nonenzymatic antioxidants ascorbate (AsA) and GSH in lettuce plants (Rios et al. 2009). As a result the plants exhibited lower production of H2 O2 . Selenite treatment was proved to enhance the toxic effects as the lettuce plants showed higher accumulation of H2 O2 and malondialdehyde (MDA) compared with the nontreated plants (Rios et al. 2009). Wheat plants treated with Se at 0.05 mM kg−1 exhibited increased activities of catalase (CAT) and peroxidase (POD). However, in agreement with our previous discussion, higher Se concentrations of 0.15 and 0.45 mM kg−1 reduced the activities of both of the antioxidant enzymes (Nowak et al. 2004). Thus at high concentrations, Se acts as a pro-oxidant and so it is necessary to optimize the application dose of Se to achieve agricultural benefits. The Se-treated wheat plants also showed a substantial rise in nitrate reductase activity during the late developmental stages. This is possibly due to the incorporation of SeCys into a NADPH-bound active site. SeCys (having greater nucleophilic power and lower pK than Cys) increased the redox potential of the associated enzymes (Nowak et al. 2004). Similarly, concentrations of 0.1 and 1.0 mg kg−1 Se reduced lipid peroxidation in ryegrass (Hartikainen et al. 2000). Xue et al. (2001) showed that Se delays senescing by stabilizing the 𝛼-tocopherol content and triggering the activity of superoxide dismutase (SOD). Cartes et al. (2005, 2010) reported increased GPX and reduced lipid peroxidation in higher plants grown in seleniferous soils. The presence of a Se-dependent GPX in plants has also been suggested. Se also affected the activities of CAT and glutathione-S-transferase in sorrel plants exposed to salt stress (Hasanuzzaman et al. 2010).

7.4 Se Ameliorating Abiotic Stresses in Plants In view of the potential antioxidative roles of Se in plants, it is evident that this nonmetal micronutrient can alleviate major abiotic stresses causing global crop losses (Banerjee and Roychoudhury 2015; Banerjee and Roychoudhury 2017b). In the following sections we have highlighted the reports that validate Se as an agent conferring multiple-stress tolerance in plants (Figure 7.2). 7.4.1

Se and Salt Stress

Kong et al. (2005) showed increased activities of POD and SOD along with the accumulation of water-soluble sugar in the leaves of Se-treated (1–5 μM) sorrel seedlings exposed to salt stress. However adverse results were observed on treatment of the seedlings with 10–30 μM Se (Kong et al. 2005). Se application at concentrations of 5 and 10 μM stimulated the growth rate and levels of photosynthetic pigments and proline in the leaves of cucumber plants subjected to salt stress. This along with reduced lipid peroxidation promoted salt tolerance in the Se-treated seedlings (Hawrylak-Nowak 2009).

7.4 Se Ameliorating Abiotic Stresses in Plants

Se

Nonenzymatic antioxidants

Enzymatic antioxidants

AsA

Proline

SOD

GPX

GSH

Sugars

CAT

POD

APX

GST

α-tocopherols

Chlorophyll damage

Protein degradation

Lipid peroxidation

Necrosis ROS

Membrane damage

Electrolytic leakage

Figure 7.2 Se alleviates oxidative stresses in plants by efficiently activating the antioxidant system. Exogenous treatment of Se triggers the accumulation of nonenzymatic antioxidants like ascorbate, glutathione, soluble sugars, proline, and 𝛼-tocopherols. The activities of antioxidant enzymes like glutathione peroxidase, catalase, superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione-S-transferase are also enhanced. This confers systemic protection against the tissue-damaging effects of reactive oxygen species.

Hasanuzzaman et al. (2010) reported that Se induced tolerance in stressed seedlings by inhibiting MDA and H2 O2 production, and by stimulating the accumulation of AsA and GSH. Proline is an essential osmolyte which equilibrates cellular homeostasis in plants exposed to stress (Roychoudhury et al. 2015). Increased proline level was observed in Se-treated soybean seedlings (Djanaguiraman et al. 2005). This indicates the existence of direct correlation between Se and proline signaling under stress conditions. Compared with chloride-induced salinity, sulfate salinity drastically inhibited Se uptake by plants. This is due to competition of sulfate with Se to be absorbed by plant roots. Such a decrease in Se uptake is rarely observed during chloride-induced salt stress (Hasanuzzaman et al. 2010). 7.4.2

Se and Drought Stress

Drought stress heralds diverse biochemical and physiological alterations in plant species (Roychoudhury and Banerjee 2016). The ameliorative roles of Se in drought stress have been less investigated. Stomatal conductance that was decreased during drought stress

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in common buckweed (Fagopyrum esculentum) plants was significantly restored after supplementation with Se. The treated plants also exhibited better water management, which resulted in higher photochemical efficiency of photosystem II (PS II) compared with the control plants exposed to stress (Tadina et al. 2007). Exogenous application of Se at concentrations of 1.0 and 2.0 mg kg−1 promoted biomass accumulation, root activity, carotenoid, chlorophyll and proline contents, and POD and CAT activities in wheat plants exposed to drought. These plants also accumulated low amounts of MDA (Yao et al. 2009; Xiaoqin et al. 2009). 7.4.3

Se Counteracting Low-temperature Stress

Crops grown at high altitudinal and latitudinal locations often succumb to low temperatures. Chilling triggers the formation of intercellular water crystals which disrupt the cellular physiology. Massive membrane damage owing to reduced fluidity also affects the integrity of the cells (Banerjee and Roychoudhury 2016a). In wheat plants exposed to cold stress, Se treatments at 1.0 mg kg−1 reduced the MDA content and ROS accumulation via an increase in anthocyanins, flavonoids, and phenolics. Activities of antioxidant enzymes like POD and CAT were also enhanced. This led to declining peroxidation of the membrane lipids (Chu et al. 2010). The study clearly illustrated the utility of Se as an agent to promote ecological adaptation of wheat seedlings to cold conditions. Exogenous application of Se (2.5–10.0 μM) in cucumber seedlings exposed to short-term chilling stress resulted in an increase in leaf proline content and decreased the rate of peroxidation in the membrane systems of the roots (Hawrylak-Nowak et al. 2010). Thus Se recharges the antioxidant machinery in plants and promotes adaptive development during chilling stress. Investigations on more plant species should be carried out to establish the ameliorative roles of this trace element. 7.4.4

Se Ameliorating the Effects of UV-B Irradiation

Stratospheric ozone forms a protective layer against atmospheric UV-B radiation, which is a serious threat to the existence of human kind. Depletion of the ozone layer owing to uncontrolled anthropogenic activities has facilitated UV-B rays reaching the Earth’s surface and caused rapid deterioration in crop and plant physiology. Exposure to such harmful radiation negatively affects photosynthesis, respiration potential and transpiration (Banerjee and Roychoudhury 2016b). The optimum amount of Se recharged the antioxidant machinery in wheat plants, which led to efficient scavenging of ROS and reduced membrane lipid peroxidation and MDA content during exposure to UV-B rays (Yao et al. 2010a,b). Se treatment even ameliorated damaging effects in the plants grown under harmful short-wavelength light. The plants exhibited higher activities of GPX and CAT. Interestingly APX activity did not increase, although the enzyme shares a common substrate with GPX and CAT (Xue and Hartikainen 2000). Se specifically increases GPX activity in plants exposed to stress and this indicates the presence of Se-dependent GPX isoform in the plant systems. Apart from GPX, Se induced CAT and SOD activities in UV-exposed lettuce and ryegrass respectively (Hasanuzzaman et al. 2010). Similar treatments under UV stress triggered the activities of POD and SOD in wheat roots (Yao et al. 2010b). Tobacco plants

7.5 Conclusion

subjected to ozone stress revealed an induction of cytosolic Cu/Zn SOD and cytosolic APX (Willekens et al. 1994). These results thoroughly prove the potential of Se to alleviate radiation and oxidative stresses in both the aerial and underground biomasses in plant species. 7.4.5

Se and Heavy Metal Stress

Heavy metal (HM) toxicity is an edaphic condition that severely deteriorates plant development by inducing necrosis via ROS accumulation (Gupta and Gupta 2017; Liu et al. 2018). The beneficial roles of Se in protecting Brassica napus from cadmium (Cd) and lead (Pb) toxicity have been verified (Wu et al. 2016). Exogenous application of 5–15 mg kg−1 Se reduced Cd and Pb accumulation in the roots and shoots. The treatment alleviated oxidative damage by scavenging ROS and protected membranes from lipid peroxidation. The plants also exhibited increased activities of antioxidant enzymes like SOD and GPX (Wu et al. 2016). Reduced production of ROS was reported in Cd-stressed marine algae after treatment with 50 μM Se (Kumar et al. 2012). As little as 2 μM Se maintained the structure and fluidity of the chloroplast membranes in rape seedlings exposed to Cd stress. Similar beneficial effects of Se were reported in the case of wheat seedlings subjected to Cd toxicity (Filek et al. 2009). A 1.5 μM Se solution alleviated Pb stress in the Vicia faba seedlings. However, 6 μM Se proved to be toxic and decreased cell viability in the seedlings (Mroczek-Zdyrska and Wojcik 2012). Exogenous application of Se also reduced the ROS levels in Phaseolus aureus (mungbean) seedlings grown in arsenic (As) contaminated medium (Malik et al. 2012). The protective roles of Se during HM stress can be attributed to the induction of the activities of antioxidant enzymes, metabolic channelization to equilibrate the cellular osmotic homeostasis, reduction in electrolytic leakage and improvements in cell integrity (Malik et al. 2012). Se also promoted the uptake of iron (Fe), which is an important constituent of the chloroplast and the photosynthetic electron chain transport (Gupta and Gupta 2017). Thus Se confers beneficial effects on plants subjected to HM toxicity. However, the alleviation strategy is similar to that in case of other environmental stresses. The antioxidant machinery is efficiently activated to restore the plant after oxidative damage.

7.5 Conclusion Se is a nonmetallic micronutrient that is beneficial to plants at very low concentrations. However, the optimum concentration varies with the plant species. At high concentrations, Se triggers oxidative stress in the tissues. Thus the antioxidant roles of Se in plants are often capricious and enigmatic. Se reportedly shuttles metabolic equivalents during abiotic stresses to activate the antioxidant machinery (Figure 7.2). This induction plays a pivotal role in alleviating ROS-induced damage in plant parts. The physiological processes like photosynthesis, respiration and photosynthetic organelles like chloroplasts are also protected by Se during harsh environmental conditions. Thus Se also appears to have adopted the nature and qualities of the Greek moon goddess “Selene” along with her name.

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7.6 Future Perspectives The chapter is a concise discussion of the origin, metabolism, and beneficial effects of Se in the plant system. The stress-ameliorative effects of Se clearly highlight that exogenous application of this trace element can confer multiple-stress tolerance in a particular plant species. This would be an economically favorable and agriculturally profitable strategy. However, the exact biochemistry of Se in correlation with S metabolism in plants is largely unknown. Metabolomic crosstalks at the global scale need to be identified to elucidate this aspect. The effects of Se on the kinetics of antioxidant enzymes need more exhaustive investigations. Future perspectives should also involve the detection of the phytohormones that act as the messengers for Se-mediated signal transduction. The crucial components of this signaling pathway are also unknown. As we have discussed, at high concentrations, Se is toxic for the system. Hence identification of putative Se exporters should be performed so that excess Se does not accumulate in the edible plant parts owing to bioconcentration. This can lead to biomagnification of Se down the food chain, causing direct harm to consumers (humans and animals).

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8 Polyamines Ameliorate Oxidative Stress by Regulating Antioxidant Systems and Interacting with Plant Growth Regulators Prabal Das 1 , Aditya Banerjee 2 , and Aryadeep Roychoudhury 2 1

Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, West Bengal, India Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata, 700016, West Bengal, India 2

8.1 Introduction Abiotic stresses like salinity, drought, heat, cold, light, and heavy metal toxicity severely affect worldwide crop production (Banerjee and Roychoudhury 2017a,b, 2018a,b), resulting in the loss of a large proportion of agricultural investments. Plants have evolved intricate molecular mechanisms to counteract suboptimal conditions. Such stress-responsive strategies include a robust phytohormone-mediated signaling system synchronized with the accumulation of multiple antioxidants and compatible solutes (Roychoudhury and Banerjee 2015, 2016; Banerjee and Roychoudhury 2016a). The antioxidants mainly scavenge harmful reactive oxygen species (ROS) like hydrogen peroxide (H2 O2 ), superoxide, and hydroxyl radicals which are formed within the cell during intense oxidative stress (Arif et al. 2016; Banerjee and Roychoudhury 2018c). Compatible solutes are a broad class of molecules that maintain the cellular homeostasis and help in equilibrating the cellular environment in the presence of stressors (Banerjee and Roychoudhury 2017c; Banerjee et al. 2016). Polyamines (PAs) are positively charged, low-molecular-weight organic molecules. They are probably the best characterized compatible solutes along with proline (Roychoudhury et al. 2015). Three major types of PAs have been reported in plants, viz., tetra-amine spermine (Spm), tri-amine spermidine (Spd) and their diamine precursor putrescine (Put) (Liu et al. 2015). Put is synthesized from ornithine or arginine by the activities of the enzymes ornithine decarboxylase or arginine decarboxylase (ADC), respectively, and thereafter by sequential activities of agmatine iminohydrolase (AIH) and N-carbamoyl Put amidohydrolase (CPA), respectively (Kusano et al. 2008). Spd synthase (SPDS) catalyzes the conversion of Spd from Put where the aminopropyl group donor is the decarboxylated S-adenosylmethionine (dcSAM). Spm synthase (SPMS) converts Spd to Spm. dcSAM is synthesized by the action of S-adenosylmethionine decarboxylase on S-adenosyl-methionine, which is again synthesized through S-adenosylmethionine synthetase or methionine adenosyltransferase from methionine (Wimalasekera et al. 2011). Thus, endogenous PA level in plants is regulated by these enzymes during growth and development as well as under stressful conditions. Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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8.2 PAs as Cellular Antioxidants 8.2.1

PAs Scavenge Reactive Oxygen Species

The ROS production in a cell is a highly regulated mechanism and is balanced by its synthesis and subsequent detoxification. In unstressed conditions, ROS is produced in the cell, but during adverse conditions their synthesis is escalated, resulting in excess ROS production and accumulation which is detrimental to the cell (Biswas and Mano 2015; Liu et al. 2018). PAs have been reported to limit ROS production in two ways. They may hinder the auto-oxidation of metals, thereby curtailing the flow of free electrons for the generation of ROS (Banerjee and Roychoudhury 2018c). They may also up regulate the antioxidant machinery of plants under stress conditions, thereby enhancing tolerance toward abiotic stress. In this regard, a number of reports have been documented where the priming of plants with PAs increased endogenous PA levels, which resulted in elevated stress tolerance (Roychoudhury et al. 2011). The exogenous Put application in drought-stressed wheat decreased membrane damage, maintained photosynthetic parameters, and increased soluble sugar, proline, and total amino acid contents along with an increase in total yield (Gupta et al. 2012). Pre-treatment with 1 mM Put significantly reduced water loss and maintained PSII efficiency in detached tobacco leaf disks subjected to polyethylene glycol (25%) treatment. Tanou et al. (2014) reported a positive effect of PAs in alleviating NaCl toxicity in Citrus aurantium by reducing the nitration of tyrosine and protein carbonylation, while increasing protein S-nitrosylation. PAs modulated the oxidative status through up regulation of genes related to the antioxidant enzymatic pathway. Exogenous application of PAs was found to ameliorate drought and salinity stress in Bermuda grass (Cynodon dactylon) by increasing the levels of antioxidant enzymes and different stress-associated proteins. Put application enhanced tolerance to high temperatures (35 ± 2 ∘ C for 4–8 h) in wheat plants (Hassanein et al. 2013). Spd application for 1 week before NaCl treatment in rice markedly enhanced grain yield and calcium content and lowered Na+ /K+ ratio (Saleethong et al. 2011). Co-application of Spd with NaCl in Panax ginseng seedlings showed an elevated antioxidant pathway, resulting in decreased peroxide and superoxide formation (Parvin et al. 2014). Spd treatment in rice seedlings ameliorated heat stress injury by elevating the activities of antioxidant enzymes and antioxidant levels (Mostofa et al. 2014). The ameliorative effects of PAs were reported earlier from soybean (Radhakrishnan and Lee 2013) and cucumber (Shu et al. 2013). Put was reported to increase the growth, biomass, carotenoids, and antioxidant enzyme activities of salinity-induced leaf tissues of Brassica juncea, in concert with a decline in H2 O2 , malondialdehyde and electrolyte leakage (Verma and Mishra 2005). Spd application resulted in an escalation of free Spd and Spm in the leaves of drought-sensitive wheat seedlings which was associated with amelioration of polyethylene glycol-mediated stress injury (Liu et al. 2015). To establish the ameliorative effects of PAs in ROS detoxification, inhibitors of PA biosynthetic enzymes were used. Application of d-arginine (d-Arg) showed a decline in endogenous PA contents which was concomitant with a rise in ROS level (Banerjee and Roychoudhury 2018c). From this work, it was clear that PAs mitigate oxidative stress by modulating the antioxidant defense system, accompanied by an alteration in redox balance and ROS detoxification (Tanou et al. 2014). A number of research works have been documented regarding the interrelation between PAs and ROS in plants (Gill and

8.3 The Relationship Between PAs and Growth Regulators

Tuteja 2010). In general, the elevation in PA level is associated with an increase in its catabolism and simultaneously the H2 O2 production increases. ROS have a dual role in cellular systems. On the one hand, they participate in signal transduction pathway to induce resistance (Moschou et al. 2012), while on the other hand, higher accumulation of ROS damages membranes, destroy chlorophyll, etc. Thus, while PAs have been ascribed a cell protection role (Tanou et al. 2014), they can also induce toxic effects to the cell by the production of H2 O2 , as evidenced in tobacco (Mano 2012). 8.2.2

The Co-operative Biosynthesis of PAs and Proline

The interrelationship between PAs and proline is very interesting and they share a common precursor, i.e. glutamate (Glu) (Verslues and Sharma 2010). Under abiotic stress conditions, PA biosynthesis is enhanced, resulting in an increased flux of Glu toward ornithine (Orn) and Arg. Orn and Arg are substrates of ornithine decarboxylase and ADC enzyme that help in Put biosynthesis. These reactions simultaneously up regulate proline levels in the cell. However, the exact mechanism is not yet fully understood. It may come directly from Glu by pyrroline-5-carboxylate synthetase or from Orn with the help of ornithine aminotransferase. Apart from proline, the 𝛾-amino butyric acid (GABA) content is also found to be related to PAs and proline. GABA biosynthesis in cell is mediated via two pathways, one from Glu by the enzyme glutamate decarboxylase and the second one from Put by diamine oxidase. Another metabolite of this pathway is nitric oxide (NO), which shares the common pathway. NO level is found to be increased during various abiotic stresses (Wimalasekera et al. 2011). The complex network of abscisic acid (ABA), NO, and PAs has found to be very important in modulating abiotic stress tolerance. PA accumulation is also regulated by ABA induction (Alcazar et al. 2010) and NO synthesis via PA degradation by PA oxidase (PAO) (Wimalasekera et al. 2011), which revealed the interrelationship between these three factors in stress tolerance.

8.3 The Relationship Between PAs and Growth Regulators PAs, being major growth regulators themselves, also interact with other plant growth regulators under challenging situations. 8.3.1

Brassinosteroids and PAs

Brassinosteroids (BRs) regulate plant growth and development during different stress responses, either alone or in association with ABA, cytokinin, auxin, salicylic acid (SA), jasmonic acid, gibberellins, and ethylene. A relationship between PA and BRs was established by the fact that epibrassinolide application ameliorates copper toxicity in Raphanus sativus (Choudhary et al. 2012) and also showed an influential role in regulating the role of PAs. Addition of a brassinosteroid analog maintained the PA level in salt-stressed lettuce plants and helped in the recovery from stressed conditions (Serna et al. 2015). 8.3.2

Ethylene and PAs

Ethylene and PAs have been found to be antagonistic to each other. PAs are associated with senescence inhibition, whereas ethylene promotes it (Kumar and Rajam 2004).

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The common substrate for both PA and ethylene synthesis is S-adenosyl methionine, which in turn is required for the biosynthesis of 1 aminocyclopropane-1-carboxylic acid (ACC), which is the precursor molecule for ethylene production. It was observed in tomato fruit that Spm controls ethylene production by blocking ACC synthase transcript accumulation (Alexander and Gierson 2002). In Arabidopsis, ethylene inhibited the activities of the enzymes ADC and SAMDC (Kumar and Rajam 2004). The correlation between ethylene-induced H2 O2 production and PAO activity was demonstrated from Arabidopsis guard cells, where elevated transcript levels of AtPAO2 and AtPAO4 genes resulted in higher activity of PAO and subsequently more H2 O2 production (Hou et al. 2013). 8.3.3

Salicylic Acid and PAs

Salicylic acid has very important role in the induction of plant defenses. However, the interrelationship between salicylic acid and PA contents has been less reported. It is also clear that SA application modulates PA metabolism in a dose-dependent manner (Wang and Zhang 2012). Furthermore, seed priming with Spm or Spd was reported to be very effective in elevating the SA level in salt-stressed wheat plants (Iqbal et al. 2006). In tobacco, SA-induced protein kinase cascade has an important function in transcriptional regulation of the genes involved in biosynthesis of Put (Jang et al. 2009). On the basis of these findings, it is evident that a connection exists between endogenous PA level and SA contents, although the exact mechanism is still elusive. 8.3.4

Abscisic Acid and PAs

Abscisic acid is considered as the universal stress phytohormone (Roychoudhury and Banerjee 2017) and an increase in endogenous ABA level owing to various abiotic stress factors is a well-known phenomenon (Banerjee and Roychoudhury 2016b). ABA confers water, salinity, and extreme temperature tolerance by inducing the expression of multiple genes related to stress defense (Danquah et al. 2014). ABA synthesis occurs in parenchyma cells or root tip cells from carotenoid precursors after induction by salt and drought (Paul et al. 2017). Following synthesis in the roots, ABA enters the xylem and is thereby transported to the leaves. This is very critical for stress adaptation, since the root is in direct contact with the rhizosphere and counteracts various stresses (Banerjee and Roychoudhury 2015). Drought activates both ABA-dependent and ABA-independent pathways. ABA induces PAO activity and thereby regulates PA catabolism (Guo et al. 2014), while PAs trigger ABA synthesis (Marco et al. 2011). Under a stressful environment, transgenic Lotus tenuis plants overexpressing ADC gene showed regulation of 9-cis-epoxycarotenoid dioxygenase gene (NCED), which encodes the primary enzyme related to ABA biosynthesis, through Put accumulation. In addition, inhibition of expression of ADC gene resulted in reduced expression of NCED3 and other ABA-regulated genes (Espasandin et al. 2014). Put was found to control the endogenous ABA concentration in Arabidopsis plants growing under low-temperature conditions (Cuevas et al. 2008). Arabidopsis plants were found to contain double genes for ADC (i.e. ADC1, ADC2), SPDS (i.e. SPDS1, SPDS2), and SPMS (i.e. SPMS and ACL5) (Panicot et al. 2002). Application of exogenous ABA was used to investigate the effect of ABA on the

8.4 Conclusion and Future Perspectives

PA biosynthetic pathway. The ABA-mediated up regulation of PA biosynthesis in Arabidopsis thaliana plants subjected to water deficit and salinity stress has been reported (Alcázar et al. 2006). Exogenous ABA application modulated the expression of the genes ADC2 and SPMS (Urano et al. 2003). Among the different PA biosynthetic genes (ADC1, ADC2, AIH, ACL5, SPDS1, SPDS2, CPA, SPMS, SAMDC1, and SAMDC2) studied from wild-type and mutated (aba2-3, abi1-1) A. thaliana, the ADC2, SPDS1, and SPMS genes exhibited maximum expression under drought stress (Alcázar et al. 2006). The ABA-responsive element or ABRE related motifs were also detected in the promoters of ADC2, SPDS1, and SPMS genes which are highly up regulated under drought conditions (Alcázar et al. 2006; Basu et al. 2014). Moreover, studies on the Arabidopsis ABA-deficient (aba2-3) and ABA-insensitive (abi1-1) mutants showed smaller increments in ADC2, SPDS1, and SPMS gene expression (Alcázar et al. 2006), indicating the possible role of ABA in transcriptional up regulation of these three biosynthetic genes under drought conditions. Moreover, endogenous Put levels were also enhanced under drought in wild-type Arabidopsis plants. The primary transcription factor associated with signal transduction during drought and osmotic stress is ABRE-binding factor (ABF) (Yoshida et al. 2015). In Poncirus trifoliata, PtrABF, which is an ABF4 homolog, interacts with the ABREs and modulates the expression of ADC gene. The up regulation of PtrABF assisted in more synthesis of ADC transcripts, resulting in the escalation of endogenous Put. In addition, ADC inhibitor application decreased the Put level (Zhang et al. 2015). All of these observations make it clear that ABF regulates PA biosynthesis by modulating the expression of ADC gene.

8.4 Conclusion and Future Perspectives Environmental stresses increase the oxidative load within the plant tissues, contributing to severe perturbations in the overall ecosystem and reducing plant yield. Agricultural pursuits are also severely hampered, causing large-scale global economic losses. In order to tackle such abiotic stresses, plants trigger the accumulation of endogenous antioxidants and compatible solutes. PAs are one such class of molecules; they effectively scavenge the toxic ROS and also interact with the signaling pathways of multiple growth regulators to contribute to stress tolerance. Exogenous application of PAs ameliorates salinity, drought, heavy metal toxicity, etc., in susceptible plant cultivars. At the molecular level, PAs interact with DNA and membranes and protect them from oxidative damage. Excerpts regarding the interactions of PAs with growth regulators like BRs, SA, and ethylene have been briefly highlighted. The elaborate association and signaling co-operation between PAs and ABA largely control systemic signaling during abiotic stress. The chapter highlights the potential of PAs as efficient alleviators of oxidative stress. Future perspectives include overexpression of PA biosynthetic genes and studying its effect on the fluctuations of BRs, SA, ethylene, and other hormones like jasmonic acid, etc. A systemic blueprint should be designed to understand the less-understood signaling synchronization among PAs and phytohormones. The impact of PAs on the epigenomic status of the plants should also be investigated, since such meta-stable alterations have been found to regulate abiotic stresses (Banerjee et al. 2017; Banerjee and Roychoudhury 2018d). In the context of the immense stress ameliorative potential, PA research is rapidly expanding among scientific communities with novel endeavors.

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Acknowledgments Financial support from the Council of Scientific and Industrial Research, Government of India through the major grant [38(1387)/14/EMR-II] to Dr Aryadeep Roychoudhury is gratefully acknowledged. The University Grants Commission, Government of India is acknowledged for providing a Junior Research Fellowship to Mr Aditya Banerjee.

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9 Abscisic Acid in Abiotic Stress-responsive Gene Expression Liliane Souza Conceição Tavares 1 , Sávio Pinho dos Reis 1,2 , Deyvid Novaes Marques 1,3 , Eraldo José Madureira Tavares 4 , Solange da Cunha Ferreira 1,5 , Francinilson Meireles Coelho 1,3 , and Cláudia Regina Batista de Souza 1 1 Universidade Federal do Pará, Belém, PA 66.075-110, Brazil 2

Universidade do Estado do Pará, Marabá, PA 68.502-100, Brazil Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Pará, Belém, PA 66.075-110, Brazil 4 Empresa Brasileira de Pesquisa Agropecuária, Petrolina, PE 56.302-970, Brazil 5 Programa de Pós-Graduação em Agronomia, Universidade Federal Rural da Amazônia, Belém, PA 66.077-830, Brazil 3

9.1 Introduction The role of the phytohormone abscisic acid (ABA) in plant resistance to abiotic stress has long been investigated. ABA is well known for its crucial involvement in controlling stomatal aperture to prevent water loss by evaporation, thus protecting against drought stress (Tuteja 2007). Water deficit induces ABA accumulation, which leads to stomatal closure (Mittler and Blumwald 2015), which decreases photosynthesis (Scandalios 1993) and increases the level of reactive oxygen species (ROS) (Carvalho 2008). In reality, the hormone does much more than that. Intriguingly, ABA is so versatile that it acts in reponse to several major sources of abiotic stress factors, such as heavy metals, high salinity, heat, cold and radiation (Vishwakarma et al. 2017). The ABA pathway is capable of distinguishing ABA resulting from the different stressors and gives the correct response (Sewelam et al. 2016). In addition, ABA responds to ROS (Mittler and Blumwald 2015), which work as secondary messengers (Sharma et al. 2012). The machinery for such a sophisticated control is not simple. With the dramatic increase in our knowledge in the -omics, transgenic technologies, systems biology, and evolution of life, our understanding of the role of ABA is growing rapidly (Bendall 1983; Orgel 1973; Hauser et al. 2011; Mittler and Blumwald 2015). Many technological breakthroughs in crop production could come in the future decades (Oh et al. 2012; Vishwakarma et al. 2017). The ABA pathway and control machinery are very complex. Recent advances are making the role of ABA easier to understand. A core pathway of ABA signaling has been discovered. It consists of three components: (i) receptor proteins (PYR/PYL/RCAR); (ii) protein phosphatases (PP2C); and (iii) protein kinases (SnRK2) (Umezawa 2011). In normal conditions, PP2C inhibits SnRK2 via direct dephosphorylation. ABA enables the receptors PYR/PYL/RCAR to bind and sequester PP2C. The kinase SnRK2 Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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Abiotic factors affecting plants

Figure 9.1 Regulation of the plant abiotic stress response modulated by abscisic acid (ABA)-mediated gene expression.

Endogenous ABA levels differentially modulated by abiotic stresses

Induction of ABA-mediated plant signal transduction pathways

Interaction of transcription factors with cis-regulatory elements responsive to abiotic stresses in promoters of plant genes

Differential expression of genes regulated in response to abiotic stresses

Modulation of plant tolerance

autophosphorylates, becoming activated, and then phosphorylates and activates downstream transcription factors (TFs) (Umezawa 2011; Sheard and Zheng 2009; Roychoudhury and Paul 2012). A major function of this core pathway is the control of abiotic stress. “Stress,” from an evolutionary view point, can be understood as a force shaping evolution and adaptation in environments with changes, and it is a feature of both the stressor and the stressed (Bijlsma and Loeschcke 2005). The most important source of stress is exposure to environmental conditions far from the evolutionary optimum ones for the organism. Extreme environments are usually regarded as being more stressful. In this chapter, we consider the regulation of ABA-mediated gene expression in response to abiotic stress. First, we analyze how the deep evolutionary base helps in understanding abiotic stress and ABA response. Then we summarize the coordinated ABA response from ABA signaling and transduction to modulate gene expression, with a major emphasis on the role of cis-acting elements and transcription factors in stress-inducible promoters. In Figure 9.1, the main steps involved in plant abiotic stress response modulated by ABA-mediated gene expression are depicted.

9.2 Deep Evolutionary Roots Crop plants are being exported to different environments, many of which impose severe selective pressure on the ancestors of modern extremophilic plants. Recent

9.2 Deep Evolutionary Roots

research on extremophilic plants is valuable to understand the evolutionary machinery that confers adaptations (Oh et al. 2012). These plants help us to understand the limits of life (McKay 2014) and how to make effective genetic improvements (Eapen and D’Souza 2005). Life as we know it requires an atom capable of scaffolding biomolecules. Carbon is the main candidate if we think of covalent bonds within the temperature range of liquid water. So, carbon could fit very well on Earth. However, carbon alone is not enough. For instance, hydrogen is important to terminate carbon chains, and heteroatoms such as oxygen and nitrogen are important to confer the reactivity required for Darwinian evolution (Benner et al. 2004). Moreover, phosphorylation involves the substitution of H for PO3 in an -OH or -NH (Kamerlin et al. 2013). When the versatile carbon is combined with these and other atoms, an incredible number of forms and functions can be designed. The second element of choice for scaffolding is silicon. It belongs to the same family of elements as carbon, and the two elements present several similarities. Both can form four bonds and can produce many complex molecules. However, silicon requires different geochemical conditions (Benner et al. 2004). Any element selected as scaffolding by the evolutionary process will have constraints. For instance, in the presence of oxygen, silicon is readily oxidized into silicon dioxide, a nonsoluble solid. Silicon-based life would require an absence of oxygen (Plaxco and Gross 2011). Carbon-based life, despite supporting O2 , is not free from oxygen-related challenges. As Orgel (1973) pointed out, “the organic compounds from which all living things are made are not stable in an oxidizing atmosphere.” This is the factor behind much of the damage caused by abiotic stresses in plants. Many stress factors increase the concentrations of ROS (Das and Roychoudhury 2014; You and Chan 2015; Singh et al. 2015; Arif et al. 2016a,b; Tripathi et al. 2016, 2017a,b; Kumar et al. 2017; Singh et al. 2017). Owing to damage caused by ROS in biomolecules, all aerobic organisms could in this sense be considered extremophiles (Schulze-Makuch and Irwin 2008). Life based on molecules almost certainly also requires a solvent (Plaxco and Gross 2011). The early Earth had water (Zahnle et al. 2010), and a temperature range partially in the range for liquid water. The conditions were thus favorable to the use of water as a solvent (Cui 2010). However, any molecule used as a solvent would impose important constraints on the resulting life form. In the case of water, temperatures outside the range for liquid water are a potential source of strong abiotic stress for nonadapted species. For example, cold weather below 0 ∘ C could freeze the water, damaging cells; extreme heat could inactivate the proteins (Plaxco and Gross 2011). Many organisms resolved the problem of solidification by lowering the freezing point of their intracellular solution with salts and other solutes (McKay 2014). In plants, excessive heat causes an imbalance of osmotic pressure. The overall major consequence of heat stress, however, is the increase in the number of ROS. ABA was shown to induce heat tolerance and to lower the damage from ROS (Hasanuzzaman et al. 2013). Water would dramatically shape the machinery of life. Prebiotic evolution is a bottomup process. Complexity increases from tiny molecules up to complex molecular beings. The primitive molecules could not move actively to find a reactant. Therefore, solvents are useful to increase the probability of those encounters (Benner et al. 2004). The solubility of metabolites in water is important to a life form using water as a solvent (National Research Council 2007), and it is conceivable that many molecules of life were screened or even selected to fit an aqueous environment (Cui 2010). That seems to be one of the reasons why the chemical group OH is so common among organic molecules central to

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metabolism (Benner et al. 2004). Electric charges are also common and useful to provide solubility in water. RNA and DNA are both very soluble because of their repeating charges (National Research Council 2007). Taking into account the above limits of life, we can understand some consequences for the evolution of life and the roles of ABA in abiotic stress. All of the advanced machinery in the ABA pathway can be traced to its evolutionary origin. That was the source of the building blocks of life, and of their reactivity and sensitivity to ROS, among other things. The prebiotic origins of life are not well understood yet (Zahnle et al. 2010). Organic compounds of life are not stable in an oxidizing atmosphere. An organic compound within an oxidizing atmosphere containing O2 will eventually convert into CO2 . On the other hand, a CO2 molecule within a reducing atmosphere containing H2 will eventually get converted into methane. If enough CO2 is added to the reducing atmosphere to consume all H2 but not all CO2 , organic compounds can be formed. The formation of organic compounds requires an atmosphere that is not totally reducing, but reducing enough to produce organic molecules (Orgel 1973). In the long story of life, the persistent selective pressure for adaptation to harmful chemical reactivity has been a major force guiding the evolution of more complex life (Plaxco and Gross 2011; Raymond and Segre 2006). It has been argued that there was little or no free oxygen. The idea is corroborated by mineral deposits of ferrous iron, which are formed under a nonoxidizing atmosphere and were found in early Precambrian times (Orgel 1973). Oxygen can be produced photochemically through UV light. However, the productivity is low and the primitive atmosphere would only contain traces of O2 (Orgel 1973). We can expect that many of the original organic macromolecules were very sensitive to oxygen (Weiss et al. 2016). Scientists seem now to be near to understanding how the original life forms and their molecules evolved. Patel et al. (2015) showed that the precursors of amino acids, ribonucleotides, and lipids can all be derived from hydrogen cyanide and its derivatives, and that all cellular subsystems could have been assembled simultaneously from common simple chemistry. The proposed geochemical scenario for the development of the reaction network involved separate streams and pools. If the hypothesis is correct, the first ancestor of all life on Earth lived in water. The necessity for water was being reinforced. Paradoxically, the toxicity of oxygen helped oxygen-users to win the battle for life via the elimination of many competitors that could not adapt to oxygen (Ebeling and Feistel 2011). This had numerous remarkable benefits for the survivors. For instance, it favored the evolution of aerobic respiration (Soo et al. 2017) and the subsequent more complex life forms (Plaxco and Gross 2011). At a biochemical level, the availability of O2 favored the evolution of new reactions and pathways, and promoted the increase in complexity of cellular biochemical networks (Raymond and Segre 2006). The principal requirement for life gave it its impulse: the use of O2 as the terminal electron acceptor has the benefit of yielding higher amounts of energy in comparison with anaerobic respiration (Scandalios 1993). Finally, the abundant free oxygen facilitated the colonization of the land (Plaxco and Gross 2011): more free oxygen results in more ozone, which increases the absorption of the damaging UV radiation (Mcconnell and Jin 2008). The ensuing aerobic respiration, therefore, occurred in the presence of ROS (Mittler 2017). There was an additional pressure: aerobic metabolism inevitably produces ROS (Sharma et al. 2012; You and Chan 2015). A long time later, the time for plants to conquer the land arrived. However, that would not be an easy fight. The new environment provided new,

9.2 Deep Evolutionary Roots

highly stressful abiotic factors for a water-dependent, carbon-based organism. Several new adaptations would be required. The plants had to find a way to prevent desiccation; a higher exposure to UV radiation would increase DNA damage by the production of ROS; there was decreased access to mineral antioxidants; there was exposure to greater variations of temperature; and the osmotic pressure was increased under saline conditions, among others (Oh et al. 2012). Sophisticated metabolic adaptations arose from this. A major challenge was the adaptation to low water availability. Water could easily vaporize, and the life forms were highly constrained by evolutionary use of it as a solvent. The biochemical evolutionary response was the PYR/RCAR ABA receptor; some of the core ABA signaling components (Hauser et al. 2011) evolved during the colonization of land. However, the creation of new machinery can be expensive. Life does the best it can with what it has. PP2C and SnRK, also part of the core, already existed—fruits of the evolutionary usefulness of phosphates. Both PP2C and SnRK were effectively re-adapted to respond to shortages in the water supply (Hauser et al. 2011). For scientists involved in biotechnology with plants, this historical fact could lead them in new directions. As pointed out by Duboule and Wilkins (1998), “As the characterization of specific genes in specific developmental systems proceeds, we will obtain better insight into the sorts of mutational events that allow preexisting genes to be used for new developmental roles.” The prevention of dehydration intensified the fight against ROS. The closure of stomata increases ROS concentration (Das and Roychoudhury 2014). Oxidative stress may be even more challenging for plants than for other eukaryotes because plants not only consume but also produce O2 (Scandalios 1993). Intriguingly, however, plants produce ROS to use them as secondary messengers. Several primordial toxins became instrumental in signaling processes of plants, including ROS, nitric oxide and hydrogen sulfide (Hancock 2017). A major discovery was that ABA activates the synthesis of H2 O2 , and H2 O2 mediates ABA-induced stomatal closure (Pei et al. 2000; Neill et al. 2002). The ROS have properties that favor the evolution of a messenger. ROS can oxidise proteins, changing their activity. Protein phosphorylation relays and transcription factors are examples (Inupakutika et al. 2016). An initial step was the evolution of a network that could keep ROS levels under control. Later, ROS could be used as signal transduction messengers (Inupakutika et al. 2016). The first use of ROS as signaling was to sense toxic levels of atmospheric O2 (Mittler 2017), and later they evolved into important regulators in plants and several other groups (Mittler 2017). The antioxidative system keeps ROS at a basal level, and deviations from this could be used for signaling (Mittler 2017; Banerjee and Roychoudhury 2017a). In a broad sense, this is why the ABA pathway uses ROS as secondary messengers. Despite the adaptations, the benefits of ROS in modern plants still depend on their concentration. Uncontrolled production of ROS seriously damages tissues and macromolecules to the point of death. Such fine-tuned control requires sophisticated defense mechanisms. The excessive ROS are scavenged by an antioxidative defense system composed of several enzymes and compounds (Noctor and Foyer 1998; Das and Roychoudhury 2014). Sometimes the defense systems may be defeated. Stress factors like drought, salinity, heavy metals, cold, and UV irradiation cause increased levels of ROS (Sharma et al. 2012; Das and Roychoudhury 2014; Singh et al. 2015; Arif et al. 2016a,b; Tripathi et al. 2016, 2017a,b; Singh et al. 2017). Interestingly, ABA is known to act toward all of those stress

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factors (Vishwakarma et al. 2017). Among the reactive toxins that life had to deal with were the metals. Much of the current toxicity in transition metals comes from oxidative stress (Enriquez and Do 2012). Before the rise of O2 , ions like Fe2+ , Mn2+ and Mo6+ were abundant in the oceans. With the rise of oxygen, soluble iron (Fe2+ ) reacted with O2 , producing insoluble iron oxides. As a result, the availability of soluble Fe2+ decreased. However, the remaining Fe2+ is dangerous in the presence of oxygen. Fe2+ reacts with H2 O2 , producing the reactive hydroxyl radical (⋅OH). A critical solution was to control the metal ions (Enriquez and Do 2012). A good example of this is the protein group ferritin (Enriquez and Do 2012). This protein resolved at once the problems of solubility and toxicity of iron in the presence of oxygen. Thousands of iron atoms are sequestered in the central cavity of the protein (Majerus et al. 2009). In aerobic soils, iron forms the insoluble ferric hydroxide and has low availability. However, under anaerobic conditions such as in submerged soils, the toxicity from ferrous iron may be important (Majerus et al. 2009). As a response, iron induces the production of ferritin. It was shown that ABA induces the biosynthesis of ferritin in maize (Lobréaux et al. 1993). However, this role of ABA may depend on the species (Majerus et al. 2009). Currently, other major sources of abiotic stress still demand attention in crops. Problems with water supply are an important cause of casualties. From a physiological view point, the mechanisms responsible for plant mortality by drought are still poorly understood (McDowell et al. 2008; McDowell 2011), but two major hypothesis have been proposed (Saiki et al. 2016). One of them is the hydraulic failure: with a dry soil and high evaporation rate, the xylem conduits and rhizosphere become filled with air, interrupting the water flow. The concurrent hypothesis is carbon starvation: the stomata close to prevent hydraulic failure, decreasing carbon photosynthetic uptake. However, the demand for carbohydrates continues, causing starvation. This mechanism seems more likely if drought is not intense enough to cause hydraulic failure but lasts enough to consume all carbon reserves (McDowell et al. 2008). In contrast, in some environments, the water may be present but difficult to use because of excessive salt content. The osmotic pressure towards the salt could dry out the organism. Several mechanisms evolved to overcome aggressive salinity. Highly halophilic archaea equilibrates the osmotic pressure with high concentrations of potassium chloride within the cells. As a result, the proteins had to evolve to properly fold under those saline conditions. Salt-adapted algae tend to equilibrate the osmotic pressure using organic molecules (Plaxco and Gross 2011). Many plants can live in or even depend on highly salty environments, and ABA is a key regulator of this stress (Park et al. 2016). Plants also found several ways to adapt to metal toxicity, and some species pushed the adaptations to an extreme level. The extremophile plant Noccaea caerulescens can grow on soils containing high concentrations of various metals and even hyperaccumulate them to extremely high levels (Lin et al. 2014). However, heavy metals are still a source of stress in many species of plants (Tripathi et al. 2012a,b; Liu et al. 2018). Human activity has increased the presence of metals and this causes losses in agriculture yield (Vernay et al. 2007). Hyperaccumulating plants are being studied to engineer plants more suitable to take up metals in phytoremediation (Eapen and D’Souza 2005). These techniques are effective and cost much less than traditional approaches because they do not require highly specialized personnel or expensive equipment (Salt et al. 1995;

9.3 ABA Chemical Structure, Biosynthesis, and Metabolism

Tangahu et al. 2011). The genetic selection of those hyperaccumulating plants for engineering will have to take ABA into account. For instance, exogenous ABA application was shown to decrease the accumulation of cadmium in Arabidopsis (Fan et al. 2014). From the above, it is clear that extremophilic plants are like treasure waiting to be found. Several natural plant species exist in almost any stressful environmental condition (Oh et al. 2012). These extremophilic plants could be key to discovering how to adapt crops to extreme conditions while maintaining productivity. As Oh et al. (2012) pointed out, “Extremophiles provide not only a model for what is possible, but for the traits that may be necessary for crops in the future.” The genome and transcriptome of many species have already been described (Oh et al. 2012). The model plant Arabidopsis thaliana alone is not enough. A deeper understanding of the ABA pathway genes in plants could lead to numerous technological breakthroughs that we cannot even conceive off today.

9.3 ABA Chemical Structure, Biosynthesis, and Metabolism ABA is a sesquiterpenoid (C15 H20 O4 ) weak acid. Its chemical structure was confirmed by chemical synthesis (Cornforth et al. 1965) and spectroscopic methods (Ohkuma et al. 1965). It has one optically asymmetric active carbon atom at C-1′ (Finkelstein and Rock 2002). The C-15 ABA skeleton is commonly found in biosynthetic precursors such as abscisic aldehyde, abscisic alcohol, and xanthoxin as well as oxidized catabolites including 8′ -hydroxy-ABA, dihydrophaseic acid and phaseic acid (Vishwakarma et al. 2017). ABA has an asymmetric carbon atom at position 1′ in the ring, resulting in the R and S enantiomers. The natural form is the S enantiomer (Cutler et al. 2010). The side chain of the ABA molecule contains two double bonds conjugated to the carboxylic acid. On exposure to UV light, biologically active 2-cis,4-trans ABA is reversibly isomerized to the inactive trans form, 2-trans,4-trans ABA (Cutler et al. 2010). Works with members of the PYR/PYL/RCAR receptor class have shown the basis of the requirements for structure and function of ABA (Hauser et al. 2011; Finkelstein 2013; Zhang et al. 2015a). These receptors bind ABA through a combination of residues with hydrophobic interactions forming a stereo-selective pocket around the side chain and the ring, a charge interaction between a conserved lysine in the receptors and the carboxyl group of the ABA side chain, and a water-mediated hydrogen bond network surrounding the side chain (Miyakawa et al. 2013; Finkelstein 2013). ABA biosynthesis, as well as concentrations of this phytohormone, is modulated in specific tissues during development or in response to changing environmental conditions, such as abiotic stresses. Several studies have identified all of the main genes for the enzymes in the biosynthesis pathway, although a great challenge is to understand how the regulation of these biosynthesis genes and the biosynthetic pathway as a whole occurs (Schwartz et al. 2003; Xiong and Zhu 2003). Several ABA-deficient mutants are related to abnormal phenotypes and have been identified with lesions at specific steps of the pathway; they have contributed to the elucidation of ABA biosynthesis (Xiong and Zhu 2003; McAdam et al. 2015). The ABA-deficient mutants identified are related to the following phenotypes: an increase in stomatal conductance, precocious germination, an ability to germinate and grow on

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media containing a high concentration of sucrose or salt and susceptibility to wilting. These mutants are also very helpful in cloning the genes that encode ABA biosynthetic enzymes (Schwartz et al. 2003). ABA biosynthesis takes place in plastids and early steps begin with MEP (2C-methyld-erythritol-4-phosphate) (Finkelstein 2013). 1-Deoxy-d-xylulose-5-phosphate synthase is the first enzyme of the MEP pathway. ABA is synthesized from the oxygenated carotenoid intermediate violaxanthin, whose synthesis is catalyzed by zeaxanthin epoxidase (ZEP) in Arabidopsis. 𝛽-Carotene conversion to ABA is made via number of enzyme-catalyzed steps (Vishwakarma et al. 2017). The enzyme ZEP catalyzes the zeaxanthin conversion to violaxanthin, a key reaction for the xanthophyll cycle and ABA biosynthesis (Vishwakarma et al. 2017). These processes are important for acclimation to environmental stress conditions, mainly light (xanthophyll cycle) and drought (ABA biosynthesis) stress. Thus, there is tissue- and stress-specific accumulation of the ZEP protein in accordance with its different functions in ABA biosynthesis and the xanthophyll cycle (Schwarz et al. 2015). Other enzymes, such as 9-cis-epoxycarotenoid dioxygenase (NCED) (rapidly induced by water stress), which can cleave both 9′ -cis-neoxanthin and 9-cis-violaxanthin, and aldehyde oxidases, related to the final step creating the carboxyl group at the end of the side chain, are also important for ABA biosynthesis (Finkelstein 2013). Vishwakarma et al. (2017) reported that the abiotic stress which entails triggering of assorted ABA biosynthetic genes corresponding to NCED, ZEP, molybdenum cofactor sulfurase, and ABA-aldehyde oxidase might be because of the calcium-dependent phosphorylation pathway (Tuteja 2007). The increase in de novo ABA biosynthesis is due to the rise in abiotic stress which plays an important role in inhibition of degradation and is thought to be stimulated by stress relief. The basal transcript level of ZEP gene in Arabidopsis can be detected in nonstressful conditions, as in tomato and tobacco. The basal transcript levels, which also cover the stress inducibility of genes, are associated with variations in the expression of ZEP genes. In addition, ABA biosynthesis is achieved after cleavage in the rate-limiting step, and thus expression of NCED genes has acquired major importance (Vishwakarma et al. 2017). Besides biosynthesis, transport by xylem or phloem, compartmentalization and degradation (by oxidation or conjugation) are also related to the modulation of endogenous levels of ABA present in the cytosol of plant cell. In fact, the endogenous ABA content is determined by the dynamic balance between catabolism and biosynthesis (regulated by ABA 8′ -hydroxylase). ABA conjugation by cytosolic UDPglucosyltransferases, or release by 𝛽-glucosidases, is important for maintaining ABA homeostasis (Leng et al. 2014). For instance, gain-of-function and loss-of-function mutant analyses have shown UGT71B6, an ABA UGT, and its two closely related homologs, UGT71B8 and UGT71B7, to play a crucial role in adaptation to various abiotic stresses and in ABA homeostasis (Dong and Hwang 2014). Xu et al. (2012) verified that a vacuolar 𝛽-glucosidase plays an major role in osmotic stress responses in Arabidopsis and ABA is produced in various organelles by organelle-specific 𝛽-glucosidases in response to environmental stresses. Finkelstein (2013) pointed out that Arabidopsis utilizes the two major pathways of ABA catabolism: (i) esterification of ABA to ABA-glucose ester; and (ii) hydroxylation of ABA at the 8′ position by P-450 type monoxygenases to give an unstable intermediate

9.4 ABA Perception and Signaling

(8′ -OH-ABA) that is isomerized to phaseic acid. ABA or its metabolites can also be inactivated by conjugation to another molecule, the glucosyl ester being the most common conjugate, which accumulates in vacuoles and the apoplast, and is relocated to the endoplasmic reticulum in response to hydration stress (Finkelstein 2013).

9.4 ABA Perception and Signaling Cells need to sense their environment and give the proper responses. The extracellular signal carrying the information arrives in different forms, and will frequently have to be converted into another form for the information to proceed. Conversions from one form of information into another are referred to as “signal transduction.” In molecular biology, this expression may also refer to the process of transmitting an extracellular signal into the cell interior so as to elicit a response (Voet et al. 2008; Alberts et al. 2010). In general, a signaling pathway consists of a receptor protein that binds to the external messenger; a mechanism to transmit the binding-event information into the cell interior; and intracellular responses that may include second messengers, kinases and phosphatases (Voet et al. 2008). Secondary messengers are molecules inside the cell that respond to the transduced signal by changing in concentration. This change in concentration transmits information within the cell (Berg et al. 2002). Pathways in response to abiotic stresses are complex. The external stress signal is first detected by cell membrane receptors, and is transduced, resulting in secondary messengers such as calcium and ROS (Mahajan and Tuteja 2005). This ultimately leads to plant adaptation to the stress (Tuteja 2007). Land plants have to detect and respond to several abiotic stress factors, such as drought. The transduction of the first, extracellular signal leading to ABA response is not well understood. At the onset of water stress in maize, for instance, the levels of sulfate increase. The ion is transported in the xylem to the shoots, inducing ABA biosynthesis and ABA-induced stomatal closure (Ernst et al. 2010; Jarzyniak and Jasi´nski 2014). If the water stress is prolonged, root ABA synthesis is induced, in response to some hydraulic change (Ernst et al. 2010). The ABA transport outward and inward of the vasculature is performed by transporter proteins (Merilo et al. 2015). The protonated form of ABA is able to diffuse through the plasma membrane, but the importance of this mechanism is debated (Merilo et al. 2015). At some point on the pathway, the ABA in the guard cells triggers the ABA stress response. This raises the question of what receptors bind ABA directly. Some hypotheses have been proposed involving GPCR type G-proteins (Cutler et al. 2010). Two membrane-localized GPCP-type G proteins were proposed to bind ABA specifically. They could be involved in some kind of signal transduction (Pandey et al. 2009). Another candidate receptor for ABA binding is the plastid-localized magnesium cheletase (ChlH) (Cutler et al. 2010). However, the role of this protein has been extensively debated because of doubtful experimental results (Cutler et al. 2010; Finkelstein 2013). Finally, there is good experimental support for the hypothesis that the proteins PYR/PYL/RCARs directly bind ABA and are part of the “core” ABA signaling components (Cutler et al. 2010; Roychoudhury and Paul 2012). The interconnections of these various factors are not currently known (Cutler et al. 2010). However, the core of ABA pathway has been well explained. In guard cells, reverse

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phosphorylation in the ABA signal network is central and antique. Those cells need a rapid response to a variety of stimuli, and the efficient control provided by phosphorylation is well suited to this (Wasilewska et al. 2008). According to Umezawa (2011), the core of ABA signaling components (PYR/PYL/RCAR, PP2C, SnRK2) “indicates that protein phosphorylation/dephosphorylation is the most important factor in ABA signaling.” Essentially, the kinase SnRK2 phosphorylates itself, while PP2C phosphatase dephosphorylates SnRK2 (Umezawa 2011; Sheard and Zheng 2009). SnRK2 is the enzyme that activates downstream transcription factors, and it does it in its phosphorylated state. The pathway is switched on if SnRK2 is phosphorylated, and off if SnRK2 is dephosphorylated. Since SnRK2 autophosphorylates (Ng et al. 2011), it is ready to activate downstream transcription factors unless it is inhibited. Under normal conditions, PP2C inhibits SnRK2 via dephosphorylation. This keeps the switch off. ABA switches the pathway on by binding the receptor PYR/PYL/RCAR, which can then bind and sequester the inhibitor PP2C. This prevents dephosphorylation of SnRK2, which can now stay on. This short piece of network enabled ABA to eventually turn into “the master stress hormone.” As previously described, ABA acts during many stress factors, such as drought, salinity, heavy metals, cold and UV irradiation (Vishwakarma et al. 2017). A reason for this versatility is that abiotic stresses caused by cold, drought, high salinity, high light levels, and heat stress all have a common consequence: a decrease in water content. As a result, some of the genes expressed in response to those stresses are the same (Tuteja 2007; Mittler and Blumwald 2015).

9.5 ABA Regulation of Gene Expression ABA plays an important role in plant response to osmotic stress. ABA transmits its message through signal transduction pathways that finally convert the initial stress signals into changes in gene expression. In the presence of ABA, the phosphatase PP2C, which negatively regulates the protein kinases, is inhibited. ABA binds to the PYR/PYL/RCAR receptors, enabling them to bind and repress PP2C. This permits the activation of the kinase SnRK2, which phosphorylates and activates downstream TFs to initiate transcription of ABA-responsive genes (Sheard and Zheng 2009; Roychoudhury and Banerjee 2017). ABA-regulated genes play a key role in responses to stress and regulating plant growth. The ABA induces several genes associated with stress response and tolerance, such as enzymes of compatible solute metabolism, a variety of transporters, enzymes that detoxify ROS, protein kinases, phosphatases, and transcription factors. In contrast, ABA represses genes associated with growth and development (Cutler et al. 2010; Fujita et al. 2011; Roychoudhury et al. 2013). ABA is also thought to play a role in the gene expression regulation of the stress-related Late Embryogenesis Abundant (LEA) proteins (Shinde et al. 2012; Huang et al. 2017) and Translationally Controlled Tumour Protein (TCTP) (Kim et al. 2012; Meng et al. 2017). Studies using heterologous expression in a host microorganism (Barros et al. 2015; Santa Brígida et al. 2014; Marques et al. 2017a; Meng et al. 2017) and plant transformation (Kim et al. 2012; Wang et al. 2015a; Yu et al. 2016a) detected the importance of such proteins in response to several abiotic stresses. However, the complete role of ABA in

9.5 ABA Regulation of Gene Expression

gene regulation of proteins related to abiotic stress tolerance and response remains to be elucidated. 9.5.1

Cis-regulatory Elements

The transcription of ABA-responsive genes is regulated by TFs that recognize and bind cis-elements in the promoter regions of their target genes (Fujita et al. 2011). These regulatory sequences for any given gene contain binding sites for a variety of factors (Cutler et al. 2010). At the end of the phosphorylation cascade, TFs are activated and bind specifically to cis-elements in the promoters of stress-responsive genes and regulate their transcription (Mukherjee et al. 2006; Wang et al. 2016a). TFs are master regulators of gene expression that can control the expression of several target genes through specific binding of the TF to the cis-acting element in the promoters of respective target genes (Nakashima et al. 2009; Roychoudhury and Banerjee 2015). The majority of the ABA-regulated genes contain a conserved ABA-responsive element named ABRE (PyACGTGG/TC) as the determinant cis-elements in their promoters (Fujita et al. 2011; Ganguly et al. 2011). ABRE, an important cis-element in ABA-responsive genes, is recognized by members of the basic leucine zipper (bZIP) transcription factor family (Cutler et al. 2010, Banerjee and Roychoudhury 2017b). Generally, more than one copy of ABRE is necessary for ABA-responsive gene expression. ABA response requires either coupling elements (CEs), which are similar to ABRE, a dehydration-responsive element (DRE), or additional copies of the ABRE (Fujita et al. 2011; Himmelbach et al. 2003; Nakashima et al. 2009). The cis-element DREs and CEs, GC-rich sequences, are bound by the APETALA2 (AP2) transcription factor family (Cutler et al. 2010; Roychoudhury et al. 2008; Roychoudhury and Sengupta 2009). DREs contain a conserved sequence TACCGACAT and are found in the promoter regions of several drought- and cold-inducible genes (Basu and Roychoudhury 2014). Similar cis-acting elements, called C-repeat (CRT) and low-temperature-responsive element, both containing a A/GCCGAC core motif, regulate cold-inducible promoters (Narusaka et al. 2003). ABRE and DRE/CRT are major cis-elements in abiotic stress response functioning in ABA-dependent and ABAindependent gene expression, respectively (Nakashima and Yamaguchi-Shinozaki 2010; Roychoudhury et al. 2013). In response to ABA, the DRE/CRT sequence also functions as a CE of ABRE in ABA-dependent gene expression. Narusaka et al. (2003) reported that ABRE and DRE are interdependent in the ABA-responsive expression and interact synergistically. The promoters of ABA-response genes also contain cis-elements such as the MYB recognition sequence (MYBRS; C/TAACNA/G) and the MYC recognition sequence (MYCRS; CANNTG), which are regulated by MYB and MYC TF families, respectively (Tuteja 2007). In addition, the NAC recognition sequence is also involved in ABA-responsive gene expression. This cis-element contains the CACG core DNA binding motif, which is recognized by the NAC TFs (Tran et al. 2004). ABRE, DRE, MYCRS, and MYBRS act as positive cis-elements, and when specifically bound to their respective TFs, work as transcriptional activators in the expression of stress-inducible genes. However, C2H2 zinc finger proteins have been reported to be negative regulators implicated in ABA-mediated gene expression under hydration stress conditions (Fujita et al. 2011). These proteins bind to A(G/C)T repeats within an EP2

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sequence that is a negative cis-element, and the STZ and AZFs act as transcriptional repressors (Sakamoto et al. 2004). 9.5.2 Transcription Factors Involved in the ABA-Mediated Abiotic Stress Response As mentioned above, TFs interact with cis-acting regulatory elements in the promoters of abiotic stress-responsive genes. In this section, information about major TFs involved in plant abiotic stress response mediated by ABA is presented. Examples of recent studies of overexpression or silencing of genes coding for TFs and their relationship with ABA sensitivity, modulating tolerance against abiotic stresses, are presented in Table 9.1. Table 9.1 Examples of genes coding for transcription factors (TFs) and their action in abiotic stress tolerance and abscisic acid (ABA) sensitivity. Overexpressed or silenced genes coding TFs

Abiotic stress tolerance and ABA sensitivity

OsbZIP71

Increased drought and salinity tolerance generated by gene overexpression; increased ABA insensitivity generated by gene silencing

Oryza sativa

Liu et al. (2014a).

OsMYB48–1

Increased drought and salinity tolerance and increased ABA sensitivity generated by gene overexpression

Oryza sativa

Xiong et al. (2014).

TaNAC29

Increased drought and salinity tolerance and increased ABA sensitivity generated by gene overexpression

Arabidopsis thaliana

Huang et al. (2015).

ONAC022

Increased drought and salinity tolerance and increased ABA sensitivity generated by gene overexpression

Oryza sativa

Hong et al. (2016).

ZmNAC55

Increased drought tolerance and increased ABA sensitivity generated by gene overexpression

Arabidopsis thaliana

Mao et al. (2016).

MYB37

Increased drought and salinity tolerance and increased ABA sensitivity generated by gene overexpression

Arabidopsis thaliana

Yu et al. (2016b).

GaMYB85

Increased drought and salinity tolerance and increased ABA sensitivity generated by gene overexpression

Arabidopsis thaliana

Butt et al. (2017).

GsNAC019

Increased alkaline stress tolerance and reduced ABA sensitivity generated by gene overexpression

Arabidopsis thaliana

Cao et al. (2017).

CaAIEF1(AP2/ERF)

Reduced and increased drought tolerance generated by gene silencing and overexpression, respectively; reduced and increased ABA sensitivity generated by gene silencing and overexpression, respectively

Arabdopsis thaliana; Capsicum annuum

Hong et al. (2017).

OsMYBR1

Drought tolerance and reduced ABA sensitivity generated by gene overexpression

Oryza sativa

Yin et al. (2017a).

Transformed plant

References

9.5 ABA Regulation of Gene Expression

9.5.2.1

bZIP Family

The bZIP TFs are key transcriptional regulators of ABA-dependent gene expression (Raghavendra et al. 2010; Banerjee and Roychoudhury 2017b). The bZIP TFs contain a conserved bZIP domain which is composed of a highly basic region for nuclear localization and specific binding of the TF to its target DNA at the N-terminus and a leucine-rich motif for dimerization at the C-terminus (Wang et al. 2016a). Probably, the bZIP TFs are ubiquitously present in the nucleus, and their transcriptional activities are positively regulated by ABA-dependent phosphorylation of the SnRK2 kinases. The bZIP TFs interact as dimers with ABRE and regulate the expression of stress-related genes in an ABA-dependent manner (Wang et al. 2016a). Most of the well-studied bZIP TFs belonging to the group A are involved in ABA signaling. Based on phylogenetic relationships, the nine members were divided into two groups: the ABI5/AtDPBF family genes (ABI5, EEL, DPBF2/AtbZIP67, DPBF4 and AREB3) are mainly expressed in seeds and appear to play important roles in seed maturation and development, whereas the AREB/ABF family genes (AREB1/ABF2, AREB2/ABF4, ABF1, and ABF3) are mainly expressed in vegetative tissues under abiotic stress conditions (Fujita et al. 2011). Initially identified on the basis of binding to ABREs in yeast one-hybrid screens, these four AREB/ABF proteins were originally identified as AREB (ABA Response Element Binding: Uno et al. 2000), and also named ABFs (ABRE binding factors) (Choi et al. 2000). AREB1/ABF2, AREB2/ABF4, and AREB3 are induced by high salt levels, dehydration, or ABA treatment in vegetative tissues (Yoshida et al. 2010). ABA-dependent phosphorylation is required for full activation of all AREB/ABF transcription factors. AREB1 activation requires multiple phosphorylations of the R-X-X-S/T sites at conserved regions, and Furihata et al. (2006) demonstrated that these modifications are performed by SnRK2-type kinases. As explained above, SnRK2 activation is induced by ABA. In Arabidopsis, AREB induces the stress responsive gene (RD29B), which encodes a LEA-like protein and presents in its promoter region two ABREs involved in the regulation of ABA-responsive expression (Tuteja 2007). As previously commented, the DRE/CRT sequence also functions as a CE in ABA-dependent gene expression and AREB/ABF interacts physically with DREB1A/ CBF3, DREB2A, and DREB2C proteins, suggesting crosstalk between elements of the ABA-independent and ABA-dependent response pathways (Lee et al. 2010). Furthermore, SnRK2s, ABRE promoter sequence, and AREB/ABF TFs are related to the DREB2A gene expression under osmotic stress conditions, suggesting complex interaction between the DREB and AREB regulon both at the gene expression level and at the protein level (Kim et al. 2011). Overexpression of AREB1 showed enhanced drought tolerance in soybean, Arabidopsis, and rice (Oh et al. 2005; Barbosa et al. 2013; Yoshida et al. 2015). Recently, Wang et al. (2016b) identified and characterized an AREB wheat gene (TaAREB3) encoding a bZIP TF. Transformed Arabidopsis overexpressing TaAREB3 presented ABA sensitivity and increased tolerance to drought and freezing. Functional analysis showed that TaAREB3 also activated the expression of RD29A, RD29B, COR15A, and COR47 under stress conditions, such as drought and freezing, by binding to their promoter regions. 9.5.2.2

MYC and MYB

ABA response also depends on other regulatory systems for gene expressions that involve MYCRS and MYBRS in their promoter regions. In Arabidopsis, the promoter

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region of a dehydration-responsive gene, RD22, contain MYCRS and MYBRS sites that act as cis elements in the ABA- and drought-induced RD22 gene expression (Abe et al. 2003). MYB TF, AtMYB2, and MYC TF, AtMYC2 (rd22BP1) bind these cis-elements and together activate the RD22 expression. These two TFs probably play roles at a late stage of the stress response because they are synthesized only after the accumulation of endogenous ABA (Mahajan and Tuteja 2005). Transgenic plants overexpressing MYC and MYB had higher sensitivity to ABA and showed improvement in osmotic stress tolerance (Abe et al. 2003). This study also revealed that AtMYC2 and AtMYB2 TFs cooperatively activate the expression of RD22. The MYC/MYB response system is somewhat slower than the bZIP–ABRE system, reflecting the need for de novo synthesis of MYB and MYC TFs (Finkelstein et al. 2002). The bZIP TF seems to work as a preformed target, whereas the expression of MYC and MYB TFs is induced by ABA and requires an upstream ABA-responsive TF (Finkelstein et al. 2002; Himmelbach et al. 2003). Thus, it has been suggested that the MYC/ MYB system regulates slow adaptive responses to hydric stress (Finkelstein et al. 2002). MYC TFs belong to the basic-helix–loop–helix (bHLH) family of TFs that have a characteristic bHLH domain. This family is characterized by the bHLH signature domain, which consists of ∼60 amino acids with two functionally different regions. The basic region, located at the N-terminal end of the domain, is involved in DNA binding, and the HLH region, at the C-terminal end, acts as a dimerization domain (Toledo-Ortiz et al. 2003). Two bHLH TFs, AtAIB and MYC2, have been related to ABA-mediated gene expression in Arabidopsis (Fujita et al. 2011). Guard cell transcriptome analysis demonstrated the presence of ABA-responsive genes with MYC binding sites in these cells (Wang et al. 2011). MYC2 has been related as the main regulator of crosstalk in biotic and abiotic stress responses and in light signaling pathways via hormone signaling pathways, including ABA (Fujita et al. 2011). The MYB TFs belong to a large family defined by a highly conserved MYB domain for DNA-binding, containing from one to four imperfect repeats (MYB repeat) at the N-terminus. In plants, most MYB proteins belong to the R2R3MYB group (Wang et al. 2016a). Several R2R3MYB genes have been shown to be the major mediators of ABA-mediated gene expression under environmental stress conditions in Arabidopsis (Fujita et al. 2011). AtMYB96 also appears to mediate ABA signaling via RD22 expression. Furthermore, MYB96 regulates drought stress response by integrating ABA and auxin signals. Overexpression of MYB96 in Arabidopsis showed improved drought resistance with reduced lateral roots (Seo et al. 2009). Seo et al. (2011) reported that MYB96 also promotes drought resistance by activating cuticular wax biosynthesis. Cominelli et al. (2008) reported that AtMYB41 gene was activated in response to salinity, desiccation, cold, and ABA. Analysis of transgenic lines that overexpress MYB41 has revealed that MYB41 controls stress responses linked to cell wall modifications including cell expansion and cuticle deposition. In addition, Lippold et al. (2009) demonstrated the involvement of AtMTB41 in the control of different cellular processes in Arabidopsis seedlings, including negative regulation of short-term transcriptional responses to hydric stress and associated modifications in primary metabolism. Jung et al. (2008) reported that AtMYB44 plays an important role in the ABAmediated signaling pathway conferring drought tolerance by regulating stomatal closure. The overexpression of this gene in transgenic Arabidopsis enhanced the sensitivity

9.5 ABA Regulation of Gene Expression

to ABA and tolerance to salt and drought stresses by suppressing the expression of genes encoding a group of Ser/Thr PP2Cs that have been demonstrated to be negative regulators of ABA signaling. In addition, the overexpression of MYB44 suppresses jasmonateresponsive gene activation (Jung et al. 2010). The hypothesis of mutual antagonistic actions between jasmonate- and ABA-mediated signaling pathways is supported by these observations (Jung et al. 2010). Overexpression of MYB44 in Arabidopsis resulted in improvement of salt and drought stress tolerance (Persak and Pitzschke 2014). In addition, salt stress-induced accumulation of destructive ROS is efficiently prevented in transgenic MYB44. As observed in Arabidopsis, the expression of the AtMYB44 gene in transgenic soybeans also exhibited significantly enhanced drought and salt stress tolerance (Seo et al. 2012). The results infer that a mechanism conferring abiotic stress tolerance is conserved in Arabidopsis and soybean. 9.5.2.3

NAC Family

The NAC (named based on its members NAM, ATAF, and CUC genes) superfamily is composed of several plant-specific transcriptional factors that share a highly conserved DNA-binding NAC domain located in the N-terminal region and a highly diversified transcriptional activation domain in the C-terminal region (Zélicourt et al. 2012; Xu et al. 2013). According to studies in Arabidopsis and Oryza sativa, there are more than 100 NAC genes divided into two groups. Some of these genes are induced by ABA in plants. In addition, some of them, when transformed in plants, are overexpressed and improve plant drought tolerance (Liu et al. 2011; Mao et al. 2014). Several studies have reported changes in tolerance of plants under several abiotic stresses when they are transformed by NAC genes (Marques et al. 2017b). Here, we present some recent advances showing these regulations. A wheat NAC was transformed and overexpressed in Arabidopsis to a better understanding of its role under various abiotic stresses. In the model plant, its involvement in tolerance to freezing, drought and salt stresses was noted. Physiological improvements included improved photosynthetic potential, water retention, and cell membrane stability (Mao et al. 2014). In a study with a NAC factor (NAC2) isolated from peanut leaves (Arachis hypogaea), improved expression in Arabidopsis was reported with increased sensitivity to ABA. NAC2 was upregulated in transgenic Arabidopsis under ABA treatment. Nevertheless, the transgenic lines presented significantly lower germination rates than the wild type under ABA treatment, as well as inhibited root growth. These results show that NAC2 plays a major role in ABA signaling. Furthermore, there was a significant upregulation in several genes related to abiotic stresses (Liu et al. 2011). Hong et al. (2016) identified a novel NAC gene (ONAC022) in rice that was induced by several environmental stresses, drought, high salinity, and ABA. When rice plants were transformed to overexpress NAC, they also showed increased tolerance to the same abiotic stresses. Higher survival ratios and better development than wild-type specimens were reported under 150 mM salt treatments, as well as less transpiration and water loss and enhanced sensitivity to ABA. Fang et al. (2015) observed that in rice a NAC protein (SNAC3) was upregulated under drought and heat stress, enhancing the tolerance of plants against these abiotic stresses. The specimens with overexpressed SNAC3 under environmental stresses upregulated five ROS-associated genes, contributing to lower levels of H2 O2 presented in transgenic plants, when compared with wild-type plants.

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There are several works reporting a secondary role of NAC factors in plants, viz. senescence control. It is known that salt stress accelerates this process, and proteins that act against these abiotic stresses are important to control senescence. NAC proteins can upregulate the genes that code these proteins. In Arabidopsis, a regulatory network between three different NAC factors was reported, with at least two different roles affecting different pathways in leaves: flavonoid and pathogen response pathways. These roles were related to improved abiotic stresses tolerance and developmental senescence regulation (Hickman et al. 2013). Wu et al. (2012), in a study with Arabidopsis, reported a hydrogen peroxide-induced NAC (JUB1) related to longevity regulation. This NAC factor scavenges hydrogen peroxide when upregulated, also enhancing the tolerance to some environmental stresses. JUB1 is the main regulator of cellular levels of H2 O2 , controlling the expression of some ROS-responsive genes. Zélicourt et al. (2012) also observed, in Medicago truncatula, the dual role of NAC proteins. A NAC factor (NAC969), when induced by nitrate treatment, increased the root development under salt stress and showed retarded nodule senescence. Therefore, this NAC protein is involved in different pathways, negatively regulating the salt-stress response in roots and nodule senescence. Lee et al. (2012) reported opposite results. They observed in transformed Arabidopsis that NTL4, a NAC factor, promoted leaf senescence under salt, drought, and heat stress. These mutant plants presented hypersensitivity to drought environment and ROS accumulation in leaves. In mutants without NTL4 expression, leaf senescence was severely delayed and salt resistance was enhanced. Furthermore, it was observed that Arabidopsis seeds were more sensitive to ABA, showing the connection between different pathways. As a transcription factor, NAC influences the expression of various other proteins. TaNAC29, a wheat NAC transcription factor, improves drought and salt tolerance in transgenic Arabidopsis (Huang et al. 2015). Another interaction between proteins against environmental stresses was reported by Xu et al. (2013): a NAC protein (ANAC096) and two ABRE proteins. All of them are transcription factors. A large genome expression analysis in Arabidopsis revealed that ANAC096 is a major regulator of ABA-responsive genes, but not alone. Plants without the three TFs had much more sensitivity to drought and other associated stresses than plants without one or two of them. The three factors, when associated, strongly enhance tolerance to dehydration and osmotic stresses. 9.5.2.4

AP2/ERF Family

The AP2/ERF (APETALA2/ethylene response factor) superfamily has a strongly conserved AP2 DNA-binding domain in plants, and is crucial to the plant physiology and development, regulating environmental stress responses like cold, heat, salt, and drought (Cao et al. 2015; Du et al. 2016). These genes are expressed in different tissues, but the highest expression levels occur in the root. They can be divided into four subfamilies and 13 subgroups, based on the number of AP2/ERF domains of each species and the presence of other DNA binding domains: AP2 (APETALA2), with two AP2/ERF conserved domains; ERF (ethylene-responsive-element-binding-factor), with a single AP2/ERF domain; DREB (dehydration-responsive-element-binding), that has also a single AP2/ERF domain; RAV (related to ABI3/VP), with one AP2/ERF domain too, but also a specific B3 motif; and other proteins (soloists) (Li et al. 2015; Dossa et al. 2016).

9.5 ABA Regulation of Gene Expression

Owing to numerous genes in this superfamily, there have been several genome-wide analyses reported during last few years. Dossa et al. (2016) made a broad analysis in sesame (Sesamum indicum), an important oil crop, under drought stress. They identified 132 AP2/ERF genes in its genome. Among them, 23 DREB genes were positively regulated under drought stress, suggesting that the DREB subfamily could be strongly related to drought stress. On the other hand, after 3 days under stress, 44% of DREB genes were downregulated. Du et al. (2016) reported in rapeseed (Brassica napus L.) 118 expressed genes under cold stress. DREB and RAV genes in general are upregulated at first 2 h under stress; then AP2 genes are expressed, reaching the highest levels after 12 h. Thirteen genes were induced by cold during all of the time of exposure, members of DRED, ERF and RAV subfamilies. Li et al. (2015) identified from the carrot whole genome 267 AP2/ERF genes: 214 ERF/DREB genes, 38 AP2 genes, 12 RAV genes and 3 soloist genes. Using qRT-PCR, the gene expression patterns of eight genes under cold, drought, salt, and heat stresses was analyzed. The results were inconclusive, with no clear patterns of up- or downregulation. In Medicago truncatula, another genome-wide analysis was made. About 123 AP2/ ERF were identified and characterized: 50 DREB genes, 48 ERF genes, 21 AP2 genes, 3 RAV genes and only 1 soloist gene. The expression patterns were assessed using qRT-PCR, transcriptome, and sequencing. A total of 87 AP/ERF genes were expressed in some tissues or under abiotic stress. Some DREB genes were reported with upregulation under cold and freezing stresses, being strong candidates for future transgenic studies (Shu et al. 2016). With a transcriptome approach, Wu et al. (2015) identified 89 AP2/ERF genes from four cultivars of a tea plant (Camellia sinensis): 45 ERF, 29 DREB, 12 AP2, 2 RAV, and 1 soloist gene. A total of five genes were chosen for further qRT-PCR analysis and were related to temperature stresses. Under cold stress, all genes were upregulated, in general. Under heat stress, the gene expression varied, with downand upregulations at different levels. Finally, a study in physic nut (Jatropha curcas L.) reported a total of 119 AP2/ERF genes, and the response of these genes in transformed rice under saline stress was analyzed. The division in subfamilies was: 98 ERF, 16 AP2, 4 RAV, and 1 soloist gene. No DREB genes were identified. Different expression was noted between tissues (seed, root, stem, and leaves) from many genes, and 38 genes were regulated by at least one environmental stress (phosphate/nitrogen starvation, salinity and drought) (Tang et al. 2016). Another work focused only on overexpression of AP2/ERF genes in transgenic plants under different stresses. Jisha et al. (2015) reported the overexpression of an AP2/ERF (EREBP1) gene in transformed rice under drought stress. The transgenic plants when compared with wild-type specimens showed enhanced drought tolerance, associated with increased levels of ABA and other molecules. These results suggested that EREBP1 upregulation activates ABA pathways, conferring enhanced abiotic stress tolerance. Mishra et al. (2015) observed an AP2/ERF factor (PsAP2) from Papaver somniferum that conferred tolerance to drought and salt stress in transgenic tobacco. The germination and growth rates and fresh weight in the transformed lines were higher than in wild-type lines. They also reported the interaction between this factor and a tobacco gene promoter in vitro, suggesting that the binding between them improves the abiotic stress tolerance. There are reports showing that AP2/ERF gene regulation also has a role in plant development. Another study of Tang et al. (2017) with physic nut reported the functional

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characterization of an AP2/ERF gene (DREB2). This gene was mainly expressed in leaves and was induced by ABA but suppressed by salt. When DREB2 was overexpressed in transformed rice, it altered the plant growth, with some plants presenting dwarfism. Furthermore, the same upregulated gene increased rice sensitivity to salt stress and possibly contributed to upregulation of salt-tolerance-related genes. Another study with the same species observed that a novel AP2/ERF (ERF2) factor improved the tolerance against some environmental stresses in transformed tobacco. It belongs to ERF subfamily and was induced by ABA, ethylene, salt, and drought, conferring tolerance to these stresses when compared with normal plants. It also upregulated several stress-related genes (Wang et al. 2015b). Finally, Yang et al. (2016) isolated a novel AP2/ERF gene (ERF1) from Stipa purpurea and produced transgenic Arabidopsis to assess its changes in expression under abiotic stress. It was induced by cold and drought stresses, reaching a maximum expression after 5 days under drought treatment and 3 h of cold treatment. 9.5.2.5

Zinc Finger Family

Zinc finger proteins are a large group of transcription factors with a zinc-binding activity of the zinc finger domain, which is formed by a zinc atom surrounded by Cys and His residues. Since this domain varies in plants, they can be classified into many categories based on the order and the number of Cys and His residues: C2 HC, C2 HC5 , C2 HC4 , CCCH, C4 , C4 HC3 , C6 , C8 and C2 H2 (Kielbowicz-Matuk 2012). C2 H2 zinc finger proteins are one of the best known and most important subgroups. They are transcription factors that were identified as the main proteins involved in plant growth and development, mainly in roots and flowers, and also in plant responses to abiotic and biotic stresses, up- and downregulated genes needed for tolerance against environmental challenges (Lawrence et al. 2014). C2 H2 refers to a domain composed of 30 amino acids, including two Cys and two His residues bound by a zinc ion. According to the number of zinc finger domains, they can be divided into three groups (Muthamilarasan et al. 2014; Liu et al. 2015). There are some broad analysis reports related to C2 H2 zinc finger family. For instance, in Arabidopis, 176 C2 H2 -type zinc finger factors have been found, according to in silico analysis; 143 are plant specific and 33 are common among eukaryotes (Englbrecht et al. 2004; Joseph et al. 2014). In poplar, about 109 genes were identified, being classified in groups according to the number of motifs: motif-rich groups (four to six motifs) and motif-poor groups (one to three motifs). Besides this classification, this study showed the high expression levels of some genes under saline, heat, and drought stresses, mainly in leaves and roots (Liu et al. 2015). Another work was based on transcriptome identification in Crocus sativus. Eighty-one zinc finger factors were found, being grouped into eight families. From the C2 H2 zinc finger family, 29 transcripts were identified. Some of these transcripts were strongly induced by saline, drought, and oxidative stresses (Malik and Ashraf 2017). In the last 5 years, several studies have linked C2 H2 genes to environmental stresses tolerance. Fan et al. (2015) reported that a C2 H2 -type zinc finger (STOP1) in Vigna umbellata is inducible by H+ and aluminum stress. With the qRT-PCR approach, this metal quickly improved STOP1 expression (within 2 h) in roots. Similar results were found under low pH values (H+ stress). This protein is arguably important both

9.5 ABA Regulation of Gene Expression

in H+ tolerance and in aluminum tolerance. Li et al. (unpublished data), working with tomato, identified a new C2 H2 -type zinc finger factor (ZF3). The expression of this gene was rapidly induced by saline stress (150 mM NaCl): the activity was noticed after just 30 min, reaching a maximum after 12 h of treatment. The ZF3 overexpression also increased ascorbic acid levels in both Arabidopsis and tomato. In foxtail millet (Setaria italica), the miRNA was used to target 15 C2 H2 transcripts. They were induced by cold, salt, and dehydration stresses, mainly in root, stem, and leaf (Muthamilarasan et al. 2014). In rice, the ZFP36, a new C2 H2 gene, was identified, and transgenic plants overexpressing and silencing this gene were produced. During overexpression, antioxidant enzymes were induced, increasing the drought and oxidative stress in these plants. On the other hand, mutants with silenced ZFP36 were more sensitive to the same kinds of environmental stress. Furthermore, it was noticed that ABA-induced proteins regulate ZFP36, showing the role of this protein in ABA signaling (Zhang et al. 2014). C2 H2 -type zinc finger genes can present spatial differences in expression. In Eucalyptus grandis, Wang et al. (2014) observed the response of seven C2 H2 genes: ZFP1–7. They noticed differential patterns of expression of some genes between tissues. Two genes were overexpressed in roots, when compared with stems and leaves. The other five did not show differences between the three genes. When exposed to environmental stresses, their expression also varied. Six genes oscillated their expression under cold treatment, with up- and downregulation. Under 200 mM salt treatment, the same six genes were induced, with another one being inhibited. In another study with Arabidopsis, it was found that the overexpression of a nuclear C2 H2 gene, ZAT18, enhanced the drought tolerance, with less water loss. When mutant plants with no ZAT18 transcripts were produced, the plant tolerance to dehydration strongly decreased. Like other groups of genes, ZAT18 was preferentially expressed in stems and leaves. A number of stress-related genes were reported to be targets of this transcription factor (Yin et al. 2017b). Zhang et al. (2016) observed in transformed Arabidopsis the role of a soybean C2 H2 gene (ZFP3) against abiotic stresses. Using a qRT-PCR approach, temporal and spatial differences in expression were reported. ZFP3 was mainly expressed in roots, leaves, and stems, with lower levels in pods and flowers. This gene was also overexpressed by ABA treatments. Interestingly, the study showed a negative role of this gene in plant tolerance to dehydration. ZFP3 may be related to the ABA-dependent pathway during response to water loss. Shi et al. (2014) observed in Arabidopsis that ZAT6, another C2 H2 zinc finger gene, was induced by cold, dehydration, salinity, and pathogen infection. Lawrence et al. (2014) identified another transcript, ZFP2, from potato that was overexpressed under potato beetle and tobacco hornworm. These studies showed the relevance of C2 H2 zinc finger gene expression against several kinds of biotic and abiotic stresses. Finally, proteins of zinc finger family have roles in plant development as well. Joseph et al. (2014) observed in Arabidopsis a C2 H2 zinc finger (ZFP3) that negatively regulates the ABA suppression of seed germination. When ZFP3 was overexpressed under ABA treatment, the transgenic plants were insensitive to ABA, also altering expression of a number of ABA-induced genes: hundreds of genes were up- and downregulated, including genes that influence light signaling. These mutants presented altered phenotypes, such as decreased fertility and development.

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9.6 Post-transcriptional and Post-translational Control in Regulating ABA Response Post-transcriptional and post-translational mechanisms can alter the transcriptome and proteome, thus adjusting transcription to certain adverse, momentary conditions, including those related to stress (Mazzucotelli et al. 2008). Post-transcriptional modifications come within the context of the processing undergone by eukaryotic mRNA molecules before they enter the translation mechanism. In the context of gene expression control, this post-transcriptional mechanism aims, among other things, to integrate the expression to the most emerging adaptive responses, altering the development and metabolism to immediate needs (Roychoudhury et al. 2008; Floris et al. 2009). This adjustment can involve environmental factors, such as nutrient availability, and, in particular, the response to biotic and abiotic stresses (Pant et al. 2009). In this scenario, RNA-binding proteins (RBPs) appear. These proteins are capable of interacting with the pre-mRNA, thus altering the abundance of transcription. They even change the location, transport, and control of translation, besides the degradation of the transcript for said purposes. This modulation of the transcriptome includes the regulation of genes integrated to hormonal regulation, such as the regulation of ABA), thus promoting modifications in the signaling of this phytohormone (Kuhn and Schroeder 2003; Lorkovi´c 2009; Ambrosone et al. 2012). Characteristically, the RBPs have in common one or more conserved RNA binding domains: RNA Recognition Motif, K homology domain, zinc-finger, double-stranded RNA-binding domain, cold-shock domain, and aspartate–glutamate–alanine–aspartate (DEAD) Box (Ambrosone et al. 2012). For example, an important domain protein (DEAD) Box would be DEAD Box RNA helicase (ZmDRH1), whose enzymatic features are involved in the de-spiralization, transport and control of RNA translation. This activity, in turn, may be affected by salt and cold stress (Gendra et al. 2004; Owttrim 2006). The mutations in subgroups of these types of proteins confer resistance to heat, osmotic shock, and salt stress independently of ABA (Kant et al. 2007). Cases of mutations, on the other hand, are not always beneficial. An example of this is the mutations provoked in another class of proteins, hyponastics leaves (hyl1), involved in the processing of RNA, which result in hypersensitivity to ABA, causing serious damage to the root growth. They also affect development and fertility in Arabidopsis (Lu and Fedoroff 2000). There are several reports of proteins involved in ABA signaling and linked to the stress response. Among them, we can quote as an example, the product of ABH1 of hypersensitivity to ABA, involved in drought tolerance (Hugouvieux et al. 2001). The SAD1 gene (supersensitive to ABA and drought 1) and CBP20 (cap-binding protein 20) are related not only to drought tolerance but also to the repression of germination by ABA induction (Kuhn and Schroeder 2003). The RNA-binding proteins (GRPs) are related to floral development and biotic and abiotic stress, thus altering RNA splicing (Wu et al. 2016; Kim et al. 2007). There are also genes, such as fiery2 (fry2), that exhibit both hypo- and hypersensitivity to ABA, depending on the stage of development, and may therefore be insensitive during the germination stage or supra-sensitive during development, and thus act on root lengthening and stress signaling during these phases (Xiong et al. 2002; Koiwa et al. 2002). An interesting group of genes related to multiple stress conditions corresponds to the Differentially Expressed Genes family (OsDEGs), whose expression in Oryza sativa

9.6 Post-transcriptional and Post-translational Control in Regulating ABA Response

reached high levels under high concentrations of ABA, especially OsDEG10, OsDEG17, and OsDEG27. Notably, the OsDEG10 gene presented the highest significance in several situations of abiotic stress, such as cold, dry, extreme absence of oxygen (anoxia), photo-oxidation, salt, and osmotic stress. Therefore, the study of this family of genes would be a promising option to counteract at once several situations of abiotic adversities (Park et al. 2009a). A more recent finding involves the RBP protein, called Stress Associated RNA-binding protein 1 (SRP1). The characteristic of this enzyme is that it has been suggested to modulate several ABA signaling genes, such as the ABI family, especially ABI2, involved in germination and stress response. SRP1 would be involved in an interconnected gene network, namely, ABI1, ABI3, ABI5, EM1, and EM6 and on other genes of ABA signaling pathways like RD29A and RD29B. The results suggest that this new protein plays a role in the pruning and cleavage of pre-mRNA, or by cooperating with RNAse enzymes in this type of process. Mutants of this new protein induced a greater response to the situations of abiotic stress, during and after germination (Xu et al. 2017). Posttranslational modification is another intricate modulation mechanism of the ABA signaling pathway and involves a dynamic ubiquitination process, being associated with many aspects of biological development and response to environmental adversities. This process consists of one or more proteins (ubiquitins) and the key protein factors (receptors) related to a wide range of signaling and modifications of the cellular environment, and which can alter positively or negatively various cellular processes of metabolism, homeostasis, and resistance to external factors (Peng et al. 2003; Chen and Hellmann 2013). Ubiquitin is a protein of 76 residues of amino acids. Specifically, it has seven lysine residues capable of binding to other ubiquitins to form polyubiquitin chains (Peng et al. 2003). Polyubiquitination is generally related to the proteolytic destruction of the target-receptor and therefore suppresses the underlying cellular function, whereas monoubiquitination usually only regulates, activates, or inhibits underlying activities (Mukhopadhyay and Riezman 2007; Hicke 2001) and involves endocytosis, membrane trafficking, and histone modification (Tian and Xie 2013). However, ubiquitin itself is unprotected. First, it needs to be activated and complexed enzymatically. In addition, three enzymes participate in this type of process. First, an E1 enzyme (ubiquitin activating enzyme) hydrolyzes an ATP molecule to build a thioester bond next to ubiquitin, then both are transferred to a second enzyme, E2 (ubiquitin conjugating enzyme). E1 is replaced by E2, which ultimately interacts with the third and last enzyme, the E3–ligase protein complex, which ultimately promotes the transfer of ubiquitin to the target protein (Hicke 2001; Vierstra 2009). It is observed, however, that the specificity of the final E3 enzyme will depend on the type of subunits present, which will confer selectivity on various types of cellular receptors. Therefore, according to the type of subunit and type of action, it involves four types of E3 ligases: Cullin-RING ligase, really interesting new gene (RING), homologous to E6-AP carboxyl terminus and U-box (Vierstra 2009). These four types can even be subdivided into multiple subgroups. The enzyme complex E3 ligase 26S protease (RPN10) involved in the protease and destruction of ubiquitinated proteins, has, in the same way, a special domain, the motif RPN10, responsible for recognizing specific substrates so that the proteosomal part (26S) can degrade (Smalle et al. 2003). One of the targets of this proteolysis is the receptor ABI5, a transcription factor embedded in the ABA signaling pathway, related to the stress response and seed germination (Finkelstein et al. 2005; Zhang et al. 2007). The ABI5 is supposed to be

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ubiquitinated by the enzyme E3 ligase ring KEEP ON GOING (KEG), and this would still occur in the cytoplasm or the trans-Golgi networks before ABI5 reaches the cell nucleus (Liu and Stone 2010). It has been suggested that the presence of ABA not only induces the proteolytic destruction of the KEG enzyme complex, but also prevents the recognition of ABI5 receptors by the proteolytic system. Generally, ABI5 factors are short-lived proteins, and thus, in the absence of ABA and/or stressful conditions, protection over ABI5 would be exempted, leading to the destruction of these factors. (Liu and Stone 2013; Stone et al. 2006; Molina et al. 2003). Something similar happens with the factors ABF1 and ABF3, which in the same way as ABI5 functions in the germination and development of seedlings, as well as in stress response, being likewise positively regulated by the presence of ABA and negatively by the ubiquitination exerted by the enzyme E3-ligase KEG (Finkelstein et al. 2005). According to Zhang et al. (2007, 2015b), factors such as ABF3 and ABI5 are the key players in the ABA signaling pathway, and thus a control over the KEG enzyme would allow the creation of more resistant and developed plants, with resistance to drought and salt stress. In addition, the nonubiquitination of ABF1, ABF3, and ABI5 factors allows them to act synergistically with other factors, as in the case of ABI3, and thus stimulate the action of ABI3 on dormancy and seed maturation (Finkelstein et al. 2005). The interaction between hormone receptors and transcription factors is one of the main forms of ubiquitination regulating accurately many aspects of ABA signaling in response to environmental stimuli. For example, receptors such as PYR1 may interact with the factor ABI3 in order to cause its ubiquitination and subsequent proteolytic destruction, thus inhibiting the underlying function of this factor. This is triggered owing, supposedly, to the interaction between the PYR1 receptor with factor ABI3 in an ABA-dependent manner, which makes ABI3 more receptive to ubiquitination, and therefore, the destruction of the latter is facilitated (Park et al. 2009b; Kong et al. 2015). Recent studies have revealed new mechanisms integrated to the ubiquitination process. Interestingly, ubiquitinated receptors on the plasma membrane, such as PYR1 and PYL4, may not be degraded by the conventional 26S proteolytic pathway, but by the endosomal route. In such a situation, the recruitment of a set of differentiated proteins called Endosomal Sorting Complex Required for Transport (ESCRT) occurs, which are complexed to the plasma membrane, directing vesicle budding (with ubiquitinated proteins included). It has been found that the machinery (ESCRT) has a FYVE1 domain protein which is required by the mechanism, and is capable of interacting with the ubiquitinated PYL4 receptor to lead it to vacuolar degradation. Mutations in this FYVE1 component present in the machinery resulted in the increase in the PYL4 receptor and, consequently, in the greater action of ABA on these proteins (Belda-Palazon et al. 2016). The whole process, in fact, corresponds to a multiple monoubiquitination cascade, which leads to the lysosomal destruction of PYL4 (Tian and Xie 2013; Hoeller et al. 2007; MacGurn et al. 2012). It is assumed that PYL4 interacts with several phosphatases in an ABA-dependent manner in order to regulate germination and development as well as in the response to abiotic stress (Pizzio et al. 2013). It is understood that the advances in themselves allow better dissection of the mechanisms involved in the modulation processes. The advances in their utility allow the reprogramming of the regulation systems for the purpose of improvement and biological suitability to the environmental constraints. In any case, it is also understood that,

9.7 Epigenetic Regulation of ABA Response

with the available knowledge, it is already possible to obtain fruitful work on the adequacy of biological systems for resistance or improvement (Finkelstein 2013).

9.7 Epigenetic Regulation of ABA Response Epigenetics can be defined as the mechanism of regulation of expression (transcription and translation) of genes that do not depend on changes in DNA bases (Franco 2017). Some epigenetic mechanisms include DNA methylation, histone modifications, and the production of microRNAs (Meyer 2015). Plant epigenetic modifications are often regulated by environmental stress, providing an important mechanism for mediation of gene–environment interaction (King 2015; Banerjee and Roychoudhury 2017c; Banerjee and Roychoudhury 2018). Several recent studies have shown the involvement of epigenetic changes in plant response to abiotic stresses, such as cold, salinity, drought, and heat (Kim et al. 2015; Abid et al. 2017). Interestingly, an increasing number of studies have demonstrated that numerous abiotic stresses may induce possible epigenetic changes that could be transmitted in a stable manner to subsequent generations that may help plants to cope with environmental constraints (Abid et al. 2017). DNA methylation is one of the best studied epigenetic mechanisms in plants. DNA methylation is a chemical modification in which the addition of a methyl group on the fifth carbon of the cytosine ring, catalyzed by DNA methyltransferase enzymes, leads to the formation of 5-methylcytidine (Corella and Ordovas 2017). In both plants and animals, cytosine is first methylated in CpG (cytosine–phosphate–guanine) dinucleotides, but in plants, methylation can occur in two other complexes, totaling three: CpG, CpNpG and CpHpH (Chen et al. 2010; Law and Jacobsen 2010), where nitrogen can be A, C, G or T and H may be A, C or T. Methylation of the DNA in the promoter regions accompanies the repression of gene expression. The effect of DNA methylation varies according to the level of methylation. Hypermethylation or excessive methylation decreases the expression of the target genes, whereas hypomethylation (the reduction of methylation) triggers an increase in gene expression (Liddle and Jirtle 2006). Recent studies suggest that ABA can regulate gene expression through DNA methylation. Gohlke et al. (2013) showed that ABA increased the DNA methylation of promoters in three ABA-repressive genes in Arabidopsis, showing the importance of DNA methylation in ABA gene regulation. In another study, Abid et al. (2017) have shown that DNA methylation is an important regulatory mechanism for fava bean response to drought and also for other environmental stresses, since drought stress induces accumulation of ABA. Modification of histones, another type of epigenetic regulation, presents a higher level of complexity and is still little studied. Acetylation of nucleic histones generally induces an “opening” of the chromatin structure and is associated with gene activation, while histone deacetylation is generally related to chromatin “compaction” and gene repression. Histone deacetylation is catalyzed by histone deacetylases (Liu et al. 2014b). Histone deacetylases can be divided into four groups: RDP3, HDA1 and SIR2, which have homology with yeast histone deacetylases; and HD2, which is plant specific (Souza 2013). Sridha and Wu (2006) studied a gene encoding a histone deacetylase HD2 in A. thaliana, called AtHD2C. Overexpression of this gene in Arabidopsis plants

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conferred a phenotype insensitive to ABA. In addition, plants with this overexpressed gene exhibited reduced transpiration and increased salt tolerance and drought stress when compared with wild-type plants. This study therefore provides evidence that the AtHD2C gene can modulate responses to ABA and abiotic stress. Zhao et al. (2016) evaluated the expression pattern and function of histone deacetylase HDA705 in rice and found that the overexpression of HDA705 decreases expression levels of gibberellin biosynthetic genes and increases the levels of ABA biosynthetic gene expression during the germination of rice seeds. This shows that HDA705 may play an important role in regulating seed germination and response to rice abiotic stress. These studies show that histone acetylation and deacetylation play a major role in the regulation of ABA responsive genes and in plant responses to abiotic stresses. The involvement of small RNAs in response to abiotic stress has received more attention in recent years. The small RNAs belong to at least two different groups: microRNAs and small interfering RNAs. It has been shown that microRNAs and interfering RNAs control gene expression during various abiotic stresses: cold, nutrient deficiency, dehydration, salinity, and oxidative stress. MicroRNAs are the smallest (20–25 bp) and the most studied. MicroRNAs are small fragments of noncoding RNA and with important regulatory function (Corella and Ordovas 2017). They regulate post-transcriptional gene expression, complementing target mRNAs causing degradation of target mRNA or translation repression (Bartel 2009). MicroRNAs can exert epigenetic regulation of gene expression by deregulation of the main epigenetic regulators, such as histone deacetylases and DNA methyltransferases (Sato et al. 2011; Banerjee et al. 2016). MicroRNA genes with altered expression in response to ABA were demonstrated in plants. In Arabidopsis, ABA regulates the expression of miR393, miR159, and miR402, whereas miR169a can be deregulated by ABA (Sunkar and Zhu 2004, Reyes and Chua 2007). Interestingly, drought treatment may decrease miR167 expression and increase the expression of PLD (phospholipase D), which is the potential target of miR167 (Wei et al. 2009). Because PLD is a major regulator of ABA response and stress signaling in plants (Guo and Wang 2012), it is likely that miR167 is related to the regulation of ABA-dependent stress signaling networks. The expression profile in rice identified 34 microRNAs whose expression is induced or suppressed by ABA treatment (Shen et al. 2010). The majority of the microRNAs responded to ABA under at least one of the stress treatments (cold, salt, or dry), suggesting that they are the key factors for ABA-related stress pathways (Shen et al. 2010). According to the current literature, epigenetic modulation through histone acetylation, DNA methylation, and microRNA action has a main role for ABA-mediated stress signaling.

9.8 Conclusion This chapter presents information about the modulation of the endogenous ABA levels and the role of this phytohormone in response to abiotic stress, related to the action of signaling molecules and transcription factors culminating in gene expression. Prospecting of novel genes responsive to ABA, identification of components yet unknown of ABA signal transduction pathway and understanding of mechanisms of crosstalk between ABA and other hormones are very important to promote better insights

References

into the generation of tolerant plants at the field level (related to their effect on plant morphological, physiological, biochemical, and molecular changes). Therefore, the knowledge of ABA-mediated endogenous defense mechanisms of plants in response to abiotic stress seems to be crucial for the future of crop plants.

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10 Abiotic Stress Management in Plants: Role of Ethylene Anket Sharma 1,2 , Vinod Kumar 2 , Gagan Preet Singh Sidhu 3 , Rakesh Kumar 2 , Sukhmeen Kaur Kohli 1 , Poonam Yadav 1 , Dhriti Kapoor 4 , Aditi Shreeya Bali 5 , Babar Shahzad 6 , Kanika Khanna 1 , Sandeep Kumar 7 , Ashwani Kumar Thukral 1 , and Renu Bhardwaj 1 1 Plant Stress Physiology Laboratory, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, 143005, India 2 Department of Botany, DAV University, Sarmastpur, Jalandhar, 144012, Punjab, India 3 Department of Applied Sciences, UIET, Chandigarh 160014, India 4 School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India 5 Department of Botany, M.C.M. DAV College for Women, Chandigarh 160036, India 6 School of Land and Food, University of Tasmania, Hobart, Tasmania, Australia 7 Department of Environmental Sciences, DAV University, Sarmastpur, Jalandhar, 144012, Punjab, India

10.1 Introduction Plants are exposed to a wide array of environmental stresses that alter their morphological, anatomical, physiological, biochemical, and molecular functioning (Hussain et al. 2013; Saud et al. 2013; Tripathi et al. 2017a). Agriculture encounters various environmental stresses that cause extensive crop loss. It has been reported that abiotic stresses lead to a 50% reduction in the average crop yield (Bray et al. 2000) which might be due to the formation of harmful reactive oxygen species (ROS) that cause oxidative stress (Shafi et al. 2009; Tripathi et al. 2017b). These abiotic stresses evoke intricate and specific responses that are executed by the plants to prevent injury and fortify their durability or survival under adverse conditions. Plants modify various mechanisms with regard to abiotic stresses that comprise alterations in cellular and molecular processes against various stresses (Bohnert et al. 1995; Roychoudhury and Banerjee 2017). In response to abiotic stresses, metabolic adaptation leads to the assemblage of many organic solutes such as sugars, betaines and proline, and free-radical scavengers, and the activation of phytohormones (Roychoudhury et al. 2008; Iqbal et al. 2011a,b; Fahad et al. 2016; Roychoudhury and Banerjee 2016). Therefore, increasing the inherent flexibility of plants toward a multitude of stresses is very important and thus understanding these processes in plants will provide knowledge to increase abiotic stress tolerance (Wilkinson et al. 2012). Phytohormones or plant growth regulators are compounds that are synthesized in low concentrations and control various cellular mechanisms in plants. These plant hormones maintain various signaling processes in plants under abiotic stresses. Recent progress in plant biology research has confirmed the role of plant hormones in Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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mitigating the detrimental effects caused by abiotic stresses (Khan et al. 2013). Various plant hormones like ethylene (Shi et al. 2012), cytokinins (Peleg et al. 2011), jasmonates (Gonzalez-Aguilar et al. 2000), auxin (Ke et al. 2015), brassinosteroids (Ali et al. 2008; Banerjee and Roychoudhury 2018a,b), and abscisic acid (Roychoudhury et al. 2009; Fujita et al. 2011) have been reported to play a pivotal role in stress signaling. Among various phytohormones, ethylene, a gaseous molecule with a simple two-carbon skeleton (C2 H4 ), is a crucial mediator of stresses in plants. It is involved in the regulation of several plant developmental processes, as well as playing an important role in abiotic stress tolerance (Abeles et al. 1992). Ethylene is a stress hormone (Cao et al. 2007) and plays a critical role in combatting abiotic stresses like salt, heat, drought, and ozone (Chen et al. 2005; Chen and Zhang 2006). It is synthesized in plants from amino acid methionine with the help of S-adenosyl methionine (SAM) (S-adenosyl-l-methionine or AdoMet) and ACC (amino acid 1-aminocyclopropane-1-carboxylic acid) (Kende 1993). Abeles et al. (1992) revealed that ethylene regulates the transcription of key genes like PR (pathogenesis-related) in response to pathogen attack, wounds, and drought stress. The promoter regions of these PR genes contain a molecular switch, a GCC-box element, identified as an important target site in plants for the ethylene signal transduction pathway (Sessa et al. 1995). Ethylene has been reported to provide plant stress responses by the expression of certain genes that are required for tolerance (Hattori et al. 2009). The transcriptional activation of these genes is regulated by various transcription factors that play a key role in adapting plants to adverse environmental conditions. Among the different transcription factors identified in plants, ethylene response factors (ERFs) have been associated with abiotic stress-triggered transcription. Various abiotic stresses have promoted ethylene synthesis in plants, thereby providing tolerance to environmental stresses (Morgan 1990; Tudela and Primo Millo 1992; Masood et al. 2012; Khan et al. 2013). Ethylene applied exogenously has been reported to protect photosynthesis in Brassica juncea plants in response to heavy metal (HM) stress (Khan and Khan 2014a,b,c). Moreover, ERF transcription factors have been reported to induce stem elongation and photosynthesis in rice under flooding stress (Hattori et al. 2009). This finding indicates a complete involvement of ethylene in photosynthesis; hence, it is essential to appraise the alteration in photosynthesis and amount of ethylene in plants encountering various environmental stresses as a connected process.

10.2 Ethylene: Abundance, Biosynthesis, Signaling, and Functions Ethylene is a simple, unsaturated hydrocarbon formed in all plant parts; however, its production rate is dependent upon the type of the tissue and its development stage. Dimitry Neljubov, in 1901, identified ethylene as a biologically active gas that induces a triple response in pea seedlings (Abeles et al. 1992). Leaf abscission, flower senescence, and fruit ripening elevate ethylene synthesis in plants. Plants can form ethylene both photochemically and enzymatically. Although it can be formed by all the organs of higher plants, its synthesis is more common in tissues undergoing senescence. Advances in research on ethylene production and function began after the introduction of gas chromatography (Burg and Stolwijk 1959).

10.3 Abiotic Stress and Ethylene Biosynthesis

In the biosynthesis AdoMet (SAM) is first produced from methionine catalyzed by SAM. After that, SAM is converted to the key intermediate ACC by the enzyme ACC synthase. Then ACC is converted to ethylene in a reaction catalyzed by ACC oxidase (Kende 1993). The SAM synthetase and ACC synthase are part of the Yang or methionine cycle that recycles the methylthio (CH3 -S) group of methionine (Adams and Yang 1979). ACC synthase bifurcates both the Yang cycle and ethylene production cycle and produces 5′ -methylthioadenosine and ACC. During the ripening of fruits, the rate of synthesis of ACC and ethylene increases many fold (Yang and Hoffman 1984). Further, stressful conditions like drought, chilling, and ozone exposure enhance ethylene biosynthesis, which is due to the increased transcription of ACC synthase mRNA (Stearns and Glick 2003). The interaction between ethylene and its receptor is responsible for plant responses under variety of abiotic stresses. The signal transduction pathway of ethylene has been evaluated in Arabidopsis thaliana on the basis of mutant analysis of the triple response of etiolated seedlings involving ethylene receptors CTR1 (CONSTITUITIVE TRIPLE RESPONSE 1), EIN2 (ETHYLENE INSENSITIVE 2), EIN3 (ETHYLENE INSENSITIVE 3) and other components (Guo and Ecker 2004). Further, in Arabidopsis five receptor genes have been identified that are divided into two subfamilies on the basis of structural differences. Receptor ETR1 (ETHYLENE RESPONSE 1) and ERS1 (ETHYLENE RESPONSE SENSOR-1) are included in subfamily I and ETR2 (ETHYLENE RESPONSE 2), EIN4 (ETHYLENE INSENSITIVE 4) and ERS2 (ETHYLENE RESPONSE SENSOR-2) belong to subfamily II (Hall et al. 2000). All of these receptors are restricted to the endoplasmic reticulum system as multimeric complexes, unlike other hormone receptors localized at the plasma membrane (Gao et al. 2008). Ethylene signaling plays a significant role in controlling plant growth under abiotic stress responses. Furthermore, variations in the amount of expression of ethylene receptors conciliate the plant responses to abiotic stresses. Ethylene modulates a broad array of responses in plants, like seed germination, expansion of cells, flowering, ripening of fruits and senescence. Additionally, ethylene has been identified as a hormone that accelerates the ripening of fruits (Nath et al. 2006). Additionally, Barry and Giovannoni (2007) identified some novel components of ethylene synthesis and transcription factors that influence ethylene production during ripening in tomato plants. Ethylene has been reported to decrease cell length and increase root width and root hair length (Le et al. 2001; Tanimoto et al. 1995). Likewise, ethylene has the ability to break dormancy and initiate seed germination (Abeles and Lonski 1969; Linkies and Leubner-Metzger 2012). Senescence of the leaf is the final phase in leaf development, followed by cell death. Ethylene has been reported to accelerate leaf senescence in plants with the help of transcription factors (Koyama 2014). This chapter summarizes the ways ethylene affects growth parameters, osmolytes, the pigment system, photosynthesis, and oxidative stress under different abiotic stresses.

10.3 Abiotic Stress and Ethylene Biosynthesis Abiotic stress conditions lead to the synthesis of ethylene by modulating the activities of ACC synthase and ACC oxidase. Water-deficit conditions are known to enhance ethylene levels in avocado, faba bean, French bean, and orange (Adato and Gazit

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1974; El-Beltagy and Hall 1974; Ben-Yehoshua and Aloni 1974; Upreti et al. 1998). The ethylene levels in plants under stress conditions may also depend upon other factors like the growth stage of plant and intensity as well as the duration of exposure to stress (Upreti et al. 2000). Cold stress causes enhanced ethylene concentrations in plants, which contributes to cold tolerance (Zhao 2014). In Capsicum, heat stress results in enhanced abscission of flower parts, which might be due to ACC accumulation and increased activity of ACC oxidase (Upreti et al. 2012). In tomato, Zhao et al. (2009) observed that ethylene levels and cold tolerance are positively related. Ethylene is also known to regulate the gene expression of plants by regulating ERF transcriptional levels under abiotic stress (Hussain et al. 2011). ERF possess the ability to bind with GCC box as well as dehydration responsive element (DRE)/C-repeat (CRT) motifs and cis-acting elements involved in the cellular response to cold/osmotic stress (Wang et al. 2004). SodERF3 is another ERF that plays a crucial role in enhancing resistance against salt and drought stress (Trujillo et al. 2008). Zhang et al. (2009) reported that genetically modified plants in which GmERF3 is overexpressed possess high tolerance to salt and drought stress. ACC biosynthesis in water-flooded roots is enhanced owing to the lower oxygen levels followed by ACC transport to upper plant parts, resulting in its conversion to ethylene (Colmer 2003). Additionally, the formation of free radicals under flooding conditions also triggers the formation of ethylene from ACC (Beltrano et al. 1997). Xu and Qi (1993) suggested that mild drought did not have a significant effect on ethylene or ACC levels; however, a swift change in the drought conditions may alter concentrations of ethylene and ACC. Sl-ERF.B.3 (Solanum lycopersicum ethylene response factor B.3) is responsible for encoding a transcription factor of ERF family and is regulated by various abiotic stresses (Klay et al. 2014). SUB1A-1 allele is also involved in the regulation of the biosynthesis of ethylene and plays an important role in the survival of plants which are fully submerged in water for long durations (Xu et al. 2006). Ethylene homeostasis is involved in freezing conditions and RARE COLD INDUCIBLE 1A (RCI1A) protein plays a crucial role by interacting with ACC synthase to confer chilling stress tolerance (Catala et al. 2014). Salinity stress positively regulates ethylene biosynthesis, which contributes to enhanced salt resistance in plants by regulating Na/K homeostasis (Lockhart 2013). Jiang et al. (2013) suggested that ethylene is helpful in the regulation of Na+ and K+ concentrations in xylem tissue under salt stress. Ethylene also crosstalks with other hormones to regulate plant metabolism under salt stress. Archard et al. (2006) suggested that, in genetically modified plants, suppression of salt sensitivity takes place by ACC synthase and ethylene helps to increase the resistance of transgenic plants against salinity.

10.4 Role of Ethylene in Photosynthesis Under Abiotic Stress Plants face different abiotic stress environments that inflict various harmful effects on the crop yield (Bray et al. 2000). Abiotic stresses induce significant harm to the photosynthetic apparatus in plants. Photosynthesis is a key process occurring in all green plants that helps to convert light energy into a usable form of chemical energy (Pan et al. 2012a,b). Baker (2008) describe photosynthesis as a multiple-step phenomenon

10.4 Role of Ethylene in Photosynthesis Under Abiotic Stress

in which sequential redox reactions occur when light energy in the form of photons is absorbed by light harvesting complexes and gets transferred to reaction centers of photosystem I and II via electrons. Abiotic stresses affect photosynthesis by changing the organelle structure and pigment concentration, and altering stomatal regulation in plants (Banerjee and Roychoudhury 2018a,b). Photosynthesis involves two main steps: (i) light reaction, in which energy in the form of light is converted to energy, which can be used by plants (ATP and NADPH), followed by oxygen evolution; and (ii) dark reaction, where products of light reactions are utilized in fixing CO2 into carbohydrates (Taiz and Zeiger 2010). The main site for photosynthesis is chloroplasts that are highly susceptible to abiotic stresses and perform a crucial role in regulating various stress responses (Biswal et al. 2008). These abiotic stresses inhibit the rate of photosynthesis by affecting stomatal conductance (Rahnama et al. 2010). Mild intensity of drought reduces photosynthetic efficiency accompanied by a decline in the stomatal conductance of green plants (Medrano et al. 2002). Various reports suggested the closure of stomata in drought stress, which inhibits transpiration of water as compared with CO2 diffusion in leaf (Sikuku et al. 2010). Dias and Brüggemann (2010) reported decreased efficiency of mesophyll cells in using CO2 in response to drought stress. Thus, the reduced photosynthesis rate under abiotic stresses is generally accredited to inhibition in mesophyll conductance and closure of stomata (Chaves et al. 2009). Similarly salinity stress also induces harmful effects on the photosynthetic machinery and metabolism in plants (Omoto et al. 2010). Wu and Zou (2009) documented that increased concentration of Na+ and Cl− ions in Pyrus betulaefolia under salinity stress damages the thylakoid membrane. Moreover, Mittal et al. (2012) observed that, in B. juncea, high salt stress inactivates electron transport and photophosphorylation in the thylakoid membrane, thereby affecting photosynthesis in plants. Likewise, Allakhverdiev et al. (2008) reported that high temperature affected the photosynthetic apparatus in plants owing to inhibition of the electron transport system. Ethylene regulates growth and development in plants. Endogenous ethylene evokes various physiological responses associated with photosynthesis by its effect on stomata or on the photosynthetic apparatus (Desikan et al. 2006; Iqbal et al. 2011a,b). The amount of ethylene and its sensitivity to plants regulates its effect on photosynthesis (Iqbal et al. 2012). Ethylene has been known to activate the signaling pathway in plants under various abiotic stress conditions (Kazan 2015). It plays a role in plants under stresses like drought (Manavella et al. 2006; Pan et al. 2012a,b; Zhang et al. 2010), cold/chilling (Chu and Lee 1989; Ciardi et al. 1997), heat (Hays et al. 2007), salinity (Xu et al. 2008), and heavy metals (Masood et al. 2012, 2016). Drought stress or water stress is indicated by depletion of water content, leaf water potential, stomatal closure, and reduced growth. Plants have developed various physiological mechanisms to counteract drought stress. Jaleel et al. (2008) documented that acute drought stress may hinder photosynthesis, interrupt metabolism, and finally cause plant death. In addition, drought stress causes considerable harm to the photosynthetic apparatus, regression of the thylakoid membrane (Kannan and Kulandaivelu 2011), and reduction of chlorophyll content (Din et al. 2011) in plants. On the contrary, the findings of Pirzad et al. (2011) suggest increased chlorophyll content under drought stress. Variation in the leaf chlorophyll content is attributed to alterations in the enzyme activities related to the chlorophyll biosynthesis (Ashraf and Karim 1991). In general, the amount of Chl b is reduced more as compared with Chl a, increasing

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the overall ratio of Chl a/Chl b (Jaleel et al. 2009). Ashraf et al. (1994) documented an increased ratio of Chl a/Chl b in a drought-resistant cultivar of wheat in contrast to a drought-sensitive cultivar, which might be due to reduced proportion of PS-II to PS-I (Estill et al. 1991). Furthermore, Zhang et al. (2011) reported damage to the PS II reaction center in response to drought stress, which might be due to the degradation of D1 polypeptide, which inactivates the reaction center of the photosystem (Zlatev 2009). These changes in the photosynthetic machinery led to the generation of ROS, which induces oxidative damage in the plant cells (Anjum et al. 2011). Many other reports have suggested the role of ethylene under drought stress (Larrainzar et al. 2014; Valluru et al. 2016). For example, Alarcón et al. (2009) demonstrated an inverse relationship between ethylene level and elongation of the root under drought stress in maize. However, mutants generated by knockout of ACC synthase enzyme showed reductions in ethylene emission and inhibition of drought-induced senescence in maize plants (Young et al. 2004). Habben et al. (2014) documented that downregulation of the ethylene biosynthetic pathway enhanced the yield in maize plants growing in water-deficit conditions. Similar reports were found in Medicago truncatula roots and nodules, where drought stress triggers the reduction in methionine levels and ultimately regulates ethylene biosynthesis (Larrainzar et al. 2014). Foliar spray of ACC in low concentrations increased the relative shoot growth rate in both drought-resistant and drought-sensitive groups of wheat plants owing to the accumulation of carbohydrates in the leaves (Valluru et al. 2016). Wang et al. (2016) reported that ethylene provides a high survival percentage in tobacco plants against drought. Iqbal et al. (2012) revealed that ethephon (a source of ethylene) application resulted in enhanced nitrate reductase activity and triggered the photosynthetic performance in two cultivars of B. juncea. Moreover, administration of norbornadiene;( which is an inhibitor in ethylene signaling) to plants which had been treated with ethephon inhibited ethylene action and suppressed the photosynthetic responses induced by ethylene (Iqbal et al. 2012). The increased photosynthesis in response to ethephon treatment might be due to the increased activity of ACC synthase enzyme (Imsabai et al. 2010). The detrimental effect of water-deficit conditions on photosynthesis can be alleviated by ethylene. Xu et al. (2007) investigated the role of Triticum aestivum transcription factor TaERF1 in mitigating drought stress and found that overexpression of TaERF1 regulated stomatal apertures and provided adaptation toward drought stress. Another study carried out by Aharoni et al. (2004) reported that AP2/EREBP family transcription factors reduced stomatal density in Arabidopsis mutant shine (shn) in response to drought stress. Quan et al. (2010) demonstrated increased expression of photosynthesis-related genes in tomato, owing to the synthesis of ERFs in response to drought stress. The ERFs induced drought tolerance in plants owing to their role in modulating the expression of stress-related genes (Cao et al. 2006a,b). Ethylene enhances the closure of stomata, which may be mediated by ROS production in stomatal guard cells (Desikan et al. (2006). Ethylene decreases the stomatal aperture in tomato, Solanum lycopersicum, Dianthus caryophyllus, and Arabidopsis (Madhavan et al. 1983; Desikan et al. 2006), and enhanced stomatal conductance in mustard (Iqbal et al. 2011a,b). Moreover, ethylene-induced tolerance to drought stress is correlated with the increased maximal quantum efficiency of photosystem-II (PSII), net photosynthetic rate and Rubisco activity (Masood et al. 2012). More than one-third of the land on Earth is affected by salinity stress (Cano et al. 1998). Salt stress negatively affects the growth of plants by inducing ion toxicity, osmotic

10.4 Role of Ethylene in Photosynthesis Under Abiotic Stress

stress, and oxidative stress (Zhu 2007). Salt stress induces the peroxidation of lipids and interrupted water and osmotic balance owing to the generation of ROS (Khan et al. 2014), which results in damage to the photosynthetic apparatus (Nazar et al. 2014). The increased level of Na ions in plants leads to the degradation of chlorophyll (Yang et al. 2011). Salt stress in plants induces changes in the chlorophyll content correlated to defective biosynthesis or increased degradation of pigment (Ashraf and Harris 2013). However, many reports have suggested that salt stress decreases the amount of glutamate and 5-aminolaevulinic acid (ALA), the precursors of chlorophyll, which induces a greater effect on chlorophyll synthesis than on chlorophyll disintegration (Santos et al. 2001; Santos 2004). Plants tolerate salinity by maintaining stability of the membrane (Sudhakar et al. 2001). Increased salinity in crop plants is related to ethylene synthesis (Arbona et al. 2005), which can positively or negatively affect the salt tolerance (Peng et al. 2014). The enzyme ACC synthase is the perfect target for modulating the process of ethylene biosynthesis in salt-stressed plants (Tao et al. 2015). Ellouzi et al. (2014) reported that Cakile maritima and Thellungiella salsuginea (halophytes) accumulated large amounts of ACC in leaves as well as roots under salt stress. Ma et al. (2012) conducted research on soybeans under salinity stress using 2-DE gel analysis and noticed that salt-tolerant genotype Lee 68 contained profuse amounts of various ethylene biosynthesis components as compared with salt-sensitive genotype Jackson. Further, Achard et al. (2008) suggested that ethylene biosynthesis promoted salt tolerance in Arabidopsis, which might be due to the interaction between ethylene and its receptor (Cao et al. 2007). Administration of ethylene or ACC augments the expression of ROS scavengers that help plants to cope with salinity stress (Peng et al. 2014). Interaction between glycine betaine (compatible solute) and ethylene has been associated with the regulation of salt tolerance in plants (Khan et al. 2012). This further prevents dissociation of PSII polypeptides, resulting in protection of oxygen-evolving complexes (Murata et al. 1992). The transcription factors of ethylene, ERFs have been reported to provide tolerance against salinity stress. Xu et al. (2007) reported increased adaptation of wheat to salt stress owing to the expression of TaERF1 in transgenic varieties, which induces enhanced chlorophyll content in response to salt stress in comparison with control plants. Further, Zhai et al. (2013) noticed increased chlorophyll and carbohydrate content and decreased malondialdehyde content in transgenic tobacco plants under salt stress owing to overexpression of ERF7 obtained from Glycine max. Khan et al. (2014) suggested that application of salicylic acid suppresses the level of ethylene; however, it maintains the formation of optimal ethylene under salt stress and this optimal ethylene inhibits oxidative stress and regulates the process of photosynthesis in salt-stressed Vigna radiata. These results were substantiated by application of ethylene precursor, ACC, in Arabidopsis under salt stress, which resulted in reduced oxidative stress and maintained optimal ethylene levels (Wang et al. 2009). Further, ethylene controls the movement of stomata, permitting more inflow of CO2 for carboxylation and enhanced photosynthesis, which might be due to enhanced carboxylation as well as water use efficiency (Iqbal et al. 2011a,b). Plants are subjected to variety of temperature fluctuations during the changing seasons. Plants subjected to high temperature stress showed decreased levels of chlorophyll synthesis (Reda and Mandoura 2011). The reduced chlorophyll synthesis is the first indicator of high-temperature stress in plastids (Li et al. 2010a,b), which might be due to the degradation of key enzymes involved in chlorophyll biosynthesis (Reda and

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Mandoura 2011). Tewari and Tripathy (1998) reported decreased activity of enzyme 5-aminolevulinate dehydratase, involved in the biosynthesis of chlorophyll in cucumber and wheat grown in the presence of heat stress. Moreover, the thylakoid membrane permeability is considered to be the most prominent heat-sensitive component of the photosynthetic apparatus (Havaux et al. 1996). The most detrimental effect of heat stress on chloroplast reactions is the deactivation of enzyme Rubisco (Sharkey 2005). Thus, plants have established some adaptive mechanisms in order to tolerate rapid changes in temperature. Heat stress in plants results in the generation of heat shock proteins (Howarth and Ougham 1993). During high-temperature stress or heat stress, heat shock proteins are accumulated in large amounts owing to the induction of oxidative stress (Schett et al. 1999). Ethylene is a signaling molecule which plays a crucial role in heat stress responses (Foyer et al. 1997). Wu et al. (1994) reported that application of ethephon, a mimic of ethylene, induces synthesis of heat shock protein APX1 in Arabidopsis, which helps in reducing heat stress-induced damage in plants. In another study, Larkindale and Knight (2002) found that ethylene along with salicylic acid, ABA and calcium protects Arabidopsis against oxidative damage induced by heat stress. Recently, Firon et al. (2012) indicated the role of ethylene in maintaining pollen quality in tomato plants under heat stress, which was attributed to the expression of ethylene-responsive genes in pollen. Khan et al. (2013) pointed out that salicylic acid interacts with ethylene signaling and influences photosynthesis in plants under heat stress. Application of salicylic acid inhibited synthesis of ethylene in response to heat stress and resulted in elevation of proline metabolism and photosynthesis in T. aestivum (Khan et al. 2013). These results correlated with the maintenance of optimal levels of ethylene by salicylic acid in plants, which caused proline production and significantly affected the photosynthetic machinery (Khan et al. 2013). However, Djanaguiraman et al. (2011) observed increased leaf senescence and reduced photosynthesis in soybean in response to enhanced ethylene production during high-temperature stress. These results are attributed to the increased activity of ACC synthase enzyme in heat-stressed conditions that restricts photochemical efficiency and carbon fixation in plants, thereby decreasing photosynthesis (Djanaguiraman et al. 2011). ERFs play a key role in mediating signaling and ethylene response on exposure to high-temperature stress. They can bind to DREs to trigger transcription of stress-responsive genes that provide tolerance against heat stress in plants (Ahammed et al. 2016). Further, Cai et al. (2015) found that endogenous application of ethylene along with jasmonates and abscisic acid induced the expression of CaWRKY6 gene in Capsicum annuum, which plays a crucial role in high-temperature stress. Cold stress, chilling or low-temperature stress significantly affects the distribution and productivity of crops (Tian et al. 2011). It adversely affects the metabolic machinery of plants, thereby reducing growth and development. Cold stress leads to enhanced levels of ROS in plants, especially H2 O2 (Cheng et al. 2007). Tewari and Tripathy (1998) observed a 90% reduction in chlorophyll synthesis in cucumber seedlings under cold-temperature stress that might be due to reduced biosynthesis of ALA involved in chlorophyll formation. Plants have developed various strategies to accommodate cold stress by activating numerous events that lead to increased cold tolerance. The ERF family of transcription factors plays a key role in regulating cold stress in plants (Chinnusamy et al. 2007). These ERF proteins bind to DRE/CRT motif present

10.4 Role of Ethylene in Photosynthesis Under Abiotic Stress

at the promoter region of genes that provide tolerance to cold stress (Lee et al. 2004). Tian et al. (2011) investigated the role of TERF2, an ERF, in providing cold tolerance in transgenic rice seedlings. The results revealed that TERF2 enhanced the concentration of osmotic solutes and chlorophyll pigment accompanied by a reduction in the levels of ROS species and malondialdehyde content in rice seedlings grown in the presence of high levels of cold (Tian et al. 2011). Similar results were reported by Zhang and Huang (2010), where overexpression of transcription factor TERF2/LeERF2 was triggered by ethylene biosynthesis and increased the cold resistance in tobacco and tomato plants. However, Shi et al. (2012) reported that cold stress impedes ethylene production in A. thaliana, which is due to the overexpression of EIN3 that negatively affects cold tolerance. Recently, Hu et al. (2017) identified 201 differentially expressing proteins in Bermuda grass (Cynodon dactylon) grown in the presence of ethylene and cold stress. They suggested that lipid peroxidation and protein metabolism are related to the ethylene-mediated cold resistance in C. dactylon. Many reports have suggested the role of glycine betaine and ethylene in providing tolerance toward cold stress (Allard et al. 1998; Quan et al. 2004a,b) by scavenging the excess electrons which are generated by the electron transport chain and increasing the rate of photosynthesis, which is due to the linkage between biosynthesis of glycine betaine and endogenous levels of ethylene (Kurepin et al. 2015). In addition to frequent abiotic stresses, HM stress has emerged as a new widespread problem for plants (Ahmad et al. 2015; Tripathi et al. 2012; Arif et al. 2016b; Tripathi et al. 2016; Singh et al. 2017). HM stress induces oxidative stress owing to excessive generation of ROS (Sharma and Chakraverty 2013; Kumar et al. 2017; Singh et al. 2015; Liu et al. 2018). Increased amounts of ROS hinder various cellular, biochemical, and physiological mechanisms in plants (Dugardeyn and Van Der Straeten 2008; Nagajyoti et al. 2010). Liu et al. (2011) observed a damaged electron transport chain and aggregation of protein complexes of photosystems upon accumulation of high amounts of Cd in leaves (Roychoudhury et al. 2012). Further, the activities of the Calvin cycle or C3 cycle enzymes were inhibited by high concentrations of Cd (Mobin and Khan 2007). High concentrations of Zn and Pb reduced stomatal conductance and downregulated the functions of PSI and PSII, thereby affecting the process of photosynthesis in Phragmites australis (Bernardini et al. 2016). Exposure to Pb decreased the leaf pigment concentration in Coronopus didymus, which might be attributed to decreased synthesis of chlorophyll or its degradation in response to HM toxicity (Sidhu et al. 2017). Metal stresses have been reported to alter the functions of the chloroplast membrane and impede the light reactions of photosynthesis (Ventrella et al. 2011). Recently, Li et al. (2015) reported reduced photosynthesis in response to HM toxicity that is correlated with the inhibition of photosynthetic efficiency (Fv/Fm) and the electron transport rate in Elsholtzia argyi. Moreover, their accumulation in the food chain can cause severe ecological and health problems (Malik 2004; Tripathi et al. 2015). Therefore, plants are engaged in various mechanisms to detoxify heavy metals and tolerate HM stress. Ethylene has been known to play a major role in reducing HM stress-induced alterations in photosynthesis owing either to the overexpression of genes which are involved in biosynthesis of ethylene (Khan et al. 2015) or variations in the expression of ethylene-responsive genes (Maksymiec 2007). Ethylene evokes its effect on photosynthesis depending upon its concentration and the sensitivity of the plant (Iqbal et al. 2012). According to Khan et al. (2015), Cd and Cu stimulated ethylene biosynthesis

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by boosting the ACC synthase activity, a key enzyme of the ethylene biosynthesis pathway. Cao et al. (2009) reported participation of EIN2, a component of the ethylene signaling pathway, in providing resistance to Pb stress in Arabidopsis. Masood et al. (2012) documented alleviation of photosynthetic inhibition in Brassica grown under different HMs like Ni, Zn, and Cd by maintaining the balance of the ethylene level, which attributed to the sulfur-induced activation of the antioxidant system by ethylene signaling (Masood et al. 2012). Several reports have suggested increased ethylene production in response to Cd stress in B. juncea, A. thaliana and T. aestivum (Masood et al. 2012; Schellingen et al. 2014; Khan et al. 2015). However, Carrió-Seguí et al. (2015) reported decreased ethylene production in A. thaliana in response to long-term Cd exposure. Khan and Khan (2014a,b,c) observed an improvement in photosynthetic attributes and PS II activity in Ni- and Zn-treated mustard plants in response to exogenously applied ethylene. The increased photosynthesis might be attributed to the enhanced activity of Rubisco after endogenous ethylene application (Khan and Khan 2014a,b,c). The intensity of sunlight is a major factor that is unsystematically altered many fold during the day time (Külheim et al. 2002). Changes in solar radiation notably affect the light reactions of photosynthesis occurring in the thylakoid membrane of chloroplast (Kirchhoff 2014). Prasil et al. (1992) pointed out that all light intensities damage the PS II photosystems; however, under high light intensity damage to photosystems is greater than the repair, which might be due to impaired D1 subunits (Ohad et al. 1984). Stomatal density is also supposed to be dependent upon light (Miyazawa et al. 2006). High light intensity, especially in the UV region, damages the photosynthesis process owing to production of ROS, which causes impairment of photosynthetic electron transport (Hideg et al. 2013; Banerjee and Roychoudhury 2016). The production of ROS in plants leads to the synthesis of ethylene that triggers the defense response to high light stress like UV-B rays (Brosché and Strid 2003). Further, Plettner et al. (2005) observed an increased amount of ethylene in Ulva intestinalis plants when exposed to high light intensities; however, exogenous application of ethylene caused reduction in the content of chlorophyll a under light stress. There are very few reports mentioning direct involvement of ethylene in mitigating light stress; however, endogenous ethylene regulates the synthesis of glycine betaine (Kurepin et al. 2015), which plays a major role in alleviating light stress. Enhanced levels of glycine-betaine against high light stress in wheat assisted in maintaining high chlorophyll content and net photosynthetic rate in plants owing to the repair of PS II (Wang et al. 2014a,b; Roychoudhury and Banerjee 2016).

10.5 Role of Ethylene on ROS and Antioxidative System Under Abiotic Stress As immobile organisms, plants are bound to face various abiotic stresses and have to regulate their growth and development accordingly. Abiotic stress enhances the generation of ROS, which can disintegrate various biomolecules like proteins, cellular membranes, DNA, etc. To neutralize the effect of ROS, plants activate their defense system, comprising antioxidative enzymes and antioxidant molecules like ascorbic acid, glutathione, and tocopherol (Arif et al. 2016a). Various phytohormones actively take

10.5 Role of Ethylene on ROS and Antioxidative System Under Abiotic Stress

part in the regulation of plant growth and development and also control the various physiological responses of plants to adverse conditions. Ethylene is a gaseous molecule that plays s role in the modulation of various cellular and molecular metabolic activities. Ethylene also have a functional role against different environmental stresses (Khan and Khan 2014a,b,c). Ethylene plays biphasic both beneficial and negative role in plants against abiotic stress conditions. It has been reported that ethylene changes root morphology under Cd stress in Arabidopsis plants by enhancing the activity of superoxide dismutase (SOD) isoenzymes (Abozeid et al. 2017). Similarly, it has been observed that exogenous application of ethylene and sulfur to mustard plants counteracted the Cd-induced toxicity by decreasing oxidative stress and enhancing the cysteine, methionine, and glutathione contents (Khan et al. 2016). Exogenous application of ethylene enhanced the tolerance of plants under salinity stress, mainly by increasing the expression of ROS scavengers (Cao et al. 2007; Peng et al. 2014). In Arabidopsis, salt stress induced EIN3/EIL1, increasing salt stress tolerance by improving ROS scavenging in an EIN2-independent manner (Peng et al. 2014). Ethylene-insensitive EIN2-1 mutant plants showed an enhanced lead uptake accompanied by a reduction in GSH content and possible crosstalk among ethylene, antioxidants, and metal chelaters (Cao et al. 2009). High transcription levels of Cu/Zn SOD2 and CAT3 resulted in higher activities of SOD and catalase (CAT) enzymes in EIN2-1 mutant plants in comparison with control plants (Cao et al. 2006a,b). Similar results were observed in Al-exposed EIN 2-1 mutants, which showed enhanced SOD and CAT activities (Zhang et al. 2014). EIN2-1, EIN3-1, and EIN4 mutant Arabidopsis leaves showed greater ascorbic acid content, suggesting a possible relationship between ethylene and the antioxidative defense system of plants (Gergoff et al. 2010). Similar results were noticed in ethylene-sensitive tomato (Alba et al. 2005). It has been suggested by Yoshida et al. (2009) that, under ozone exposure, ethylene enhances de novo synthesis of glutathione in A. thaliana plants, providing protection for the leaf. Exogenous application of ethephone enhanced glutathione levels in B. juncea plants under Cd toxicity (Masood et al. 2012). Similar findings were reported in Ni- and Zn-treated B. juncea plants, where application of ethephone enhanced the glutathione accumulation and helped alleviate HM toxicity (Khan and Khan 2014a,b,c). Moreover, ethylene-induced glutathione accumulation was also recorded in Arabidopsis plants exposed to Al and Cd and L. chinense exposed to Cd (Zhang et al. 2014; Guan et al. 2015; Schellingen et al. 2015). In genetically modified tobacco plants, overexpression of ERF genes from L. chinense showed increased expression of glutathione synthesis genes, leading to increased Cd tolerance (Guan et al. 2015). Additionally, in Pb-treated Arabidopsis, EIN2 is crucial in providing metal tolerance by enhancing glutathione concentration (Cao et al. 2009). Conversely, ethylene has been reported to decrease the activity of antioxidative enzymes and enhance the accumulation of ROS. In rice plants, under salinity, OsMPK3 and OsMPK6 activities upregulate ethylene signaling. Ethylene helps in the accumulation of ROS under salinity stress. Accumulation of ROS depends on the reduced activities of peroxidase (POD) and glutathione reductase (Li et al. 2014). ERFs are also involved in controlling ROS production and signaling in other plants. The transcription factor AP2/ERF multigene family is associated with the signaling pathways of ROS and ethylene under abiotic stress. ERF1has been reported to reduce the expression of genes like SOD and POD in Tamarix hispida under drought and high-salinity conditions,

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thus resulting in increased ROS levels by reducing their scavenging capacity (Wang et al. 2014a,b). Transcriptome analysis of rice seedling roots treated with Cr(VI) showed ethylene biosynthesis, signaling, and ROS level modulations as a part of the Cr signaling pathway (Huang et al. 2014; Trinh et al. 2014). The ctr1-1 mutant showed reduced ascorbic acid levels (Gergoff et al. 2010). Ethylene signaling also regulates the biosynthesis of ascorbate followed by its accumulation in tomato leaves (Mazzorra Morales et al. 2014). Under water stress, EIN 3-1 mutants showed delayed enhancement of tocopherol, which is an important molecule to enhance the tolerance of plants to many abiotic stresses (Cela et al. 2009). Application of ethylene action inhibitor steroid sulfatase to Cd-stressed plants showed a decrease in Cd-induced toxicity symptoms like ROS accumulation and retarded leaf growth (Maksymiec 2011). Similarly, Schellingen et al. (2014) noticed overexpression of ERF1, ETR2, and ACO2 under Cd exposure, while enhanced levels of ethylene during stress led to a reduction of leaf biomass in A. thaliana. Together these reports show that induction of ethylene under abiotic stress may cause both beneficial and harmful effects on plants. These results propose a complex and biphasic role of ethylene under abiotic stress, which depends on its endogenous levels.

10.6 Conclusion Overall, ethylene plays a crucial role in regulation of plant growth and development under various abiotic stress conditions. Its exogenous application to plants under environmental stress conditions results in recovery of growth, photosynthesis, and reduction in oxidative stress accompanied by enhanced antioxidative defense potential of plants.

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11 Crosstalk Among Phytohormone Signaling Pathways During Abiotic Stress Abhimanyu Jogawat National Institute of Plant Genome Research, New Delhi, 110067, India

11.1 Introduction Plants face various challenges to their survival such as abiotic stresses (heavy metals, wounding, drought, heat, cold, water-logging, and salinity) and biotic stress (bacteria, fungi, viruses, nematodes, and insects) (Tuteja and Sopory 2008; Suzuki et al. 2014; Singh et al. 2015; Arif et al. 2016a,b; Zhu 2016; Tripathi et al. 2016, 2017a,b; Singh et al. 2017; Liu et al. 2018), because they are sessile organisms. During their evolutions, plants have developed robust stress signal perception and transduction mechanisms for enduring successfully under extremely unfavorable situations. There are various plant hormones (phytohormones) which play pivotal roles in developing tolerance toward such harsh conditions. The sensing of abiotic stress(es) activates the signal transduction pathways which cooperate with downstream phytohormone signaling pathways to respond or adapt to the onset of a particular abiotic stress (Dolferus 2014). Hormone signal transduction pathways crosstalk and act conversely or antagonistically to defend plants from abiotic and biotic stresses (Fraire-Velázquez et al. 2011; Nguyen et al. 2016). Combined phytohormone signaling constructs a sophisticated signaling network that results in tolerance or adaptation under a stressful environment. Individually, abiotic stress-related hormone transduction networks such as abscisic acid (ABA), gibberellins/gibberellic acid (GA) and ethylene (ET) play important roles during cold, salinity, and drought stresses (Fujita et al. 2006; Tuteja and Sopory 2008), with ABA playing the central role (Roychoudhury and Paul 2012). Crosstalk among the signaling networks of ABA, GA, ET, auxins, cytokinins (CTs), salicylic acid (SA), jasmonic acid (JA) and ET further improves the tolerance of plants against multiple abiotic stresses (Huang et al. 2012; Golldack et al. 2014). For instance, seed dormancy and germination events are regulated by the two key phytohormones ABA and GA, depending on the plant’s responses to environmental stress signals (Leung and Giraudat 1998; Gray 2004; Verma et al. 2016). Sophisticated crosstalk among phytohormone signaling networks synergistically improves the response of plants to abiotic stresses and improves tolerance (Ruberti et al. 2012). Phytohormone signaling networks not only crosstalk with each other; they have also been reported to crosstalk with other signaling modules such as the calcium signaling module and mitogen-activated protein kinase (MAPK) cascades during an abiotic stress encounter (Ludwig et al. 2005; Roychoudhury et al. 2013; Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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Roychoudhury and Banerjee 2017). As a result, the action of phytohormone crosstalks leads to the regulation of the important genes involved in the biosynthesis or signaling of other hormones. There are some overlapping responses of different phytohormones which make plants tolerant to multiple stresses. Moreover, the phytohormone crosstalk also alters and regulates the expression of stress-responsive genes. In this chapter, we sum up the crosstalk between important phytohormones such as ABA, GA, ET, auxins, and CTs under an abiotic stress environment that strengthens defenses. ABA will be taken as the central phytohormone for better understanding. Here, we also try to analyze physiological changes and the gene expression regulated by crosstalk between different phytohormones under abiotic stress conditions, specifically drought, cold, and salinity stresses. This chapter will further improve our understanding of phytohormonal crosstalk during abiotic stress conditions, which will subsequently further widen our knowledge for crop improvement.

11.2 Phytohormone Crosstalk Phenomenon and its Necessity There are various defensive phytohormonal signaling pathways in plants, such as SA, ET, and JA (Tuteja and Sopory 2008), and growth-regulating phytohormonal pathways such as auxins, ABA, GA, and CTs. Under abiotic stress, these pathways also crosstalk with themselves (Knight and Knight 2001). To survive under abiotic stresses, plants have to maintain their health and growth as well as effectively minimizing the harsh effects of stress (Mittler 2006). Thus, the defense phytohormones need to crosstalk with the growth-regulating phytohormones to achieve these goals (Kohli et al. 2013). The response to a particular abiotic stress is not the result of a single phytohormone, rather it is the result of crosstalk between two or more. Crosstalk among phytohormones may be positive or negative in sense of the regulation and effect on each other (Pieterse et al. 2009). Apart from stress responses, hormonal crosstalk is also needed in regulation of plant development and growth (Munné-Bosch and Müller 2013).

11.3 Various Phytohormonal Crosstalk Under Abiotic Stresses for Improving Stress Tolerance 11.3.1

Crosstalk Between ABA and GA

Many hormones including GA regulate abiotic stress tolerance of plants via the involvement of crosstalk with ABA signaling (Skubacz et al. 2016). Both ABA and GA are important hormones whose levels regulate the decision between dormancy and germination (Peng and Harberd 2002). GAs crosstalk with other phytohormones in order to maintain growth and development under drought, salinity, and cold stress (Verma et al. 2016). It is known that antagonistic crosstalk between GA and ABA is regulated by DELLA proteins (Jiang and Fu 2007). This mechanism enables plants to escape harsh abiotic stress conditions by maintaining seed dormancy. As a survival strategy, this crosstalk results in delayed germination to protect seeds from the harmful effects of abiotic stresses. Production of ABA INSENSITIVE 5 (ABI5), a basic leucine zipper transcription factor that contains abscisic acid responsive element

11.3 Various Phytohormonal Crosstalk Under Abiotic Stresses for Improving Stress Tolerance

(ABRE) and is reportedly accumulated for growth, slows down under abiotic stress conditions. This growth inhibition is carried out by regulating stress adaptation genes such as LATE EMBRYOGENESIS ABUNDANT (LEA) genes under such conditions (Skubacz et al. 2016; Banerjee and Roychoudhury 2017). LEA proteins play important role in abiotic stress tolerance of conditions such as cold, salt, drought, and heat. In contrary, GAs work in the opposite manner. GAs escalate seed germination when there are optimum conditions. GAs suppress the activity of ABA to enable successful germination. Some DELLA proteins are regulated by GAs which negatively affects ABA signaling and remove the growth-diminution effects of ABA. Some DELLA proteins are regulated by ABA for suppressing GA signaling to halt growth under an abiotic stress environment (Peng and Harberd 2002). In Arabidopsis, there are a total of five DELLA proteins present, namely GA INSENSITIVE (GAI), REPRESSOR OF GA1-3 (RGA), RGA-LIKE1 (RGL1), RGL2, and RGL3. RGL-2 is the key repressor protein of GA signaling (Tyler et al. 2004). Additionally, Liu et al. (2016) has shown that three Arabidopsis NUCLEAR FACTOR-Y C (NF-YC) homologs, NF-YC3, NF-YC4, and NF-YC9, regulate the GA–ABA crosstalk-mediated seed-germination mechanism. Further, they mentioned that these NF-YCs interact with the DELLA protein RGL2. Furthermore, the NF-YC–RGL2 complex, by targeting ABI5, modulates genes related to germination and dormancy (Liu et al. 2016). Thus, GA and ABA are pivotal phytohormones that decide the choice between seed dormancy and germination during abiotic stress conditions. 11.3.2

Crosstalk Between GA and ET

GA signaling components, DELLA proteins, have been found to be involved in ET signaling during salt tolerance. Plant mutants having disrupted quadruple-DELLA proteins have shown improved root growth even in salinity stress (Achard et al. 2008a,b; Alvey and Boulton 2008). The results clearly indicated that salt stress suppresses root growth by a DELLA protein-mediated mechanism. In other findings, ET signaling also affects the root growth via the DELLA mechanism. Therefore it is clear that both GA and ET regulate root growth by crosstalking with each other (Achard et al. 2006; Achard and Genschik 2009; Golldack et al. 2014). DELLA and the CTR1-dependent ET response pathways crosstalk, downstream of the EIN3, for improving abiotic stress tolerance (Achard et al. 2006). In other studies, expression of ET-responsive CBF1/DREB1B transcription factor has been observed to show cold stress tolerance, via activating DELLA proteins (Achard et al. 2006; Huang et al. 2009; Thomashow 2010; Movahedi et al. 2012). Therefore, the crosstalk between GA and ET is the result of regulation of DELLA proteins which make the plants successful for tolerating cold, drought, and salt stresses. 11.3.3

Crosstalk Between ABA and ET

The gaseous phytohormone ET crosstalks with ABA during abiotic stresses (Fujita et al. 2006). ET-mediated root growth inhibition needs ABA (Ma et al. 2014). Similarly, antagonistic crosstalk between these two hormones has been reported to regulate shoot growth when roots encounter hard soil (Hussain et al. 2000). ET inhibits ABA signaling (Tanaka et al. 2005; Harrison 2012). ABA affects ET biosynthesis genes such as ethylene

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response factor 11 (ERF11), acyl-CoA synthetase 5 (ACS5), and 9-cis-epoxycarotenoid dioxygenase (NCED) (Jiang and Joyce 2003; Zhang et al. 2009; Li et al. 2011). ABA affects the ET by synthesis by regulating key ET biosynthesis genes. A mutant enhanced response to ABA3 (era3) with increased sensitivity to ABA has been shown to be related to ETHYLENE INSENSITIVE2 locus which confirmed that ET is a negative regulator of ABA, whereas ET signaling positively affects ABA action in the absence of ET (Ghassemian et al. 2000). At a molecular level, ABA regulated DREB transcription factors which belong to ethylene responsive (ERF) family of transcription factors known to be induced by ET (Agarwal et al. 2006; Lata and Prasad 2011). For breaking dormancy, ET intervenes with ABA signaling (Matilla and Matilla-Vázquez 2008) and for regulation of stomatal closure, ABA also need to crosstalk with ET signaling under drought and salt stress (Tanaka et al. 2005; Neill et al. 2008). Integration of ABA and ET signaling has shown to play a role in the improvement of salinity stress tolerance (Amjad et al. 2014). In a study, it was shown that ET receptors ETR1 and ETR2 have roles in stress signaling which are independent of ET signaling. Both ETR1 and ETR2 modulate ABA signaling for regulating germination under salinity stress (Wilson et al. 2014). Thus, the crosstalk between ABA and ET is also important in maintaining the hormonal level of each other for finalizing decisions on growth, dormancy, fruit ripening, stomatal closing, etc. under conditions of abiotic stress (Arc et al. 2013; Dolferus 2014).

11.3.4

Crosstalk Between ABA and Auxins

ABA and auxins work antagonistically for regulating developmental processes such as growth, differentiation, calcium level, and pH level of plant cells (Gehring et al. 1990; Daminato et al. 2013; Sun and Li 2014). For instance, A SHATTERPROOF-like gene is regulated by auxin and ABA antagonistically for controlling fruit ripening (Daminato et al. 2013). ABSCISIC ACID INSENSITIVE3 (ABI3) is an auxin-regulated, ABRE-based transcription factor which plays important role in regulating seed dormancy (Liu et al. 2013). During abiotic stress, the crosstalk between ABA and auxin assists the survival of seeds. During water stress, auxin transport is modulated by ABA for maintaining root growth in the root tip (Xu et al. 2013). Under drought stress situations, an R2R3-type MYB transcription factor, MYB96, has been shown to modulate stress tolerance by integrating ABA and auxin signaling, which further results in the regulation of some auxin metabolism-related GH3 genes (Seo et al. 2009). Two Cys2/His2 zinc-finger proteins AZF1 and AZF2 have been reported to suppress ABA-repressive and auxin-inducible genes during salinity stress, which indicates regulatory crosstalk (Kodaira et al. 2011). In a recent study, WRKY46 was shown to regulate lateral root development during salinity stress via regulating ABA signaling and auxin balance (Ding et al. 2015). Additionally, ASCORBATE PEROXIDASE6 (APX6) has been identified as a crosstalk-mediating enzyme between ABA, auxin, and reactive oxygen species for protecting plants from abiotic stress (Chen et al. 2014). Induced activity of ABA was found in auxin-primed seed, which rescued plants under salt stress as a result of crosstalk between these hormones (Fahad et al. 2015). Thus, the crosstalk of ABA and auxin has major role in the improvement of abiotic stress tolerance of germinating seed as well as developing plants.

11.4 Conclusion and Future Directions

11.3.5

Crosstalk Between ET and Auxins

Auxins are one of the major hormones in plant development and have been shown to play an important role during stress survival. Along with ET, auxins control root development during abiotic stresses such as salinity and drought (Muday et al. 2012). Both of the hormones strengthen the root system in such a way to enable plants to survive under abiotic stresses. In general, NAC2 transcription factor acts downstream of ethylene and auxin signaling pathways and plays a role in abiotic stress response and lateral root development for better survival in various plants (He et al. 2005; Hao et al. 2011; Nuruzzaman et al. 2013; Shan et al. 2014). Recently, ethylene-insensitive Never ripe (Nr) and auxin-insensitive diageotropica (dgt) tomato mutants were identified. In further studies, it was observed that the crosstalk between ET and auxins is important in maintaining Cd stress tolerance in tomato via communication between root and shoot parts (Alves et al. 2017). Under abiotic stress, auxin-triggered ethylene modulates ABA biosynthesis and growth inhibition, which leads to the rescue of plants from such conditions (Hansen and Grossmann 2000). Therefore, it is clear from several studies that there is pivotal crosstalk between ET and auxins for improvement of abiotic stress tolerance. 11.3.6

Crosstalk Between ABA and CTs

Individually, ABA and CTs play important roles in stress tolerance, mainly during salinity stress. Additionally, there also exists crosstalk between ABA and CTs specifically under stress conditions (Verslues 2016). Cytokinin histidine kinase receptors are known as ARABIDOPSIS HISTIDINE KINASEs (AHKs) in Arabidopsis. Under salt, drought, and cold stress, AHKs have been proved to be important regulators (Tran et al. 2007; Jeon et al. 2010; Kumar and Verslues 2015). AHK1 has been shown to be a positive regulator of ABA signaling, whereas AHK2 and AHK3 are negative regulators of ABA signaling (Tran et al. 2007; Kumar and Verslues 2015). Functional analysis of CT-deficient plants has proven that CTs negatively modulate salt and drought stress signaling linked to induced ABA sensitivity. It was also observed that CT biosynthesis genes such as ISOPENTENYL-TRANSFERASEs and CYTOKININ OXIDASES/ DEHYDROGENASES were suppressed by ABA (Nishiyama et al. 2011). In drought stress, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINs negatively regulate expression of ABA-responsive genes. CT catabolism-related enzymes CYTOKININ OXIDASEs are also negatively regulated by ABA. During heat stress, CT and ABA balance has been observed to be an important factor for developing kernels in maize (Cheikh and Jones 1994). CT oxidase has also been reportedly affected by CT and ABA under abiotic stress (Brugière et al. 2003). Therefore, ABA crosstalks with CTs under abiotic stress for maintaining CT homeostasis and stress tolerance (Kumar and Verslues 2015; Li et al. 2016).

11.4 Conclusion and Future Directions In plants, the complex but specific crosstalk networks among phytohormones enable them to grow as well as survive under any kind of stress environment. It seems that all phytohormones need to crosstalk among themselves to maintain their balance levels (Figure 11.1). In a study, it was reported that inhibition of GA biosynthesis affects the

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Abiotic stresses (Salinity, drought, cold etc.)

ROS, NO, Ca+2

ROS

Plant Cell

ABA biosynthesis NCED ABA2

AHK2, AHK3

ET biosynthesis ACO DREBs ACS JA DELLAs ET JAZs GA biosynthesis

ABA

SA Auxin

CKX1, CKC2, CKC3, CKC4

CT

ABA

GA GID1 DELLA

JA ARR1

SHY2

RGL2

RGL2

ABA Signaling PIN1, PIN3, PIN7 Export

GA

GA

ERF1, EREBs

Seed germination growth

Auxin

ARRs

Auxin signaling

ABA ABI3, Responsive ABI5 genes

ABI3

Abiotic Stress Responses

Figure 11.1 Phytohormonal crosstalks in abiotic stress environment. Upon perceiving abiotic stress signals, mainly abscisic acid (ABA) biosynthesis occurs. ABA crosstalks with other phytohormones either by affecting their biosynthesis or by interfering with their signaling pathways. Therefore, there are a lot of crosstalk points intercepting each other at different points under abiotic stress, which leads to overall abiotic stress adaptation in plants.

References

levels of auxins, cytokinins, ABA, and ET in rice (Izumi et al. 1988). Therefore, there is possibility of crosstalks at multiple levels between more than two phytohormones which may be complex, yet specific and precise. In a recent study, ABA-induced root elongation was found to be affected by ET and auxins. By using inhibitors of ethylene and auxin pathway-related genes, it was revealed that ABA-mediated growth occurs by crosstalk among ET and auxins. It was concluded that low ABA-mediated growth stimulation occurs by ethylene-independent pathway which in turn crosstalks with auxin signaling and the PIN2/EIR1-mediated auxin-efflux-dependent pathway. On the other hand, high ABA-mediated growth inhibition follows ethylene-dependent pathway crosstalk with auxin signaling and the AUX1-mediated auxin-influx dependent pathway (Li et al. 2017). In Arabidopsis thaliana, mutant etr1-2 metabolic pathways of major phytohormones such as ABA, auxin, cytokinin, and gibberellin have been found to be altered during seed dormancy, moist-chilling, and germination processes (Chiwocha et al. 2005). Under salinity pressure, the root growth and development of the plant are regulated by ABA signaling through crosstalk with other growth hormones such as auxin, CT, and ET (Liu et al. 2017). For better understanding of crosstalk networking in plant cells, plant scientists are using robust and high-throughput techniques. In the coming years, the whole network of phytohormonal signaling during multiple abiotic stresses will be revealed which will further help improve the abiotic stress tolerance of crops for better yield and survival under such conditions.

Acknowledgements The author acknowledges the financial support from SERB-National post doctoral scheme, the Government of India.

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12 Plant Molecular Chaperones: Structural Organization and their Roles in Abiotic Stress Tolerance Roshan Kumar Singh, Varsha Gupta, and Manoj Prasad National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India

12.1 Introduction Being sessile, plants are continually challenged by environmental stresses, which are primarily classified as biotic (pathogens and herbivores) and abiotic. Among these, abiotic stresses are prevalent throughout the globe, and include factors such as temperature, salinity, water deficit, waterlogging, etc. Owing to these stresses, plant productivity is rapidly declining, and abiotic stresses have led to a reduction in yield of crop plants by 50% (Wang et al. 2004). A report by the Food and Agriculture Organization of the United Nations (FAO) suggests that only 3.5% of the total cultivable land area is not affected by any environmental stress (FAO 2011). This will lead to a tremendous increase in the price of many important agricultural crops. For instance, the land available for rice cultivation worldwide amounts to 130 million hectares, of which 30% exhibits saline conditions, 20% is subjected to drought conditions and 10% experiences low temperature (15 ∘ C or below) (Korres et al. 2017; Ghosh et al. 2016); such conditions will result in reduced productivity in such areas, hence they are becoming a major threat to food security. When a plant is challenged by abiotic stress, a network of cellular responses is triggered, which help it to combat the stress. One such system, protecting the plants from stress-induced damage, is governed by heat shock proteins (HSPs) and heat shock factors (HSFs) (Al-Whaibi 2011). HSFs are transcriptional activators that recognize the stress signals and result in the transcription of HSP-encoding genes (Reddy et al. 2014). These HSPs are also referred to as molecular chaperones, and are stress-responsive proteins that accumulate at higher levels to protect other proteins from being dysfunctional. The rapid increase in the level of HSPs upon exposure to various stresses indicates their importance in stress response. The HSPs were discovered by the Italian scientist Ferruccio Ritossa in 1962 while studying the type of nucleic acid synthesized during the puffing of chromosomes of Drosophila melanogaster after exposure to heat stress. These proteins are present in normal conditions, also playing a role in maintaining protein homeostasis by assisting the folding–refolding of naïve proteins, and the disaggregation, import, and proteolytic degradation of other proteins. These proteins are highly conserved, widespread, and known to be ubiquitously present in all living organisms, from bacteria to plants to Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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human. In eukaryotic organisms, various proteins of HSP/chaperones are located in both the cytoplasm and cellular compartments, including nucleus, chloroplast, mitochondria, and endoplasmic reticulum (ER). The HSPs belong to six major classes on the basis of the approximate molecular mass and nature of function: small heat shock proteins (sHSPs), HSP40 (J-proteins), HSP60 (chaperonins), HSP70 (DnaK), HSP90, and HSP100 (Clp proteins) (Table 12.1). In addition to these major families, the presence of some other proteins with chaperone functions has been reported, such as GrpE protein assisting in preventing heat-induced protein aggregation in Escherichia coli (Wu et al. 1996) and calnexin/calreticulin and protein disulfide isomerase playing an important role in protein folding in the endoplasmic reticulum. These proteins function in a cooperative manner by forming a molecular network to maintain the proteome homeostasis. They do not covalently bind to target unfolded/misfolded, aggregated substrate proteins and do not form part of the final native proteins. A major myth surrounding HSPs is that they are induced only upon heat stress; various studies indicate the upregulation of these genes in conditions such as freezing temperature, salinity, drought, light, and oxidative and heavy metal stress (Timperio et al. 2008; Singh et al. 2016). This indicates that HSP chaperones are integral members of stress-responsive cascade and therefore, considering their importance, genome-wide studies have been performed in several model crops such as Arabidopsis, rice, and foxtail millet (Sarkar et al. 2009; Guo et al. 2008; Singh et al. 2016). Table 12.1 Summary of molecular size, existing members, cellular location and function of major families of plant heat shock proteins (HSPs).

Family

Molecular size (kDa)

Chaperones

Location

Function

HSP100

100–104

HSP100, Clp

Cytosol, chloroplast, mitochondria

Helps in resolubilization of heat-inactivated protein aggregates, protein folding, degradation of proteins unable to refold

HSP90

82–90

HSP90, Grp94/gp96

Cytosol, nucleus, ER, chloroplast, mitochondria

Regulation of receptor, role in signal transduction, refolds and maintains proteins

HSP70

68–75

HSP70, BiP/Grp78

Cytosol, nucleus, ER, chloroplast, mitochondria

Helps in proper folding of nascent polypeptide chain, role in protein translocation, refolding of denatured proteins

HSP60

—

Cpn60

—

Assist in refolding and prevent aggregation of proteins, role in assembly of Rubisco in chloroplast, helps in protein degradation

HSP40

35–54

HSP40

Cytosol, ER

Essential co-chaperone of HSP70 proteins, regulates its ATPase and substrate release activity

sHSP

16–30

HSP20

Cytosol, ER, chloroplast, mitochondria

Prevent aggregation and heat inactivation of proteins, acts as co-chaperone for HSP70 and HSP100

ER, Endoplasmic reticulum.

12.2 Classification of Plant HSPs

12.2 Classification of Plant HSPs 12.2.1

Structure and Functions of sHSP Family

The sHSPs are a family of molecular chaperones of diverse size and molecular mass ranging from 16 to 43 kDa. An important feature of sHSPs is their ability to form dimeric or large oligomeric structures under normal cellular conditions. The number of subunits in oligomers varies greatly among different sHSPs of even the same organism (Banerjee and Roychoudhury 2018). The individual monomer of sHSP is characterized by a well-conserved C-terminal 𝛼-crystallin domain of ∼90 amino acids flanked by a hypervariable N-terminal region and C-terminal extension (Figure 12.1a). Recent reports suggest the presence of I–X–I, I/V–X–I/V, or an I/V/L–X–I/V/L motif in the C-terminal part of sHSP sequences (Poulain et al. 2010). The sHSP16.9 from wheat formed a barrel-shaped structure assembled from two hexameric disks with a total of 12 monomers (Poulain et al. 2010). The chaperone activity of these proteins is ATP-independent (Basha et al. 2013). Among the six families of molecular chaperones (sHSPs, HSP40, HSP60, HSP70, HSP90, and HSp100), sHSPs are the most abundant. On an average, there are one to two present in Archaea while all higher plants have at least 20 sHSPs in the cytoplasm and various cellular compartments, including semiautonomous organelles—the chloroplast and mitochondria. Many genome-wide studies have identified the number of sHSP encoding genes in various plant species, such as 39 in rice (Ouyang et al. 2009), 163 in wheat (Muthusamy et al. 2017), 51 in soybean (Lopes-Caitar et al. 2013), 42 in tomato (Yu et al. 2016), and 37 in foxtail millet (Singh et al. 2016). All plant sHSPs are encoded by the nuclear genome. In higher plants, on the basis of cellular localization, sHSPs are categorized into 11 subfamilies, namely CI-CVI, MTI, MTII, ER, CP, and

(a)

Signal peptide

Folded protein

N-Terminal

HSP20 domain

C-Terminal

Denatured protein

sHSP complex with denatured proteins

Disaggregated proteins

Heat activation

(b)

sHSP oligomers

sHSP dimers

Figure 12.1 Structural organization and mode of action of small heat shock proteins (sHSP): (a) domain structure of sHSP; and (b) mechanism of sHSP-mediated protein disaggregation.

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PX. Subfamilies I–VI localize to the cytoplasm/nucleus, MTI and MTII occur in the mitochondria, and ER, CP and PX localize to endoplasmic reticulum, chloroplast, and peroxisome, respectively (Waters 2013). The expansion of the plant sHSP gene family reflects a molecular adaptation to various stress conditions that are exclusive to the plants. In addition to heat stress, the expression of sHSPs in plants is also upregulated in other abiotic stress conditions as well as being restricted to certain developmental stages including embryogenesis, seed germination, pollen development, and fruit maturation (Sun et al. 2002; Al-Whaibi 2011; Koo et al. 2015). When wheat chloroplast-localized TasHSP26 was overexpressed in Arabidopsis, transgenic plants showed tolerance to high temperature, and accumulated more photosynthetic pigments, higher biomass and seed yield than the wild type (Chauhan et al. 2012). The antisense Arabidopsis plants were sensitive to heat (even nonlethal heat) shock, and accumulated less biomass and lower seed yield under normal growth conditions. Another important function of these proteins is the degradation of improperly folded proteins. The sHSPs cannot refold non-native proteins but bind through hydrophobic interaction to partially folded or denatured proteins, hence preventing their aggregation (Al-Whaibi 2011; Sun et al. 2002) (Figure 12.1b). Recent studies showed that the sHSP purified from the plant system binds to the denatured protein and facilitates subsequent refolding in cooperation with ATP-dependent chaperones such as HSP40/HSP70 and HSp100/ClpB complexes (Mogk et al. 2003; Muthusamy et al. 2016). 12.2.2

Structure and Functions of HSP60 Family

The HSP60 proteins are sometimes called chaperonins. They play a crucial role in assisting numerous newly synthesized and/or newly translocated multimeric proteins to achieve their native topology. This class of chaperones is most conserved and ubiquitously present in the plastids, mitochondria, and cytoplasm of all eukaryotes. They are abundant in both mitochondria and chloroplast, even in the absence of stress. The bacterial homolog of HSP60 is known as GroEL. The chaperonins are further subdivided into two subfamilies on the basis of structural organization: group I chaperonins occur in bacteria (GroEL) and endosymbiotic organelles such as chloroplast (Rubisco binding protein) and mitochondria (HSP60). The group II chaperonins contain “chaperonins containing t-complex polypeptide 1” (TCP1) and are found in the archaea and cytosol of the eukaryotic system (Wang et al. 2004). Both the classes of chaperonins form high-molecular-weight (∼800 kDa) oligomeric complexes. Group I chaperonins consist of 14 identical protomeric subunits of 57 kDa organized into a two-tier ring structure capped with 10 kDa GroES subunit on the top. On the other hand, group II chaperonins are organized into hetero-oligomeric ring complexes of eight to nine subunits per ring. The capping structure of the central cavity of the ring is composed of apical domain of the polypeptides forming the ring structure. However, the basic structures of the peptides forming both groups of chaperonins are identical, consisting of the equatorial domain, followed by a middle hinge region and an apical domain (Figure 12.2a). The overall method of function of both groups is identical but differs in the substrate encapsulation process. In group I chaperonins the GroES/HSP10 subunit functions as a detachable lid that binds in an ATP-dependent manner; on the other hand in group II chaperonins, the lid is formed by the protrusion of the ring structure of the complex which moves upward to open in a substrate-accepting

12.2 Classification of Plant HSPs

Equatorial domain

(a)

Middle hinge domain

Apical domain

Misfolded polypeptide HSP10

HSP10

ATP

ATP

ADP + Pi Native protein

HSP60 complex

Association of misfolded polypeptide to hydrophobic protein binding site of SHP60

Release of folded polypeptide from HSP60 subunit

Chaperone function of group I HSP60 Misfolded polypeptide Apical protrusion ADP + Pi

HSP60 complex

(b)

Association of misfolded polypeptide to hydrophobic protein binding site of SHP60

Native protein Release of folded polypeptide from HSP60 subunit

Chaperone function of group II HSP60

Figure 12.2 Structural organization and mode of action of HSP60: (a) domain structure of HSP60; and (b) mechanism of protein folding mediated through group I and group II HSP60.

state and closes in the ATP-bound state of the complex (Xu and Sigler 1998; Spiess et al. 2004). The catalytic chamber of chaperonins provides an optimum environment that enables the proper folding of complex multimeric proteins into their native state in an ATP-dependent manner (Vierling 1991) (Figure 12.2b). Functional characterization of chaperonins in plant response to abiotic stress and development has been carried out in several independent studies. They play an important role in the folding of many plastid proteins such as Rubisco (Boston et al. 1996). A mutant species of Arabidopsis for chloroplast chaperonins Cpn60𝛼 gene fails in chloroplast development and, successively, in the proper development of embryo and seedling (Apuya et al. 2001). Similarly, transgenic Nicotiana plant expressing antisense Cpn60𝛽 resulted in abnormal phenotypes including retarded growth, plant stunting, leaf chlorosis, altered distribution of photoassimilates, and delay in flowering (Zabaleta et al. 1994). Group II cytoplasmic chaperonin has been shown to assist in the proper folding of architectural proteins such as actin and tubulin (Gutsche et al. 1999). The deletion of the chloroplast chaperonin-encoding gene LEN1 leads to cell death in Arabidopsis, resulting in the establishment of systemic acquired resistance without pathogen attack (Ishikawa et al. 2003). 12.2.3

Structure and Functions of the HSP70 Family

Molecular chaperones belonging to 70 kDa (HSP70) have been extensively studied and are a well-characterized HSP family in bacteria as well as the eukaryotic system. In prokaryotes, the HSP70 homolog is known as DnaK, HscA, and HscC protein which is present under regular growth condition and induced by elevated temperature. HSP70

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was one of the first higher organism genes cloned and characterized in many species (Lindquist 1986; Lindquist and Craig 1988). Structurally, HSP70 consists of two conserved functional domains—an N-terminal ATPase domain of 44 kDa that hydrolyzes ATP to ADP and a peptide-binding domain of ∼25 kDa at the C-terminus that can transiently interact with the short linear peptide of folding intermediate (Figure 12.3a). ATPase and substrate binding domains are connected through a conserved linker region. The peptide-binding domain consists of a variable helical region, acts as a lid over the substrate binding cavity, and regulates the interaction with the substrate (Lund 2001). The substrate binding and release are coupled to the ATPase activity of HSP70, which requires the assistance of co-chaperones. HSP40/J-proteins and Hip are essential for the HSP70 system, and acts as co-chaperone to achieve the purpose of HSP70 complex and nucleotide exchange factors (Figure 12.3b). The lid formed by peptide binding domain remains open at the ATP bound state of HSP70 and folding peptides bind and release rapidly. During the ADP-bound state of HSP70, the lid remains close, and substrate proteins are tightly bound to the peptide binding domain (Mayer et al. 2001). The role of plant HSP70 is the prevention of the accumulation and aggregation of newly synthesized proteins and their correct folding during their translocation (Sung et al. 2001). They are localized in cytoplasm, chloroplast, mitochondria, and ER. The ER-localized HSP70 homologs are also called binding protein (BiP) or glucose-regulated protein. There seems to be cooperation in the activities of the HSP70 and sHSP families of proteins as studied in Pisum sativum (Al-Whaibi 2011). These chaperones work in unison with their co-chaperones like DnaJ/HSP40 and GrpE. Also, HSP70 and sHSP are primarily involved in protecting the plant cell against the detrimental effects of abiotic stress by facilitating the degradation of unstable proteins by targeting them to lysosomes or proteasomes (Hartl 1996). A recent study in Arabidopsis thaliana suggests the role of HSP70 in the stroma of chloroplast for the differentiation of germinating seeds and their tolerance to heat stress. A few members of HSP70 family are involved in controlling the biological activity of regulatory proteins and so might act as negative repressors of HSF-mediated transcription (Morimoto 1998). It was reported that the interaction between HSP70 and

Substrate binding domain

ATPase domain

Lid domain

Linker domain

(a)

HSP70 HSP70

NEF

ATP

ADPNEF + Pi

AT P Folded polypeptide

40

HSP

(b)

40 HSP

Protein synthesis

NEF

Figure 12.3 Structural organization and mode of action of HSP70: (a) domain structure of HSP70; and (b) mechanism of co-translational protein folding mediated through HSP70 and HSP40.

12.2 Classification of Plant HSPs

HSF inhibits the trimerization of HSF and thereby prevents the binding of HSF to heat shock element (HSE). Thus, the HSF-regulated transcriptional activation of heat responsive genes is repressed (Kim and Schöffl 2002). They are also involved in the modulation of various signal transducers like protein kinase A, protein kinase C, and protein phosphatase (Ding et al. 1998). Therefore, it is possible that the HSP70 family of proteins might play diverse roles in modulating signal transduction pathways under normal growth condition as well as abiotic stress. 12.2.3.1

DnaJ/HSP40

HSPs of 40 kDa (commonly known as HSP40 or DnaJ) play an important role in HSP70 function. They are expressed in almost all organisms from bacteria to higher plants. HSP40 shows much higher structural and sequence divergence than HSP70 within the cell. All HSP40/DnaJ proteins possess a highly conserved “J-domain” of ∼70 amino acids in an 𝛼-helical region, which is the signature sequence of this family (Figure 12.4). It contains four 𝛼-helices where helices II and III are tightly packed in an antiparallel orientation. The loop connecting these two helices contains a functionally crucial and highly conserved HPD motif that is a characteristic feature of J-domain (Walsh et al. 2004). The J-domain of HSP40 interacts with HSP70, and HPD tripeptide is essential for regulating its ATPase activity (Fan et al. 2003; Walsh et al. 2004). The J-domain is followed by a G/F region, a zinc finger motif and a C-terminal domain (Figure 12.4a). The G/F region is rich in glycine/phenylalanine and acts as a linker region between the J-domain and zinc finger motif. It is required for stability of the J-domain during interaction with HSP70 (Rajan and D’Silva 2009). The central region is cysteine-rich zinc finger motif that contains four CXXCXGXG repeats in two separate clusters, each cluster associated with a zinc ion. This region is crucial for sequestering the denatured peptide and their modulation through HSP70 (Walsh et al. 2004). The C-terminal region is comparatively less conserved and essential for dimerization and chaperone function. J-proteins can be classified into four groups on the basis of domain organization (Rajan and D’Silva 2009). Group I J-proteins contains all of the domains found in HDP40/DnaJ, such as J-domain, G/F region, zinc finger domain, and C-terminal domain. Group II proteins lack the zinc finger motif whereas Group III proteins lack both G/F region and zinc finger motif. Group IV J-proteins are similar to the group III proteins with the J-domain toward the C-terminal region but avoid HPD motif in their peptide sequence. Instead of the HPD motif, they possess a less conserved DKE motif and the J-proteins containing this are termed J-like proteins (Rajan and D’Silva 2009). HSP40/DnaJ together with GrpE are often called a co-chaperone of HSP70. They can regulate the housekeeping and stress-related function of HSP70 by assisting in ATP hydrolysis and substrate binding through its J-domain (Figure 12.3b). In addition to their co-chaperone function, HSP40/DnaJ proteins also actively associate with the misfolded/ unfolded substrate peptides to prevent their aggregation (Christen and Han 2004). Thus,

J domain

G/F motif

Figure 12.4 Domain organization of HSP40.

Zinc finger domain

C-Terminal

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12 Plant Molecular Chaperones: Structural Organization and their Roles in Abiotic Stress Tolerance

HSP40/DnaJ and HSP70/DnaK proteins may bind to the same substrate polypeptide to form a ternary complex and result in cis-interaction of the J-domain and ATPase domain of HSp70. The chaperone cycle begins with the association of unfolded polypeptide with either ATP-bound HSP70 or with HSp40/DnaJ. The J-domain of HSP40/DnaJ stimulates ATPase activity of HSP70 by transiently interacting with it. The hydrolysis of ATP results in the conversion of HSP70 to the ADP-bound state which stabilizes its association with the client polypeptide. Nucleotide exchange factors exchange ADP with ATP, leading to dissociation of the bound substrate and hence driving HSP70 for another cycle of activity (Laufen et al. 1999; Craig et al. 2006). The repeated cycle of chaperone action of HSP70/DnaK with its associated co-chaperones HSP40/DnaJ ensures the proper folding disaggregation of substrate protein (Craig et al. 2006). Thus, HSP40/DnaJ plays an important role in cellular physiology. 12.2.4

Structure and Functions of HSP90 Family

The HSP90 chaperones are distinct from other classes of molecular chaperones, and are ∼80–94 kDa in size. They are abundantly present (constituting 1–2% of total cell protein) in nonstressed cells and upregulated during stress (Buchner 1999). They play an important role in signaling protein function and trafficking. They assist the folding of a wide range of signal transduction proteins including protein kinases, steroid hormone receptors, and transcription factors. It has been postulated that they play a role in morphological evolution and stress adaptation in Arabidopsis (Wang et al. 2004). Like other classes of HSPs, they are also localized in cytoplasm, chloroplast, endoplasmic reticulum (Grp94), and mitochondria (TRAP1). In yeast, the cytoplasmic HSP90 is commonly known as Hsc82/Hsp82. HSP90 requires ATP hydrolysis to carrying out its function. All of the members of HSP90 family from bacteria (HtpG) to eukaryotes share a conserved protein architecture and mode of function. HSP90 peptide contains the highly conserved ATP-binding domain and a structurally flexible middle domain followed by a C-terminal dimerizing domain to which different co-chaperones and substrate proteins bind (Figure 12.5a). A divergent charged sequence links the N-terminal ATP-binding domain and middle domain. The functional homodimer form of HSP90 forms a circular structure in the ATP-bound state to stabilize the conformational flexibility of substrate proteins to mature into their native state. HSP90 chaperone differs from other HSPs as it does not act on nascent protein for folding, but associates with a substrate protein, which is in a near-native conformation at a later stage of protein modulation (Young et al. 2001) (Figure 12.5b). The role of HSP90 in signal transduction is highly studied. It plays an important role in maintaining functional conformation of receptors for steroid hormones, signal transducers, cell cycle regulators, many tyrosine and serine/threonine kinases, and proteins like calcineurin and nitric oxide synthase (Echeverria and Picard 2010). It also plays a role in the assembly and maintenance of 26S proteasome, which is the central protein complex for cellular protein degradation (Imai et al. 2003). A study in A. thaliana indicates that cytoplasmic HSP90 negatively regulates the HSFs in the absence of heat, but under elevated temperature, the inhibitory effect of HSP90 is terminated temporarily to initiate HSF function (Yamada et al. 2007). Expression of HSP90 is regulated and responds to heat, cold, and salinity stresses (Wang et al. 2004). The role of HSP90 has also been elucidated in biotic stress in Arabidopsis, two species of Nicotiana

12.2 Classification of Plant HSPs

ATPase domain Middle domain Charged sequence

HS P4 0

HSP90 cochaperone

HSP90 open state

Protein folding through HSP70 complex

Substrate binding domain /dimerization domain

ADP + Pi

ATP

HS P4 0

(a)

HSP90 dimer

HSP90 cochaperone HSP90 dimer state with nearnatively foldded substrate

HSP90 cochaperone

Folded protein

(b)

Figure 12.5 Structural organization and mode of action of HSP90: (a) domain structure of HSP90; and (b) mechanism of protein folding mediated through HSP90.

(Nicotiana tabacum and Nicotiana benthamiana) and rice where cytoplasmic HSP90 confers pathogen resistance by modulating the R protein, which is the pathogen signal receptor (Hubert et al. 2003; Liu et al. 2004; Thao et al. 2007). 12.2.5

Structure and Functions of HSP100 Family

The HSP100/Clp family of chaperones is member of the large AAA+ ATPase family which are involved in reactivation of aggregated proteins by resolubilization and degradation of irreversibly misfolded proteins in an ATP-dependent manner (Bösl et al. 2006; Kim et al. 2007). Their presence under normal growth conditions is not required but is induced many fold under extreme harsh environmental stress (Hong and Vierling 2001). The crystal structure of HSP100/Clp protein reveals the presence of an N-terminal domain, the first nucleotide binding domain (NBD-1) and a middle domain (M domain) followed by a second nucleotide binding domain (NBD-2) (Figure 12.6a). This family is further divided into two classes based on the number of nucleotide-binding domains. The first class contains two nucleotide-binding domains (or ATP-binding domains) while the second contains only one nucleotide-binding domain. The protomers of HSP100 assemble to form a three-tiered hexameric ring-shaped structure. ATP binding to the N-terminal domain of HSP100 stabilizes its oligomeric state to facilitate interaction with the substrate. Similar to other HSPs, the HSP100 family of chaperones is constitutively expressed in plants, but their expression is regulated in response to heat, cold, dehydration, salinity, or dark conditions (Wang et al. 2004). Other than their role in providing adaptation to harsh conditions, some specific members of HSP100/Clp protein deliver a housekeeping function that is crucial for the development and maintenance of chloroplast (Lee et al. 2007). The mechanism of the disaggregation function of HSp100/Clp for rescuing proteins involves the collaboration of another ATP-dependent chaperone system, the HSP70/DnaK. Both chaperone systems work synergistically to refold/degrade substrate proteins that each can remodel separately (Figure 12.6b). The precise sequence of

229

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12 Plant Molecular Chaperones: Structural Organization and their Roles in Abiotic Stress Tolerance

(a)

N-Terminal

Middle domain

Nucleotide-binding domain-I

Nucleotide-binding domain-II

C-Terminal

40 70

HSP70/ HSP40

HSP100 70 40

sHSP-bound aggregated polypeptide

(b)

HSP70/HSP40 associated protein aggregate

70 40

70

40

ATP

sHSP dimers Association of HSP70/HSP40 and HSP100 with protein aggregate Threading and extraction of substrate polypeptide

Chaperone mediated re-folding of peptide

Figure 12.6 Structural organization and mode of action of HSP100: (a) domain structure of HSP100; (b) mechanism of protein folding mediated through HSP100 with the assistance of sHSP, HSP70, and HSP40.

events for protein remodeling has not been defined yet. It can be considered that the HSP100/Clp protease solubilizes the heat-denatured aggregated proteins and releases them to the HSP70/DnaK system for refolding (Goloubinoff et al. 1999). At the same time, it is possible that the HSP70/DnaK chaperone machinery acts initially on the aggregated proteins to direct the process of disaggregation by assisting the sorting of polypeptides for further refolding and presenting the unstructured polypeptide that fails to refold to the HSp100/Clp for degradation.

12.3 Regulation of HSP Expression in Plants Various mechanisms control the regulation of HSP expression in plants. The central regulators that trigger the transcription of HSP-encoding genes are the HSF proteins. HSFs are transcription factors located in the cytoplasm in an inactive state and are activated under stress conditions. Under normal unstressed conditions, HSFs are maintained in an inactive monomer state, and upon the occurrence of stress, they are converted into a transcriptionally active trimer state. HSFs recognize the conserved palindromic motif (5′ -AGAAnnTTCT-3′ ) known as HSE conserved upstream of heat-stress responsive genes in all higher organisms (Bienz and Pelhan 1987). Despite the variation in size and sequence, HSF structure and function are conserved throughout the organisms. Structurally, HSF is composed of five domains each having a particular function: (i) DNA-binding domain (DBD) for binding to HSE; (ii) oligomerization domain (HR-A/B) for trimer formation; (iii) Nuclear Localization Signal and Nuclear Export Signal for nuclear import and export, respectively; (iv) activator motif (AHA motif ) for recruiting RNA polymerase machinery; and (v) repressor domain for transcriptional repression. The AHA motif is reported to be rich in aromatic (F, Y, W), hydrophobic (V, L, I), and acidic (D, E) residues (Nover et al. 2001). The DBD domain situated in the N-terminal region of the HSF peptide is highly conserved in all kingdoms and is composed of four antiparallel 𝛽-sheets tightly packed against a bundle of three 𝛼-helices. The core of this domain is formed by a hydrophobic helix–turn–helix motif that is essential for HSE

12.4 Crosstalk Between HSP Networks to Provide Tolerance Against Abiotic Stress

recognition (Harrison et al. 1994). The adjacent HR-A/B region, formed from hydrophobic heptad repeats, is connected through DBD by a flexible linker. The helical coiled-coil structure formed from heptad repeats of HR-A/B motif is responsible for oligomerization of HSFs (Peteranderl et al. 1999). Based on the differences in insertions in the oligomerization (HR-A/B region) domain, plant HSFs are grouped into three classes: HSF-A, HSF-B, and HSF-C. The HR-A/B region in plant HSFs is similar to that in nonplant HSFs while the class A and class C HSFs have 21 and 7 amino acids insertions, respectively. Class B HSFs differ from the classes A and C by the absence of this additional amino acid insertion. HSF-B and HSF-C are also characterized by the absence of AHA motif, which is present in class A HSFs. The class A HSFs are primarily involved in the activation of HSPs and responses to environmental stimuli, while class B HSFs, owing to the absence of an activator motif, serve as repressors of gene expression (Yang et al. 2014). Binding of HSF A to the HSE activates transcription of HSPs and transcriptional relay of HSFs, including HSFA2, A3, The HSF classes B and C are devoid of the activator AHA motif that is essential for the transcriptional regulation of HSF A. Therefore, class B and C HSFs are considered as inhibitory HSFs. HSFB1 and HSFB2b have been reported to repress the expression of HSPs during stress recovery in Arabidopsis (Ikeda et al. 2011). The HSFA1 is a central regulator of HSP gene expression, plant development, and abiotic stress responses. The transcription factors HSFA1a, b, d, and e are expressed constitutively in Arabidopsis and act as a positive regulator of heat stress responsive genes. They are responsible for initiating the acquisition of basal thermotolerance (Yoshida et al. 2011). In another study in Arabidopsis, the different triple mutants HSFA1a, b, d; HSFA1a, b, e; HSFA1b, d, e; and HSFA1a, d, e as well as quadruple mutant HSFA1a, b, d, e revealed specificity for different stress tolerance. The triple mutant HSFA1a, b, d and quadruple HSFA1a, b, d, e mutant are highly sensitive to even moderately high temperature. The HSFA1b, d, e mutant was unable to grow under salt stress. The results indicated that all HSFA1s (HSFA1d and HSFA1e with more preference) are associated with osmotic stress tolerance. HSFA1b and HSFA1d are involved in providing tolerance to oxidative stress. The quadruple mutant HSFA1a, b, d, e showed a drastic effect on seed development with the abortion of more than 20% of the seeds (Liu and Charng 2013). All of these aberrations were partially or fully resolved upon overexpression of HSFA2. It is sufficient to provide tolerance against heat, salt, osmotic stress, and combined heat, high light conditions, and oxidative stresses (Ogawa et al. 2007). Thus, it can be concluded that HSE’s function is not only to regulate HSP expression, but also activate multiple signal transduction pathways to provide tolerance against abiotic stress and regulate normal cellular development under nonstress conditions.

12.4 Crosstalk Between HSP Networks to Provide Tolerance Against Abiotic Stress The protective response to abiotic stress leads to the activation of various HSPs/ chaperones which work coordinately or synergistically to form a network of chaperone machinery. Individual HSPs can also be involved in other stress-related signaling events to acquire tolerance in plants. Thus, there is crosstalk between different HSPs of the same or other classes to prevent cellular damage and to re-establish proteome

231

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12 Plant Molecular Chaperones: Structural Organization and their Roles in Abiotic Stress Tolerance

homeostasis (Jacob et al. 2017). Abiotic stress usually causes protein dysfunction and degradation. To maintain proteins in functional forms, sHSPs first bind to non-native polypeptides and prevent their aggregation. It has been demonstrated in vitro that sHSPs provide a reservoir of the misfolded substrate to the HSP70/HSP100 for subsequent refolding or degradation of the substrate that cannot be refolded. The cooperation between sHSP, HSP70, HSP40, and HSp100 was confirmed in vitro in several studies ̇ (Zwirowski et al. 2017). Another array of events proposed that HSP100/Clp protein can efficiently resolubilize protein aggregates, the solubilized protein is then refolded by the cooperation of HSP70/DnaK protein and the final refolding of nearly native proteins can be completed by the assistance of HSP60 chaperone family (Ben-Zvi and Goloubinoff 2001). In addition to the interaction of various HSPs within the chaperone network, several studies indicated that they also interact with the other stress-response mechanism. A study performed in E. coli (Diamant et al. 2001) showed that some osmolytes such as trehalose, glycerol, proline, glycine, and betaine could also act as a chemical chaperone that suppresses the aggregation of the denatured substrate and assists in the refolding of unfolded proteins. Heat shock proteins and osmolytes can act synergistically to protect cellular protein degradation from osmotic stress (Viner and Clegg 2001). Other than osmolytes, proteins of HSp70 and HSP90 chaperone and their co-chaperones were shown to interact with a wide variety of signaling molecules such as hormone receptors, cell-cycle and cell-death receptors, tyrosine, and serine/threonine kinases, indicating that HSPs might play an important role in cellular signal transduction cascade (Nollen and Morimoto 2002). Although the majority of these studies are carried out in the non-plant system, similar interactions might operate in plants. In Arabidopsis, under high light conditions, several HSPs were shown to induce and be involved in reducing the adverse effect of oxidative stress in addition to their chaperone function (Rossel et al. 2002).

12.5 Genetic Engineering of HSPs for Abiotic Stress Tolerance in Plants A large number of plant species have been genetically engineered for some desired traits including tolerance to abiotic stress, resistance to biotic stress, increase in yield, and nutritional properties. Different approaches are used to generate transgenic abiotic stress-tolerant plants. One of the major approaches of producing genetically engineered plants is the overexpression of a key molecular chaperone in plants, which activates the stress-specific molecular response. Rice transgenic plants overexpressing sHSP HSP17.7 confer thermotolerance and resistance to UV-B stress (Murakami et al. 2004). In another study, cytosolic sHSP17.8 from Rosa chinensis was introduced into E. coli, yeast, and Arabidopsis for constitutive expression (Jiang et al. 2009). Recombinant E. coli- and yeast-expressed RcHSP17.8 demonstrated enhanced viability under heat, salinity, and oxidative stress. Transgenic Arabidopsis plants showed resistance to heat, salt, drought, and oxidative stress. Overexpression of maize cytosolic class I sHSP ZmHSP16.9 in tobacco conferred heat and oxidative stress tolerance by increased seed

12.5 Genetic Engineering of HSPs for Abiotic Stress Tolerance in Plants

Table 12.2 Summary of different classes of HSP overexpression in plants (recently) and resulting phenotype with respect to abiotic stress tolerance. Gene

Plant

Stress tolerance

Reference

CsHSP17.7, CsHSP18.1, CsHSP21.8

Arabidopsis

Increases heat and cold tolerance

Wang et al. 2017

PtHSP17.8

Arabidopsis

Tolerance to heat and salt stresses

Li et al. 2016

AsHSP17

Arabidopsis

Enhanced sensitivity to heat and salt stress

Sun et al. 2016

OsHSP18.2

Arabidopsis

Improved seed vigor, longevity and improved germination and seedling establishment under abiotic stress

Kaur et al. 2015

OsHsp17.0, OsHsp23.7

Rice

Enhances drought and salt tolerance

Zou et al. 2012

GmHSP40.1

Arabidopsis

Cell death and accelerated senescence

Liu and Whitham 2013

BcHSP70

Tobacco

Tolerance to heat stress

Wang et al. 2016

MuHSP70

Arabidopsis

Tolerance to heat, cold, drought, salinity and oxidative stress

Masand and Yadav 2016

EaHSP70

Sugarcane

Increases drought and salinity tolerance

Augustine et al. 2015

GmHsp90

Arabidopsis

Tolerance to multiple abiotic stress

Xu et al. 2014

AtHsp90.2, AtHsp90.5, AtHsp90.7

Arabidopsis

Enhances plant sensitivity to salt and drought stresses

Song et al. 2009

germination rate, root length and antioxidant enzyme activity in a transgenic plant compared with wild type (Sun et al. 2012). These observations suggest that sHSPs trigger a regulatory network which provides tolerance to a wide range of unfavorable conditions (Table 12.2). Genetically engineered Arabidopsis plants constitutively expressed Trichoderma harzianum HSP70 gene, which conferred tolerance to heat and other abiotic stresses (Montero-Barrientos et al. 2010). The high level of fungal HSP70 accumulation in Arabidopsis plants negatively regulates HSF transcriptional activity by preventing the expression of HSFs and blocking the synthesis of new HSPs. The overexpression of cytosolic AtHSP90.3 gene in Arabidopsis makes the plant more sensitive to heat stress; the seed germination rate was low with higher seedling mortality rate but better tolerance to Ca+2 (Xu et al. 2010). From this study, it can be concluded that overexpression of AtHsp90.3 might have altered the usual homeostasis of Ca-binding proteins and disturbed the normal Ca signaling pathway, thus producing transgenic seeds and seedlings that are more sensitive to heat stress. In another similar kind of study where three AtHsp90 isoforms, cytosolic AtHsp90.2, endoplasmic reticulum-located AtHsp90.7, and chloroplast-located AtHsp90.5 were overexpressed in Arabidopsis, it resulted in reduced plant tolerance to drought and salt stress with lower germination rate and fresh weight, but improved tolerance to high Ca concentration (Song et al. 2009). A brief effect of overexpression of various classes of HSPs on abiotic stress in different plant species has been summarized in Table 12.2.

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12.6 Conclusion Plants must be protected in a variety of stressful conditions, including high temperature and salt, drought, chilling, and oxidative environments, which directly affect structure and function of cellular proteins. In response, plants produce a number of cellular molecules that protect the plant cells from being impaired and help to sustain the normal cell physiology. Production of heat shock proteins is one of the ancient response mechanisms. Different families of HSPs synthesized by plants are present abundantly during unfavorable conditions but are also a major component of unstressed plant cells. They are found in multiple cellular compartments including cytoplasm, nucleus, and endoplasmic reticulum, as well as semiautonomous organelles, mitochondria, and chloroplast. The proteins of HSPs are highly structurally and functionally conserved throughout the living kingdom. Plant HSPs belonging to different classes are homologous to HSPs of prokaryotes and other eukaryotes. The diversity and abundance of small-molecular-weight HSPs are unique to plants. Individual members of each family of HSP/chaperone perform a specific function; the interaction between different HSPs/chaperones constitutes a chaperone network which plays a central role in maintaining cellular protein homeostasis. The HSP/chaperone network determines the fate of the non-native or denatured protein under extreme or normal growth condition. In fact, individual HSP members also act as regulatory molecules and participate in stress-induced signal transduction, stress sensing, and activation of abiotic stress-related genes. Furthermore, many transgenic plants constitutively expressing HSP show tolerance to abiotic stress(s). Other than a key component of the stress response, HSPs/chaperones play an essential role in protein folding, assembly, translocation, and degradation in growing conditions. Research is ongoing to investigate the fine tuning of the HSP/chaperone network and its inter-relationship with other cellular components.

Acknowledgements Roshan Kumar Singh acknowledges the research fellowship received from Council of Scientific and Industrial Research, Government of India.

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Hubert, D.A., Tornero, P., Belkhadir, Y. et al. (2003). Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein. EMBO J. 22: 5679–5689. Ikeda, M., Mitsuda, N., and Ohme-Takagi, M. (2011). Arabidopsis HsfB1 and HsfB2b act as repressors of the expression of heat-inducible Hsfs but positively regulate the acquired thermotolerance. Plant Physiol. 157: 1243–1254. Imai, J., Maruya, M., Yashiroda, H. et al. (2003). The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J. 22: 3557–3567. Ishikawa, A., Tanaka, H., Nakai, M., and Asahi, T. (2003). Deletion of a chaperonin 60 beta gene leads to cell death in the Arabidopsis lesion initiation 1 mutant. Plant Cell Physiol. 44: 255–261. Jacob, P., Hirt, H., and Bendahmane, A. (2017). The heat-shock protein/chaperone network and multiple stress resistance. Plant Biotechnol. J. 15: 405–414. Jiang, C., Xu, J., Zhang, H. et al. (2009). A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana. Plant Cell Environ. 32: 1046–1059. Kaur, H., Petla, B.P., Kamble, N.U. et al. (2015). Differentially expressed seed aging responsive heat shock protein OsHSP18.2 implicates in seed vigor, longevity and improves germination and seedling establishment under abiotic stress. Front. Plant Sci. 6: 713. Kim, B.H. and Schöffl, F. (2002). Interaction between Arabidopsis heat shock transcription factor 1 and 70 kDa heat shock proteins. J. Exp. Bot. 53: 371–375. Kim, H.J., Hwang, N.R., and Lee, K.J. (2007). Heat shock responses for understanding diseases of protein denaturation. Mol. Cells 23: 123–131. Koo, H.J., Park, S.M., Kim, K.P. et al. (2015). Small heat shock proteins can release light dependence of tobacco seed during germination. Plant Physiol. 167: 1030–1038. Korres, N.E., Norsworthy, J.K., Burgos, N.R., and Oosterhuis, D.M. (2017). Temperature and drought impacts on rice production: an agronomic perspective regarding short- and long-term adaptation measures. Water Resour. Rural Dev. 9: 12–27. Laufen, T., Mayer, M.P., Beisel, C. et al. (1999). Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc. Natl. Acad. Sci. U.S.A. 96: 5452–5457. Lee, U., Rioflorido, I., Hong, S.W. et al. (2007). The Arabidopsis ClpB/Hsp100 family of proteins: chaperones for stress and chloroplast development. Plant J. 49: 115–127. Li, J., Zhang, J., Jia, H. et al. (2016). The Populus trichocarpa PtHSP17.8 involved in heat and salt stress tolerances. Plant Cell Rep. 35: 1587–1599. Lindquist, S. (1986). The heat-shock response. Annu. Rev. Biochem. 55: 1151–1191. Lindquist, S. and Craig, E.A. (1988). The heat-shock proteins. Annu. Rev. Genet. 22: 631–677. Liu, H.C. and Charng, Y.Y. (2013). Common and distinct functions of Arabidopsis class A1 and A2 heat shock factors in diverse abiotic stress responses and development. Plant Physiol. 163: 276–279. Liu, J.Z. and Whitham, S.A. (2013). Overexpression of a soybean nuclear localized type-III DnaJ domain-containing HSP40 reveals its roles in cell death and disease resistance. Plant J. 74: 110–121. Liu, Y., Burch-Smith, T., Schiff, M. et al. (2004). Molecular chaperone Hsp90 associates with resistance protein N and its signaling proteins SGT1 and Rar1 to modulate an innate immune response in plants. J. Biol. Chem. 279: 2101–2108.

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13 Chloride (Cl− ) Uptake, Transport, and Regulation in Plant Salt Tolerance DB Shelke 1,2,# , GC Nikalje 1,3,6,# , TD Nikam 1 , P Maheshwari 4 , DL Punita 4 , KRSS Rao 4 , PB Kavi Kishor 5 , and P. Suprasanna 6,7 1

Department of Botany, Savitribai Phule Pune University, 411 007 Pune, India Department of Botany, Amruteshwar Arts, Commerce and Science College, Velha, 412213 Pune, India 3 Department of Botany, R.K. Talreja College of Arts, Science, and Commerce, Ulhasnagar, 421003, Thane, India 4 Center for Biotechnology, Acharya Nagarjuna University, 522 510 Guntur, India 5 Department of Genetics, Osmania University, 500 007 Hyderabad, India 6 Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, 400 085 Mumbai, India 7 Homi Bhabha National Institute, Mumbai, 400 095, India 2

13.1 Introduction Increasing soil salinity in the form of NaCl is one of the most important abiotic stress factors affecting agriculture worldwide. Salt-affected soils are categorized into saline, saline-sodic, and sodic, depending on abundance of salt, types of salt, amount of Na+ present and soil alkalinity. Na+ is the common factor in both types and is present along with Cl− , sulfate, calcium, and magnesium in saline soils and with molybdate and carbonate in sodic soils. Among the world’s salt-affected areas, 397 Mha are saline and 434 Mha are sodic (FAO 2008). Excessive irrigation without proper drainage, climate change, rising sea levels, and underlying rocks rich in harmful salts are the factors responsible for elevating salt levels. If these environmental problems continue, there is a possibility of a gradual decrease in the amount of available agricultural land of up to 50% in the future (Wang et al. 2003). Among the various salts, Na+ and Cl− are the most dominant in soil, constituting 50–80% of soluble salts from the majority of saline soils (Rengasamy 2010). Hyperosmotic stress, ionic imbalance, and toxicity are important responses after plants are exposed to salinity. Ion homeostasis assumes importance for plant metabolic processes and functioning (Tripathi et al. 2015; Arif et al. 2016). Na+ -related salt tolerance research has been extensively conducted than that of Cl− in cultivated crops (Teakle and Tyerman 2010). Plants differentially respond to Na+ and Cl− ions and possess separate transport systems and associated genetic machinery. It has thus become important to study the individual effects of Cl− and Na+ ions on physiological and biochemical aspects of plant growth and development. In rice and soybean, toxicity was more pronounced by Na+ and Cl− (Kumar and Khare 2016; Shelke et al. 2019). Sudden increases in the concentrations of Na+ and Cl− will # Equal contribution

Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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have a negative impact on plant metabolic processes and subsequently on growth (Tavakkoli et al. 2011). However, Cl− is considered as an essential micronutrient as it acts as a co-factor in photosynthesis, stomatal regulation, and regulation of enzyme activities in the cytoplasm. It is involved in the maintenance of turgor pressure, which stabilizes membrane potentials and regulates pH (Xu et al. 2000; White and Broadley 2001; Franco-Navarro et al. 2016). Both Na+ and Cl− ions are metabolically toxic to plants at higher concentrations in the cytoplasm. In some plants like faba bean, barley, Lotus and Chrysanthemum, the root or shoot concentration of Cl− rather than Na+ is positively correlated with salt tolerance (Rajendran et al. 2009; Tavakkoli et al. 2010; Guan et al. 2012), implying that Cl− acts as an osmoticum. In many salt-tolerant plants, their salt tolerance is correlated with efficient control of Cl− transport and exclusion. Dang et al. (2008) carried out several field trials and underlined the major contribution of Cl− ions in soil salinity as compared with Na+ and its role in growth and yield reduction. In faba bean, NaCl stress resulted in decreased growth and photosynthesis (Tavakkoli et al. 2010). Tavakkoli et al. (2011) studied the additive effects of Na+ and Cl− ions on four barley cultivars, namely Barque73, Clipper, Sahara, and Tadmor, in both soil and hydroponic culture. The authors found that, in NaCl stress, there was greater reduction in water potential, photosynthesis, net transpiration (T), and stomatal conductance (gs) than the individual effects of Na+ and Cl− ions. The same pattern was followed for shoot dry weight and cumulative plant water use efficiency in the above-mentioned barley cultivars. NaCl showed a marked effect on the four key chlorophyll fluorescence parameters Fv/Fm, ΦPSII, qP, and NPQ, but the effects varied depending on the genotypes. Recently, in rice, Khare et al. (2015) showed that additive effects of NaCl are more severe than the individual effects of Na+ and Cl− ions. NaCl generated more reactive oxygen species (ROS) than individual ionic effects, which ultimately resulted in cell death and higher antioxidant enzyme activities, but an imbalance in nonenzymatic antioxidants (Roychoudhury et al. 2008; Roychoudhury and Ghosh 2013). The toxic effects of Na+ ions and its contribution in final productivity are well studied in crop plants, while there is meager information available on the impact of Cl− ions. Hence, there is a need to understand regulatory mechanisms which control Cl− accumulation, toxicity, and root to shoot movement. This information will aid our knowledge of Cl− homeostasis in plants and about anion channels/transporters and their regulation during salt stress tolerance.

13.2 Sources of Cl− Ion Contamination The major deposition of Cl− in soil is in the form of Cl− , the only stable oxidation state of Cl− which is the monovalent anion (Bohn et al. 1979). Both natural and anthropological factors are responsible for Cl− deposition in soil. In natural systems, Cl− deposition is due to precipitation, rainwater, sea spray, and rock erosion. Cl− deposition in soil also depends on anthropogenic factors such as irrigation water, dust, air pollution and fertilizer applications. There are almost 2000 naturally occurring organochlorines present in the marine and saline environment which are responsible for Cl− deposition (Fleming 1995). Rainwater originating from salt water is an important source for Cl− deposition and its concetration varies greatly from 11 μM to 8.5 mM (Xu et al. 2000). Precipitation deposits 0.1 kg of Cl− ha−1 soil away from sea and between 1 and 100 kg ha−1 year−1 closer to the seashore (Oberg 1998). Organic chloride deposition is not well documented, but some estimates says that in plants, organic Cl− content (0.01–0.1 mg g−1

13.3 Role of Cl− in Plant Growth and Development

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Table 13.1 Source of contamination, roles, deficiency, and toxicity cause owing to Cl− ions. Source of Cl− contamination

Precipitation Rainwater Sea spray Rock erosion Irrigation water Dust Air pollution Fertilizer application Water pollution Natural organochlorines

Functional role

Deficiency symptoms

Toxicity effects

Increased dry matter of plant Opening and closing of stomata Essential for photosynthesis Osmoregulation Turgor maintenance Regulation of enzyme activity Improved leaf water balance Improved water use efficiency Lower stomatal conductance Leaf cell expansion Guard cell growth Charge balance

Reduced leaf growth Wilting of leaf Chlorosis Bronzing Necrosis Stunted root growth Decreased fruit number and size Suppressed lateral organ development

Ion toxicity Nutrient imbalance Impact on mineral uptake and translocation Inhibition of plant growth and development Enhanced lipid peroxidation Membrane damage ROS production Reduced photosynthetic pigments Impaired synthesis of chlorophyll Chlorophyll degradation Decreased transpiration Decreased photosynthesis Decreased crop yield and quality Inhibited gas exchange Changes in leaf apoplastic pH Reduction in quantum yield of photosystem II

dry weight) and Cl− deposition range between 0.04–0.4 kg ha−1 year−1 in Swedish forest ecosystem soil (Oberg 1998). Cl− concentrations in saline irrigation water range from 2 to 30 mM, and even irrigation with water of low salinity can easily deposit 1000 kg Cl− ha−1 year−1 (Xu et al. 2000). In some circumstances, this may exceed all other forms of Cl− deposition. In one study, it was shown that the Cl− concentration of the upper 30 cm of soil was 0.25 mM for untreated plots and 0.73 mM for those receiving 169 kg of fertilizer ha−1 (Parker et al. 1983), indicating that fertilizers are also responsible for Cl− deposition. Cl− deposition in soil also occurs owing to organochlorines such as artificial sweeteners, pesticides, and herbicides (xenobiotics). The inputs of chlorine in soil have been increasing on a daily basis owing to an increase in environmental constraints; therefore a focus on inputs is equally important to improve soil health (Table 13.1).

13.3 Role of Cl− in Plant Growth and Development Cl− ions act as micronutrients and play an important role in plant growth at 3 μmol g−1 dry weight (Kirkby and Marschner 2012). They are essential for opening and closing of stomata in higher plants. The fluxes of potassium (K+ ) and accompanying anions such as malate and Cl− in plant species like Allium cepa are essential for stomatal functioning, while in the absence of Cl− , stomatal opening is inhibited (Marschner 1995). It was observed that Cl− is responsible for the Hill reaction in photosystem II (PSII) for O2 evolution. In spinach chloroplast, an external Cl− supply increased O2 evolution, suggesting that Cl− plays a fundamental role in the water-splitting system of PSII besides manganese (Ball et al. 1984). It acts either as a bridging ligand for stabilization of the

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oxidized state of manganese or as a structural component of the associated (extrinsic) polypeptides (Critchley 1985). Several anions can counterbalance the positive charges of cations and are part of the osmoregulatory function (Barbier-Brygoo et al. 2011). Cl− is an osmotically active solute in the vacuole, functioning in turgor maintenance and osmoregulation and in cytoplasm it regulates enzyme activities. Recently, it was observed that Cl− plays a role in regulating leaf osmotic potential and turgor to improve leaf water balance in tobacco. It lowers the stomatal conductance, which results in lower water loss, greater photosynthetic, and integrated water use efficiency. Cl− deficiency causes reduced leaf growth and wilting, followed by chlorosis and bronzing, which leads to necrosis. Root growth becomes stunted, the number and size of fruits decrease, and the development of lateral organs is suppressed (Marschner 1995). Burdach et al. (2014) found that anion channel blockers could diminish the indole-3-acetic acid-induced maize coleoptile growth. This indicates that Cl− plays a role in the indole-3-acetic acid-induced growth of maize coleoptile segments. Cl− also acts as a specific signaling molecule and stimulates leaf cell growth, including guard cells (Franco-Navarro et al. 2016).

13.4 Cl− Toxicity Direct and excessive entry of Cl− into plant cells inhibits mineral uptake and results in nutrient imbalance and ion toxicity (Chen et al. 2007; Wu et al. 2013). Under high-salt conditions, inhibition of plant growth and development is observed. Ionic imbalances owing to Cl− accumulation result in enhanced lipid peroxidation, membrane damage, and increased production of ROS like singlet oxygen, superoxide radicals (O2 − ), hydrogen peroxide (H2 O2 ), and hydroxyl radicals (OH) (Wang et al. 2003). An increase in the concentration of Cl− reduces photosynthetic capacity owing to nonstomatal effects; there is also impaired synthesis of chlorophyll (Chl), enhanced chlorophyll degradation, and a reduction in the actual quantum yield of PSII electron transport which is associated with both photochemical quenching and the efficiency of excitation energy capture (Tavakkoli et al. 2010). Woody perennial crops (Vitis sp. Citrus sp., Persea americana) and legumes (Glycine max, Vicia faba) accumulate more Cl− than Na+ in their leaves. This chloride accumulation leads to decreased transpiration, photosynthesis, crop yield and quality, and ultimately plant death (Teakle and Tyerman 2010; Storey and Walker 1999; Brumos et al. 2009; Brumós et al. 2010; Gong et al. 2011; Moya et al. 2003; Tregeagle et al. 2010; Fort et al. 2013; Luo et al. 2005; Tavakkoli et al. 2010). The decrease in plant biomass and chlorophyll content, along with an increase in malondialdehyde content, proline content, and electrolyte leakage under Cl− stress, were observed in Chrysanthemum (Guan et al. 2012). The decrease in plant growth under Cl− was also observed in cucumber (Huang et al. 2015). Na+ is metabolically more toxic than Cl− but these plants have better adaptation to Na+ . They excrete sodium in a higher proportion than chloride; therefore, chloride is more toxic. Poncirus trifoliate, below 100 mM salt treatment, translocates Na+ into the woody tissues of the roots and the basal portion of the stem. In contrast, leaves accumulate high Cl− at only 25 mM NaCl treatment (Walker 1986). The responses of two cultivars of soybean, Nannong 1138-2 and Zhongzihuangdou-yi, to Na+ and Cl− ions were studied by Luo et al. (2005). Their major findings suggest that

13.5 Interaction of Soil Cl− with Plant Tissues

the leaves of both cultivars are more susceptible to Cl− than Na+ and the roots played crucial role in salt stress tolerance. These plants have the ability to withhold Cl− ions and minimize toxicity to leaves. Cl− reduces the growth and water use efficiency if its concentration is in the range of 4–7 mg g−1 for sensitive species and 15–50 mg g−1 for tolerant species (Xu et al. 2000; White and Broadley 2001). Many important cereals, vegetables, and fruit crops are susceptible to Cl− toxicity during cultivation and this is a major constraint to horticultural production on irrigated or saline soils (Xu et al. 2000). In addition, the exposure of Cl− to roots restricts gaseous exchange by indirect long-distance signal, which causes an increase in apoplastic pH of leaf. This results in redistribution of abscisic acid (ABA) in leaves and closure of stomata (Geilfus et al. 2015). The uptake and accumulation of Cl− in shoot vacuoles inhibits the uptake of nitrogen by competing with nitrate transporters (Cubero-Font et al. 2016; Qiu et al. 2016; Glass and Siddiqi 1985). The nitrate and Cl− share the same or different transporter proteins and play an important role in balancing charge and regulating turgor (Li et al. 2016a,b,c). The high Cl− /NO3 − ratio causes growth reduction (Qiu et al. 2016), necrosis (Xu et al. 1999) and reduced subsequent yield (Baby et al. 2016).

13.5 Interaction of Soil Cl− with Plant Tissues 13.5.1

Cl− Influx from Soil to Root

High Cl− stress in the root zone results in the reduction of water potential and causes water deficit, phytotoxicity, and nutrient imbalance, as well as affecting the uptake and transport of nutrients (Munns 2002). Variation in the content of soil Cl− ion leads to stress in the plant root. Cl− moves from soil to the vascular system by both symplastic and apoplastic pathways. The Cl− anion does not form complexes readily and shows little affinity (or specificity) in its adsorption to soil components. Thus, Cl− movement within the soil is largely determined by water flows. These fluxes are regulated by the Cl− content of the root because Cl− is mobile within the plant (White and Broadley 2001). Ion fluxes in the roots are limited by ion exchange because plasma membrane has high ion selectivity. The anion and cation selectivity are independent of each other while the interior contents are more combined in nature. Auxin is one of the plant growth regulators that induce the Cl− transport system in the cell plasma membrane, regulating an abrupt increase in chloride uptake (Olga et al. 1998). During high concentrations of salt in soil, the tonoplast shows both passive and active transport. An electrogenic Cl− /2H+ symporter is present in the plasma membrane of root hair cells and Cl− channels mediate either Cl− influx or Cl− efflux. The biochemical and electro-physiological evidence shows that Cl− channels mediate Cl− fluxes in either direction across the tonoplast (White and Broadley 2001). Nevertheless, flux localization studies with controlled conditions are required to ascertain whether chloride transport capability is an intrinsic property of the bare regions or a general property of the membrane as a whole, with very sensitive dependence. 13.5.2

Mechanism of Cl− Efflux at the Membrane Level

A NaCl-induced efflux of Cl− has been observed in several plant species, for example, in the halophyte Diplachne fusca (Bhatti and Wieneke 1984) and in glycophytes like

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barley (Yamashita and Matsumoto 1996; Britto et al. 2004), Sorghum (Boursier and Lauchli 1989) and Arabidopsis (Lorenzen et al. 2004). Cl− efflux in Chara sharply decreases when external Cl− is replaced with sulfate or benzene sulfonates (Hope et al. 1966). Populus euphratica under 100 mM NaCl exhibits significant Cl− efflux of about 400–1200 mM from the apex part and shows salt tolerance, while sensitive genotypes failed to do so, which marks the importance of chloride efflux in the tolerance of this species (Sun et al. 2009). The reduction of Cl− and K+ efflux may arise from decreased permeability of the tonoplast upon transport of Cs+ to the vacuole. The Cs+ inhibition of Cl− and cation fluxes does not appear to be directly linked to respiratory inhibition. The flux effects of Cs+ are evidently direct on the permeability of the cell membrane. The effects of Ca2+ are much like those of Cs+ in reducing Cl− flux rates and the rate of internal equilibration, but do not inhibit respiration. This is consistent with the observation that Ca2+ reduces the influx and efflux of both Na+ and K+ in barley roots and affects barley root cell structure (Jackson and Edward 1966). Earlier studies have shown that Cl− efflux in the acid region is three times greater than that of the alkaline region. The simplest explanation for the doubling of Cl− efflux with the Ca2+ solution is that the efflux is purely passive (Goldman 1943). It has been observed that Cl− efflux is stimulated by an increased concentration of external Cl− (Britto et al. 2004; Sun et al. 2009) and the Cl− efflux can approach 90% of the influx in barley (Britto et al. 2004). There is a need to screen the genotypes that have efficient Cl− efflux mechanisms to mitigate salt tolerance. 13.5.3

Differential Accumulation of Cl− in Plants and Compartmentalization

Differential accumulation of Cl− in shoot tissues of tolerant species and understanding their regulation are crucial in the development of crop plants with high salinity tolerance. Studies have shown that net Cl− loading into the root xylem is lower in grapevine genotypes that have lower shoot Cl− accumulation. At the root level, Cl− transport across different cell types from the epidermis cortex to the xylem could affect the total flux of Cl− to the shoot. The salt-tolerant genotypes of Citrus, grapevine, and Lotus with low shoot Cl− actually have higher root Cl− concentrations compared with more sensitive genotypes (Storey and Walker 1999), suggesting more efficient compartmentation of Cl− in root vacuoles in the tolerant genotypes. The compartmentation of Cl− is a very important aspect of salt tolerance, such as leaf to leaf gradient which protects younger leaves or other cells of same plant part. To avoid the toxicity in mesophyll cells, which affects photosynthesis, plants accumulate Cl− in leaf epidermis. When tolerant and sensitive genotypes of barley are compared, tolerant plants show effective Cl− exclusion from mesophyll cells (Huang and Van Steveninck 1989), indicating that mesophyll cells must be protected against Cl− toxicity. The salt gland or bladder also contributes to intracellular compartmentation and about 20% of leaf Cl− is excreted from the salt glands of Leptochloa fusca at 100 mM NaCl (Jeschke et al. 1995). Thus, plants have evolved sophisticated mechanisms to avoid Cl− toxicity. Also, halophyte and glycophyte species exhibit varying abilities to accumulate Cl− ; for example, halophytes accumulate high Cl− concentrations in their tissues (340–475 mM) compared with glycophytes (7–70 mM). Different parts of the same plant also display differences in Cl− accumulation; for example, older leaves deposit high Cl− than younger ones (Cram 1976; Greenway and Munns 1980). This helps younger leaves to grow rapidly with

13.6 Electrophysiological Study of Cl− Anion Channels in Plants

low transpiration levels for minimal recycling and continued deposition of Cl− into older leaves, which is an important mechanism for salinity tolerance in glycophytes. In contrast to this, Cl− content in all the leaves of halophytes such as L. fusca, Atriplex hastate and the glycophyte Beta vulgaris was the same (Greenway and Munns 1980; Jeschke et al. 1995). This perhaps needs to be elucidated using mutants. Floral tissues and fruits generally have low contents of Cl− ; however, this depends on the availability of Cl− (Levy and Shalhevet 1990; Xu et al. 2000). Seeds from Cl− -tolerant soybean varieties contained 100 μg g−1 , but susceptible varieties contained 682 μg g−1 (Parker et al. 1983). In grapevine, the vacuoles of root endodermis have 50% lower Cl− than inner cortex and pericycle under 25 mM NaCl (Storey et al. 2003). The efficient sequestration of Cl− in leaf vacuoles is associated with the salt tolerance of some avocado rootstocks (Xu et al. 2000) and lupin cultivars (Van Steveninck et al. 1982). Schachtman and Thomas (2003) found that the salt-tolerant grapevine genotype showed low shoot Cl− content and 20% more vacuolar Cl− in pericycle cells than grapevine salt-sensitive genotypes. This implies that the pericycle could play a role in Cl− transport to the shoot. Root tips contain lower concentrations of Cl− than mature roots (Scott et al. 1968; Storey and Walker 1987). Thus, the compartmentalization of excess Cl− may contribute to elevated salt tolerance but more studies are needed to elucidate the mechanism.

13.6 Electrophysiological Study of Cl− Anion Channels in Plants Thermodynamic criteria determine the mechanism of movement of Cl− ion transported across the membrane. Voltage and concentration are the principal driving forces for Cl− movement across the membrane and make up the Cl− electrochemical gradient. Passive transport occurs when Cl− moves in the direction of its electrochemical gradients, while its transport is mediated by either channel or carrier, resulting in facilitated diffusion. Cl− transport occurs with the help of ATP hydrolysis (primary active transport) against the electrochemical gradient or the mechanism of symport and antiport. This suggests that, under nonsaline conditions, there is active Cl− influx across the plasma membrane of root cells though H+ /Cl− symport and efflux across the plasma membrane is mediated by anion channels. In saline environment, Cl− influx across the plasma membrane is passive. By using an ion-selective electrode, it is noted that root-hair cells of the plasma membrane have an electrogenic Cl− /2H+ symporter (Felle 1994). Cl− fluxes across the tonoplast are passive and mediated by anion channels, and Cl− is loaded into the xylem down its electrochemical gradient. The tonoplast has slightly negative membrane potentials, because of which there is two to three times greater Cl− concentration in the vacuoles. Existing evidence suggests that a Cl− /H+ antiporter mediates Cl− influx to the vacuole either through biochemical or electrophysiological means (Shuvalov et al. 2015), and also that Cl− channels mediate Cl− fluxes in either direction across the tonoplast. Shone (1968) observed that Cl− was transported to the xylem against its electrochemical gradient even at lower concentrations of Cl− in the external medium. Thus, Cl− movement to the xylem requires an active transport step. From studies on changes in the Cl− electrochemical potential across a maize root, Dunlop and Bowling (1971) concluded that there was passive Cl− movement from the cells of the xylem parenchyma

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to the xylem vessels. Assuming that Cl− movement across the root to the xylem is symplastic, active Cl− transport across the plasma membrane could account for this phenomenon. Similarly, under saline conditions, passive Cl− transport to the xylem may occur as Cl− influx across the plasma membrane of root cortical cells becomes passive. It was shown that the cytoplasmic concentration of Cl− increases within minutes after a rise in the external Cl− concentration (Felle 1994). In barley root protoplasts, the Cl− permeability of protoplasts increased during 200 mM NaCl concentration exposure owing to the presence of Cl− channels (Yamashita and Matsumoto 1996). If Cl− influx is active, then the opening of a Cl− -permeable channel in nonsaline conditions favors the passive efflux. The plasma membranes of stomatal guard cells having Cl− channels are the most important because they serve the specific role of stomatal closure through anion efflux. In V. faba guard cells, there is an involvement of two different anion channels for stomatal closure, which show selectivity for Cl− . This is rapidly activated upon depolarization of the membrane potential, resulting in a short-term anion efflux. Also, there are slowly activating Ca2+ -dependent anion channels that make long-term anion efflux and depolarization of guard cells for stomatal closure. Such voltage-dependent channels are located in all the plant membranes including plasma membrane, tonoplast, endoplasmic reticulum, leaf mesophyll cells, hypocotyl/coleoptile cells, root cells and suspension cultures, and mitochondrial and chloroplast membranes (Hedrich 1994; Tyerman and Skerrett 1999; Krol and Trebacz 2000). Protoplasts from epidermis and cortical cells have shown the presence of outward rectifying depolarization-activated anion channels (OR-DAACs) in wheat, maize, Arabidopsis, and lupin (Skerrett and Tyerman 1994; Pineros and Kochian 2001; Diatloff et al. 2004; Zhang et al. 2004). These channels show similar features between species but differ in the rates of activation and rectification. Two studies have examined these channels specifically in the context of high salinity (Skerrett and Tyerman 1994; Tyerman et al. 1997). The S-type channel is a candidate for elevated anion efflux that occurs under salinity, since high salinity will tend to acidify the cytoplasm (Martinez and Lauchli 1993; Felle 1994). This channel has also been identified in root hair cells after desiccation, which depolarized the membrane potential (Dauphin et al. 2001). Thus, outward rectifying channels appear to be important for anion efflux. Thus, activation of a Cl− -permeable channel in saline conditions could be useful for reduction in Cl− net influx if modulated at the gene or protein level. Variability among Cl− accumulation was observed in different genotypes of crop plants (Tavakkoli et al. 2010, 2011; Khare et al. 2015; Shelke et al., 2017, 2019). If such studies are carried out, this may help to identify species with better salt stress tolerance for subsequent use in breeding programs. Net xylem loading of Cl− , intracellular compartmentation and efflux from root cells appear to be the key aspects that contribute to salt tolerance in some plant species (Teakle and Tyerman 2010).

13.7 Channels and Transporters Participating in Cl− Homeostasis Channels are the integral membrane proteins that form aqueous pores and facilitate ion fluxes. These pores participate in anion uptake, turgor maintenance, osmoregulation, catalysis of the rapid release of anions, electrical excitability, stabilization of membrane

13.7 Channels and Transporters Participating in Cl− Homeostasis

potential, and calcium-activated Cl− movement, which are important for intracellular signaling and signal propagation (Skerrett and Tyerman 1994; Dieudonne et al. 1997). Different gene families, namely slow anion channel and associated homologs (SLAC/SLAH), aluminum-activated malate transporter (ALMT), CLC gene families, and cation, chloride co-transporters (CCC) have been known to encode chloride channels or transporters (Negi et al. 2008; Vahisalu et al. 2008; De Angeli et al. 2009). The ATP-binding cassette (ABC) transporters, chloride conductance regulatory protein, and nitrate transporter 1/peptide transporter (NPF) proteins (Suh et al. 2007; Brumós et al. 2010; Li et al. 2016a) also play role as chloride channels or transporters. Among these, both CCC and CLCc have been found to be crucial for Cl− homeostasis and NaCl stress tolerance in plants (Colmenero-Flores et al. 2007; Jossier et al. 2010) (Figure 13.1). 13.7.1

Slow Anion Channel and Associated Homologs

Anions like nitrate, sulfate, malate, citrate, and chloride contribute to the osmotic strength of the plant cells and may regulate cell turgor. Stomata are closed when exposed to abiotic stress and the process is mediated by the release of ions from guard Guard Cell

Mesophyll Cell

AtCLCa AtALMT9 AtCLCc

AtCLCg

Xylem Vessels AtNPF 2.5 Active Cl- influx

Passive Cl- influx

Passive Cl- efflux

Epidermis

Endomembranes AtCCC VviCCC GmSALT3

AtCLC c?

AtNPF2.4 AtNPF7.3? AtSLAH1 AtSLAH3 AtALMT12?

AtALM T9

Vacuole

Cortex

Vacuole

AtNPF7.2 OsCCC?

Stele

Figure 13.1 Diagrammatic presentation of channels and transporters associated with chloride homeostasis in plants. Source: Reproduced with permission from Li et al. (2016c).

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cells (Kim et al. 2010). The guard cell SLAC or SLAH is thought to be responsible for anion loading of the xylem in roots. SLAC1 is expressed strongly in guard cells and the protein, consisting of some 556–557 amino acids with a predicted 10 membrane spanning helices, was localized in the plasma membrane (Negi et al. 2008; Vahisalu et al. 2008). Protoplasts isolated from guard cells showed that SLAC1 mutants had increased accumulation of malate, fumarate, Cl− , and K+ , consistent with a deficiency in anion release from the guard cells (anion release activates K+ release). The most direct indication of a close link of the gene with S-type of anion channel was that patch-clamped protoplasts from mutants (including a T-DNA mutant) had no activity of the S-type channel when this is normally activated by ABA or Ca2+ (Vahisalu et al. 2008). On the other hand, neither R-type channel nor Ca2+ channel was affected (Vahisalu et al. 2008). The SLAC1 mutants lack stomatal response not only to increased CO2 , but also sensitivity to ozone (Negi et al. 2008; Vahisalu et al. 2008). The S-type anion currents have been detected in xylem, companion cells and, cortical cells, besides guard cells (Frachisse et al. 2000; Kim et al. 2010). Thomine et al. (1997) speculated that these currents could be associated with turgor regulation and cell expansion. Such an occurrence of anion currents in cells other than guard cells may indicate the role of these currents in loading of anions to the xylem sap. In addition to SLAC1, four SLAC1 homologs (SLAHs) have been identified in Arabidopsis (Negi et al. 2008; Vahisalu et al. 2008), but there are nine orthologs in rice (Barbier-Brygoo et al. 2011). The stomatal closure deficiency of the slac mutant line was complemented with AtSLAH1 and AtSALH3 but AtSLAH2 was unable to complement the mutant phenotype (Negi et al. 2008). SLAH 1, 2, and 3 were expressed in roots, with SLAH1 showing strong expression in the root vascular cylinder, making it a candidate for the S-type anion channels in root xylem parenchyma (Negi et al. 2008). Out of the four SLAHs, SLAH3 is expressed in guard cells (Hedrich 2012) and the xylem-pole pericycle from the root vasculature (Cubero-Font et al. 2016), but SLAH3 predominantly conducts nitrate in chloride-based media guard cells expressing the SLAH3 gene (Geiger et al. 2011; Hedrich 2012). The heterodimerization of SLAH1/SLAH3 causes SLAH3-mediated chloride efflux from pericycle cells into the root xylem vessels. Guard cell SLAC is activated by calcium-dependent protein kinases CPK23 and CPK21 (Hedrich 2012) or alternatively calcium-independent SnRK2.6 kinase activates both guard cell SLAC and quickly activating anion conductance (QUAC) (Imes et al. 2013). In Arabidopsis, AtSLAH3 interacts with AtCPK20 in the pollen tube (Gutermuth et al. 2013). Among CPKs, differential expression of VvCPK20 between grapewine rootstocks indicates its involvement with regulation of VvSLAH3 (Henderson et al. 2014). In addition, VvSnRK2.6 and VvSnRK2.7 showed differential expression between grapevine rootstocks, which implicates involvement of ABA signaling in Cl− exclusion from roots (Henderson et al. 2014). This elucidation in other plants will help in understanding long-distance Cl− transport. In roots, ABA and high cytosolic calcium inhibit xylem-quickly activating anion conductance (X-QUAC) (Kohler and Raschke 2000; Gilliham and Tester 2005).

13.7 Channels and Transporters Participating in Cl− Homeostasis

In Arabidopsis, AtSLAH1 anion channel plays an important role in controlling root-to-shoot Cl− transport (Qiu et al. 2016; Cubero-Font et al. 2016). However, not much is known about the other members of SLAC/SLAH family and it would be interesting to learn more about their involvement in Cl− translocation during stress conditions. The phylogenetic tree of SLAC/SLAH is shown in the Figure 13.2. 13.7.2

QUAC1 and Aluminum-activated Malate Transporters

The ALMT family is specific for plants and no relatives have been observed so far in bacteria and yeast. Gruber et al. (2010) first identified a gene encoding the R-type (for rapid) channel or QUAC (for quick anion channel) HvALMT1 in barley that transports organic anions. This transporter conducts malate in plants (Raman et al. 2005; Gruber et al. 2010), but when mutated, no malate formation was observed in plants (Sasaki et al. 2010). Meyer et al. (2010) observed that AtALMT12 represents an ABA-activated R-type anion channel required for stomatal movement in Arabidopsis guard cells, and interestingly, AtALMT12 is also found in root stelar cells (Sasaki et al. 2010). Likewise, Sasaki et al. (2010) noticed that the closing of stomata requires a homolog of an ALMT. Also, mutants lacking QUAC1 displayed reduced R-type currents. It is not known at the moment if ALMT family members encode additional QUACs in higher plants and if they play any role in stomatal movement and stress tolerance. However, ALMT1 and QUAC1 share many features, like malate-permeable channel (Hoekenga et al. 2006; Meyer et al. 2010). It is interesting to examine Cl− accumulation of the aluminum (Al) tolerant wheat varieties under salinity, because if Al-activated malate transporter1 from Triticum aestivum (TaALMT1) is partially active without Al, it may contribute to Cl− fluxes across the plasma membrane. However, its function in Cl− transport or salt stress tolerance is not known. Furthermore, the selectivity of TaALMT1 for malate over Cl− is rather complex. At low external Cl− , the transport is more selective for malate than for Cl− (30 : 1), while at high external Cl− , the channel becomes less selective (1 : 1) (Pineros et al. 2008). This has implications for salinity effects, because at high external Cl− , the reversal potential will shift in a negative direction and the channel would potentially become equivalent to the OR-DAACs observed in wheat roots, and capable of transient inward flux of Cl− . If Al stress co-occurred with salinity, the ability of the root tips to excrete malate may be reduced because of the shift in reversal potential to more negative potentials combined with the depolarization under salinity, and the possible competition between Cl− and malate at the cytoplasmic face of the channel (Teakle and Tyerman 2010). Under stress conditions, when concentrations of Cl− increase, ALMT gets activated; this is negatively regulated by a nonprotein amino acid 𝛾-amino butyric acid (GABA) (Ramesh et al. 2015). Another member of ALMT family, ALMT9, plays a key role in long-distance translocation of salt ions and is expressed in root and shoot vasculature (Baetz et al. 2016). It is also interesting to note if homologs of ALMT1 carry any anion other than malate. The phylogenetic tree of ALMT family members is shown in Figure 13.3.

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13 Chloride (Cl− ) Uptake, Transport, and Regulation in Plant Salt Tolerance XP 013586286.1 BoSLAC1 XP 009148588.1 BrSLAC1 XP 013742612.1 BnSLAC1 NP 563909.1 AtSLAC1 XP 010476220.1 CsSLAC1 XP 010553483.1 ThSLAC1 XP 010108084.1 MnSLAC1 XP 015901593.1 ZjSLAC1 XP 012476007.1 GrSLAC1 XP 011029367.1 PeSLAC1 XP 002509647.1 RcSLAC1 XP 012087058.1 JcSLAC1 XP 012567411.1 CaSLAC1 XP 015963713.1 AdSLAC1 XP 004300729.1 FvSLAC1 XP 008238713.1 PmSLAC1 XP 009373337.1 PbSLAC1 XP 008373862.1 MdSLAC1 XP 004147985.1 CsSLAC1 XP 008448932.1 CmSLAC1 XP 006477326.1 CsSLAC1 XP 010054795.1 EgSLAC1 XP 010277436.1 NnSLAC1 XP 010676623.1 BvSLAC1 XP 015636891.1 OsSLAC1 XP 003581553.1 BdSLAC1 XP 004976557.1 SiSLAC1 XP 008780343.1 PdSLAC1 XP 011071099.1 SiSLAC1 XP 009765224.1 NsSLAC1 XP 009601617.1 NtSLAC1 XP 004245686.1 SlSLAC1 XP 015085801.1 SpSLAC1 XP 006363742.1 StSLAC1 XP 006858025.2 AtSLAC1 KMZ58505.1 ZmSLAC1 XP 012701267.1 SiSLAH2 XP 014754268.1 BdSLAH2 XP 015160475.1 StSLAH2 AEE85415.1 AtSLAC1 KHN19952.1 GsSLAH3 XP 006587259.1 GmSLAH3 AED93247.1 AtSLAC1 XP 006490606.1 CsSLAH2 XP 006589515.1 GmSLAH2 XP 014512596.1 VrSLAH2 XP 014512595.1 VrSLAH2 XP 015622010.1 OsSLAH1 XP 004967317.2 SiSLAH1 AEE33945.1 AtSLAH1 XP 010418209.1 CsSLAH1 AEE33943.1 AtSLAC1 XP 010533799.1 ThSLAH1 KHG00717.1 GaSLAH1 XP 012092365.1 JcSLAH1 XP 010099185.1 MnSLAH1 XP 011096154.1 SiSLAH1 XP 015578243.1 RcSLAH1 KMZ56819.1 ZmSLAH1 XP 014499396.1 VrSLAH1 XP 014499394.1 VrSLAH4 CBJ19440.1 CcSLAC XP 002280770.1 VvSLAH1 XP 006473900.1 CsSLAH4 EOY14904.1 TcSLAC1 XP 014632605.1 GmSLAH1 KHN45897.1 GsSLAH1 XP 003523559.2 GmSLAH4 CDW59851.1 TtSLAC1

13.7 Channels and Transporters Participating in Cl− Homeostasis

Figure 13.2 SLAC/SLAH phylogenetic tree. The dendrogram indicates the degree of similarity between SLAC proteins of different plant species including monocots and dicots. Six distinct clades were noticed, indicating their independent evolution. The sequences were collected from the NCBI database. An alignment of known full-length proteins was performed using Clustal W as part of the MEGA6 software package programs and the phylogenetic tree was constructed using the neighborjoining method. Species prefixes: At, Arabidopsis thaliana; Cc, Citrus clementina; Ga, Gossypium arboretum; Mn, Morus notabilis; Os, Oryza sativa; Rc, Ricinus communis; Zm, Zostera marina; Jc, Jatropha curcas; Si, Sesamum indicum; Gs, Glycine soja; Vv, Vitis vinifera; Th, Tarenaya hassleriana; Cs, Camelina sativa; Va, Vigna angularis; Tc, Theobroma cacao; Cs, Citrus sinensis; St, Solanum tuberosum; Sp, Solanum pennellii; Bd, Brachypodium distachyon; Si, Setaria italic; Bn, Brassica napus; Bo, Brassica oleracea; Zm, Zostera marina; Ca, Cicer arietinum; Gr, Gossypium raimondii; Cs, Cucumis sativus; At, Amborella trichopoda; Fv, Fragaria vesca; Pe, Populus euphratica; Bv, Beta vulgaris; Sl, Solanum lycopersicum; Nn, Nelumbo nucifera; Eg, Eucalyptus grandis; Ns, Nicotiana sylvestris; Nt, Nicotiana tomentosiformis; Pb, Pyrus bretschneideri; Br, Brassica rapa; Pd, Phoenix dactylifera; Cm, Cucumis melo; Md, Malus domestica; Pm, Prunus mume; Ad, Arachis duranensis; Zj, Ziziphus jujuba; Tt, Trichuris trichiura.

13.7.3

Plant Chloride Channel Family Members

Chloride channel proteins were identified for the first time by Miller and White (1980) from a torpedo fish and named CLC-0. Later, these proteins were found to be very widespread and present in both prokaryotic and eukaryotic species (Miller 2006). Studies on plant functional analysis has indicated that there are several members of CLC family that take part in anion transport. In Arabidopsis and tobacco, CLC channels were cloned (Hechenberger et al. 1996; Lurin et al. 1996) and were shown to have 10–12 transmembrane domains with N- and C-termini located in cytoplasm. The conserved sequence motifs (GxGxPE, GKxGPxxH, PxxGxLF) are located in the cytoplasmic loops between transmembrane domains D2–D3 and D5–D6, and are thought to contribute to anion selectivity (Barbier-Brygoo et al. 2000). There are nine CLC members in humans (De Angeli et al. 2009), and seven each in Arabidopsis (Marmagne et al. 2007) and rice (Diedhiou and Golldack 2006). The AtCLC mRNA is induced by the addition of NO3 − in nitrogen-starved plants (Geelen et al. 2000). This suggests the involvement of CLC in the adaptation to excess NO3 − . Since a single amino acid substitution can change selectivity from NO3 − to Cl− (Bergsdorf et al. 2009), this raises the possibility that other members of the CLC family might actually be Cl− /H+ antiporters. Comparison of rice genotypes that exclude Cl− , for example, in the salt-tolerant variety Pokkali or varieties that accumulate Cl− (IR-29), showed that there were differences in the expression of OsCLC1 (Diedhiou and Golldack 2006). Under salt stress, OsCLC1 from IR-29 was repressed, while Pokkali showed induction in leaves and roots. The root induction was particularly strong and expression was located to the xylem parenchyma and phloem (Diedhiou and Golldack 2006). This could be a response to perturbed NO3 − homeostasis rather than to high Cl− concentration. Nakamura et al. (2006) found that OsCLC1 and 2 are located on the tonoplast. In soybean, GmCLC1 and GmNHX1 (Na+ /H+ antiporter) are both localized to the tonoplast and transcripts are increased by NaCl (125 mM) treatment and dehydration (Li et al. 2006). When both GmNHX1 and GmCLC1 were expressed separately in tobacco BY-2 cells, the cells were more tolerant to salt (100 mM NaCl), but not to dehydration. The GmCLC1 cells overexpressing the gene accumulated more Cl− in the vacuole compared with cytoplasm (Li et al. 2006). The GmCLC1 gene overexpressed in Arabidopsis resulted in low Cl− accumulation in shoots and subsequently enhanced salt tolerance. When this gene was overexpressed in hairy roots of soybean

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13 Chloride (Cl− ) Uptake, Transport, and Regulation in Plant Salt Tolerance ACG38733.1 ZmALMT1 XP 014661334.1 SiALMT1 ABY52957.1 ScALMT1 AAZ22853.1 AetALMT1 AAZ22852.1 TaALMT1 ACJ15441.1 HvALMT1 EMS50055.1 TuALMT1 BAN78903.1 HlALMT1 KVH90543.1 CcALMT XP 014495025.1 VrALMT8 NP 001237989.1 GmALMT XP 003553620.1 GmALMT8 XP 003539851.1 GmALMT2 XP 014493437.1 VrALMT2 EFH65952.1 AlALMT1 AEE28289.1 AtALMT1 NP 001289915.1 CsALMT1 BAE97280.1 BnALMT Q9SJE8.2 ALMT2 Q9SJE8.2 AtALMT2 Q9XIN1.1 AtALMT7 XP 002520759.1 RcALMT14 XP 014495658.1 VrALMT14 XP 015161287.1 StALMT2 AET22417.1 CmALMT AET22398.1 CsALMT XP 015058071.1 SpALMT2 XP 003556423.1 GmALMT10 XP 002518887.1 RcALMT10 XP 002527763.1 RcALMT2 Q9SRM9.1 AtALMT8 Q9C6L8.1 AtALMT4 GAQ81186.1 KfALMT Q9LS46.1 AtALMT9 XP 014516643.1 VrALMT9 XP 003532498.1 GmALMT9 XP 002529135.1 RcALMT9 Q9SHM1.1 AtALMT6 XP 014512654.1 VrALMT4 XP 002532651.1 RcALMT9 XP 010236888.1 BdALMT9 Q9LPQ8.1 AtALMT3 XP 006574876.1 GmALMT4 Q93Z29.1 AtALMT5 XP 006662090.1 ObALMT Q9LS23.1 AtALMT13 Q9LS22.1 AtALMT14 XP 014520912.1 VrALMT12 XP 002519839.1 RcALMT12 AEE83973.1 AtALMT12 XP 003535771.2 GmALMT XP 003555193.1 GmALMT14 XP 003555195.1 GmALMT12 XP 014512012.1 VrALMT12 Q3E9Z9.1 AtALMT11 O23086.2 AtALMT10

13.7 Channels and Transporters Participating in Cl− Homeostasis

Figure 13.3 Aluminum-activated malate transporter (ALMT) channel phylogenetic tree shows six clades. The dendrogram indicates the degree of similarity between ALMT proteins of different plant species. The sequences were collected from the NCBI database. An alignment of known full-length proteins was performed using Clustal W as part of the MEGA6 software package programs and the phylogenetic tree was constructed using the neighbor-joining method. Species prefixes: Aet, Aegilops tauschii; Ta, Triticum aestivum; Hv, Hordeum vulgare; Zm, Zea mays; Al, Arabidopsis lyrata; At, Arabidopsis thaliana; Sc, Secale cereal; Tu, Triticum urartu; Hl, Holcus lanatus; Sb, Sorghum bicolor; Os, Oryza sativa; Bn, Brassica napus; Cc, Cynara cardunculus; Gm, Glycine max; Cm, Citrus maxima; Cs, Citrus sinensis; Ob, Oryza brachyantha; Rc; Ricinus communis; St, Solanum tuberosum; Kf, Klebsormidium flaccidum; Cs, Camelina sativa; Sp, Solanum pennellii; Bd, Brachypodium distachyon; Si, Setaria italica; Vr, Vigna radiata.

(whole plant), the plants trapped more Cl− in the roots which led to low shoot Cl− content and low relative electrolyte leakage (Wei et al. 2016). In contrast to rice and soybean, the ortholog of Arabidopsis CLCd in Citrus leaves was not differentially expressed between the Cl− -accumulating and -excluding genotypes under saline conditions (Brumos et al. 2009). This points out that variation exists for CLCd expression in different genotypes. Recently, the Cl− /H+ antiporter activity was found in the Golgi-enriched membrane fractions (Shuvalov et al. 2015). It was speculated that Cl− /H+ antiporters may be involved in the regulation of cytoplasmic Cl− by vesicular trafficking from cytoplasm to the vacuoles by endosomes, and they may be derivatives of Golgi membrane.

13.7.4

Phylogenetic Tree and Tissue Localization of CLC Family Members

To look into the phylogenetics of CLC family members, we have collected CLC sequences from NCBI database, and an alignment of known full-length proteins was performed using Clustal W as part of the MEGA6 software package programs. Phylogenetic tree was constructed using the neighbor-joining method which revealed that there are four major clades (Figure 13.4). In Arabidopsis, CLC family members are expressed in all tissues. Using green fluorescent protein, De Angeli et al. (2009) found that AtCLCa was localized to the tonoplast in Arabidopsis. On the other hand, AtCLCe and AtCLCf were co-localized with the thylakoid membrane of chloroplasts and early cis-Golgi subcompartment and trans-Golgi cisternae, respectively (Marmagne et al. 2007; Qun-dan et al. 2009). Von der Fecht-Bartenbach et al. (2007) and Guo et al. (2014) found out that AtCLCd is targeted to the trans-Golgi network. Recently, the chloride channel-like AtCLCg has been characterized and is expressed in mesophyll cells, hydathodes and phloem (Nguyen et al. 2016). Their work revealed that AtCLCg is involved in chloride homeostasis during NaCl stress in Arabidopsis thaliana. Both AtCLCc and AtCLCg are crucial players for imparting tolerance to excess chloride, but the functions are not redundant. It appears that these two genes are part of the regulatory network controlling chloride sensitivity in plants. AtCLCg appears to be localized in the tonoplast and is highly expressed in mesophyll cells (Nguyen et al. 2016). The localization of other CLC proteins and their precise functions during stress, however, are not clear. These studies indicate that different CLC proteins have different tissue-specific functions and their transport mechanisms may also vary (Figure 13.5).

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13 Chloride (Cl− ) Uptake, Transport, and Regulation in Plant Salt Tolerance KHG13135.1 GaCLC-b XP 012445644.1 GrCLC-b XP 006470044.1 CsCLC-b EEE84906.1 PtCLC-a EEF50918.1 RcCLC XP 012070534.1 JcCLC-b AEE77275.1 AtCLC-b XP 013598232.1 BoCLC-b AED94613.1 AtCLC-a XP 013717092.1 BnCLC-a XP 013592026.1 BoCLC-a NP 001267676.1 CsCLC-b NP 001236494.1 GmCLC XP 014509989.1 VrCLC-b XP 013451406.1 MtClC XP 004513192.1 CaCLC-b XP 006604142.1 GmCLC-b XP 014491244.1 VrCLC-b XP 011098867.1 SiCLC-a XP 011097296.1 CLC-b XP 006338691.1 StCLC-a XP 006357190.1 StCLC-b XP 015066133.1 SpCLC-b XP 003576525.1 BdCLC-a XP 006664534.1 ObCLC-a NP 001183936.1 ZmCLC XP 004962545.1 SiCLC-a EMT27046.1 AetCLC-c EMS50378.1 TuCLC-c ADW93911.1 HvCLC XP 006652320.1 ObCLC-c XP 012703146.1 SiCLC-c XP 014757006.1 BdCLC-c EMS67360.1 TuCLC-c EMT28663.1 AetCLC-c EMS55037.1 TuCLC-c AIY56605.1 AhCLC XP 004512644.1 CaCLC-c KHG08138.1 GaCLC-c XP 013458724.1 MtCLC1 ADK66979.1 VvClC1 XP 006349289.1 StCLC-c XP 015055840.1 SpCLC-c XP 011087632.1 SiCLC-c AED95868.1 AtCLC-c XP 013701993.1 BnCLC-c XP 006470992.1 CsCLC-c XP 012066731.1 JcCLC-c ERP62990.1 PtCLC-c GAQ87178.1 KfCLC XP 006590397.1 GmCLC-d XP 014520999.1 VrCLC-d XP 012574363.1 CaCLC-d ADW93910.1 TaCLC EMT29473.1 AetCLC-d XP 010231301.1 BdCLC-d XP 004982102.2 SiCLC-d AAO19370.1 OsCLC-d XP 006650436.1 ObCLC-d KVI01687.1 CcCLC XP 006359908.1 StCLC-d XP 015087944.1 SpCLC-d XP 011071571.1 SiCLC-d EOY05626.1 TcCLC isoform 1 EOY05628.1 TcCLC isoform 3 EOY05627.1 TcCLC isoform 2 EOY05630.1 TcCLC isoform 4 XP 012452606.1 GrCLC-d XP 006489358.1 CsCLC-d XP 012069593.1 JcCLC-d AED93540.1 AtCLC-d AGZ89689.2 BrCLC-d XP 013599188.1 BoCLC-d ABO97926.1 OlCLC XP 005649139.1 CsCLC AEE86511.1 AtCLC-e XP 013596552.1 BoCLC-e KHG02756.1 GaCLC-e XP 014524297.1 VrCLC-e XP 004491649.1 CaCLC-e XP 010232203.1 BdCLC-e EMT19807.1 AetCLC-e EMS57808.1 TuCLC-e KVI10281.1 CcCLC XP 013592212.1 BoCLC-f AEE33275.1 AtCLC-f XP 013692267.1 MnCLC-f KHG22539.1 AtCLC-f XP 012434332.1 GrCLC-f XP 015082747.1 SpCLC-f XP 010235261.2 BdCLC-f XP 004973814.1 SiCLC-f

13.7 Channels and Transporters Participating in Cl− Homeostasis

Figure 13.4 CLC channel phylogenetic tree shows four clades. The dendrogram indicates the degree of similarity between CLC proteins of different plant species. The sequences were collected from the NCBI database. An alignment of known full-length proteins was performed using Clustal W as part of the MEGA6 software package programs and the phylogenetic tree was constructed using the neighbor-joining method. Species prefixes: Ah, Arachis hypogaea; Mt, Medicago truncatula; Tc, Theobroma cacao; Rc, Ricinus communis; Cs, Coccomyxa subellipsoidea; Ol, Ostreococcus lucimarinus; At, Arabidopsis thaliana; Ga, Gossypium arboretum; Cs, Cucumis sativus; Cc, Cynara cardunculus; Br, Brassica rapa Os, Oryza sativa; Gm, Glycine max; Zm, Zea mays; Ob, Oryza brachyantha; Cs, Citrus sinensis; Kf, Klebsormidium flaccidum; St, Solanum tuberosum; Sp, Solanum pennellii; Bd, Brachypodium distachyon; Si, Setaria italic; Vr, Vigna radiate; Bn, Brassica napus; Bo, Brassica oleracea; Hv, Hordeum vulgare; Ta, Triticum aestivum; Vv, Vitis vinifera; Ca, Cicer arietinum; Gr, Gossypium raimondii; Jc, Jatropha curcas; Pt, Populus trichocarpa; At, Aegilops tauschii; Tu, Triticum urartu; Si, Sesamum indicum.

PPi

Cl– Cl– sequestration

H+

A

NRT1 NAXT1

PPi

PYL/RCAR

RE AB

CPK

SnRK

Cl–

Cl–

Cl– sequestration H+ Cl– Vacuole

2H+

Cl–

Cl–

Cl–

Stretch activated Hyperpolarization activated

Cl–

ATP

H+

ROS Cl–

Cl– Cl–

Ca2+

NRT1 H+

Cl–

ALMT1 SLAH

GABA

ALMT1

Cl–

Cl–

2Pi

AB

Cl–

CLC

Cl–

Cl–

Xylem

Cl–

Cl–

Photosynthesis

CLC

H+

ALTMT

Vacuole

2Pi

ROS

CAX

Cl–

Leaf cell

Cl–

X-IRAC CCC

Cl–

Root cell

Figure 13.5 Possible molecular mechanisms of Cl− influx, efflux, reduced net xylem loading, and compartmentalization.

13.7.5

Cation, Chloride Co-transporters

It has been predicted that xylem ion concentrations may be controlled by active retrieval of Cl− from the xylem stream (Colmenero-Flores et al. 2007; Munns and Tester 2008). A Na+ : K+ : 2Cl co-transporter has been identified in Arabidopsis and named AtCCC (Colmenero-Flores et al. 2007). Evidence for the role of AtCCC in controlling Cl− loading/unloading at the xylem/symplast boundary was based on: (i) AtCCC expression

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found in xylem parenchyma cells using GUS constructs; and (ii) Cl− concentrations of an Arabidopsis atccc mutant. Chloride homeostasis was not noticed in Atccc plants under high salt stress, and mutants accumulated higher levels of Cl− in shoot tissues and lower amounts in roots than the wild-type plants (Colmenero-Flores et al. 2007). Colmenero-Flores et al. (2007) analyzed CCC mutant lines and showed involvement of AtCCC in long-distance Cl− transport. Thus, CCCs are known to regulate ion concentration in the root xylem as demonstrated by Flowers and Colmer (2008). Previous studies have suggested that Cl− transport in the xylem is an electroneutral process (Kohler and Raschke 2000) that is accompanied by permeable cations (Lorenzen et al. 2004), which suggests that CCC could be a feasible candidate gene for xylem retrieval of Cl− under salt stress. Comparison of gene expression in leaves between two Citrus genotypes that differ in Cl− exclusion in response to elevated Cl− revealed that the Citrus AtCCC ortholog recorded an increased expression over time (12 weeks) in the Cl− -accumulating genotype (Carrizo citrange), but not in the Cl− -excluding genotype (Cleopatra mandarin) (Brumos et al. 2009). This indicates that CCC in Citrus may not be associated with Cl− exclusion under NaCl stress. Expression of AtCCC in Xenopus laevis oocytes indicated its bonafide role in Na+ : K+ : Cl− cotransport (Colmenero-Flores et al. 2007). Later, Kong et al. (2011) reported OsCCC, which is localized to the root cells of Oryza sativa. However, these results have been questioned by Teakle and Tyerman (2010), indicating that further clarifications are necessary to determine the role of CCCs in conferring exclusion of shoot Cl− and salt stress tolerance. Recently, Henderson et al. (2014) isolated and characterized CCCs from grapevine and Arabidopsis. Their findings suggested that it is a member of Na+ –K+ –2Cl− co-transporter class. The expression in ccc knockout mutants resulted in normal phenotype and decreased shoot Cl− and Na+ levels compared with wild-type plants. This CCC is localized to the Golgi and trans-Golgi network and indirectly influenced the transport of ions and salt stress tolerance in plants (Henderson et al. 2014). Chen et al. (2016) demonstrated that OsCCC1 co-transport is involved in cell elongation by regulating ion Cl− , K+ , and Na+ homeostasis to maintain cellular osmotic potential. The work of Saleh and Plieth (2013) revealed another important aspect of Cl− accumulation in plants. They discovered that internal calcium impacted the transport of chloride. A calcium-activated chloride channel (CaCC) blocker, anthracene-9-carboxylic acid (A9C), caused Cl− accumulation, as observed by Saleh and Plieth (2013) in root cells of A. thaliana during salt stress, and is controlled by both internal and external calcium levels. 13.7.6 ATP-binding Cassette Transporters and Chloride Conductance Regulatory Protein ABC transporter family members control the movement of ions in guard cells. In Arabidopsis and rice, these transporters play diverse roles including auxin transport and xenobiotic and metal detoxification (Davies and Coleman 2000; Rea 2007). They are located either in the plasma membrane or in tonoplast. Hedrich et al. (1990) and Schroeder and Keller (1992) were the first to discover both Rapid-type (R-type) and Slow-type (S-type) anion channels in V. faba guard cells. Multidrug-resistance protein 4 (MRP4) is expressed in primary roots and it regulates S-type anion channel activity in guard cells (Suh et al. 2007). It is a member of

13.7 Channels and Transporters Participating in Cl− Homeostasis

ABC family and is upregulated by salt stress (Suh et al. 2007). This indicates the role of MRP4 in Cl− transport and it is essential to validate its function in roots. In animal cells, ICln (CLNS1A) acts as an anion channel (Ritter et al. 2003) in artificial membranes. A microarray study showed that the homolog of ICln in Arabidopsis (AT5G6290) has no role in salt stress (Winter et al. 2007), but in Citrus root stock, CcICln showed differential expression and diverse Cl− exclusion abilities, which suggests involvement of CcICln in Cl− transport (Brumós et al. 2010). 13.7.7

Nitrate Transporter1/Peptide Transporter Proteins

Li et al. (2016a) determined the first protein, AtNPF2.4, in Arabidopsis, which is directly involved in transport of Cl− in root xylem. This protein is localized in plasma membrane and its promoter specifically regulates expression in the stellar cells of roots. The overexpression of AtNPF2.4 caused about a 23% increase in shoot Cl− content. In Xenopus, it executed the efflux of Cl− at similar membrane potentials as in plant stele (C-120 mV). The ascribed currents for AtNPF2.4 were pH independent and channel-like but not like the major conductance that is thought to be involved in xylem loading of Cl− and NO3 − X-QUAC (Gilliham and Tester 2005; Kohler et al. 2002; Li et al. 2016a,b,c). Arabidopsis may lack X-QUAC-type channels (Gilliham and Tester 2005; Kohler et al. 2002). The silencing of AtNPF2.4 reduced by only 20–30% shoot Cl− concentration (Li et al. 2016a,b,c); this confirms that Cl− loading in Arabidopsis xylem is a multigenic trait (Gilliham and Tester 2005). It is a member of nitrate excretion transporter (NAXT) and the closest relative of the AtNPF2.5 homolog. It is localized to the plasma membrane of root cortical cells and mediates Cl− efflux (Li et al. 2016b). AtNPF2.5 is salt inducible and may encode for a transporter which is involved in shoot Cl− exclusion (Li et al. 2016b). Both AtNPF2.4 and AtNPF2.5 are involved in Cl− transport and this suggests that other members of the root-specific NAXT family may be involved in this root chloride excretion. One more NPF gene family member, AtNPF7.3, was identified to have a role in Cl− transporter and is regulated by salt stress. This protein is a stele-specific anion channel, which is involved in the loading of NO3 − directly into the xylem of root (Lin et al. 2008). Salinity severely downregulates expression of AtNPF7.3, which may be related to the antagonism between shoot Cl− and NO3 − accumulation (Chen et al. 2012). 13.7.8

Chloride Channel-mediated Anion Transport

Mechanosensitive channels of the small conductance-like (MSL) transporter family possess 10 members in Arabidopsis (Haswell et al. 2008). Out of these, MSL9 and MSL10 showed root-specific expression and are localized on plasma membrane. They are Cl− permeable and have 10 Pa stretch-activated channels which are observed in Arabidopsis root cortical cells (Haswell et al. 2008). The quintuple mutants of MSL4, MSL5, MSL6, MSL9, and MSL10 did not show growth inhibition under high salt conditions (up to 250 mM NaCl), suggesting that these channels may not be directly involved in salt stress. Voltage-dependent anion channel, identified for the first time in Pennisetum glaucum, present on the outer membrane of mitochondria (Clausen et al. 2004; Lee et al. 2009; Desai et al. 2006), is involved in expression under high salt, cold, and salicylic acid, but not by ABA. While these transporters were downregulated in Cl− -accumulating Citrus species, they showed constant expression in the salt-excluder genotype Cleopatra (Brumos et al. 2009).

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13.7.9 Possible Mechanisms of Cl− Influx, Efflux, Reduced Net Xylem Loading, and its Compartmentalization Prevention of Cl− accumulation in shoots is important for reducing Cl− toxicity by alteration in the transport process. Decreasing influx minimizes the uptake of Cl− from the root epidermis to cortex. Generally Cl− enters root cells by secondary active uptake or passively under high salinity (Skerrett and Tyerman 1994). The AtSLAH1 and AtSLAH3 play a major role in Cl− metabolism. The expression of AtSLAH1 is downregulated by salinity and ABA treatment while AtSLAH3 showed differential behavior (Cubero-Font et al. 2016). ABA prevents the loading of Cl− in root xylem but it has no effect on Cl− uptake (Gilliham and Tester 2005). This results in the restriction of Cl− in root under salt stress and contributes to the exclusion of salt from shoots. Like ABA, post-translational signals also regulate the loading of Cl− in xylem. In soybean, ROS is associated with exclusion of Cl− (Ren et al. 2012). The ALMT activity is inhibited by GABA and ATP (De Angeli et al. 2016). The CLC activity is also regulated by ATP and may influence the storage capacity of roots for Cl− and therefore disturbs Cl− delivery to xylem (De Angeli et al. 2016). In the promoter region of AtNPF2.4, the ABA-responsive elements were recognized (Li et al. 2016a) and it is interesting to know its role in Cl− homeostasis. Compartmentalization of toxic Cl− ions in to vacuoles to reduce the cytoplasmic toxicity occurs in many cells. The sequestration of Cl− in vacuoles of root affects long-distance transport of Cl− from root to shoot (Storey and Walker 1999). Sequestration minimizes net xylem loading, which is a major hurdle in shoot Cl− exclusion and involves increased active retrieval and reduced passive loading. Shoot compartmentalizes Cl− in the leaf epidermis and protects mesophyll cells and ultimately photosynthesis (Huang and Van Steveninck 1989).

13.8 Conclusion and Future Perspectives Prevention of accumulation of toxic Cl− ions in shoots is the key to reducing its toxicity. This can be achieved by altering the transport process of Cl− ions. The root stellar cells and their transporters act as gatekeepers for shoot Cl− accumulation. Among them, NPF and SLAH proteins are reported to play a role in the modulation of long-distance transport of Cl− ions. These transporters are targeted for improving Cl− exclusion and salt tolerance in crops. Also, AtALMT9 and CCCs are endomembrane transporters which have shown involvement in long-distance transport of Cl− along with CLC transporters. Methods for direct experimental measurements of Cl− fluxes and concentrations in vacuoles of intact plants have so far not been established, although some estimates have been made using X-ray microanalysis (Hajibagheri and Flowers 1989), intracellular ion-sensitive microelectrodes (Felle 1994), tracer compartmental analysis (Britto et al. 2004), or Cl− sensitive fluorescent probes (Lorenzen et al. 2004). There is a need to develop efficient methods to measure the Cl− translocation in phloem and xylem (Cubero-Font et al. (2016). Advanced methods and techniques have to be in place to better understand Cl− homeostasis in plants. Plant responses under Cl− stress have not been intensively investigated at their influx, efflux, accumulation, and compartmentation. Studies based on transcriptome, proteome, metabolome, biochemical, physiological, and hormone modulation against Cl− homeostasis will

References

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14 The Root Endomutualist Piriformospora indica: A Promising Bio-tool for Improving Crops under Salinity Stress Abhimanyu Jogawat 1 , Deepa Bisht 2 , Nidhi Verma 2 , Meenakshi Dua 3 , and Atul Kumar Johri 1 1

National Institute of Plant Genome Research, New Delhi, 110067, India School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India 3 School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, 110067, India 2

14.1 Introduction The root endomutualist Piriformospora indica was isolated from the roots of two desert plants, Prosopsis juliflora and Zizyphus nummularia, found in Thar Desert of Rajasthan, India. The natural habitat of P. indica and its host plant experiences severe drought and hot climatic conditions with a range of temperature between 45 and 50 ∘ C (Verma et al. 1998; Varma et al. 1999). This remarkable fungus possesses most of the beneficial characteristics reported for arbuscular mycorrhiza (AM) fungi (Varma et al. 1999). It has been established as the model endophytic fungus for studying and exploring molecular, biochemical, physiological, and biotechnological aspects during interaction with the host plant, which was previously not possible with AM fungi. Thus, P. indica has boosted the plant–fungal association-related research to a new level. Efforts have been made to use P. indica in the improvement of crops under abiotic stress (Kumar et al. 2012) and for nutrient enrichment (Johri et al. 2015) of the host plant. In this overview, we focus on recent advances in research associated with P. indica and host plant association under salinity stress, which leads to physiological, biochemical, and molecular changes in host plants to combat salinity stress.

14.2 P. indica: An Extraordinary Tool for Salinity Stress Tolerance Improvement Its nonspecificity of interaction makes P. indica an extraordinary endomutualist. The surprisingly broad host range enables it to infest dicots as well as monocots, including model plant Arabidopsis thaliana (Verma et al. 1998, 2001; Sahay and Varma 1999; Peškan-Berghöfer et al. 2004; Pham et al. 2004; Sherameti et al. 2005; Shahollari et al. 2005, 2007; Waller et al. 2005; Kumar et al. 2009; Oelmüller et al. 2009; Yadav et al. 2010; Jogawat et al. 2013). The colonization of P. indica has been observed to be intercellular or intracellular (Peškan-Berghöfer et al. 2004). It has been shown to increase Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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abiotic stress tolerance, biotic stress tolerance, nutrient uptake, and photosynthetic yield (Waller et al. 2005; Kumar et al. 2009; Yadav et al. 2010; Jogawat et al. 2013; Johnson et al. 2014; Murphy et al. 2015). It has been studied under salinity stress with various host plants such as rice (Jogawat et al. 2013, 2016; Bagheri et al. 2013), barley (Waller et al. 2005; Ghabooli et al. 2013; Ghabooli 2014; Ghaffari et al. 2016), and A. thaliana (Vahabi et al. 2015; Abdelaziz et al. 2017). Various studies have shown that its association modulates the host plant milieu to combat prevailing stress. The biochemical, molecular, and physiological changes occurring during colonization enhance the defense mechanism of the host plant to overcome stresses. The mechanism of salt stress tolerance by the host plant has been studied in detail, specifically in barley and rice. P. indica may be a new hope for improving crops under high salinity. It is expanding new horizons in the area of fungus-induced plant modulations and regulations, specifically in model plant A. thaliana. It is revealing various molecular aspects of plant–fungus interaction which were been impossible employing AM fungi.

14.3 Utilization of P. indica for Improving and Understanding the Salinity Stress Tolerance of Host Plants Approximately 7% of land has been recorded to have salinity stress, which is a major challenge to achieving optimum crop yield (Mahajan and Tuteja 2005). Salinity stress interferes with efficient nutrient uptake through roots. Even under extreme conditions, P. indica has been reported to confer abiotic stress tolerance to plants by improving growth and rescuing them from the detrimental effects of such stress conditions (Waller et al. 2005; Jogawat et al. 2013; Johri et al. 2015; Gill et al. 2016). As an initial study, we reported a beneficial interaction between P. indica and rice under salt stress up to 300 mM NaCl. We showed that P. indica induces growth, photosynthetic pigments, and proline accumulation in rice under salt stress and further increased its salt tolerance (Jogawat et al. 2013). In barley, its colonization not only resulted in plant growth promotion, but also increased crop yield and salinity stress tolerance (Waller et al. 2005). Its association elevates antioxidative capacity via activation of the glutathione–ascorbate cycle in host plants (Baltruschat et al. 2008; Kumar et al. 2012; Bagheri et al. 2013; Ghaffari et al. 2016). Moreover, as a bio-regulator, it has been reported to activate various genes involved in stress acclimation, metabolism, and antioxidant system in host plant (Waller et al. 2005; Baltruschat et al. 2008). Moreover, it also confers cadmium and arsenic tolerance in plants (Hui et al. 2015; Mohd et al. 2017). Recently, Arabidopsis and Medicago truncatula were also studied with P. indica under salinity stress (Abdelaziz et al. 2017). Thus, P. indica has proven as rescuer of the growth arrest under salinity stress for plants.

14.4 P. indica-induced Biomodulation in Host Plant under Salinity Stress The complexity of salinity stress mechanism is well known among scientists who are trying to develop salinity stress-tolerant crop varieties. The association of P. indica was found to affect different biochemical processes such as metabolic activity, fatty acid

14.4 P. indica-induced Biomodulation in Host Plant under Salinity Stress

composition, lipid peroxidation, and metabolic heat production as well as molecular processes such as salinity stress tolerance genes in host plants (Baltruschat et al. 2008; Jogawat et al. 2016). In a study, P. indica-colonized salt-sensitive plants were shown to develop salinity stress-tolerant characteristics such as increased heat emission and ethane production. Salt-induced osmostress leads to enhanced accumulation of harmful reactive oxygen species (ROS). The increased level of ROS induces osmostress signaling that exhibits a response to stress so as to adapt to or combat such conditions (Banerjee and Roychoudhury 2017). Thus, P. indica-colonization activates the ROS scavenging antioxidant system which further helps plants to combat salinity stress (Harrach et al. 2013; Bagheri et al. 2013). In fennel (Foeniculum vulgare) and Thymus vulgaris plants, it reduces salt stress-induced lipid peroxidation and oleic acid composition in host cells (Dolatabadi et al. 2011a,b). In another proteomic analysis of barley, under salt stress of 300 mM NaCl, P. indica has been shown to modulate ion accumulation by increasing the foliar potassium (K+ )/sodium (Na+ ) ratio, which is considered to be a reliable indicator of salinity stress tolerance. Further, it also induces calcium (Ca2+ ) accumulation under such conditions. About 51 proteins were identified which belonged to different functional categories such as photosynthesis, cell antioxidant defense, protein translation and degradation, energy production, signal transduction, and cell wall arrangement (Alikhani et al. 2013). It was further shown that the P. indica association induces a systemic response to salinity stress by altering the physiological and proteome responses of the host plant (Alikhani et al. 2013). The symbiotic association between P. indica and wheat plants was reported to improve growth parameters under salinity stress. Also, the uptake of water and photosynthetic pigment contents and proline accumulation in wheat seedlings were increased in P. indica-inoculated wheat seedlings (Zarea et al. 2012). In another study, it was found that P. indica helps rice plants during high salt stress and resulted in an increase in root and shoot lengths, dry weight, and total chlorophyll contents as compared with the noncolonized plants (Jogawat et al. 2013; Bagheri et al. 2013). P. indica association improved salt stress resistance by increasing dry weight, shoot length, ion content (Na+ , K+ , Ca+2 ), sugars, and free amino acids. Moreover, P. indica-inoculated plants exhibited higher ion ratios of K+ /Na+ and Ca+2 /Na+ (Ghabooli 2014). In rice plants, the biochemical parameters such as proline content, the rate of lipid peroxidation, and Na+ and K+ ion concentrations were increased in P. indica-colonized plants under salinity stress (Bagheri et al. 2014). Similarly, in the case of Nicotiana tobacum, the salinity tolerance gene (osmotin promoter binding protein) OPBP1 and pathogenesis-related (PR) protein genes such as PR-1a, PR2, PR3, and PR5 were found to be upregulated. Further, P. indica-colonization was found to be associated with malondialdehyde and proline contents, and plasma membrane permeability (FeiQiong et al. 2014). In a recent study using integrated ionomics, metabolomics, and transcriptomics under high salinity (300 mM NaCl) in P. indica-colonized barley plants, 14 metabolites and ions, and 391 differentially expressed genes were identified at 300 mM NaCl in P. indica-colonized plants, which conferred tolerance to salinity stress. It was observed that the major and minor carbohydrate metabolism, nitrogen metabolism, and ethylene biosynthesis pathway might be playing a role in systemic salt-tolerance in leaf tissue induced by P. indica (Ghaffari et al. 2016). Additionally, salinity stress response genes and the other metabolic regulatory systems were identified in P. indica-colonized barley plants, using a combination of the transcriptomic, metabolomic, and ionomic approaches. The

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identified genes were increased in the sense of differential expression in barley from 254 to 391 with increased salt concentration from 0 to 300 mM NaCl in P. indica-colonized conditions compared with noninoculated barley plants. The levels of auxin, ethylene, and brassinosteroid were also shown to be increased in the 300 mM condition. Two genes associated with trehalose metabolism, viz., trehalose phosphate phosphatase and trehalse-6-phosphate synthase were upregulated at severe salt stress of 300 mM NaCl (Ghaffari et al. 2016). Triose phosphate isomerase, one of the proteins involved in carbohydrate metabolism, was found to be upregulated in salinity stress (Ghaffari et al. 2016). Another protein, thaumatin-like protein, was also found to be associated with tolerance against biotic as well as abiotic stress. Thaumatin-like protein responded to salt stress by displaying osmotic adaptation under stress conditions (Singh et al. 1987; Ghaffari et al. 2016). Thus P. indica moderates salt tolerance by affecting cell membrane maintenance, cell milieu steadiness, and osmolyte accumulation, and decreasing the level of membrane lipid peroxidation in the host plant under salt stress conditions. The literatures review clears the role of P. indica by affecting various mechanisms and metabolic pathways, which leads to biomodulation of the host for combating salinity stress. In short, P. indica colonization forces the readiness of plant system to combat upcoming stress effectively.

14.5 Activity of Antioxidant Enzymes and ROS in Host Plant During Interaction with P. indica In rice plants, the activities of antioxidant enzymes such as catalase, peroxidase, superoxide dismutase, and polyphenol oxidase were observed to increase in P. indica-colonized plants under salinity stress (Bagheri et al. 2014). In barley, upregulation of peroxidases at 300 mM NaCl in the P. indica-colonized plants was reported, which is fundamental for antioxidative defense systems. Moreover, it was also reported that ROS are involved in the interaction of P. indica within barley plants. Here, the fungus senses and uses the redox system of the plant in establishment as well as maintenance of its colonization (Alikhani et al. 2013; Ghaffari et al. 2016). In addition to the regulation of ions, the regulation of some salinity stress-responsive proteins was also reported in barley under severe salinity stress. Polyamine oxidase, a protein in barley which is involved in stress responsive pathway, was also reported to be affected by salt stress (Ghaffari et al. 2016). In maize plants, polyamine oxidase was reported to be involved in the elongation and overall growth of the leaf during salt stress conditions (Rodríguez et al. 2009). A P. indica-mediated salinity tolerance mechanism was found to be linked strongly to an increase in antioxidants in barley, which attenuates the NaCl-induced lipid peroxidation, metabolic heat efflux, and fatty acid desaturation in barley plants (Baltruschat et al. 2008). In Arabidopsis, P. indica significantly elevated the amounts of ascorbic acid and increased the activities of antioxidant enzymes during salinity stress conditions (Vadassery et al. 2009a).

14.6 Role of Calcium Signaling and MAP Kinase Signaling Combating Salt Stress Ca2+ has been reported to be crucial for maintaining the ionic balance, as well as being a secondary messenger for activating various signal transduction pathways. In addition,

14.7 Effect of P. indica on Osmolyte Synthesis and Accumulation

it has vital roles in plant growth, carbon assimilation, nourishment, and water transport (Roychoudhury and Banerjee 2017). In barley plants, P. indica also induces calcium (Ca2+ ) accumulation under salinity stress conditions in its host (Alikhani et al. 2013). Interestingly, cell wall extract from P. indica (PiCWE) is also reported to promote the growth of seeding of A. thaliana. PiCWE also increased the Ca2+ level mainly in roots of transgenic A. thaliana as well as those of N. tabacum (Vadasserry et al. 2009b). Further, microarray analysis revealed that some genes of the Ca2+ signal transduction pathway were also upregulated in P. indica co-cultivated plants. Moreover, the increased level of glutamate receptor gene GLR2.5 and cyclic nucleotide gated channels CNGC-10 and -13 were also stimulated in PiCWE-treated plant roots. Two of the MAP kinases, MPK3 and MPK6, were also found to be activated in Arabidopsis roots treated with PiCWE (Vadassery et al. 2009). Taken together, it can be stated that an increase in the Ca2+ level in P. indica-colonized plants helps to defend the plant effectively against salinity stress (Vadassery et al. 2009; Alikhani et al. 2013). Thus, the rise in Ca2+ level may have a key role in stress signal transduction and maintenance of ionic balance in P. indica-colonized plants.

14.7 Effect of P. indica on Osmolyte Synthesis and Accumulation P. indica increased the biomass of shoots and roots, photosynthetic pigments, total soluble proteins, relative water content, free proline content, and antioxidant enzyme activity of inoculated rice plants and the lipid peroxidation was decreased with an increase in the level of osmolytes such as polyamines and amino acid proline in colonized plants (Jogawat et al. 2013; Bagheri et al. 2013). This increase in polyamine content is due to the upregulation of methionine synthase in colonized plants, which plays a crucial role in the biosynthesis of polyamines and ethylene (Peškan-Berghöfer et al. 2004). Similarly, the rise in trehalose level also increases tolerance of different abiotic stresses as it acts as an osmolyte and helps in protein and membrane stabilization (Bianchi et al. 1993; Drennan et al. 1993). Compatible solutes such as proline have hydroxyl radical scavenging activity, which can neutralize the detrimental effects of salinity stress during the P. indica-colonized stage (Smirnoff and Cumbes 1989). Under salinity stress, P. indica-symbiosis triggered the accumulation of different osmolytes, which has been reported in various studies in different plants (Table 14.1). Thus, the Table 14.1 Piriformospora indica-induced accumulation of osmolytes in host plant under salinity stress. Host plant

Osmolyte

Salt stress (NaCl)

Reference

Rice

Proline, polyamines, protein content

200–300 mM

Jogawat et al. 2013; Bagheri et al. 2013

Wheat

Proline

400 mM

Zarea et al. 2012

Barley

Sugars and free amino acids

300 mM

Ghabooli 2014

Trehalose

300 mM

Ghaffari et al. 2016

Tomato

Proline

100 mM

Al-Absi and Al-Ameiri 2015

Tobacco

Proline

300 mM

FeiQiong et al. 2014

273

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14 The Root Endomutualist Piriformospora indica

P. indica + Salinity stress Signals

Proline Free Amino Acids

Sugars Trehalose

Polyamines

Plant

Salinity Stress Tolerance, Growth Diminution Rescue, Decreased Chlorosis, Decreased ROS Activity

Figure 14.1 Osmolyte accumulation in host plants during Piriformospora indica and salinity stress. Upon salinity stress, P. indica-colonized plants are found to synthesize and accumulate osmolytes such as free amino acids, proline, sugars, trehalose, polyamines, etc., which protect plants from deteriorating effects of salinity stress.

increased concentration of osmolytes owing to P. indica-association provides strength to the host plant to combat salinity stress effectively (Figure 14.1).

14.8 Salinity Stress Tolerance Mechanism in Axenically Cultivated and Root Colonized P. indica In the first attempt at exploring the potential of P. indica, it was observed that P. indica itself can tolerate up to 600 mM NaCl. Further, the 36 salinity tolerance-related genes from 400 mM NaCl-grown P. indica were isolated utilizing overexpression in Escherichia coli under high salinity pressure (Gahlot et al. 2015). From these salinity tolerance-conferring genes, a cyclophilin A-like protein (PiCypA) was isolated and functionally characterized. The transgenic expression of PiCypA increased the salinity tolerance of the plants (Trivedi et al. 2013; Gahlot et al. 2015). The overexpression of PiCypA also improved the salinity tolerance of E. coli and tobacco plants (Trivedi et al. 2013, 2014; Gahlot et al. 2015). From the same set of genes, a fatty acid desaturase gene was also cloned and characterized (Shekhawat 2015). In real-time polymerase chain reaction analysis, upregulation of six genes, i.e. cyclophillin, stearoyl-CoA desaturase, thiamine pyrophosphate-binding domain-containing protein, BCL-2 associated athanogene, 3-like protein, and cytochrome P-450, and 60S ribosomal protein genes was shown under prolonged salinity stress (Gahlot et al. 2015), whereas

14.8 Salinity Stress Tolerance Mechanism

only two genes, i.e. sphingolipid C9-methyltransferase-like protein (PiSLC9M) and cytochrome P450-like protein (PiCP450) were found to be upregulated in wild-type P. indica during colonization with the rice plant under 0.5 M NaCl as compared with the nonsalinity-treated wild-type P. indica (Jogawat et al. 2016). In addition to the above, the osmoregulatory and osmoadaptive MAP kinase cascade [high osmolarity glycerol (HOG) pathway] was also observed to be activated during axenic as well as colonized conditions in P. indica. Upregulation of most of the HOG pathway genes was observed upon osmostress (Jogawat et al. 2016). The central player of this MAP kinase pathway, i.e. PiHOG1, was also found to be regulated upon salinity stress via phosphorylation. It was shown to regulate other salinity stress-conferring genes of P. indica (Figure 14.2; Jogawat et al. 2016). Upon osmostress, PiHOG1 is phosphorylated in P. indica. Na+ –K+ ATPase PiENA1 was a major gene along with PiHOG1, which was induced to a great extent upon osmostress. PiHOG1 plays role in multistress (i.e. salt, heat, and oxidative) tolerance, glycerol accumulation, morphology, and growth during functional expression in the heterologous system Saccharomyces cerevisiae. Moreover, the role of PiHOG1 in P. indica was dissected in different salt conditions. PiHOG1 knock down (KD) P. indica was found to be impaired in osmo-adaptation and demonstrated a slow-growth phenotype in osmostress conditions. This also resulted in a reduced P. indica colonization of rice roots under nonsalt and salinity stress conditions. Thus, the role of PiHOG1 in salinity stress tolerance in P. indica–rice association was investigated (Jogawat et al. 2016) under 200 mM NaCl stress conditions with a salt-sensitive rice variety Oryza sativa L. cv “IR-64.” Apart from this, the transcriptional activity of 11 HOG pathway homolog genes and 21 salt tolerance-conferring genes was found to be induced in wild-type P. indica. The same plethora of genes was found to be downregulated in PiHOG1-KD P. indica with and without host under nonsalt stress and salt stress conditions. The putative HOG pathway activity (Table 14.2) has been observed as necessary for osmoresponsive salt tolerance genes and PiHOG1 seems to regulate not only the expression of putative HOG pathway genes, but also salt tolerance-conferring genes in axenic as well as in colonized conditions (Figure 14.2). The decreased and delayed phosphorylation of PiHOG1 MAP kinase was observed in the case of KD-PiHOG1 fungus during axenic culture as well as during the rice root colonized stage (Jogawat et al. 2016). Homologs of the transcription factors which are reported to be regulated by HOG1 in S. cerevisiae under osmostress have been found along with some putative genes in the P. indica genome (Table 14.3). Knockdown of PiHOG1 also affected P. indica-colonization of rice plant roots after 15 days post-inoculation, which is reduced by up to 30% under 200 mM salinity stress conditions compared with the usual P. indica colonization which is reduced by up to 20% at 15 days post-inoculation under the same stress conditions. The usual and basic pattern of chlamydospore arrangement in roots was converted into the clumps of chlamydospore upon PiHOG1 downregulation. The penetration of the hyphal tube also seemed to be affected in KD P. indica strain as the chlamydospores were mainly observed at the surface area of roots (Jogawat et al. 2016). Thus, the HOG pathway and other salinity stress tolerance genes may be playing an important role in the P. indica-mediated salinity stress tolerance of the associated plants.

275

Salinity Stress

Host Plant’s Root

Rhizosphere SO4–2

Mg+2 Free amino acids

SOS1

Polyamines Free proline Na+

H+

?

Fe+3

? PiSTE20

Vacuole PO4–2

Osmosensors PiYPD1 PiCDC42

?

?

PiSSK2

PiPBS2

Metabolism Protein syntheis

Nutrients flow PiHOG1 PiHXT5

Defensins Phytoalexins Osmotins PR genes Antioxidants SAR mechanism

Hexose sugars

+ ? NH4 PiAMT1

PO4–2 PiPT

Mg+2 Ca+2

PiPT

Na+ Vacuole

NH4+

SRTFs

?

Fe+3

?

Mg+2

?

SO4–2

?

Zn+2

Metabolism Protein synthesis

SRGs 2K+ Salinity tolerance Responses

3Na+ PiENA1

Figure 14.2 Association of P. indica with host plant root for improving salinity stress tolerance. P. indica provides nutrients to the host plant even in harsh conditions, which helps the plants to grow better (Yadav et al. 2010; Gahlot et al. 2015; Rani et al. 2016; Jogawat et al. 2016).

14.9 Conclusion

Table 14.2 HOG1 pathway homologs in P. indica genome and similarity to yeast members.

P. indica

Saccharomyces cerevisiae

Query cover (%)

Identity (%)

Total score

e-Value

Accession no.

ANA76492.1

PiHOG1

HOG1

79

79

600

0.0

PiPBS2

PBS2

49

59

397

1e − 130

CCA68314.1

PiSTL1

STL1

83

29

200

2e − 56

CCA70422.1

PiSHO1

SHO1

52

50

122

3e − 15

CCA74511.1

PiSLN1

SLN1

32

36

232

2e − 15

CCA73434.1

PiSTE20

STE20

59

49

494

4e − 163

CCA71165.1

PiCDC42

CDC42

67

55

239

9e − 81

CCA68283.1

PiSTE11

STE11

66

56

414

4e − 101

CCA69216.1

PiYPD1

YPD1

95

24

55.5

5e − 12

CCA75105.1

PiSSK2

SSK2

75

31

570

5e − 176

CCA67810.1

PiGPD

GPD

59

44

310

2e − 102

CCA69572.1

PiPTC1

PTC1

87

55

212

1e − 50

CCA73455.1

PiPTC2

PTC2

62

48

271

5e − 84

CCA72082.1

PiPFK26

PFK26

51

41

313

5e − 96

CCA77980.1

Table 14.3 HOG pathway-regulated putative salinity stress responsive transcription factors of P. indica. Gene in Saccharomyces cerevisiae

P. indica putative gene accession no.

Query cover (%)

Identity (%)

e-Value

Msn2

CCA67162 CCA72342

7 15

50 34

6e − 14 7e − 09

Msn4

CCA70948 CCA70297

17 10

32 33

17e − 10 6e − 09

Sko1

CCA73588 CCA69511.1

14 10

35 35

9e − 08 0.014

Hot1

CCA68510.1

6

35

6.1

Smp1

CCA69719 CCA66959

14 14

59 40

4e − 21 7e − 11

Skn7

CCA66665.1

50

61

2e − 36

Yap1

CCA70182

18

47

9e − 09

14.9 Conclusion The association of AM fungi started to support the evolution of terrestrial plants 400 million years ago, but plants still need this association for their betterment (Rodriguez and Redman 2008). Unlike AM fungi, axenically cultivable P. indica has a broad host spectrum which can be utilized to understand this evolutionary role of AM

277

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fungi (Newman and Reddell, 1987). The association of P. indica with the model plant Arabidopsis has revolutionized the research toward this goal. The journey started with the exploration of various beneficial effects of this magical fungus on various hosts including economical, medical, and ornamental plants. This model fungus is revealing various aspects of molecular mysteries beneath beneficial fungus and host plant association day by day. The advantageous colonization with Arabidopsis has strengthened our understanding of how a beneficial fungus can modulate the host milieu to tolerate various abiotic as well as biotic stresses. Researchers have succeeded in finding various pathways which P. indica is affecting for successful colonization under normal as well as stress conditions using global analysis tools such as proteomics, transcriptomics, and metabolomics with various host plants, i.e. Arabidopsis, barley, and chinese cabbage. They have also enriched our knowledge of the various mechanisms by which P. indica modulates the plant system for successfully rescuing the severe effect of various abiotic as well as biotic stresses. P. indica association mainly affects the defense system in such a way that the host plant attains “defense readiness” for better survival under such threats. Researchers are also trying to understand why some of the endophytes are meant to be beneficial or some are harmful. In conclusion, the use of P. indica to help plants may give new hope to saline agriculture as well as crop improvement.

Acknowledgments The author would like to thank Professor Atul Kumar Johri for the guidance and motivation for preparing the manuscript. This work is supported by a research fellowship from CSIR and SERB-National post doctoral fellowship from the Government of India.

Conflict of Interest There is no conflict of interest.

References Abdelaziz, M.E., Kim, D., Ali, S. et al. (2017). The endophytic fungus Piriformospora indica enhances Arabidopsis thaliana growth and modulates Na+ /K+ homeostasis under salt stress conditions. Plant Sci. 263: 107–115. Al-Absi, K. and Al-Ameiri, N. (2015). Physiological responses of tomato to inoculation with Piriformospora indica under osmotic stress and chloride toxicity. Int. J. Agric. For. 5: 226–239. Alikhani, M., Khatabi, B., Sepehri, M. et al. (2013). A proteomics approach to study the molecular basis of enhanced salt tolerance in barley (Hordeum vulgare L.) conferred by the root mutualistic fungus Piriformospora indica. Mol. BioSyst. 9: 1498–1510. Bagheri, A.A., Saadatmand, S., Niknam, V. et al. (2013). Effect of endophytic fungus, Piriformospora indica, on growth and activity of antioxidant enzymes of rice (Oryza sativa L.) under salinity stress. Int. J. Adv. Biol. Biomed. Res. 1: 1337–1350.

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15 Root Endosymbiont-mediated Priming of Host Plants for Abiotic Stress Tolerance Abhimanyu Jogawat 1 , Deepa Bisht 2 , and Atul Kumar Johri 2 1 2

National Institute of Plant Genome Research, New Delhi, 110067, India School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

15.1 Introduction Worldwide, abiotic stresses result in more than 50% of crops being lost annually (Bray et al. 2000). Root symbiosis is the naturally occurring strategy from which plants receive the benefit of improved survival under extreme situations such as nutrient deficit and abiotic and biotic stresses (de Zelicourt et al. 2013). There are various microorganisms which can establish beneficial interactions with plants, specifically with plant roots, such as fungi, bacteria, cyanobacteria and in some cases algae (Werner 1992). The symbiosis of terrestrial plants with ecto- or endo-mycorrhiza was estimated to have evolved approximately 450 million years ago (Pirozynski and Malloch 1975; Remy et al. 1994; Rodriguez and Redman 2008; Newsham et al. 2009; Martin et al. 2016). Arbuscular mycorrhizal fungi (AMF) are widespread and mostly associated with vascular plants. AMF belong to the Glomeromycota division of fungi. They form arbuscules (small tree-like structures) in plant cells, thus their name (Parniske 2008). The interactions of these beneficial microbes with plant roots help in various functions such as nutrient uptake and growth promotion of host plants (Marschner and Dell 1994; Landeweert et al. 2001; Vessey 2003). Moreover, these beneficial symbionts promote the performance of host plants in adapting to and successfully tolerating biotic and abiotic stresses (Rodriguez et al. 2004; Rodriguez and Redman 2008; Pozo et al. 2010; Smith et al. 2010; Grover et al. 2011; Singh et al. 2011; Nadeem et al. 2014; Gupta et al. 2017; Etesami and Beattie 2017). The mechanism of alteration of plant physiology by microbes for better survival under stressful environments is termed as biomodulation, bioregulation or bio-priming for stress tolerance (Rodrigues and Rodrigues 2014; Matsubara et al. 2013). For this reason, these microbes are often called biomodulators or bioregulators. The beneficial microbes are also sometimes called probiotics because of their role in protecting plants from harmful microbes (Shenderov 2011; Berlec 2012). In several studies, mycorrhizal fungi (ericoid, ecto-, and vascular) and endophytic bacteria have been reported to improve the uptake of various nutrients, such as nitrogen, phosphate, ammonium, nitrate, potassium, sulfate, copper, zinc, and iron (Marschner and Dell 1994; Smith et al. 2003; Bucher 2007; Parniske 2008; Bonfante and Genre 2010; Johri et al. 2015). Moreover, the symbionts, specifically fungi Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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and endo-rhizobacteria, intervene with the antioxidant ascorbate–glutathione cycle, ion homeostasis, phytohormone signaling, calcium signaling, MAP kinase signaling network, and other stress signaling systems for bio-priming of host plants for defense readiness and tolerance against abiotic as well as biotic stresses. In a study, it was stated that AM fungi could also help plants by modulating rhizospheric microbes in natural ecosystems (Barea et al. 1997). Various studies have been shown to induce different stress-responsive genes during these symbioses, which subsequently helps host plants to combat salinity, temperature, oxidative, heavy metal, water, and drought stress. The symbionts activate the reactive oxygen species (ROS) detoxifying enzyme system and also increase the biosynthesis and accumulation of compatible osmolytes (Schutzendubel and Polle 2002; Al-Karaki et al. 2004; Wu et al. 2006, 2010; Hildebrandt et al. 2007; Kohler et al. 2009; Evelin et al. 2009; Gamalero et al. 2009; Zhu et al. 2010a; Hamilton et al. 2012). Symbiosis-stimulated levels of osmolytes such as sugars, polyamine, and amino acids help the host plant during salinity and drought stress (Rodriguez et al. 2008; Redman et al. 2011). During symbiosis, the strengthening of defenses against stresses is a result of oxidative stress protection, which is performed by upregulating the antioxidant system, photosynthesis systems, and osmolyte accumulation by symbionts. It has been stated that symbionts prime host plants before abiotic stress (Sánchez-Díaz et al. 1990; Harrison 2005; Pozo and Azcón-Aguilar 2007; White and Torres 2010, Zhu et al. 2010a; Ruiz-Sánchez et al. 2010; Redman et al. 2011). In this chapter, we will review the symbiosis-mediated priming of the host plant in different types of symbioses, specifically bacterial and fungal, for the survival of host plants as well as the importance of such priming for improvement of crop yield under various abiotic stress environments.

15.2 Bacterial Symbionts-mediated Abiotic Stress Tolerance Priming of Host Plants Several reports have revealed that the rhizospheric bacteria and other symbiotic bacteria help crop plants tolerate and combat abiotic stresses (Yang et al. 2009; Dimkpa et al. 2009; Selvakumar et al. 2012). Bacteria form two kinds of symbiosis with host plants, i.e. Actinorhiza and legume–rhizobium symbiosis. These symbioses are much needed for nitrogen-fixation specifically in legume plants (Pawlowski and Sprent 2007). Gram-positive rhizobia have been proved to be effective biocontrol agents in plant growth promotion and bioremediation activities (Francis et al. 2010). Actinorhizal symbiosis has been found to improve the survival of older plants in contaminated soils (Roy et al. 2007; Diagne et al. 2013). In another study, alder trees (Alnus viridis ssp. Crispa, Alnus glutinosa) and shrubs with actinorhizal symbiosis were assessed for adaptability in fluctuating nitrogen conditions and under heavy metal (As, Se, or V) stress. It was found that shoot to root biomass ratios were significantly improved in the presence of the symbiont Frankia sp. (Bélanger et al. 2011). In Casuarina glauca, symbiosis with N2 -fixing bacteria Frankia has been reported to increase osmotic stress tolerance (Batista-Santos et al. 2015). In a comparative study between plant growth promoting rhizobia (PGPR) and AM fungi, it was observed that PGPR effectively induce ROS scavenging antioxidant enzymes for improving the tolerance of lettuce to salt stress (Kohler et al. 2009). Under water stress, both PGPR and AM fungus

15.2 Bacterial Symbionts-mediated Abiotic Stress Tolerance Priming of Host Plants

combinations altered peroxidase and catalase activities to alleviate the oxidative damage imposed under water stress (Kohler et al. 2008). In Solanum tuberosum, PGPR has been reported to stimulate abiotic stress tolerance by altering the expression of ROS-scavenging enzymes and photosynthetic performance (Gururani et al. 2013). Some PGPR strains, i.e. Burkholderia cepacia SE4, Promicromonospora sp. SE188, and Acinetobacter calcoaceticus SE370, have been evaluated under salinity and drought stress with cucumber plants, and it was observed that these PGPRs improved biomass and chlorophyll contents, increased water potential, decreased electrolytic leakage and sodium ion concentration, and increased potassium and phosphorus under these abiotic stresses. PGPR treatment reduced the harmful activities of catalase, peroxidase, polyphenol oxidase, and total polyphenol. Furthermore, PGPRs increased stress defense phytohormones salicylic acid and gibberellins (Kang et al. 2014). PGPR mitigates drought stress by the process of rhizobacterial-induced drought endurance and resilience, which includes various processes like modifications of phytohormonal levels, bacterial exopolysaccharides, antioxidant defense, and also those which are allied with metabolic adjustments like accumulation of compatible organic solutes like sugars, amino acids, and polyamines. In addition to the above, accumulation of heat shock proteins (HSPs), volatile organic compounds, and dehydrins is also important to make plants tougher against drought stress (Kaushal and Wani 2016). Burkholderia phytofirmans strain PsJN was reported to alleviate drought stress when colonized with wheat (Naveed et al. 2014a) and maize (Naveed et al. 2014b), and in Arabidopsis it mitigates salt stress (Pinedo et al. 2015). PGPRs were also reported to improve barley and oat plant growth by enhancing the level of indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase under salinity stress (Chang et al. 2014). A total increase in biomass and chlorophyll content and decreased level of proline were found in “Micro-Tom” tomato plants colonized with Streptomyces sp. strain PGPA39 (Palaniyandi et al. 2014). The abiotic stress alleviation and growth promotion by PGPR was associated with IAA, salicylic acid, and gibberellin signaling pathways (Bent et al. 2001; James et al. 2002). Thus, PGPRs seem to modulate phytohormone levels and signaling to prime host plants for combating abiotic stresses. In heavy metal stress, plant growth-promoting bacteria (PGPB) have been found to be beneficial in improving plant tolerance (Gamalero et al. 2009). PGPB (Pseudomonas putida, Pseudomonas sp., and Bacillus megaterium) isolated from dry environments have been shown to improve drought tolerance in host plants by producing IAA under drought stress. Further, it was shown that B. megaterium was the most efficient PGPB under drought stress conditions (Marulanda et al. 2009). PGPRs have been reported to harbor ACC deaminases which stimulate the production of stress phytohormone ethylene in host plants under abiotic stresses (Saleem et al. 2007; Bal et al. 2013). In another study in pepper, PGPR Bacillus licheniformis induced stress proteins such as specific genes of Cadhn, VA, sHSP, and CaPR-10 under drought stress. It was also observed that the PGPR alleviated drought stress by overproducing auxin and ACC deaminase (Lim and Kim 2013). In the case of legume–rhizobia symbiosis, any kind of abiotic stress such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, or pesticides, was found to restrain the growth and symbiotic benefits of most rhizobia. Some rhizobia were found to be tolerant to abiotic stresses. Therefore, legume–rhizobium symbiosis could be a solution for improving soil fertility and reclaiming arid soils (Zahran 1999). Abiotic stresses such as drought, nutrient

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deficiency, and aluminum toxicity affect N2 fixation in legume–rhizobium symbiosis, hence there is need to engineer or develop genetic legume breeds for successfully utilizing this symbiosis in agriculture (Valentine et al. 2010). For sustainable agriculture, PGPR, PGPB, and rhizobial symbiosis can be utilized for better productivity in severe environmental conditions in the near future by various means of genetic engineering and high-throughput techniques.

15.3 AM Fungi-mediated Alleviation of Abiotic Stress Tolerance of Vascular Plants Fungal symbioses help plants improve their growth, survival, and health under abiotic stresses in natural ecosystems (Singh et al. 2011). AM association with vascular plants has been reported to have evolved around 500–450 million years ago. Terrestrial plants used AM fungi to their advantage to thrive in a fluctuating environment (Rodriguez et al. 2004; Martin et al. 2016). AM fungi belong to the Glomeromycota division of fungi, and they are widely found in association with various plant roots (Schüβler et al. 2001). AMF help plants by modulating their physiological, biochemical, and molecular status. For instance, trehalose turnover affected by AM symbiosis helps plants tolerate abiotic stress (Ocón et al. 2007). AMF has been specifically found to improve salinity tolerance via various changes in host plants (Porcel et al. 2012; Evelin et al. 2009). Mycorrhizal symbiosis improves the tolerance of diverse plants such as banana and tomato under salinity stress (Yano-Melo et al. 2003; Hajiboland et al. 2010). In lettuce plants, it has been shown that AM symbiosis affects strigolactone production and further improves salt stress tolerance (Aroca et al. 2013). In AM-associated maize plants, salt stress tolerance alleviation was shown to be related to the higher accumulation of soluble sugars in roots (Feng et al. 2002). In Jatropha curcas, biomass production was improved by AM fungi under salinity stress. AM association improved plant growth and leaf relative water content by modulation of lipid peroxidation, solute accumulation (proline and sugars), and photosynthetic pigments (Chl a and b) of Jatropha (Kumar et al. 2010). In the case of Glycine max, association of the AM fungus Glomus etunicatum modulates mineral nutrients (P, K, Zn), proline, and carbohydrate concentrations, and the growth of soybean during salt stress. Further, AM fungus pre-treated with salt stress showed better performance in salt stress alleviation (Sharifi et al. 2007). AMFs have been shown to help water uptake by Lactuca sativa plants under drought stress (Marulanda et al. 2003). AM fungi Glomus coronatum, Glomus constrictum, or Glomus claroideum with B. megaterium increased the drought tolerance of maize plants (Marulanda et al. 2009). Association of AM fungus Glomus intraradices has been shown to stimulate the health and productivity of tomato plants in field conditions with drought stress. It was observed that AM-associated plants efficiently take up nitrogen and phosphate even in drought stress conditions. Moreover, AM-associated plants accumulate higher amounts of ascorbic acid under drought stress (Subramanian et al. 2006). In another study, AM fungi have been demonstrated to alter the glutathione reductase activity in roots and nodules of AM-associated soybean plants, which resulted in decreased oxidative damage to biomolecules and rescued the plants from drought stress (Porcel et al. 2003). In Phaseolus vulgaris, AM symbiosis regulates root hydraulic properties and plasma membrane aquaporins and improves the performance of the host

15.4 Other Beneficial Fungi and their Importance in Abiotic Stress Tolerance Priming of Plants

plant under drought, cold, or salinity stresses (Aroca et al. 2007). Plasma membrane aquaporin genes have been shown to improve tolerance to drought in AM-associated soybean and lettuce plants. AM-associated plants managed drought stress adaptation by downregulating the expression of the PIP genes, whereas non-AM-associated plants failed to do so (Porcel et al. 2006a,b; Jahromi et al. 2008). Another study related to drought tolerance with AM fungus G. etunicatum and Pistacia vera plants showed that P, K, Zn, Cu, N, and Ca contents, soluble sugars, proteins, flavonoid, and proline contents were higher in AM-associated plants. Further, peroxidase enzyme activity was also found to be modulated in AM-associated drought-stressed plants. It was suggested that AM increases the drought tolerance of host plants by modulating the contents of osmolytes and minerals, and antioxidant enzyme activity (Abbaspour et al. 2012). Under water stress, PGPR (Pseudomonas mendocina) with AMF, G. intraradices or Glomus mosseae modulated antioxidant enzyme (superoxide dismutase, catalase, and peroxidase) activities, phosphatase, and nitrate reductase activities, and solute accumulation in L. sativa during water stress. PGPR and G. intraradices also stimulated nitrate reductase and phosphatase activities as well as proline accumulation during drought stress. Furthermore, peroxidase and catalase activities were also shown to be stimulated under drought stress (Kohler et al. 2008). In one study, AMFs were shown to affect photosynthesis, water status, lipid peroxidation, and antioxidant enzyme activity of maize plants for improving their high- and low-temperature stress tolerance (Zhu et al. 2010a,b, 2011). AM fungi also beneficially affected plants under heavy metal stress (Gamalero et al. 2009). In Pisum sativum, AMF G. intraradices association not only improved biomass, but also altered the genes related to Cd detoxification such as metallothionein, 𝛾-glutamylcysteine synthetase, glutathione (GSH) synthetase, homoglutathione synthetase, glutathione reductase, and the phytochelatin precursor GSH. Further, it was also shown that Cd stress dialed down the accumulation of GSH/homoglutathione and increased thiol groups in roots. Furthermore, a heat-shock protein gene, chitinase gene, and a chalcone isomerase gene was also observed to be upregulated, which suggested induction of a heavy metal chelation pathway by AM fungus during Cd stress, leading to tolerance improvement (Rivera-Becerril et al. 2005). Understanding of stress tolerance priming by AMF is narrowed down by limitation with AM symbiosis. Because of the difficulty of axenic culture and genetic engineering, the molecular mechanism beneath biomodulation has not been determined. The various studies on AM-like fungi have paved the way to understanding the possible mechanism of bio-priming by fungal symbionts.

15.4 Other Beneficial Fungi and their Importance in Abiotic Stress Tolerance Priming of Plants Beneficial endophytic fungi other than AMF are also present in the rhizosphere. There are various endophytic and ectophytic symbiotic fungi which have been reportedly shown to affect plant health and performance. For instance, Trichoderma harzianum mitigates drought stress in rice genotypes by upregulating aquaporin, malondialdehyde, and dehydrin genes (Pandey et al. 2016). Application of T. harzianum to Indian mustard (Brassica juncea) provides tolerance against salinity stress and also improves nutrient uptake, enhances osmolyte and antioxidant accumulation, and decreases

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Na+ uptake (Ahmad et al. 2015). The dark septate endophytic fungi (DSEs) are being intensively researched because of their axenic cultivability and capability of colonizing even nonmycorrhizal plants of the Brassicaceae, which also includes model plant Arabidopsis thaliana (Jumpponen and Trappe 1998; Jumpponen 2001; Mandyam and Jumpponen 2005; Rodriguez et al. 2009; Singh et al. 2011). Various studies have shown that the association of other AM-like fungi and DSEs is also involved in the alleviation of abiotic stresses in various plants (Yuan et al. 2010; Newsham 2011). Specifically, the members of the order Sebacinales, mainly Piriformospora indica and Sebacina vermifera, have been shown to improve the abiotic and biotic stress tolerance of their host plants, including monocots, dicots and eudicots (Selosse et al. 2007; Weiß et al. 2011, 2016; Varma et al. 2013). 15.4.1 Piriformospora indica: A Model System for Bio-priming of Host Plants Against Abiotic Stresses Root symbiont P. indica, with its exceptionally broad host range, was discovered in the extreme environment of Thar Desert, India (Verma et al. 1998). Since then P. indica has been shown to be involved in the defense priming of host plants under abiotic as well as biotic stresses (Kumar et al. 2012; Johnson et al. 2014; Gill et al. 2016; Nath et al. 2016). In diverse host plants such as Aloe vera and Medicago truncatula, P. indica inoculation has been shown to elevate salt tolerance (Sharma et al. 2017; Li et al. 2017). At a molecular level, P. indica association has been mainly reported to enhance the salinity stress tolerance of important crop plants such as rice, wheat, and barley (Waller et al. 2005; Baltruschat et al. 2008; Zarea et al. 2012; Jogawat et al. 2013, 2016; Alikhani et al. 2013; Bagheri et al. 2014). For salinity stress tolerance, priming P. indica modulates ROS signaling, antioxidant enzymes activities, lipid peroxidation, osmolyte contents, stress tolerance genes, photosynthetic activity, ER stress signaling, and the growth of host plants (Waller et al. 2005; Baltruschat et al. 2008; Qiang et al. 2012; Jogawat et al. 2013, 2016; Alikhani et al. 2013; Bagheri et al. 2014; Ghabooli 2014; Gahlot et al. 2015; Al-Absi and Al-Ameiri 2015; Abdelaziz et al. 2017; Li et al. 2017). Recently, a P. indica-mediated mechanism of salt stress alleviation has been shown in Arabidopsis, which involve modulation of Na+ /K+ homeostasis (Abdelaziz et al. 2017). In drought stress also, P. indica improved the performance of host plants such as Arabidopsis, maize, barley, Chinese cabbage and other important plants by various modulations at the gene regulation, physiological, and biochemical levels (Sherameti et al. 2008; Vadassery et al. 2009; Sun et al. 2010; Hosseini et al. 2017; Tyagi et al. 2017; Xu et al. 2017; Hussin et al. 2017). In maize plants, P. indica elevates drought stress-related genes and the antioxidant system to improve drought tolerance (Xu et al. 2017). In a proteomic study, Ghabooli and others revealed the molecular mechanisms for alleviating water stress during P. indica–barley interaction (Ghabooli et al., 2013). In the case of heavy metal stress tolerance, P. indica also showed an incredible potential to protect host plants from the toxic effects of heavy metals such as cadmium and arsenic (Hui et al. 2015; Shahabivand et al. 2017; Mohd et al. 2017). In the last 5–10 years, the host plant–P. indica model system revealed a complex mechanism of fungal symbiosis-mediated growth promotion strategies as well as the stress priming of a large range of host plants under abiotic and biotic stresses. Day by day, P. indica is revealing many mysteries beneath root–fungal symbiosis which could not have been possible with AM fungi research.

15.5 Implication of Transgenes from Symbiotic Microorganisms in the Era of Genetic Engineering and Omics

15.4.2 Fungal Endophytes, AM-like Fungi, and Other DSE-mediated Bio-priming of Host Plants for Abiotic Stress Tolerance Various kinds of fungal endophytes from different subdivisions of fungi excluding AMFs can be placed in this section. In many plants, DSEs have shown their role in different kinds of abiotic stresses. In maize plants, DSEs have been shown to alleviate heavy metal stress (Li et al. 2011; Wang et al. 2016, He et al. 2017). A fungal endophyte Epichloë coenophialum improves the health of plants under environmental stresses such as cold, drought, and heavy metals (Arachevaleta et al. 1989; Malinowski and Belesky 2000). Associations of endophytic fungi such as Paecilomyces formosus, Phoma glomerata, and Penicillium minioluteum have been shown to improve plant growth via altering antioxidant activities of flavonoids, glutathione, catalase, peroxidase, and polyphenol oxidase, and phytohormones levels such as abscisic acid, jasmonic acid, salicylic acid, gibberellins, and IAA under salinity and drought stress (Khan et al. 2011, 2012, 2015; Waqas et al. 2012). In a report, it was shown that pretreatment of soybean seeds with endophytic fungus could also be helpful in alleviating salinity stress (Radhakrishnan et al. 2013). Fungal symbionts also modulate plants for tolerating heat stress (Redman et al. 2002; Hubbard et al. 2012). Endophyte Neotyphodium lolii generates nutrient, heavy metal and drought stress tolerance of various species of grass, like Lolium perenne (Ravel et al. 1997; Monnet et al. 2001; Hesse et al. 2003). In grass ecosystems, fungal endophytes are ubiquitious and have been known to alleviate multiple abiotic stresses of grasses and other plants (Bacon 1993; Schardl et al. 2004; Kuldau and Bacon 2008). These grass endophytes secrete bioprotective alkaloids which also protect plants from biotic stresses (Bush et al. 1997). Fungal endophytes have ecological significance and their diversity and abundance change depending on vegetation and ecosystems such as forest trees, grasses, and crop plants (Müller and Krauss 2005; Sieber 2007; Moricca and Ragazzi 2008; Rodriguez et al. 2009; Linaldeddu et al. 2011).

15.5 Implication of Transgenes from Symbiotic Microorganisms in the Era of Genetic Engineering and Omics Plant growth-promoting endophytes such as PGPRs, PGPBs, AMF, AM-like fungi, and DSEs have been of intense interest to plant scientists for sustainable agriculture under abiotic and biotic stresses (Beckers and Conrath 2007; Kapoor et al. 2013; Coleman-Derr and Tringe 2014; Vardharajula et al. 2017). The utilization of supergenes from symbiotic microbes for the generation of stress-tolerant crop plants has already begun, and plant scientists are paving the way for future super crop plants which can thrive in extreme environments merely being affected in their yields. In a study, chitinases of fungal origin have been shown to improve the resistance of transgenic tobacco plants to biotic and abiotic stresses (de las Mercedes Dana et al. 2006). In the era of high-throughput sophisticated research methodologies, researchers have started looking for strategies involving symbiosis. In G. intraradices, an effector protein has been identified which promotes a symbiotic relationship with host plants (Kloppholz et al. 2011). By modulating the soil microbiome, plant health and productivity can be improved (Chaparro et al. 2012). A 14–3–3 protein-encoding gene from AMF G. intraradices has been identified which might regulate drought stress tolerance during this symbiosis (Porcel et al. 2006a,b).

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By using such supergenes, the goal of super crops can be achieved. AM association affects jasmonate biosynthesis in barley, which may affect the abiotic stress tolerance of the host plant (Hause et al. 2002). In recent years, AM-like fungus P. indica has emerged as a potential biotechnological tool for priming of crop plants for abiotic stress tolerance (Oelmüller et al. 2009). From P. indica, a cyclophilin gene PiCypA has been isolated, and its overexpression in tobacco and Brassica has shown improved stress tolerance (Trivedi et al. 2013). In a study, 36 salt tolerance genes have been isolated and identified using the cDNA library of P. indica, overexpressed in Escherichia coli under high salt stress (Gahlot et al. 2015). Such genes from P. indica may be utilized for generating super crops. There is a huge potential for genetic engineering in this field where symbiotic supergenes can be utilized for generating super crops for sustainable agriculture in stressed fields.

15.6 Conclusion and Future Perspectives The symbionts prime host plants in such a manner that they can efficiently not only tolerate abiotic stresses but also perform better under such conditions. All types of symbionts act somewhat similarly. In common, they modulate growth parameters, antioxidant systems, phytohormone levels, osmolyte contents, and the expression of stress signaling genes of host plants under abiotic stress and prime them for better performance under stress environments (Figure 15.1). In the era of growing population and decreasing agriculture land, there is an urgent need to achieve sustainable agriculture. By utilizing the growth-stimulating and defensive nature of plant root symbionts, Abiotic stress Symbiotes

ROS

Antioxidant system H2O H2O2 GSH

Growth Auxin and GA inhibition

ROS/Ca+2 Sensors?

GSSG Growth promotion Related genes

NADP+

NADPH

Abiotic stress signaling Plant Cell ?

? Phytohormones Jasmonic acid SA ABA

Stress responsive Transcription factors Abiotic Stress Tolerance Gene activation

Other substrates Osmolyte biosynthesis and accumulation

Figure 15.1 Overview of symbiosis associated priming of a plant cell for abiotic stress tolerance. Upon abiotic stress, symbionts make changes in the cells of host plants in such a way that they will be primed for abiotic stress defense readiness. Symbionts intervene with reactive oxygen species signaling, calcium signaling, phytohormone levels, and their signaling. Thus by doing so, symbionts rescue the growth diminution of host plants owing to abiotic stress and also protect them from toxic effects of abiotic stress such as oxidative damage.

References

crop yield losses caused by abiotic as well as biotic stress can be reduced to some extent. The immense potential of symbionts is yet to be utilized efficiently. It is very important to select, screen, and apply such endosymbionts to crops, so that they can help to overcome the productivity limitations under different abiotic stress conditions. By combining the high-throughput techniques and genetic engineering, super crops with efficient symbiosis with super organisms may be generated in the near future which can tolerate severe environmental conditions with minimum loss of crop yields.

Acknowledgements AJ acknowledges the financial support from SERB-National post doctoral scheme, the Government of India.

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16 Insight into the Molecular Interaction Between Leguminous Plants and Rhizobia Under Abiotic Stress Sumanti Gupta 1,* and Sampa Das 2,* 1 2

Department of Botany, Rabindra Mahavidyalaya, Champadanga, Tarakeswar, Hooghly, 712401, West Bengal, India Division of Plant Biology, Bose Institute, P1/12, CIT Scheme, VIIM, Kolkata 700054, West Bengal, India

16.1 Introduction 16.1.1

Why is Legume–Rhizobium Interaction Under the Scientific Scanner?

Is rapid urbanization becoming more of a bane than a boon? The answer to this question is given by two parallel schools of thought with no common point of conjunction. However, one inevitable consequence of urbanization is population pressure that is leading to a perpetual decrease in the share of fertile farm lands, causing food shortages. The only promising solution put forward by plant researchers and breeders is to increase the production of sustainable high-yielding crops that can make up for the shortfall in food for the increasing population (Abdelrahman et al. 2017). Abiotic and biotic stresses are the main factors of environmental stress and cause severe damage to plant health at every level, including morphological, biochemical, and molecular, and thus ultimately reduce the quality and yield (Atkinson and Urwin 2012; Zhu 2016; Gimenez et al. 2018). In order to protect crops from various abiotic and biotic stresses, the use of chemical fertilizers is on the increase. However, the random use of chemicals has raised severe concerns about safeguarding health and ecosystems (Irfan et al. 2017). Under this scenario, biofertilizers have emerged as safe and eco-friendly alternatives. The need for nitrogen fertilizers is great, as nitrogen is one of the most important biomolecules required for plant growth and development (Chang et al. 2009). Although atmospheric nitrogen content amounts to nearly 78%, the usable amount is limited, since plants and other organisms lack the ability to convert molecular nitrogen to an assimilable form. Thus, agriculture has been reliant on the use of industrial nitrogen fertilizers that account for approximately 50% of fossil fuel used for their production. Moreover, not only does combustion of fossil fuel increase the release of greenhouse gases but leaching of nitrogen fertilizers also leads to the eutrophication of waterways (Crutzen et al. 2007). Thus, reducing the dependence on chemical fertilizers and exploring inexpensive and safe alternative nitrogen inputs is urgently needed. Production of biological nitrogen fertilizers solely depends on the process of biological nitrogen fixation. Few diazotrophic archaebacteria and eubacteria have the unique * Corresponding authors

Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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capacity to convert molecular nitrogen into ammonia that can be readily absorbed by the plants. These 𝛼-proteobacteria belonging to Rhizobiaceae phylogenetic clade possess a nitrogenase enzyme complex that help them fix atmospheric nitrogen. However, these rhizobia (including the genera Azorhizobium, Allorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium) fix atmospheric nitrogen by entering into a symbiotic relationship with plants belonging to the family of Leguminosae where they provide the plant with absorbable nitrogen in exchange for energy obtained from the plant photosynthates (Ferguson et al. 2010). Members of the Leguminosae family include important crop legumes that are a rich source of food and biofuel. They are the third largest group of angiosperms and the second largest group of food crops following cereals (Gepts et al. 2005). They are capable of fixing approximately 200 million tons of nitrogen annually (Peoples et al. 2009). Their ability to fix nitrogen symbiotically makes the legumes advantageous over any other plant species. Legumes are referred to as “green manure” and used widely in agronomic practices during crop rotation (Talgre et al. 2012). Meticulous and methodical understanding of the nitrogen fixation process is needed for developing better performing green manures for the future. However, how these manure legumes deal with ever-changing environmental factors is not clearly understood. In addition, understanding of many aspects of symbiotic interaction, especially the perception of Nod factors (NFs) by the symbiotic host legume, rhizobial behavior in planta, the host metabolic signaling during symbiosis, and interaction of the symbiotic host with other attacking pathogens during abiotic stress situations, has received very little attention. All of the above aspects shall be dealt in brief in the following sections of the chapter.

16.2 Legume–Rhizobium Interaction Chemistry: A Brief Overview The symbiotic interaction between host legume and Rhizobium is unique and intricate, and takes place following several sequential molecular events. This section will discuss the major events that take place during symbiotic association (Figure 16.1). 16.2.1

Nodule Structure and Formation: The Sequential Events

Nodule formation is initiated when the host legumes release phenolic isoflavonoids in the rhizosphere that are recognized by a specific set of rhizobacteria. Although perception of host phenolic compounds by rhizobia is believed to be grossly specific, some nonspecific interactions involving a broad range of host legumes are reported as well (Pueppke and Broughton 1999). Following recognition of host isoflavonoids, the bacterial NodD proteins are activated, resulting in the induction of nod genes (nodulation genes) that synthesize lipochitooligosaccharide-containing NFs (Peck et al. 2006). NFs are sensed by the host plant, starting the process of symbiosis. The apical region of the emerging root hair is the initial target for rhizobial infection. The thinner cell wall of the nascent root tip helps rapid rearrangement of the cell cytoskeleton, thus allowing easy penetration of the infecting rhizobia. Attachment of rhizobia with root hairs initiates root hair deformation followed by cortical cell divisions (Mathews et al. 1989). Rhizobia enter the host through the deformed root hair that encapsulates a few differentiating bacteria. These encapsulated microcolonies contain large quantities of NFs and

16.2 Legume–Rhizobium Interaction Chemistry: A Brief Overview

JA

KAPP1 /KAPP2

AON Pathway in Leaf phloem parenchyma

SD1

LRR-RLK

ABOVE GROUND 2

Specific rhizobia perceive root secreted flavonoids.

UNDER GROUND

1 Roots secrete flavonoids

LysM-RLK

4 Root hair deformation and curling LRR-RLK Initial anticlinal cortical cell divisions

HMGR

Xylem transport

SIP1

6

Calcium spiking

Nuclear

5

CCaMK/CYCLOPS CEKBEKUS KPG EKF1 NIN

NSP1/NSP2 ERN1

ENOD expression Ethylene, JA, SA, ABA, ROS

CK1

EPIDERMAL CELL Infection thread progression in outer cortex and then in inner cortex; formation of nodule primordia

3

Nucleoporins

Bacterial infectien Cation channels Periclinal cell divisions and formation of infection thread in root interior

Rhizobia released NFs are recognized by root hair LysM RLK/ epidermal LRR RLKs

Q-CLE jung distance signal

Cytosol HK

SIGNALING

7 NSP1/NSP2

Rhizibia invasion in nodule primorida, bacteroid differentiation Nodule maturation and formation of NITROGEN FIXING NODULES

Nuclears

8

NIN

ENOD expression

AXU, GA, BR ABA

CELL DIVISION

9

Cytosol

CORTICAL CELL

Figure 16.1 Integrated schematic diagram showing the molecular events occurring during nodule formation and nitrogen fixation. Source: Adapted and modified from Ferguson et al. 2010. The sequential steps indicate the events of nodule formation and nitrogen fixation. The networks within epidermal and cortical cells highlight the early signaling taking place inside the cell while the sequential steps occur in synchrony. The interconnection of the autoregulation of nodulation pathway with cell division is also shown. The red dots indicate the steps that are likely to be modified in the presence of stress-causing agents.

cell wall-degrading enzymes. These enzymes cause degradation of host cell wall leading to increased turgor pressure and resulting in forward movement of the plant-derived infection thread filled with dividing rhizobia (Gage 2004). The entering and proliferating rhizobia goes on constantly producing NFs that lead to mitotic divisions of cortical cells, eventually forming the nodule primordium. The radial position of the primordium

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is controlled by the positional gradients of ethylene close to the xylem radial cells and far from the phloem (Lohar et al. 2009). The infecting bacteria are endocytosed from the infection thread to the cell cytoplasm where they are surrounded by a plant-derived membrane, i.e. the “peribacteroid membrane,” finally resulting in a “symbiosome.” Inside the symbiosome, the rhizobial cells differentiate into nitrogen-fixing “bacteroids” (Udvardi and Day 1997). The mature nodule is confined to four zones, i.e. meristematic zone, infection zone, nitrogen fixation zone, and senescent zone. Nitrogen is converted to ammonia which enters the host via synthesis of glutamine and glutamate by glutamine synthase and glutamate synthase, respectively (Roth and Stacey 1989). Well-regulated exchange of nitrogen byproducts takes place from the symbiosomes to the host interior and photosynthates in the form of malate from the host interior to symbiosomes across the peribacteroid membrane (Udvardi and Day 1997). The key enzyme responsible for nitrogen fixation is nitrogenase, which requires a low-oxygen environment for its optimum functionality within the nitrogen-fixing zones of the nodule. On the other hand, the microaerobic environment of the nodule is taken care of by a special oxygen-binding protein, the leghaemoglobin (Downie 2005). Leghaemoglobin acts in regulating the production of respiratory energy (16ATP) that helps in providing the driving force for nitrogenase needed to convert molecular nitrogen to ammonia (Jones et al. 2007). Two major types of nodules are formed in legumes—determinate and indeterminate. They differ mainly in the presence of a persistent meristematic region in case of indeterminate forms. The site of initial cell division is the inner root cortex in the case of indeterminate nodules, while it is the outer or middle cortex for determinate ones. The shape is cylindrical and branched in the case of indeterminate ones, while it is spherical for determinate ones. The number of bacteroids is one per symbiosome and of relatively low viability for indeterminate nodule, whereas multiple bacteroids per symbiosome with high viability are found in determinate forms. Indeterminate forms occur in temperate zones in species such as Medicago, clovers, and pea, while determinate ones predominate in subtropical and tropical regions in species like soybean, bean, Lotus, and Pongamia (Ferguson et al. 2010) (Figure 16.1). However, how the diversity in structure of nodules across different geographic locations and different species is controlled remains to be identified. 16.2.2

Nod Factor Signaling: From Perception to Nodule Inception

Nod factor perception by the root hair cells is believed to be the first crucial phenomenon of symbiotic interaction. The current model predicts the presence of two receptor-like kinases (RLKs) located on root cell epidermis that bind to the NF. These receptors have been identified from Lotus, Pisum, Medicago, and soybean (Limpens et al. 2003; Arrighi et al. 2006; Indrasumunar et al. 2009). These NF receptors contain intracellular kinase domain, transmembrane domain, and an extracellular LysM domain that binds to extracellular NFs which resemble the peptidoglycan-like chemical constituents of any bacteria (Steen et al. 2003). Another RLK located in the plasma membrane of the infection thread as found in Medicago sativa contains extracellular leucine-rich repeat (LRR) and intracellular serine/threonine kinase domain (Limpens et al. 2003). Studies indicate that LysM RLK has a role in the Nod factor signaling cascade, whereas LRR RLK has a role in initiating bacterial infection events (Limpens et al. 2003).

16.2 Legume–Rhizobium Interaction Chemistry: A Brief Overview

Following NF binding to the above receptors, rapid influx of calcium ions takes place coupled with depolarization of chlorine and potassium ions in root hairs. Calcium oscillations known as “calcium spiking” are associated with the activation of ion channel proteins and nucleoporins. Calcium spiking is perceived downstream by a calciumcalmodulin-dependent protein kinase (CCaMK). The root hair actin cytoskeletal structure starts to alter (Oldroyd and Downie 2004). These phenomenal changes are externally manifested by root hair deformation and root curling. CcaMK activates several transcription factors (TFs) downstream such as nodulation signaling pathway 1 (NSP1) (Smit et al. 2005), NSP2 (Kaló et al. 2005), Ets2 repressor factor required for nodulation (Middleton et al. 2007), and nodule inception (NIN) (Borisov et al. 2003), all of which activate the early nodulation genes (ENOD) in the root epidermis. Studies on Lotus sp. indicated the involvement of LjCYCLOPS in assisting CCaMK to activate NSP1 (Smit et al. 2005). In parallel, another TF, namely SyM interacting protein (SIP1), was found to bind to the promoter region of NIN and regulate bacterial infection in Lotus sp. In addition, U box protein CERBERUS and TF ethylene response factor 1 (ERF1) were also found to localize in the epidermal nucleus and regulate bacterial infection (Zhu et al. 2008; Asamizu et al. 2008). Nodule organogenesis is achieved by the synchronization of signals across several layers in depth in planta. The perception signal of NF on epidermis is instantly transduced to cortical regions reported by rapid cytoskeletal rearrangements in the pericycle (Ferguson et al. 2005). Calcium spiking and activation of downstream CCaMK/ CYCLOPS in epidermis trigger the generation of a mobile messenger, the cytokinin that is sensed by the cytokinin receptor. The receptor has a histidine kinase domain and is located on the cortical cell membrane. Activation of cytokinin receptor leads to the induction of the same set of TFs, such as NSP1, NSP2, and NIN, which help in the positive expression of ENOD genes and control further cell division in the inner layers of the cortex, thus producing nodule primordium (Murray et al. 2007) (Figure 16.1). 16.2.3 Reactive Oxygen Species: The Crucial Role of the Mobile Signal in Nodulation Generation of reactive oxygen species (ROS) is an inevitable event in the metabolic life of any organism as well as byproduct of any interaction. It has roles from destroying to guarding individual cells under specific circumstances. ROS include singlet oxygen (1 O2 ), superoxide anion (O2− ), hydrogen peroxide (H2 O2 ), and hydroxyl radical (OH⋅ ) (Das and Roychoudhury 2014; Banerjee and Roychoudhury 2017). These molecules, being toxic, can destroy the primary constituents of the cell if accumulated in large quantities. However, they also act as secondary messengers and transmit developmental signals or prime the host during stressful conditions (Pitzschke et al. 2006; Anjum et al. 2015). Interestingly, ROS have been found to have an important role during nodule formation and NF signaling. ROS help in root hair curling and formation of an infection thread by strengthening the cross walls by oxidative crosslinking (Peleg-Grossman et al. 2007). In the semiaquatic legume Sesbania rostrata, where the Rhizobium enters through the natural fissures of the root, ROS help in nodule initiation (D’Haeze et al. 2003). ROS efflux and NADPH oxidase transcripts were found to be downregulated in Medicago truncatula during early interaction of NFs with the host, suggesting

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a differential mechanism unlike plant pathogen interaction that perhaps aids the entry of the foreign bacteria into the host legume. (Lohar et al. 2007). ROS accumulation was almost absent in the nitrogen-fixing zones of the nodule. On the other hand senescent zones showed a strong presence of ROS molecules. Lower content of leghemoglobin is marked by low accumulation of ROS, indicating leghemoglobin to be a candidate for oxidative damage (Gunther et al. 2007). In addition, antioxidant machinery was found to be active in mature nodules. Glutathione and homoglutathione were found to control nodule number and nodulin expression (Muglia et al. 2008). Thioredoxin also controls nodule numbers and regulates endosymbiosis of the rhizobia (Lee et al. 2005). Ascorbate peroxidase helps in the detoxification of ROS by creating an oxygen barrier and regulating the function of nitrogenase in mature nodules (Gunther et al. 2007). Taken together, all of the above findings indicate a special spatiotemporal regulation of ROS during nodule formation, nitrogen fixation, and senescence of nodules. Even then, many gaps still remain in the understanding of the role of ROS during symbiosis that shall be filled after detailed experimentation in the future (Figure 16.1). 16.2.4

Phytohormones: Key Players on All Occasions

Phytohormones are important molecules that regulate metabolism and stress. Auxin is believed to be the most important hormone regulating root development. Since nodulation events take place in roots, it is obvious that auxin has a role in the modulation of symbiosis. Local disruption of polar auxin transport (PAT) was found to be associated with the emergence of the primordial nodule in Medicago. In addition, PAT was also found to be linked with the development of indeterminate nodules (Subramanian et al. 2006). Cytokinin was found to be the key molecule required for initiating nodule organogenesis by controlling endosymbiosis, but its role in progression of the infection thread was elusive (Murray et al. 2007). Studies also indicated a link between auxin and the cytokinin regulation pathway, as PAT was modified in the presence of cytokinin in Medicago (Plet et al. 2011). However, the exact connection between the two still remains unsolved in symbiotic situations. Ethylene, the abundant hormone in legume roots, is referred to as the negative regulator of nodulation, as it represses nodule organogenesis and arrests the growth of the infection thread (Guinel and Geil 2002). In addition, ethylene was found to control nodule number and positioning (Ding and Oldroyd 2009). In S. rostrata, ethylene in combination with gibberellins and H2 O2 regulates the hypersensitive response in infection pockets and promotes crack entry of rhizobia (Groth et al. 2010). Similar to ethylene, abscisic acid also negatively regulates nodule development (Ding and Oldroyd 2009) while brassinosteroids positively regulate nodulation (Ferguson et al. 2005). Jasmonic acid is believed to have a role in regulating the autoinhibition of nodulation through the NARK pathway (nitrate reductase metabolic pathway) (Miyahara et al. 2008) (Figure 16.1). 16.2.5

Autoregulation of Nodulation: The Self Control from Within

Many external and internal factors regulate the autoregulation of nodulation (AON) pathway involving long-distance signaling from root to shoot and vice versa. AON is initiated during nodule development by a root-derived signal “Q” that has been identified as CLAVATA/ESR-related CLE peptide (Okamoto et al. 2009). Q travels to the

16.3 Role of Abiotic Stress Factors in Influencing Symbiotic Relations of Legumes

shoot via xylem after inoculation with rhizobia, where it is perceived by AON LRR RLK having a serine/threonine kinase domain. Studies have identified several downstream components of AON signaling such as kinase-associated protein phosphatases, KAPP1 and KAPP2, regulating the NARK signal transduction pathway of Glycine max (Miyahara et al. 2008). The Q signal perception leads to the production of novel shoot-derived inhibitor that travels from the shoot via phloem and reaches the root where it blocks the signals regulating mitotic divisions of cell. Shoot-derived inhibitor depends on the NARK pathway for its biosynthesis (Lin et al. 2009). Interestingly, another Q molecule similar to rhizobia-derived CLE peptides has been identified in root tissue in response to upregulation of the expression of nitrates. The nitrate-induced Q only differs from the rhizobia-derived Q in their inability to travel long distances (Okamoto et al. 2009). However, the AON pathway and its regulation need further investigation for more precise conclusions (Figure 16.1).

16.3 Role of Abiotic Stress Factors in Influencing Symbiotic Relations of Legumes Any biotic interaction involves the close association of two partners under specified environmental conditions. However, any slight change in the external atmosphere is likely to bring alterations in the behavioral pattern of both of the interactors. This section will emphasize the behavioral changes that are caused in root-colonizing rhizobia as well as the colonized legume host under abiotic stress situations such as drought, salinity, temperature stress, pH imbalance, heavy metal stress, and nutrient depletion. It will also shed light on the defense strategic mechanisms opted for by the symbiotic host when dealing with pathogenic organisms and during the presence of abiotic stress agents. 16.3.1 How Do Abiotic Stress Factors Alter Rhizobial Behavior During Symbiotic Association? Drought stress has been reported to induce the accumulation of chaperonins and heat shock proteins along with enzymes regulating the energy metabolism in the rhizobial partner of M. truncatula, while the host showed a decline in the amount of nitrogen-fixing enzymes (Larrainzar et al. 2009). Alteration in temperature (both low and high) brings about alteration in soil conditions as well as the behavior of soilinhabiting microorganism. In addition, soil temperature variations result in reduced populations of rhizobia, thus affecting the nodule infection process. In addition, low temperature ranges were also reported to affect the bacteroid differentiation in Rhizobium leguminosarum bv trifolii. On the contrary, high temperature affects the survival and persistence of rhizobia in the rhizosphere (Andrés et al. 2012). Saline soils also affect the growth and development of the rhizospheric microorganism as a whole. Changes in cell morphology and structure of extracellular polysaccharide (EPS) and lipopolysaccharide (LPS) are found to be associated with salinity in rhizobia (Ventorino et al. 2012). Growth of Sinorhizobium sp. was directly affected under saline conditions, probably by induced oxidative stress situations, leading to an imbalance of ROS (Arora et al. 2006). Optimum pH ranging from neutral to slightly acidic is effective from the normal growth of rhizobia. Low pH was reported to affect nodC gene expression in

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peanut microsymbiont (Angelini et al. 2003). pH alters the EPS and LPS morphology of the rhizobium (Vriezen et al. 2007). Accumulation of heavy metals in the soil as a result of indiscriminate use of chemical fertilizers causes toxicity to the microsymbiont. Nickel increases hydrogenase activity in bacteroids (El Hilali 2006). Rhizobium is reported to have developed a unique heavy metal-sequestering mechanism by producing huge amounts of EPS and LPS. Additionally, forming complexes with metal ions, transforming toxic forms to less toxic ones, methylation, precipitation, and chelations with S-rich metallothioneins and glutathione are the other metal-detoxifying mechanisms adopted by the microsymbiont (Gusmao et al. 2006). In addition, rhizobia also secrete proteins that adhere to metals and store them in periplasmic space, keeping the important cytoplasmic zones free for vital metabolic reactions (Pajuelo et al. 2011). 16.3.2

Abiotic Agents Modulate Symbiotic Signals of Host Legumes

Studies on the root nodule proteome of M. truncatula during drought stress have shown downregulation of enzymes involved in symbiotic nitrogen fixation as well as nitrate assimilation (Larrainzar et al. 2009). Nodule-specific methionine synthase isoform and sulfur metabolism were found to be linked to nodule development during water-deficit conditions. Ethylene biosynthetic pathways were also found to be downregulated (Larrainzar et al. 2014). On the other hand, lipoxygenase pathway-modulating lipid metabolism and jasmonate pathway biosynthetic genes were found to be abundant in nodules during drought-stressed conditions. The main aim during the water-deficit condition was probably to allocate maximum reserves in osmolyte production and carbon partitioning from starch to sugar in order to maintain the “stay green” state of the phenotype (Staudinger et al. 2016). Under low-temperature, low-pH and saline conditions, the NF perception of the legume host was altered, which was examined in G. max during infection with Bradyrhizobium japonicum. The plants exposed to the stress agents showed root hair deformation that was distinctly different from that of wild plants. On addition of external Nod factors, the effects of chilling stress and low pH stress were completely alleviated. However, plants previously exposed to salinity stress showed no effects (Duzan et al. 2004). High temperature causes a drastic decrease in the amount of nitrogen fixation and carbon fixation rates, probably owing to the retarded nitrogenase activity (Aranjuelo et al. 2007). Salinity affects the oxygen diffusion barrier that leads to a decline in nitrogenase activity on the one hand, while on the other hand, nodule carbon metabolism is affected by the inhibition of sucrose synthase (Ben Salah et al. 2009). Soil pH is a factor that is altered with salinity and drought, as essential nutrients such as calcium, magnesium, phosphorus, and molybdenum become limiting in dry acidic soil. This brings about nutrient depletion, thus affecting the growth of symbiotic hosts. In addition, acidic soils accumulate large amounts of heavy metals such as aluminum and manganese, which become toxic for plants and rhizobia as well. G. max and Medicago have unique capacities to lower the soil pH and regulate the net proton flow during symbiosis. However, those lacking such low cation exchange properties showed reduced quantities of fixed nitrogen (Andrés et al. 2012). In recent times, studies have revealed the role of micro ribonucleic acids in regulating the abiotic stress during symbiosis (Mantri et al. 2013). However, the process of unearthing the facts relating to authentic conclusions has only just begun.

16.4 Conclusion: The Lessons Unlearnt

16.3.3 Abiotic Stress Agents as Regulators of Defense Signals of Symbiotic Hosts During Interaction with Other Pathogens Studies indicated that the features of the early stages of interaction for both symbiotic and pathogenic organisms show a great degree of overlap by activating similar sets of plant defense responses. However, at later stages, the defense responses are found to be downregulated in the case of symbiosis (Zamioudis and Pieterse 2012). Apart from providing abiotic stress tolerance, symbiotic association is also believed to prime the legume host against biotic stress factors. However, research involving mixed inoculants (both symbiotic and pathogenic) is scarce. Studies on M. truncatula co-inoculated with Sinorhizobium meliloti and pathogenic oomycota Aphanomyces euteiches showed relatively less induction of pathogen-specific calmodulin 2, antioxidant defense responses, pathogen response proteins, Kunitz proteinase inhibitors, lectin, and proteins related to carbohydrate metabolism, compared with plants inoculated with only pathogenic oomycota A. euteiches (Schenkluhn et al. 2010). During symbiosis in Pisum sativum, the microsymbiont significantly increased the levels of proteins involved in the phytoalexin pisatin pathway upon attack with pathogenic fungi Didymella pinodes (Desalegn et al. 2016). Subsequent work with microsymbiont and pathogen in P. sativum revealed superimposition of genotypical traits linked with symbiosis and/or pathogenesis, indicating a regulatory link between the contrasting molecular responses (Turetschek et al. 2016). Complementary studies have also reported the presence of legume R genes (resistant genes) in recognition of symbionts (Samac and Graham 2007). However, this juvenile field of study needs lot more experimental analyses to attain maturity.

16.4 Conclusion: The Lessons Unlearnt Legume–rhizobium interaction is important and interesting as it reveals of one of nature’s matchless interchemistries. The above sections explained some important aspects of the interaction, but in the process unearthed several queries the drive future research on nodulation biology go ahead. Some of thes questions are: 1. Is the rationale of genetic diversity between rhizobial strains infecting a single species of legume host plant explained? Genetic knowhow regarding the “pangenome” of rhizobial species, the “core genes” as well as the “accessory genes” of a particular strain or individual bacteria is insufficient. Comparative genomics and transcriptomics help in the identification of genomic linkage groups and regulons that contribute to symbiotic processes (Galardini et al. 2011). Thus, in order to make the nitrogen fixation process more efficient, better understanding of the genetic setup of the rhizobial strains and their contribution toward the symbiotic fixation process is necessary. 2. ROS act as mobile messenger transmitting signals, but can the contrasting role of ROS as regulators of HR (hypersensitive response) during pathogen invasion on the one hand and inducers of symbiotic root curling on other hand be explained during simultaneous pathogen attack and symbiosis? 3. Ethylene is known as a stress hormone. How can the virtual borderline role of ethylene between symbiosis and defense during rhizobial entry be delineated? 4. How is the AON pathway functional during abiotic stress situations?

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5. Are the gross generalizations made taking in to consideration the studies conducted with model legumes like Medicago sp. and Lotus sp. applicable for all crop legumes and their symbiotic rhizobial partners? Instead more case studies involving crop legume hosts and their symbiotic bacterial partners should be performed. 6. Is it possible to predict the fine tuning (alterations of biotic and abiotic features) of the underground rhizospheric microenvironment under laboratory conditions? The answer today is possibly “no.” However, such a negative affirmation will probably be contradicted by more scientific studies where statistically significant models of rhizospheric microenvironment shall emerge. Thus, if meticulously explored, the study of symbiosis could not only lead to the invention of better performing biofertilizers applicable in future agronomic practices, but also be able to create an immense number of next-generation nonlegume crop plants with nitrogenfixing capabilities. The ever-growing field of bioinformatics and bioengineering designing innovative biological tools should make the task much easier in future.

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17 Effect of Nanoparticles on Oxidative Damage and Antioxidant Defense System in Plants Savita Sharma 1 , Vivek K. Singh 2 , Anil Kumar 1 , and Sharada Mallubhotla 1* 1 2

School of Biotechnology, Shri Mata Vaishno Devi University, Katra, 182320, J&K, India School of Physics, Shri Mata Vaishno Devi University, Katra, 182320, J&K, India

17.1 Introduction Nanotechnology is the science and technology of small items, in particular, items that are less than 100 nm in size. One nanometer is 10−9 m or about three atoms long. For comparison, a human hair is about 60–80 000 nm wide. Scientists have discovered that materials at small dimensions, small particles, thin films, etc., can have significantly different properties than the same materials at a larger scale. There are thus endless possibilities for improved devices, structures, and materials if we can understand these differences, and learn how to control the assembly of small structures. Nanotechnology is often described as an emerging technology, one that not only holds promise for society, but is also capable of revolutionizing our approaches to common problems. The value of nanoparticles (NPs) in many technological areas is very high because of their versatile properties. Today some NPs are already being used commercially. For example, some companies are using TiO2 NPs in sunscreen lotions because they provide transparency to a sunscreen, and are believed to be less toxic than the organic molecules currently used as UV absorbers in many sunscreen formulations. These particles are also found in sporting equipment, clothing, and telecommunications infrastructure. The future of nanotechnology is boundless, and some of the items that exist today were a topic of science fiction a decade ago and have the potential to transform our society very quickly. The term nanotechnology was coined by Taniguchi, a researcher at the University of Tokyo, Japan. NPs exhibit completely new or improved properties based on specific characteristics such as size, distribution, and morphology (Murphy et al. 2005). Nanoparticles fall into three major groups: natural, incidental, and engineered. Naturally occurring nanomaterials such as volcanic ash, ocean spray, magnetotactic bacteria, mineral composites, and others exist in our environment. Incidental NPs, also referred to as waste particles, are produced as a result of some industrial processes. The third category of NPs is engineered NPs (ENPs); these are particles associated with nanotechnology. ENPs are subclassified by the type of basic material and/or use into metals, semiconductors, metal oxides, nanoclays, nanotubules, and quantum dots. Within each category * Corresponding author

Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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the shapes, sizes, and surface coatings further determine the structure and function of these molecules. Each such material has been specifically designed for a function, such as fullerene C60 , which is used for fuel cell applications. Very little is known about how the ENPs interact with the environment. Nanotechnology has direct beneficial applications for medicine and the environment, but like all technologies it may have unintended effects that can adversely impact the environment, both within the human body and the natural ecosystem. While taking advantage of this new technology for health, environmental, and sustainability benefits, scientists still need to examine its environmental and health implications. For the interaction of biological systems with their external and internal environment for survival, growth and reproduction; the ENPs have varied technological applications in industrial, medical, and agricultural products. The considerable quantity of ENPs released into the ecosystem has brought about serious environmental concerns owing to their possible toxic effects in living organisms, particularly in plants (Rico et al. 2011; Miralles et al. 2012). They can induce modifications in the physiological and biochemical processes of plants that may have implications on their growth and seed production. This concern has brought about fast growing research interest in plant-ENP interactions. Plants are often exposed to environmental stresses in their life cycle. These stresses lead to the production of reactive oxygen species (ROS) in plant tissues. When the level of ROS exceeds the defense mechanisms, a cell is said to be in a state of “oxidative stress.” Plants actively produce ROS as signaling molecules to control processes such as programmed cell death, pathogen defense and abiotic stress response, and these ROS are unavoidable byproducts of the oxygenic photosynthesis. Oxidative stress arises from an imbalance in the generation and utilization of ROS. However, despite their potential for causing harmful oxidation, it is now well established that ROS are also powerful signaling molecules that are involved in the control of plant growth and development as well as priming acclimatory responses to stress stimuli (Foyer and Noctor 2009). The ability of a plant to overcome the effect of the oxidative stress and to sustain its productivity may be related to the scavenging of stress-induced toxic oxygen species, such as superoxide radical (O2⋅ − ), perhydroxy radical (HOO⋅), hydrogen peroxide, hydroxyl radical (⋅OH), peroxy radical (ROO⋅), and singlet oxygen (O2⋅ ) (Krieger-Liszkay 2005). Because of the multifunctional roles of ROS, it is necessary for cells to control their level tightly to avoid any oxidative injury so as they do not completely eliminated. Plants, however, possess an impressive array of defense mechanisms against oxidative stress, including enzymatic and nonenzymatic antioxidant systems distributed in cell organelles (Rio et al. 1998). Superoxide dismutase (SOD), catalase, peroxidase, and the ascorbate glutathione cycle enzymes are examples of enzymatic antioxidative defense systems while nonenzymatic antioxidants include water-soluble (ascorbate, glutathione, phenolic compounds, flavonoids) and lipid-soluble (tocopherol, carotenoids, lycopene) metabolites. Biochemical studies have shown that ENPs influence antioxidative enzyme activity (Navarro et al. 2012; Song et al. 2012; Zhao et al. 2012), photosynthetic processes (Perreault et al. 2010; Mohammed et al. 2011), oxidative stress (Begum et al. 2011; Wang et al. 2011; Oukarroum et al. 2012), and DNA expression (Kumari et al. 2011; Landa et al. 2012) in plants. However, further studies are still needed to draw a comprehensive picture of plant-ENP interactions at the biochemical or molecular level. The new era drugs are NPs of polymers, metals, or ceramics, which can combat conditions like cancer and fight human pathogens

17.2 Engineered Nanoparticles in the Environment

like bacteria (Kshirsagar et al. 2006; Sharon et al. 2007a,b). Thus it is of great interest to synthesize the materials from naturally occurring fibrous materials (Jagadale et al. 2007; Sharon et al. 2007a,b). Biosynthesized nanomaterials have been effectively controlling various endemic diseases with lesser adverse effects. Plants contain abundant natural compounds such as alkaloids, flavonoids, saponins, steroids, tannins, and other nutritional compounds. These natural products are derived from various parts of the plant, such as leaves, stems, roots, shoots, flowers, bark, and seeds. Recently, many studies have proved that the plant extracts act as a potential precursor for the synthesis of nanomaterials in nonhazardous ways. Since the plant extract contains various secondary metabolites, it acts as a reducing and stabilizing agent via various bio-reduction reactions to synthesize novel metallic NPs. Nonbiological methods (chemical and physical) are used in the synthesis of NPs, and demonstrate a serious hazard and high toxicity for living organisms. In addition, the biological synthesis of metallic NPs is inexpensive, involves a single step and is an eco-friendly method; plants have been used successfully in the synthesis of various NPs via green chemical reactions.

17.2 Engineered Nanoparticles in the Environment The increasing use of ENPs in industrial and household applications will very likely lead to the release of such materials into the environment. Although the number of commercial and manufactured products containing NPs is growing and novel NPs are continually being developed, only a few materials are currently used in a large number of products or in high volume. Therefore, only a small subset of nanomaterials are currently being released or will likely be released into the environment in coming decades. These include silver, titanium dioxide, zinc oxide, silica, and carbon based nanomaterials (single walled carbon nanotubes (SWCNTs), multiwalled carbon nanotubes (MWCNTs) and fullerenes). These nanomaterials are the main focus of studies within the current eco-nanotoxicology literature. It should be noted that this list of materials is not exhaustive and other materials in the future may be released in high volumes. There are various entry points for engineered nanomaterials into the environment, including direct application to an environmental compartment (either intentionally or through unintentional product degradation), and waste-water treatment plant effluent and sludge (Mueller and Nowack 2008; Gottschalk et al. 2010). As on date, it is difficult to estimate the relevant concentrations of NPs that will be released at any given time. Some of the difficulty in predicting concentrations is the result of limited data on current and its future prevalence in commercial products (Klaine et al. 2008; Batley et al. 2012). Additionally, transformations of nanomaterials, such as dissolution, agglomeration, sedimentation or change of surface moieties; could greatly affect the pathway and extent of environmental release of such materials. A number of risk assessment efforts have been made to model and calculate predicted environmental concentrations of NPs with the current understanding of NP transformations and fate (Mueller and Nowack 2008; Gottschalk et al. 2010; Praetorium et al. 2012), along with some experimental approaches to examine NP fate under natural conditions (Kiser et al. 2009; Westerhuff et al. 2011). It is clear that real-time measurement of NP is important for advancements capable of dealing with complex matrices, a large NP concentration range and evolving primary NP characteristics.

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17.3 Nanoparticle Transformations From the literature, it is clear that NPs are transformed from their original, synthesized state no matter the type, amount, or pathway. Bulk production of NPs often leads to their indiscriminate release in nature through industrial wastewaters (Brunner et al. 2006; Owen and Handy 2007). Thus soil and water contamination with NPs has become an important environmental issue. Transformations are the result of myriad processes, including aggregation/agglomeration, redox reactions, dissolution, exchange of surface moieties, and reactions with bio-macromolecules. These dynamic transformations in turn affect the transport, fate and toxicity of NPs in the environment, making it critical to understand and characterize these transformations (Maurer et al. 2009). Trends in NP aggregation, surface molecule transformations and speciation/dissolution under environmental conditions will be the focus in the near future. The size of NPs is an important determinant of reactivity, transport and toxicity. While toxicology studies commonly characterize the primary particle size, typically using electron microscopy, NPs tend to interact with environmental systems as aggregates. Light scattering techniques are most commonly employed to study the stability of NPs in solution or as an aerosol. While systematic studies of aggregation of ENPs in soil have not been completed, one technique used to study NP aggregates absorbed onto soil particles is scanning electron microscopy (Kim et al. 2012). Within solutions, there are some notable aggregation trends observed no matter what type of nanomaterial is present. Besides ionic strength, the presence of natural organic matter (NOM) and other biomacromolecules (e.g. extracellular polymeric substances) plays a key role in determining the aggregation state of NPs. NOM is a ubiquitous and poorly defined, component of environmental systems consisting of high-molecular-weight humic and fulvic acids resulting from plant and animal material decomposition that readily absorb onto the highly reactive surface of nanomaterials (Chowdhury et al. 2013). Other molecules, like extracellular polymeric substances (i.e. bacterial secretion containing polysaccharides and proteins), cause an increase in NP aggregation rate, while, still other molecules like cysteine, a component of proteins and NOM, cause an initial increase in the aggregation rate but not in the long-term aggregate size. Understanding aggregation is critical for characterizing the transport of NPs through environmental compartments, for example, less aggregation yields lower rates of sedimentation and greater mobility. In addition, understanding the interaction of NPs under natural conditions (e.g. salinities, pH, molecular species) enables a better assessment of exposure and transport. These aggregation studies bring to light the importance of the NP surface and localized environment around that surface for the transformation of the material. As described above, NOM is of particular importance in this respect because of its pervasiveness throughout the environment. While it clearly plays a role in the aggregation dynamics, NOM is itself dynamic, with an undefined molecular structure and a variety of reactive moieties. Therefore, its adsorption onto NP surfaces may aid transformations beyond aggregation, such as surface reduction, where NOM can reduce ionic metals at an NP surface to increase NP size. Other molecules at the NP surface, such as fatty acids (Rudolph et al. 2012), can also influence NP transformation. In addition to organic molecules (NOM, proteins, carbohydrates), potentially toxic metal ions also have the ability to adsorb onto the NP surface, increasing the transport and toxicity effects of

17.3 Nanoparticle Transformations

metal atoms but also prompting the use of NPs in remediation of potentially toxic metal pollutants. Beyond adsorption of molecules/atoms, the transformation of the nanomaterial itself into other species plays a role in the toxicity assessment. For example, the dissolution of silver NPs (Ag(0) to Ag+ ), is responsible for the antimicrobial nature of Ag NPs, (Xiu et al. 2012). Understanding this speciation, studied primarily using atomic spectroscopy, is important for assessing eco-nanotoxicity. However, the surface of Ag NPs, in addition to surface adsorption of NOM and other macromolecules, is susceptible to reaction with oxygen and sulfur atoms, making it unlikely that Ag(0) is the primary species at the NP surface (Levard et al. 2012). Likewise, free dissolved Ag+ is unlikely to be present in large concentrations, as many naturally occurring compounds have a propensity to complex Ag+ . Scientists working in this field must resist over simplifying conclusions regarding speciation as it will certainly invalidate translation of their results into real, complex environments. Other NPs using metal ions (e.g. Au, ZnO, and CuO) (Mudunkotuwa and Grassian 2011) experience similar speciation either in the dissolution to ions or chemical reactions that, in turn, could affect other physicochemical changes in the NPs and ultimately NP fate and toxicity. Greater attention to NP speciation is necessary within the nanotoxicity literature in order to identify nanospecific toxicity. The importance of the NP state for subsequent transport and toxicity, is complicated by the interplay between different dynamic NP transformations (Figure 17.1) that necessitates careful, time-dependent in situ characterization of NPs in environmentally relevant conditions. In plants NPs interact with them and this results in uptake and accumulation that affect their fate and transport in the ecosystem. Moreover, they could remain attached to the plant surface and impart physical and chemical damage to the plant organs. Usually they enter through the lateral root junction into the root system and reach the xylem through the cortex and the pericycle (Dietz and Herth 2011). Owing to the specific size of the cell wall, entry of NPs into the plant can be stopped by the cell wall (Fleischer et al. 1999). However, the NPs that have a size range within the cell wall pore size could effectively cross the cell wall and reach the plasma membrane (Navarro et al. 2008). The rate of entry depends on the size and surface properties of NPs and indeed, the smaller NPs can enter plant cells easily. In contrast, larger NPs, being unable to enter the cells, cannot affect the cell metabolic pathways (Verano et al. 2014). Larger NPs can only penetrate through the hydathodes, flower stigmas, and stomata. The mechanism of interaction between NPs and plants could be chemical or physical. Chemical interactions involve the production of ROS, disturbance of ion cell membrane transport activity (Auffan et al. 2008), oxidative damage (Foley et al. 2002), and lipid Figure 17.1 Illustration of the dynamic transformations that nanoparticles undergo in the body or the environment (black arrows) and the interplay (gray arrows) between these transformations. Source: adapted with permission from Jones et al. (2013), American Chemical Society.

surface reactions

sorption NOM

O2

M+ M+ M + aggregation

salinity/pH

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peroxidation (Kamat et al. 2000). Following entry into the plant cells, NPs after mixing behave as metal ions and react with sulfhydryl and carboxyl groups and ultimately alter the protein activity. However, while conducting an engineered nanomaterials (ENMs) mediated ecotoxicity studies, much attention needs to be paid to various artifacts which often lead to misinterpretations of results (Petersen et al. 2014). These potential factors include toxic impurities in ENMs, their proper storage and dispersion in testing medium. Moreover, ENMs exert indirect toxicity which affects plant growth and development through nutrient depletion with the passage of time and estimation of ENM dispersal in organisms. In addition, ENMs face different changes (viz. settling, dissolution, agglomeration, etc.) during the exposure period, which is difficult to measure accurately. Owing to their increased surface area and properties, ENMs readily adsorb organic molecules and inorganic ions from the nutrient medium, resulting in indirect toxicity symptoms including chlorosis and wilting. Moreover, during ENM exposure, organic acids in plant root exudates decrease the pH of the media, thus altering nutrient supply and ENM properties (Marschner 1995). Inefficient exploration of the influence of these factors can imply an inappropriate explanation of phytotoxicity and ultimately an incorrect assessment of the impact of ENMs (Petersen et al. 2014).

17.4 Plant Response to Nanoparticle Stress Plant growth and development are controlled by internal regulators that respond to environmental conditions. In nature, plants seldom find optimal quantities of essential factors required for maximal growth and productivity. Therefore, most often, the “physiologically normal type of plant” is an exception in nature. Under suboptimal environmental conditions, plants show perturbations in their biochemical and physiological processes. Redox reactions, involving electron exchange, are one of the most vulnerable physico-chemical processes affected by fluctuations in the external environment. Therefore, even under seemingly favorable environmental conditions, a plant continuously produces ROS. Accumulation of these ROS is potentially harmful for the growth and development of plants. Thus, occurrence of oxidative stress, through the generation of free radicals, is an inevitable by-product of normal plant metabolism (Sharma et al. 2012). Generally, these ROS are continuously reduced and detoxified by an extensive antioxidant system. However, the detoxification process consumes essential cellular resources in terms of energy, carbon skeleton, and loss of nitrogen as NH3 . Therefore, any treatment that can help reduce the ROS would prove beneficial in improving the overall plant growth and productivity. It has been shown that plants produce natural mineralized NPs, which are required for growth (Wang et al. 1999). Specific reports are now available on the biosynthesis of such NPs by plants. However, ectopic use of ENPs is one of the most recent advances in the field of agriculture biotechnology. Studies indicate that treatment of plants with a mixture of nano SiO2 and TiO2 can enhance the activities of specific enzymes and could be used for improving seed germination and seedling growth. It has been suggested that nano-SiO2 treatment could be related to increased strength, resistance to disease, and thus, increased yield in rice. However, the mode of action for these NPs has not yet been established. Metal NPs, by virtue of having extremely large surface area to volume ratios and an ability to engineer electron exchange, can develop favorable interactions with various biomolecules in a cell. Silver

17.4 Plant Response to Nanoparticle Stress

NPs are among the most likely candidates for modulating the redox status of plants, because of their ability to support electron exchange with Fe2 + and Co3 + . The increasing application of NPs is directly related to their release in the environment and plants are particularly relevant in consideration of eco-nanotoxicity based on their interaction with air, soil and water, all of which may contain ENPs (Melissa et al. 2013). NPs with different compositions, sizes, and concentration physical/chemical properties have been reported to influence the growth and development of various plant species with both positive and negative effects. It was reported that MWCNTs markedly influenced tomato seed germination and seedling growth by upregulating stress-related gene expression (Khodakovskaya et al. 2009). In Arabidopsis, Al2 O3 NPs were reported to be least toxic as compared with zinc oxide, iron oxide, and silicon oxide NPs (Lee et al. 2010). A previous studies highlighted the toxic effects of NPs on algae (Sadiq et al. 2011). NPs like titanium oxide, zinc oxide, cerium oxide, and silver NPs were deposited on the surface of the cell as well as in the organelles, which resulted in oxidative stress to the cell through the induction of oxidative stress signaling (Buzea et al. 2007). In Cucurbita pepo, the effect of silver, copper (Cu), zinc oxide, and silicon NPs indicated that seed germination was unaffected by these NPs and their counterpart bulk materials; however, Cu NPs reduced root length compared with the control and plants treated with bulk Cu powder (Stampoulis et al. 2009). In rice, ZnO NPs, but not titanium oxide caused deleterious effects on the root length at early growth stages. It was indicated that the root growth of Triticum aestivum was affected by different concentrations of the alumina NPs (Madvar et al. 2012); however, NPs did not affect the seed germination, shoot length and dry biomass. In rice seedlings, nano-CuO treatment led to an increase in activity of antioxidant enzymes and elevated Malondialdehyde (MDA) concentration (Shaw and Hossain 2013). A similar experiment on the nano-CuO modulated photosynthetic performance and antioxidative defense system in Hordeum vulgare demonstrated restriction in root and shoot growth with decreased photosynthetic performance index (Shaw et al. 2014). Moreover, nano-CuO mediated DNA damage and plant growth restriction were reported in radish (Raphanus sativus) and ryegrass (Lolium perenne, Lolium rigidum) (Atha et al. 2012). Changes in enzyme activities, ascorbate and free thiol levels resulting in higher membrane damage and photosynthetic stress have been documented in shoots of germinating rice seedlings on exposure to very high concentrations of cerium oxide NPs (Rico et al. 2013). Generation of ROS and reactive nitrogen species and H2 O2 upon exposure to Ag and ZnO ENPs in duckweed (Spirodela punctuta) suggest the toxicity of Ag and ZnO NPs predominantly was caused by both the particulates and ionic forms (Thwata et al. 2013). Among the various metal NPs, much attention has been paid to Ag NPs owing to their characteristic physiochemical and biological properties compared with the massive bulk material (Sharma et al. 2009). The Ag NPs have broad applications as an essential component in various products like household, food and industry because of their bactericidal and fungicidal properties (Tran et al. 2013). Compared with silver-based compounds, Ag NPs, with increased surface area available for microbe interaction, are reported to be more toxic to bacteria, fungi, and viruses. Like other metal ions, Ag NPs can also induce oxidative stress in bacteria, animals, and algae as well as higher plants (Jiang et al. 2012). However, the impact of Ag NPs on plants largely depends on various factors such as plant species, growth stage of plant, composition and concentration of the NPs, and the experimental setup (temperature, treatment period, media composition, method of exposure, etc). Nano silver is one of the most

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extensively studied NPs and the toxicology has been examined in various crops (Kumari et al. 2011; Jiang et al. 2012). Although exposure of Ag NPs is reported to be detrimental for plant growth, some studies have demonstrated the growth enhancing properties of Ag-NPs in Brassica juncea (Sharma et al. 2012), Eruca sativa, wetland plants (Yin et al. 2012), Phaseolus vulgaris and Zea mays (Salama 2012). An investigative study revealed the chromotoxic effects of Ag NPs on mitotic cell division in root-tip cells of Allium cepa (Kumari et al. 2011). Moreover, Ag NPs interact with the membrane proteins and activate signaling pathways, which leads to inhibition of cell proliferation (Gopinath et al. 2010). Perusal of all of these nanotoxicity studies over the past decade reveals that plant response to NPs stress has been evaluated extensively in various crops, largely at physiological and biochemical levels. Rather, less focus has been given to the study of the plant NPs interface at transcript level. Microarray based gene expression analysis of Arabidopsis thaliana roots on exposure to ZnO NPs, TiO2 NPs, and fullerene soot indicates that the underlying mechanisms of phytotoxicity are highly specific to the NP (Landa et al. 2012). An advanced method by amalgamating genetic, photothermal, and photoacoustic strategies for highly sensitive detection of NPs in different parts of tomato plants, most importantly the reproductive organs has been designed (Khodakovskaya et al. 2009). Total gene expression analysis of tomato leaves and roots exposed to carbon nanotubes (CNTs) revealed upregulation in the stress and water channel related genes. A separate study demonstrated selective root growth in maize upon exposure to single walled carbon nanotubes (SWCNTs). Transcriptional analysis suggests that nanoparticle root cell interaction selectively modulates gene expression in seminal roots, thus affecting relative root growth and development. Similar to transcriptome analysis, only limited numbers of studies have emphasized the effects of NPs stress on plants at proteome level. Owing to the growing commercial interest in nanomaterials, modest research efforts have been invested in evaluating the potential adverse effects of these ENMs. The sheer multiplicity of the physico-chemical parameters of nanomaterials such as size, shape, structure, and elemental constituents, makes the investigation of nanoparticle mediated toxicity include oxidative stress, inflammation, genetic damage, inhibition of cell division and cell death (Stone et al. 2007; Nam and Lead 2008). Most work to date has suggested that ROS generation (which can be either protective or harmful during biological interactions) and consequent oxidative stress are frequently observed with NM toxicity (Nel et al. 2006). The physicochemical characterization of NPs including particle size, surface charge, and chemical composition is a key indicator for the resulting ROS response and nanoparticle induced injury since many of these nanoparticle intrinsic properties can catalyze the ROS production. Nanoparticle mediated ROS responses have been reported to orchestrate a series of pathological events such as genotoxicity, inflammation, fibrosis, and carcinogenesis. For instance, CNT-induced oxidative stress triggers cell signaling pathways, resulting in increased expression of pro-inflammatory and fibrotic cytokines (Li et al. 2010). Some NPs have been shown to activate inflammatory cells such as macrophages and neutrophils which can result in the increased production of ROS (Zhang et al. 2003). Other NPs such as titanium dioxide (TiO2 ), zinc oxide (ZnO), cerium oxide (CeO2 ), and silver NPs have been shown to deposit on the cellular surface or inside the subcellular organelles and

17.5 Generation of Reactive Oxygen Species (ROS)

induce oxidative stress signaling cascades that eventually result in oxidative stress to the cell (Buzea et al. 2007). The mechanism for ROS generation is different for each NP and to date the exact underlying cellular mechanism for ROS generation is incompletely understood and remains to be elucidated. Most of the metal-based NPs elicit free radical-mediated toxicity via Fenton-type reactions, whereas mitochondrial damage plays a major role in CNT-mediated ROS generation. However, it is inaccurate to assume that ROS generation is a prerequisite to NP-induced toxicity since a few studies have reported the direct toxicity of NPs without causing ROS (Wang et al. 2010). Nevertheless, ROS generation is a major event during nanoparticle induced injury that needs to be thoroughly characterized in order to predict nanoparticle induced toxicity. In some plants studied, high concentration of ENPs decreased the oxidative stress by increasing the antioxidant defense system of the plant (Cyren et al. 2013).

17.5 Generation of Reactive Oxygen Species (ROS) The chemical reactivity of a molecule is dependent upon the conformation of electrons on the outer shell. This conformation determines the ease with which the molecule can accept or donate one or more electrons. When a molecule has an unpaired or odd number of electrons in its atomic structure, it is referred to as a free radical, which is relatively unstable and, therefore, very reactive. ROS, key signaling molecules during cell signaling and homeostasis, are reactive species of molecular oxygen. ROS constitute a pool of oxidative species including superoxide anion (O2 − ), hydroxyl radical (OH⋅), hydrogen peroxide (H2 O2 ), singlet oxygen (1 O2 ), and hypochlorous acid (HOCl). ROS are generated intrinsically or extrinsically within the cell. In contrast to atmospheric oxygen, ROS are capable of unrestricted oxidation of various cellular components and can lead to the oxidative destruction of the cell (Mittler 2002). When a single electron is added to O2 , it becomes the superoxide molecule (O2 ), which is a free radical with an unpaired electron. Other molecules, such as hydrogen peroxide (H2 O2 ), are not necessarily free radicals, but are certainly very reactive and H2 O2 is formed when the superoxide radical accepts another electron and two hydrogen ions (2H+ ). A combination of H2 O2 with O2 − results in the formation of the hydroxyl (OH) radical, the most toxic free radical in biological systems (Figure 17.2). Some of the endogenous sources of ROS include mitochondrial respiration, inflammatory response, microsomes, and peroxisomes, while ENPs, environmental pollutants, act as exogenous ROS inducers. Physiologically, ROS are produced in trace amounts in response to various stimuli. Free radicals occur as essential byproducts of mitochondrial respiration and transition metal ion-catalyzed Fenton-type reactions. Inflammatory phagocytes such as neutrophils and macrophages induce oxidative outburst as a defense mechanism toward environmental pollutants, tumor cells, and microbes. A variety of nanoparticle including metal oxide particles induce ROS as one of the principal mechanisms of cytotoxicity (Risom et al. 2005). NPs have been reported to influence intracellular calcium concentrations, activate transcription factors, and modulate cytokine production via generation of free radicals (Li et al. 2010; Huang et al. 2010).

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·OH Fenton reaction (Fe, Cu) & Haber–Weiss reaction Electron transport chain in mitochondria and chloroplast

O2

e NAD(P)H oxidase (membrane); xanthine oxidase (cytosol); and others

e– H+

·HO2 hydroperoxyl

e–

H2O2 hydrogen peroxide

SOD e H+

·O–2 Superoxide

CAT, APX, GSH, peroxidase

H 2O

Figure 17.2 Metabolic pathway of reactive oxygen species in plants. Source: adapted with permission from Praduman et al. 2014.

17.6 Nanoparticle Induced Oxidative Stress An abundance of ROS can have potentially damaging biological responses resulting in oxidative stress phenomena. It results from an imbalance between the production of ROS and a biological system’s ability to readily detoxify the reactive intermediates or repair the resulting damage. To overcome the excess ROS response, cells can activate enzymatic and non enzymatic antioxidant systems (Sies 1991). The hierarchical model of oxidative stress was proposed to illustrate a mechanism for NP-mediated oxidative stress (Li et al. 2008; Huang et al. 2010). According to this model, cells and tissues respond to increasing levels of oxidative stress via antioxidant enzyme systems upon nanoparticle exposure. During conditions of mild oxidative stress, transcriptional activation of phase II antioxidant enzymes occurs Nuclear factor erythroid 2 related factor 2 (Nrf2) induction. At an intermediate level, redox-sensitive mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain enhancer of activated B cells (NF-𝜅B) cascades mount a pro-inflammatory response. However, extremely toxic levels of oxidative stress result in mitochondrial membrane damage and electron chain dysfunction, leading to cell death. Some of the key factors favoring the prooxidant effects of engineered nanomaterials include either the depletion of antioxidants or the increased production of ROS. Perturbation of the normal redox state contributes to peroxide and free radical production that has adverse effects on cell components including proteins, lipids, and DNA (Huang et al. 2010). Given its chemical reactivity, oxidative stress can amount to DNA damage, lipid peroxidation, and activation of signaling

17.6 Nanoparticle Induced Oxidative Stress

networks associated with loss of cell growth, fibrosis, and carcinogenesis (Knaapen et al. 2004; Buzea et al. 2007). Besides cellular damage, ROS can result from interactions of NPs with several biological targets as an effect of cell respiration, metabolism, ischemia/reperfusion, inflammation, and metabolism of various nanomaterials. Most significantly, the oxidative stresses resulting from occupational nanoparticle exposures as well as experimental challenge with various NPs lead to airway inflammation and interstitial fibrosis (Donaldson et al. 2004). NPs of varying chemical composition such as fullerenes, CNT, and metal oxides have been shown to induce oxidative stress. The key factors involved in nanoparticle induced ROS include: (i) prooxidant functional groups on the reactive surface of NPs; (ii) active redox cycling on the surface of NPs owing to transition metal-based NPs; and (iii) particle cell interactions (Knaapen et al. 2004). From a mechanistic point of view, we discuss the sources of ROS based on the physicochemical parameters and particle–cell interactions. Several studies demonstrate the significance of the reactive particle surface in ROS generation (Vallyathan and Shi 1997). Free radicals are generated from the surface of NP when both the oxidants and free radicals are bound to the particle surface. Surface-bound radicals such as SiO and SiO2 present on quartz particles are responsible for the formation of ROS such as OH and O2 (Knaapen et al. 2004). Ambient matter such as ozone and nitrogen dioxide (NO2 ) adsorbed on the particle surface is capable of inducing oxidative damage (Buzea et al. 2007). Reduced particle size results in structural defects and altered electronic properties on the particle surface, creating reactive groups on the NP surface. Within these reactive sites, the electron donor or acceptor active sites interact with molecular O2 to form O2 , which in turn can generate additional ROS via Fenton-type reactions (Nel et al. 2006). For instance, NPs such as Si and Zn with identical particle size and shape lead to diverse cytotoxicity responses owing to their surface properties. ZnO, being more chemically active than SiO2 , leads to increased O2 formation, resulting in oxidative stress. Free radicals are either directly bound to the NP surface or may be generated as free entities in an aqueous suspension. Dissolution of NP and subsequent release of metal ions can enhance the ROS response. For instance, aqueous suspensions of quartz particles generate H2 O2 , OH⋅, and O2 (Vallyathan and Shi 1997). Apart from surface-dependent properties, metals and chemical compounds on the NP surface accelerate the ROS response. Transition metals including iron (Fe), copper (Cu), chromium (Cr), vanadium (V), and silica (Si) are involved in ROS generation via mechanisms such as Haber–Weiss and Fenton-type reactions. Fenton reactions usually involve a transition metal ion that reacts with H2 O2 to yield OH⋅ and an oxidized metal ion. For example, the reduction of H2 O2 with ferrous iron (Fe2 + ) results in the formation of OH, which is extremely reactive and toxic to biological molecules. Cu and Fe metal NP have been reported to induce oxidative stress (O2 and OH) via Fenton-type reaction, while the Haber–Weiss-type reaction involves a reaction between oxidized metal ion and H2 O2 to induce OH⋅ (Thannickal and Fanburg 2000). NP including chromium, cobalt, and vanadium can catalyze both Fenton and Haber–Weiss-type reactions (Volko et al. 2006). Glutathione reductase, an antioxidant enzyme, reduces metal NP into intermediates that potentiate the ROS response. In addition, some metal nanoparticle (Ar, Be, Co, and Ni) promote the activation of intercellular radical-inducing systems such as the MAPK and NF-𝜅B pathways (Smith et al. 2001). The prooxidant effect of NPs, ROS are also induced endogenously where the mitochondrion is a major cell target for NP-induced oxidative stress. Once NPs

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gain access to the mitochondria, they stimulate ROS via impaired electron transport chain, structural damage, activation of the nicotinamide adenine dinucleotide phosphate (NADPH)-like enzyme system, and depolarization of the mitochondrial membrane. For instance, cationic polystyrene nanospheres induce O2 mediated apoptosis in murine macrophages based on their ability to target mitochondria (Xia et al. 2006). Cellular internalization of nanoparticle has been shown to activate immune cells including macrophages and neutrophils, contributing to ROS/RNS (Risom et al. 2005). This process usually involves the activation of NADPH oxidase enzymes. In vivo particle exposures such as silica activate the rich pool of inflammatory phagocytes within the lung, causing them to induce oxidative outburst. NPs with smaller particle size are reported to induce higher ROS, owing to their unique characteristics such as high surface to volume ratio and high surface charge. Particle size determines the number of reactive groups/sites on the NP surface (Stone et al. 1998). The pulmonary responses induced by inhaled NP are considered to be greater than those produced by micron-sized particles because of the increased surface area to particle mass ratio (Donaldson et al. 2010). Larger surface area ensures that the majority of the molecules are exposed to the surface rather than the interior of the nanomaterials (Nel et al. 2006). Accordingly, nano-sized SiO2 and TiO2 and MWCNT induce greater numbers of ROS as compared with their larger counterparts. Additionally, a study with cobalt/chromium NP exposure demonstrated particle size-dependent ROS-mediated genotoxicity (Raghunathan et al. 2013). Findings from several studies have pointed out that ROS generation and oxidative stress occur as an early event leading to nanoparticle induced injury. Oxidative stress corresponds with the physicochemical reactivity of NPs including metal-based particles as well as the fibrous CNT. Oxidative stress related to NPs exposure involves mitochondrial respiration, mitochondrial apoptosis, activation of the NADPH oxidase system, alteration of calcium homeostasis, and depletion of antioxidant enzymes, all of which are associated with tissue injury. NP-driven ROS response contributes to activation of cell signaling pathways, inflammatory cytokine and chemokine expressions, and specific transcription factor activation. Activation of these cellular mechanisms is closely associated with the transcription of genes involved in inflammation, genotoxicity, fibrosis, and cancer. Thus, the pathological consequences observed during nanoparticle exposure could be attributable to ROS generation. It is essential to incorporate these adverse biological responses as a screening tool for the toxic effects of NPs. For instance, over expression of antioxidant enzymes is indicative of the mild oxidative stress, whereas mitochondrial apoptosis occurs during conditions of toxic oxidative stress. The hierarchical model of ROS response provides a scale to gauge the adverse health effects upon nanoparticle exposure. A nanoparticle exposure study must collectively involve rigorous characterization of NPs and assign in vitro and in vivo oxidative stress markers as toxicity end points as a predictive paradigm for risk assessment (Li et al. 2008; Shvedova et al. 2012).

17.7 Antioxidant Defense System in Plants Plants possess very efficient scavenging systems of enzymatic and nonenzymatic antioxidative defense systems that can protect cell from oxidative damage. The antioxidant enzymes include SOD, peroxidase, catalase (CAT), ascorbate peroxidase

17.8 Conclusion

Table 17.1 Reactive oxygen species (ROS) scavenging system in plants. Scavenging system

Localization

Primary ROS

Superoxide dismutase

Chl, Cyt, Mit, Per, Apo

O2 –

Ascorbate peroxidase

Chl, Cyt, Mit, Per, Apo

H2 O2

Catalase

Per

H2 O 2

Glutathione peroxidase

Cyt

H2 O2 , ROOH

Peroxidases

CW, Cyt, Vac

H2 O2

Thioredoxin peroxidase

Chl, Cyt, Mit

H2 O 2

Ascorbic acid

Chl, Cyt, Mit, Per, Apo

H2 O2 , O2 −

Glutathione

Chl, Cyt, Mit, Per, Apo

H2 O2

Tocopherol

Membranes

ROOH, 1 O2

Chl

1

Caretenoids

O2

Source: Le et al. (2016).

(APX) and glutathione reductase, while the nonenzymatic antioxidants include water-soluble (ascorbate, glutathione and flavonoids) and lipid-soluble (-tocopherol, -carotene) metabolites. In plant cells, specific ROS-producing and scavenging systems are found in different organelles such as chloroplasts, mitochondria, and peroxisomes (Table 17.1). ROS-scavenging pathways from different cellular compartments are coordinated (Mittler, 2002) and three classes of SOD have so far been reported based on the metal co-factor. These are dinuclear Cu/Zn-SOD, mononuclear Fe-SOD, and Mn-SOD (Hassan and Scandalios 1990). The Cu/Zn-SOD has a central role in scavenging toxic oxygen radicals. It is the major form in leaves and is responsible for 65–80% of the total activity (Halliwell 1987). Mn-SOD is localized in mitochondria, whereas Fe-SOD is localized in chloroplasts. Cu/Zn-SOD is present in three isoforms, which are found in the cytosol, chloroplast, and peroxisome and mitochondria. In Triticum aestivum the activities of key antioxidant enzymes including, SOD, CAT, and APX were analyzed in the presence of alumina NPs and it was reported that increases in the concentration of NP increased the SOD activities. CAT enzyme activity was significantly decreased with increase concentrations of NPs whereas APX activity significantly decreased in the presence of NPs (Madvar et al. 2012). In B. juncea it was demonstrated that the presence of silver NPs in the growth media can improve the growth of seedlings by improving its antioxidant status, and optimized use of silver NPs can modulate oxidative stress (Sharma et al. 2012). The free radical scavenging property of silver NPs was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and it was showed that, with increasing concentration of synthesized silver NPs, the percentage of inhibition increases. Thus synthesized silver NPs offer effective antioxidant protection from free radicals.

17.8 Conclusion Most of the studies reported to date have focused on the effect of NPs on seed germination and few describe the biotransformation of these particles in food crops. The possible

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transmission of these into the next generation of plants exposed to NPs is unknown. Therefore, it is an urgent requirement to further elucidate the effects of NPs in plants in order to characterize their uptake, phytotoxicity, and accumulation. As this is an innovative and scientific growth area with exponential production, more information is needed concerning the impacts of these NPs in the environment, particularly in plant performance. Improper handling and disposal of nanoparticle containing wastes could result in environmental contamination, harmful effects on plants and plant-associated soil. At sublethal concentrations, NPs variably modify the production of bacterial secondary metabolites involved in plant growth and productivity. The negative effects of NPs on plant development and metabolism depend on the size, concentration, and chemistry of NPs. In addition, prolonged NP treatments trigger excess formation of ROS, resulting in severe oxidative burst. The ROS unbalances the cellular redox system in favour of oxidized forms, resulting in oxidative damage to cellular components – lipids, proteins, and nucleic acids. Moreover, ROS-like singlet oxygen directly or by means of secondary radicals attacks photosynthetic apparatus component proteins such as D1 protein of photosystem II (PSII) and causes its degradation. To scavenge and neutralize these toxic radicals, plants have evolved complex antioxidant defense mechanism comprising both enzymatic and nonenzymatic networks. Among the various metal oxide NPs, nano-TiO2 is by far the most well-studied NP whose toxicity has been tested in different crop systems. Antioxidant enzymes have long been considered as the first line of defense against ROS generation; however, their actions need to be complemented by that of other ROS scavenging systems during severe stress conditions. These scavengers may either inhibit the generation or reduce the level of ROS once they are formed. Prolonged NP treatment triggers oxidative burst and nano-stress-induced changes in antioxidant enzymes and these activities strongly indicate disruption of ROS/antioxidant balance. Although ROS are proposed to be responsible for the negative effects of inhaled NPs, the toxicity mechanism of the NPs in plants has not yet been clearly understood. However, it is probable that the production of ROS is responsible for inducing nanotoxicity. Antioxidant enzymes are involved in scavenging of the ROS and these enzymes are more active in the presence of NPs; hence, in order to determine the probability mechanism of nanotoxicity, the activity of several antioxidant enzymes should be assessed. Nanoparticle treatment induces the activities of specific antioxidant enzymes, resulting in reduced ROS levels. Moreover, higher concentrations of NPs in the environment are attributed to the higher activities of antioxidant enzymes in plants, which protect them from oxidative damage.

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18 Marker-assisted Selection for Abiotic Stress Tolerance in Crop Plants Saikat Gantait 1,2 , Sutanu Sarkar 1,2 , and Sandeep Kumar Verma 3 1 Crop Research Unit, Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal 741252, India 2 Department of Genetics and Plant Breeding, Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal 741252, India 3 Institute of Biological Science, SAGE University, Kailod Kartal, Indore, Madhya Pradesh 452020, India

18.1 Introduction Plant breeding of modern-era could be considered both as a science and as an art. There are numerous techniques which have been exercised since medieval times that modify the traits of interest so as to develop preferred characteristics in plants. “Selection,” differentiation amid divergence, is the ancient approach to the advancement of plants. The knowledge of inheritances transformed the selection procedure, removing speculation, simplifying it and formulating it more effectively. Recent plant breeders act in accordance with the typical techniques for the induction of variation, selection from the variable population, and eventually growing cultivars to be available for the growers (Acquaah 2012). Over time, plant breeding has experienced a paradigm shift in approach in terms of the use of cutting-edge scientific technologies that are sustainable and evolving day by day. Plant breeding performs a vital role in enhancing the production of crops but its efficiency is challenged by the hostile and unpredictable variation in climate/environment as well as numerous abiotic and biotic stress factors, such as insects, pests, pathogens, drought, salinity, and excess of moisture, cold, heat, etc. With respect to the present crop production patterns and increasing populations in combination with the antagonistic influence of a changing climate, the selection of commercial and viable characteristics such as stable biotic and abiotic stress tolerance, in addition to effective nutrient and water utilization, ought to be the key interest (Mackill et al. 1999; Slafer et al. 2005). In the past few decades, exhaustive efforts have been made to enhance various grain and pulse cultivars under adverse agro-climatic conditions. Nonetheless, the existence of stress (abiotic/biotic), variation in climatic conditions, enormous growth in the human population, and diminishing environmental reserves, particularly water resources for farming, have obstructed plant breeding scientists from evolving superior germplasms having greater production efficiency (Lateef 2015). To counter such impediments, there is a demand for innovative tools, involving marker-assisted selection (MAS) united with a proficient and explicit phenotyping approach to augment crop Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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yield and productivity to a greater extent. Molecular breeding tools such as MAS offer a strategy for quicker progress involving precise selection of plant genotypes with the ability to withstand unfavorable situations.

18.2 Reaction of Plants to Abiotic Stress Owing to flexibility in growth and development based on physiology and cellular metabolism, sessile plants are well adapted to divergent and alterable edaphic or climatic situations. A variable environment that negatively influences cellular homeostasis and eventually damages normal plant health and growth could be referred to as abiotic stress, which involves excess of water or its insufficiency, toxicity of alluminum/ chlorine/cadmium/ferrous/sodium ions, ferric/nitrogen/phosphorus/sulfur/zinc ion scarcity, temperature thresholds, and tropospheric ozone. The phases of stress are usually categorized as chronic or transient stress. The stress associated with initial vegetative phases decelerates plant cell division but may not significantly decrease overall crop yield, while the stress associated with reproductive phases may substantially reduce yield (Mansouri-Far et al. 2010). Abiotic stresses frequently arise in combination (for example, drought and heat or ozone and heat) or in series (such as flooding followed by drought); hence, sustainability of yield under inconstant conditions necessitates improvement in several mechanisms. Crop yield will increase if losses triggered by abiotic stress are curtailed (Mickelbart et al. 2015). Plants are immobile organism and they do not have the systems to evade unfavorable environments. However, as a result of successive evolutionary changes, the genesis of distinctive and complex reactions to abiotic stress has occurred. Upon exposure to abiotic stress, plants immediately respond with the reception of a particular signal following a string of incidents that result in such feedback. Then modifications in expression of genes and concurrent metabolic alterations induce signald to produce explicit and organized reactions inside the plant (Shao et al. 2007; Agrawal et al. 2010). Such arrays of transformations signify the exertion of this immobile organism to prevail over the hostile condition and adapt under stress (Altman 2003). Endurance under adverse conditions encourages any organism to evolve with systems of evasion, resistance, and/or tolerance. Plants that acquire the ability to withstand a certain condition can, over time, survive such conditions without damage. As an example, Anastatica hierochuntica and numerous species of the genus Selaginella are termed ‘resurrection plants’ since they have the ability to survive and recover from prolonged intervals of moisture stress. Another system of resilience to desiccation is osmolyte accretion and modifications in the metabolic process (Bouchabke et al. 2008). For the induction of tolerance in plants to specific adverse environmental conditions there are several mechanisms adopted by plants in terms of defensive approaches. For instance, the life cycle of plant is sped up at the onset of moisture stress and such acceleration in developmental stages induces early flowering. This mechanism can be considered as an excellent strategy that plants can adopt spontaneously. Several cereals, pulses and oilseed crops from dry climatic zones were enhanced using this strategy to produce better yield potential and added value with the aid of contemporary breeding approaches, which assisted the crops to evade damage from dry seasons (Des Marais and Juenger 2010).

18.3 Basic Concept of Abiotic Stress Tolerance in Plants

Evasion is a mechanism that protects plants from any type or intensity of stress situation (Madlung and Comai 2004). The system of escaping particularly from moisture stress by a plant is an instance of plant evasion mechanisms. In such a situation, when plants are exposed to drought conditions, regulation of the collection and use of water occurs spontaneously by means of adjustment of physiological activities inside roots and leaves. Controlling the movement of stomata is a frequently adopted mechanism to evade unwanted loss of water (Buckley et al. 2003). Yet, in spite of such procedures, declines in photosynthesis and plant development can occur. Usually, plants that are incompetent to adapt to a particular environment are susceptible to that environmental condition (Wang et al. 2003). During the reaction against abiotic stress, phenotypical, structural, and functional modifications might arise in plants. Such modifications are chiefly influenced by the level of flexibility of the plant while managing different environmental conditions. Such modifications can influence the developmental stages of plants, their economic yield, nutritional profile, metabolic status, and so on (Altman 2003). Hence, abiotic stress being encountered by food crop plants remains an issue for food security and the global economy.

18.3 Basic Concept of Abiotic Stress Tolerance in Plants Environmental stress is an unfavorable situation being faced by crop plants that negatively influences growth and development, eventually decreasing the yield potential. Such stress might be due to abiotic or biotic agents. Bacteria, fungi, virus, weeds, etc., cause biotic stress. On the other hand, physical and chemical agents in surplus or deficit quantities result in abiotic stress. For instance, waterlogging and drought stress occur when the amount of water is excess or deficient, respectively. Similarly, extreme temperature, excessive saline or alkaline soil, and excess heavy metal, mineral or other phytotoxin concentrations in soil, are some major abiotic stresses considered threats to economic crop production. Owing to their sessile character, plants safeguard themselves during their encounters with such hostile environments through modified or stimulated expression of genes. Additionally, transformed metabolic activities at the cellular level or induced natural resistance systems at the anatomical and/or functional levels could also play significant role. The first mechanism is characterized as “sensitive stress tolerance,” while the other mechanism is described as “stress resistance.” Such endurance against stress remains typically exclusive for a particular plant genotype or species and has been acquired in the course of its coevolution with stress-inducing factors through transformations in genetic configuration or via triggering of inactive or preexisting resistance genes (Panigrahi et al. 2013). Plants display stress tolerance or stress avoidance via acclimatization processes that have developed throughout natural selection. The attributes which support greater production during persistant abiotic stress exhibit efficient protection across the species once the plants have adjusted to analogous environments. Genetic diversity, which facilitates the improvement of consistent yields, exists in the germplasm of crops and their wild ancestors. Utilization of such biological deviation for crop improvement has usually been without information on functional genes and correlated biological mechanisms (Mickelbart et al. 2015).

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18.4 Genetics of Abiotic Stress Tolerance Usually, endurance or tolerance against abiotic stress is controlled by multiple alleles followed by the effect of genotype × environment synergy. Hence, resistance to abiotic stress is considered typically as having a quantitative and polygenic character based on the constant genetic deviation, relative heritability, and interation with a changing environment (Geiger and Heun 1989). Such a multifaceted resistance of plants against a wide range of biotic agents besides abiotic stress factors necessitates an insight into how the mechanism of modification works at a genetic level to transform the natural plant population into a resistant one. There are three key aspects that influence the mechanism behind genetic modification to a great extent: (i) the degree of genetic influence on a resistant characteristic and its heritability; (ii) the possibility of variation in the resistance characteristic (i.e. genetic drift) induced by coincidence; and (iii) natural selection, which might work on the resistance characteristic (Simms and Rausher 1992). Hereafter, to determine the mechanism of the above-mentioned issues and to design a breeding program for abiotic resistance, the mapping of simple inherited trait loci and quantitative trait loci (QTL), including candidate gene loci, needs to be performed. At present, basic genetic determinants can be recognized via QTL mapping, association mapping, and screening by recurrent selection (reviewed by Takeda and Matsuoka 2008). The latest comprehensive molecular description of QTLs and the linked genes which assist survival under stress has empowered more convenient gene transmission into modern-day crop germplasm, ensuring molecular markers are allied with the vital genes at the time of breeding. Improvement in the detection of the gene that defines the attribute has directed insights into particular biological systems, and the translation of information to other species (Mickelbart et al. 2015). As established from the available research reports, plants’ endurance against abiotic stress could be considered as a kind of genetic regulation wherein the affected plant acts either as a direct competitor of the given stress situation for its normal growth and development or interacts with the environment to thrive under stress. For the elucidation of the genetic origin of the plant’s endurance against stress situations, terminologies like “vertical” or qualitative and “horizontal” or quantitative resistance have been used by plant breeders alongside “monogenic” and “polygenic” resistance. “Vertical” resistance is effective to combat a limited number of biological races of a certain pathogen, whereas “horizontal” resistance signifies endurance against a high number of races. In addition, the genetic response amid the affected plants and the races of the same plant pathogen is different for vertical resistance, as established by Van der Plank (1963). It has been reported that vertical resistance is usually controlled by a certain gene (which is represented by merely a single pair of alleles). On the other hand, horizontal resistance, being polygenic in nature, is regulated by multiple genes (nonspecific for resistance); these genes exist in normal plants and are involved in the usual regulation process that is associated with the expression of resistance (Van der Plank 1968). Even though the association of one gene with vertical and multiple genes with horizontal resistance is evident in numerous studies, according to Bergamin Filho et al. (1995), this is not considered to be a universal theory. However, adverse results might be observed if horizontal resistance is selected in the presence of monogenic vertical resistance that might be followed up by the development of genes with vertical resistance in high frequencies (Parlevliet 1989).

18.5 Fundamentals of Molecular Markers and Marker-assisted Selection

The outcomes of horizontal resistance are quantifiable and multidimensional. In plants, horizontal resistance having polygenic inheritance displayed superior stability in comparison with vertical resistance having monogenic inheritance (Van der Plank 1968).

18.5 Fundamentals of Molecular Markers and Marker-assisted Selection One of the primary restraints on the breeding method is that the evaluation of the value of distinctive lines and specific plants has to be centered on their morphology. This has been documented for a long time to decrease the competence of breeding methods, and in several instances, to delay the development of improved varieties. Consequently, methodical exploration for clearly scorable markers that could be utilized for consistent subsidiary screening for sought characteristics was needed. This exploration commenced with phonotypical characteristics and was ultimately directed to the development of DNA-based molecular markers. Nonetheless, the development of DNA (or molecular) markers in the 1980s laid the groundwork for the efficacy of indirect screening policies in plant breeding. Furthermore, these markers facilitated the documentation and mapping of the genes of interest and effective indirect screening for the target traits. While DNA markers have diverse uses in crop breeding, the most favorable one for cultivar development is marker-assisted breeding (MAB). Molecular markers have been comprehensively utilized for the tagging and mapping of genes and QTLs presenting resistance to biotic and abiotic stresses. Such resources have also been employed for the selection of germplasms, fingerprinting, and MAB in crops. Although molecular markers are effective in crop improvement systems, especially for map construction in several crops, several kinds of DNA markers including AFLP, CAPS, DArT, ETS, ISSR, RAPD, RFLP, single nucleotide polymorphism (SNP), and simple sequence repeats (SSR) are frequently used for this purpose (Doveri et al. 2008). Nevetheless, apart from these widely used markers, SCAR and STS, associated with a particular gene or QTL, are of great use for successful MAS in any crop (Shan et al. 1999; Sanchez et al. 2000; Sharp et al. 2001; Collard and Mackill 2008; Kumar et al. 2011). Establishing the marker-to-trait links is the key criterion for MAB. Confirmed connections amid target traits/genes and molecular markers are conventionally established on genetic mapping experiments, and this is vital to check that such links are steady among the breeding lines and mapping populations. According to Xu et al. (2003), to ensure an effective MAB, markers must either co-segregate or be connected with the characteristic of interest, with a gap of 35 ∘ C temperature (Liu et al. 2012).

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However in V. amurensis 35 ∘ C did not significantly influence the photosynthesis rate (Pn) but a severe decrease in Pn was observed at 40 ∘ C and 45 ∘ C accompanied by an high substomatal CO2 concentration and low stomatal conductance (Luo et al. 2011). The phytohormone salicylic acid increases heat tolerance in young grape plants by maintaining Ca2+ homeostasis without destroying the chloroplast structure (Wang and Li 2006). Experimenting with stable isotope labeled tracer, Mori et al. (2007) proved degradation of anthocyanin accumulation in V. vinifera berry skin at high temperatures. Lijavetzky et al. (2007) used a highly efficient re-sequencing approach and SNPlexTM technology and precisely estimated short-range linkage disequilibrium by providing accurate nucleotide diversity in coding regions; the identified SNPs could be used as molecular markers for cultivar discrimination, genetic divergence analysis, and linkage mapping. Using Affymetrix oligonucleotide microarray and qRT-PCR, Liu et al. (2012) performed transcriptome analysis of V. vinifera and identified heat stress or recovery-responsive genes and transcription factors, and elucidated the role of small heat shock proteins, such as ascorbate peroxidase and galactinol synthase in grape thermotolerance. Xu et al. (2014) found a high level of heat tolerance in many wild grape species and also in hybrids between V. labrusca and V. vinifera, although most cultivated species exhibited high heat susceptibility. “NimbleGen 090818 Vitis 12X (30 K) microarrays” were deployed to track the transcriptomic variances of heat stress throughout the day and night in the green and ripening grape berries (Rienth et al. 2014). The OJIP test is done to assess plant sensitivity to stress for exploration of variation in photosystem II photochemical activities; it is recommended as a rapid, sensitive, and convenient technique for assessing high-temperature injury in grapevine.

18.7 Marker-assisted Selection for Drought Tolerance Almost 65% of the world’s population will experience water shortage by the year 2025. Drought can be explained as a condition in crop cultivation systems when plants are unable to fulfill the water needed for their transpiration. Drought is a leading stress factor, influencing plant growth and reproduction, and ultimately, plant breeding against such stress is a challenge to scientists with a view to developing water stress avoidance or tolerance. A secure food supply in the twenty-first century will depend on high-yielding stable cultivars with improved drought resistance. MAS is being considered one of the key options to achieve this (Nezhadahmadi et al. 2013; Tuberosa 2012). We will highlight the situation with maize, chickpea, oilseed Brassica and coriander in the sections below. 18.7.1.1

Maize (Zea mays)

Delays in silking and successive rises in the anthesis-silking interval (ASI) can cause huge yield loss in drought-stressed maize. Linkage analysis under well-watered and watered conditions between expression of flowering period, ASI, plant height and 153 loci of RFLPs, AFLPs and SSRs was conducted and diverse QTLs were detected for female flowering time and ASI in maize (Sari-Gorila et al. 1999). Under drought stress, maize hybrids usually yield better than cultivars, suggesting heterosis as the basis of stress resistance (Bruce et al. 2002). Ribaut and Ragot (2007) explained the presence of stress intensity-dependent genetic regulation in maize for drought tolerance. Ziyomo

18.7 Marker-assisted Selection for Drought Tolerance

and Bernardo (2013) suggested secondary traits like ASI, leaf senescence, and ears per plant as useful selection parameters for grain yield at water deficit. Messmer et al. (2011) identified 32 QTLs for relative content of leaf chlorophyll (CL) and 25 QTLs for plant senescence (SEN) specifically on chromosomes 2, 4, and 10, suggesting the involvement of a few genes that influence plant senescence and chlorophyll metabolism. On the basis of phenotypic data they suggested that high CL and low SEN are valuable characteristics for selection in favor of grain yield during water stress conditions. Zhu et al. (2011) also identified 4, 7, 6, 4, and 4 QTLs for ASI, plant height, ear height, grain yield, and ear development, respectively, during extreme late water stress. Avramova et al. (2016) used a noninvasive screening technique for drought phenotyping in hybrid maize and found a reduction in projected leaf area, rate of leaf elongation, length and width of the matured fourth and fifth leaf; reduced rate of leaf cell division significantly affected water use efficiency, chlorophyll fluorescence, and photosynthesis. Overall root length, rooting depth, width, and weight were also decreased owing to drought, as revealed by detailed root growth analysis. Based on transcriptome analysis, Min et al. (2016) elucidated the dynamic mechanisms behind drought tolerance in maize seedlings in drought stress as they found varying numbers of differentially expressed genes in the two RILs from ph4CV (drought-tolerant) × F9721 (drought-sensitive). 18.7.1.2

Chickpea (Cicer arietinum)

In India, chickpea is predominantly cultivated in post kharif season on residual soil moisture. Chickpea is a highly preferred pulse in drought-prone areas and terminal drought reduces yield. Hence drought-associated traits play an important role in breeding for drought tolerance. Ulemale et al. (2013) identified some physiological indices for drought tolerance in chickpea, viz. relative leaf water content, chlorophyll stability index, proline accumulation, chlorophyll content, nitrate reductase activity, and membrane injury index; these parameters could be beneficial for selection of drought-tolerant genotypes of chickpea. Talebi et al. (2013) identified higher concentrations of relative leaf water content, chlorophyll, carotenoid, and K+ /Na+ ratio among the drought-tolerant chickpea genotypes. The drought response index shows significant positive associations with some seed yield-associated traits like crop growth rate, harvest index and sink capacity (rate of partitioning or p); p is a key trait for drought breeding as the association between p and drought response index becomes intensified during post-flowering severe drought (Kashiwagi et al. 2013 and Krishnamurthy et al. 2013). Combination of superior p and crop growth rate with proper phenology was recommended for the best selection strategy to boost terminal drought tolerance in chickpea (Purushothaman et al. 2016). An extensive phenotyping of 20 drought component traits was performed and a separate genetic map, saturated with 241 loci and 168 loci, was constructed for the two mapping populations of chickpea. Nine QTL clusters containing 45 robust main-effect QTLs and 973 epistatic QTLs were identified; cluster 5 (QTL-hotspot) of 7.74 Mb size with 48% robust main-effect QTLs for 12 several drought-tolerance traits present on CaLG04 exhibits high potentiality in chickpea drought breeding. The QTL-hotspot contains seven SSR markers, which are considered to be very useful (Varshney et al. 2013, 2014). Kale et al. (2015) executed fine mapping of QTL-hotspot and found 23 genes by gene enrichment analysis and they split it into two subregions, namely QTL-hotspot_a and QTL-hotspot_b comprising 15 and 11 genes, respectively, as revealed by the QTL analysis. From the qRT-PCR analysis,

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they identified four key candidate genes in the QTL-hotspot for drought tolerance. Jaganathan et al. (2015) refined a 1.4 cM region in the QTL-hotspot and identified 164 main-effect QTLs containing 24 original QTLs; furthermore 49 SNP markers were also identified in that region, which were transformed into CAPS and dCAPS markers. A total of 10,996 high-quality drought-responsive expressed sequence tags (ESTs) were generated from eight different root tissue cDNA libraries of chickpea by expression and functional genomics (Varshney et al. 2009). Jain and Chattopadhyay (2010) assessed gene expression in response to water deficit that might assist in finding advantageous genes in chickpea to enhance tolerance against drought stress. 18.7.1.3

Oilseed Brassica

The descending order of drought tolerance among the Brassica species is rapeseed (B. napus), Indian mustard (B. juncea), B. carinata, and B. rapa. Richards and Thurling (1979a) demonstrated that accumulation of proline and stability of chlorophyll in leaf, as well as germination at depleted osmotic potentials, could be utilized for rapeseed breeding selection. The same group (Richards and Thurling 1979b) also recommended joint selection under drought for yield, seeds per pod, 1000-seed weight, and harvest index instead of direct selection for yield only. Early flowering and growth inhibition are adaptive responses to drought (Chaves et al. 2003). Franks (2011) suggested that B. rapa plants prefer escaping drought rather than avoiding it as they flower early and exhibit low water use efficiency as well when water stressed. Hence selection on the basis of early flowering will be rewarded in B. rapa. Relative vigor index and leaf wilting index are extensively utilized for high-throughput screening of drought tolerance during germination and seedling phases (Li et al. 2012; Yang et al. 2007). In drought-tolerant mustard, a high level of of osmatic adjustment and proline content, and greater movement of proline biosynthetic enzyme was recorded by Phutela et al. (2000). On the other hand, Alikhan et al. (2010) observed that the osmotic potential, relative water, and potassium levels were decreased and proline accumulation was increased under drought in rapeseed. With the help of Solexa Illumina array, Yu et al. (2012) identified 1092 drought-responsive genes in B. rapa, and merely 61 genes out of those were engaged in water- and osmo-sensing-responsive pathways. Some drought tolerance-associated genes of oilseed Brassica species have been reported, such as BrERF4, BnLAS, AnnBn1, BnLEA4-1, and BrECS, which could be utilized for breeding (Zhang et al. 2014). 18.7.1.4

Coriander (Coriandrum sativum)

Coriander is a medicinal as well as spice crop and it is sensitive to water deficit. Ghamarnia and Daichin (2013) conducted an experiment on it to estimate the effects of water stress on multiple factors and found 3.54% and 2.30% reduction in seed and oil yield, respectively, per unit of water stress. In breeding coriander for drought tolerance the main focus must be on increase in its water use efficiency. Khodadadi et al. (2017) established significant negative genetic association of fruit yield with early flowering, and with number, diameter, dry mass, and volume of root, and also with the assimilated loading of root and shoot. On the other hand, higher fruit yield was obtained with high chlorophyll and relative water content of leaf, and also with high assimilated loading of fruits. Hence those parameters could be taken for screening of drought-tolerant high-yielding coriander genotypes.

18.7 Marker-assisted Selection for Drought Tolerance

18.7.2

Marker-assisted Selection for Salinity Tolerance

Being the most typical abiotic stress on plants, salinity confers a negative influence on crop yield by affecting growth and the use of land becomes restricted (Turan et al. 2012). In many cases the quality of water is deteriorated by the presence of high concentrations of salt. Worldwide soil salinity significantly reduces crop yields, affecting around 20% of irrigated land and 6% of the total land (Unesco Water Portal 2007; Qadir et al. 2014). NaCl is the most common salt; hence saline soils are defined by measuring the ion osmotic pressure that is equal to 40 mM NaCl or ≥0.2 MPa (USDA-ARS 2008). Uptake of the necessary K+ ion is inhibited by high Na+ concentration. Salt toxicity also generates ROS that will cause cellular oxidative damage. When plants are exposed to salinity, roots sense Na+ and subsequently cellular signaling is initiated and this might take few minutes to few days (Roy et al. 2014); leaf expansion inhibition as well as stomatal closure are noticed at this time (Munns and Termaat 1986). In the next few days to few weeks, the shoot-ion concentration reaches a toxic level, resulting in reduced yield or even death by early senescence of older leaves (Munns and Tester 2008). Halophytes can withstand salinity whereas the glycophytes die, and the majority of crops belong to the second category. Fita et al. (2015) suggested an idea to create saline agriculture by domesticating new halophilic crops into conventional agriculture with a view to feeding the rapidly increasing population. In the following sections we can see how MAS plays an important role in generating halophytes from the glycophytes in the cases of rice, greengram, oilseed Brassica, and tomato. 18.7.2.1

Rice (Oryza sativa)

Owing to salinity, vast rice-cultivating areas in South and Southeast Asia remain fallow or suffer extremely low yields. Yeo et al. (1990) evaluated low shoot Na+ concentration, compartmentation of salt in older leaves, and plant vigor as salt stress tolerance parameters in rice. Regarding yield of rice, plant vigor could be an avoidance mechanism rather conferring tolerance. Bonilla et al. (2002) reported Saltol to be flanked by two RFLP markers (C52903S and C1733S) and two microsatellite markers (RM23 and RM140). Saltol is a major salt tolerance QTL in chromosome 1 controlling low Na+ absorption, high K+ adsorption, and a low Na+ /K+ absorption ratio. Pokkali rice is a salt-tolerant genotype and to characterize Pokkali-derived QTLs for salinity tolerance during the seedling stage, Thomson et al. (2010) detected multiple Pokkali alleles at Saltol by developing RILs and near isogenic lines (NILs). Saltol is being transmitted into seven common Indian rice culrivars (ADT 45, CR 1009, Gayatri, MTU 1010, PR 114, Pusa 44, and Sarjoo 52); for background selection the in-house designed 50 K SNP chip was utilized (Singh et al. 2016). In an F2 population raised from of IR36 (india) × Jiucaiqing (japonica), dual QTLs were identified for root Na+ /K+ ratio on chromosomes 2 and 6 (Yao et al. 2005). The corresponding QTL for the highest Na+ uptake, Na+ /K+ , and regulation of K+ /Na+ were reported earlier, namely QNa, QNa:K and SKC1/OsHKT8, respectively. The first one was mapped on chromosome 1 and the other two on chromosome 4 (Flowers et al. 2000; Singh et al. 2001; Ren et al. 2005). Lin et al. (2004) found the eight QTLs, the two major QTLs qSNC-7 and qSKC-1, with a very large effect on Na+ and K+ levels in shoot and root respectively, explained larger phenotypic variance related to salt tolerance in rice. Lang et al. (2001) identified four QTLs for tissue Na+ /K+ ratio and one QTL each for Na+ and K+ uptake.

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Ahmadi and Fotokian (2011) identified 14 QTLs for shoot and root Na+ /K+ ratio and Na+ and K+ content on different rice chromosomes. Among them, QKr1.2 on chromosome 1, for root K+ content, explained ∼30% variation regarding salt stress tolerance in rice. Other than QTL Saltol chromosome 1, Islam et al. (2011) identified two new QTLs, SalTol8-1 and SalTol10-1, on rice chromosomes 8 and10, respectively, analyzing F2 from IR61920-3B-22-2-1 (high salt stress tolerant line) × BRRI-dhan40 (medium salt stress tolerant line). SalTol1-1, SalTol8-1 and SalTol10-1 were flanked by primer pairs RM8094–RM3412, RM25–RM210, and RM25092–RM25519 respectively. In normal conditions the ascorbate peoxidase is more abundantly synthesized in Pokkali rice than in the salt-sensitive cultivar (Salekdeh et al. 2002). Platten et al. (2013) suggested plant vigor as one of the key determinants of salt tolerance in rice and identified seven major and three minor alleles of OsHKT1;5 from the salt stress-tolerant O. sativa and O. glaberrima accessions; the highest leaf Na+ exclusion was conferred by the Aromatic allele and the least by the Japonica allele. There is a relation of root cap cells to exclusion of Na+ , as observation under scanning electron microscope revealed the proliferation and extension of root cap tissues to the basal part of the root tip owing to salinity stress (Rahman et al. 2001; Ferdose et al. 2009). Moradi and Ismail (2007) investigated the effects of salinity on photosynthesis, chlorophyll fluorescence, and ROS scavenging systems at the seedling stage and the reproductive stage in rice and found tolerant genotypes exhibiting relatively higher photosynthetic function. The morpho-physiological traits like high vigor, increased shoot/root biomass, lesser shoot Na+ accumulation, and lesser shoot Na+ /K+ ratio were linked to salinity tolerance in rice (Sexcion et al. 2009). The reduced shoot area in the salt-stressed rice from the control was clearly observed by a nondestructive image-based phenotyping protocol (Hairmansis et al. 2014). Using 1 H-NMR spectroscopy Nam et al. (2015) detected five conserved salt-responsive metabolic markers of rice roots; owing to salt stress, three of them, viz. sucrose, allantoin and glutamate, accumulated, whereas the other two, i.e. glutamine and alanine, reduced. Bulked segregant assessment with 50 K SNP chip recognized 5021 polymorphic loci and 34 QTL regions (Tiwari et al. 2016). Seven rice landraces, namely Akundi, Ashfal, Capsule, Chikirampatnai, Jatai Balam, Kalarata, and Kutipatnai, showing notably lesser shoot Na+ levels, were selected. They possessed salt tolerance genes as revealed by diversity analysis using 376 SNP markers, along with allelic diversity at the major QTL Saltol (Rahman et al. 2016). From the QTL mapping in the IR36 × Pokkali F2 population using 111 polymorphic SSR markers, six QTLs associated with salt tolerance were identified on chromosomes 2, 3, 7, and 8 (Khan et al. 2016). A linkage map based on 148 F2 individuals of indica rice derived from Gharib (salt-tolerant) × Sepidroud (salt-sensitive) was constructed, and 41 QTLs for 12 functional attributes under salinity stress were identified to be allocated on all rice chromosomes (Ghomi et al. 2013). Bizimana et al. (2017) used 194 polymorphic SNP markers and identified 20 new QTLs (LOD > 3) on chromosomes 1, 2, 4, 6, 8, 9, and 12 while dealing with an IR29 (salt sensitive) × Hasawi (salt tolerant) mapping population. A total of 18 and 32 salt injury score QTLs at seedling stage were identified exercising SSR and SNP markers in introgression lines (ILs) of Pokkali × susceptible Bengal high-yielding cultivar. 18.7.2.2

Mungbean (Vigna radiata)

Salinity stress causes huge reductions in yield by reducing seed germination, root and shoot lengths, fresh weight, and seedling vigor in mungbean (Saha et al. 2010).

18.7 Marker-assisted Selection for Drought Tolerance

Reduction in carotenoids and chlorophyll content resulting in enhanced chlorosis, and necrosis in mungbean are caused by salt injury (Wahid and Ejaz 2004) and also photosynthesis is hampered by the entry of excessive salt into the transpiring system (Neelam and Dwivedi 2004); nodule formation as well as nitrogen fixation by roots are also affected by salt stress, leading to reduced leghemoglobin content (Balasubramanian and Sinha 1976). However, Naher and Alam (2010) reported lesser nodule number without change in nodule size owing to increased salinity. NaCl stress produces a pronounced deleterious effect on roots rather than shoots (Saha et al. 2010). Misra and Gupta (2006) found increased levels of proline and glycine betaine in roots and shoots by subjecting NaCl stress to the seedlings of a tolerant mungbean cultivar. There is an enhanced level of total soluble carbohydrates in the salt-tolerant cultivar as repoted by Misra and Dwivedi (1995). Increased activities of antioxidant enzymes, catechol-peroxidase and catalase with an increase in NaCl concentrations might confer a remedy to overcome the cellular toxicity of NaCl in mungbean seedlings (Roychoudhury and Ghosh 2013). At a high salinity level, mungbean plant postpones the pod ripening, which could reduce pod shattering (Sehrawat et al. 2013). Chankaew et al. (2014) identified a single major QTL conferring salt tolerance in Vigna marina subsp. Oblonga in an F2 population from V. luteola × V. marina subsp. oblonga. V. marina is a salt-tolerant wild mungbean that naturally grows on beaches in the tropics and subtropics worldwide; it is also known as beach cowpea. In Bangladesh V. marina is used as a dual-purpose cultivar. It exhibits photosensitivity and enormous vegetation (HanumanthaRao et al. 2016). It can be utilized in salinity breeding in mungbean. Sehrawat et al. (2014) developed eight novel microsatellite SSR markers specific to candidate genes involved in salt tolerance and found great allelic variation in inter-specific as well as intra-specific mungbean genotypes. 18.7.2.3

Oilseed Brassica

Owing to salinity stress, osmotic stress and water deficit occur, enhancing ABA synthesis in shoots and roots in the plants (He and Cramer 1996). The oil-producing Brassica species, including both diploids and amphidiploids, are moderately salt tolerant. From different studies it has been proved that the amphidiploid species B. carinata, B. juncea, and B. napus show superiority over the diploid species, B. campestris, B. nigra, and B. oleracea, as evident from various studies (Ashraf et al. 2001; Purty et al. 2008). Chakraborty et al. (2016) found B. napus to be most salt tolerant followed by B. juncea and B. oleracea on the basis of initial root growth assay and viability staining; they reported a strong correlation between K+ retention ability in roots and salinity stress tolerance in Brassica species and here the root plasma membrane potential and cytosolic K+ /Na+ ratio both play a key role. Previously, Kumar et al. (2009) and Chakraborty et al. (2012) reported the existence of an efficient SOS pathway as a major factor for higher K+ /Na+ ratio determining the salinity stress tolerance of B. juncea genotypes. Earlier, Hayat et al. (2011) screened salt-tolerant genotypes of B. juncea based on photosynthetic attributes and recommended Varuna as the best cultivar, which could successfully be grown in Indian soils in the range 2.8–5.6 dS m−1 salinity. Yusuf et al. (2008) reported decreased nitrate reductase activity owing to salinity in B. juncea. Sharma et al. (2015) performed transcriptome sequencing of control and salt-stressed seedlings in a salt-tolerant B. juncea var. CS52 and observed many transcription factors associated with ROS detoxification. Comparative expression profiling of key salt stress-responsive

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genes revealed their constitutive expression in the tolerant variety under normal growth conditions. CS52 is an Indian mustard variety adapted to saline (6.0–8.5 dS m−1 ) and sodic soils (pH 9.3) and it was developed at Central Soil Salinity Research Institute, India. The study of Sharma et al. (2015) confers a valuable resource of salinity-tolerance mechanisms in Brassica genotypes that will help in trait improvement. By mapping to ESTs onto the 42 327 predicted transcripts, they found 52.6% novel unigenes as compared with EST data available for B. juncea and constituent genomes. In CS52, maximum proline accumulation with minimum levels of H2 O2 , electrolyte leakage and malondialdehyde contents were observed (Joshi et al. 2011). The genomes of B. rapa and B. napus were resequenced (Shi et al. 2014) and there is a huge amount of primer information available for Brassica SSRs (microsatellite) which have been shown to be applicable within and between different Brassica species. 18.7.2.4

Tomato (Solanum lycopersicum)

Most commercial cultivars of tomato can tolerate saline conditions up to 2.5 dS m−1 without any impact on fruit yield. Therefore, selection and breeding for salt-tolerance in tomato are needed for the production of tomato in the soils above the threshold level of salinity. Performing a salinity assay, Singh et al. (2012) were able to screen the tolerant genotypes of tomato on the basis of the least-affected as well as earliest-germinating tomato seedlings. They may be used as a future source of salinity tolerance for tomato breeding. Owing to salt stress, the ratio of dry weight of root over shoot is increased, indicating a greater allocation of assimilates into root than shoot. The increased ratio of Na+ /K+ owing to salinity stress indicates that tolerant tomato plants possess enhanced K+ concentration (Singh et al. 2012); the genotypes with low Na+ /K+ ratio can be utilized in future breeding for salinity tolerance in tomato (Dasgan et al. 2002). Although the level of salt tolerance changes during different growth stages (Foolad 2004), breeders should focus initially on the most sensitive plant stages. QTL analysis of salt tolerance with the help of two populations of 135 RILs from S. lycopersicum × S. pimpinellifolium, show clustering of salt-tolerance related QTLs governing several trait sets, among them fruit weight has significant correlations with fruit number, dried weight of stem, and water-use efficiency (WUE) (Cuartero et al. 2006). The recommendations from their study were: “to breed genotypes tolerant to high (200 mM) levels of salinity, leaf-tissue tolerance should be employed, while in breeding for tolerance at medium salinity (100 mM) levels, Na+ transport to shoot should be employed”; for the genotypes with high WUE less irrigation water will be required, which means that less salt will be incorporated into the soil. Owing to the highest heritability, leaf area should be considered for breeding salt tolerance in tomato (Cuartero et al. 2002). Bretó et al. (1994) evaluated F2 populations from crosses concerning S. lycopersicum and two wild relatives, S. pimpinellifolium and S. galapagense S. Darwin & Peralta and found 43% of marker loci to be linked to QTLs for salt tolerance. Maggio et al. (2007) were able to identify a sharp increase in ABA levels in the tomato shoot and root at a specific electrical conductivity value of ∼9.6 dS m−1 . The following physiological modifications occur in the tomato plant: (i) stomatal sensitivity becomes reduced in response to ABA; (ii) variable partitioning of Na+ ions occurs amid young and mature leaves; and (iii) a radical improvement of the root-to-shoot ratio occurs. Co-localization of QTLs on chromosome 6 in the two IL libraries indicated tomato crop-specific conservation of this locus and flanking markers of the QTLs were also suggested by

18.7 Marker-assisted Selection for Drought Tolerance

Li et al. (2011). Some wild accessions of tomato viz., S. pimpinellifolium, S. peruvianum, S. cheesmaniae, S. habrochaites, S. chmielewskii and S. pennellii showed the specified extent of salt tolerance (Foolad and Lin 1997). Using an F2 population from one of the S. pennellii accessions, five salt tolerance loci were identified during germination stage (Foolad et al. 1997). In response to salt stress, seven QTLs for better seed germination, three steady QTLs triggering salt tolerance throughout the vegetative growth and other fruit-related QTLs were identified (Monforte et al. 1999; Foolad 2004). Villalta et al. (2007) reported co-localization of 49 QTLs for 19 characteristics under salt stress in two populations originating from S. pimpinellifolium and S. cheesmaniae; except for chromosome 9, these QTLs were allocated around 11 chromosomes. 18.7.3

Marker-assisted Selection for Low Temperature Tolerance

About 57% of global land area is affected by low temperature stress, which is one of the key restricting elements to crop production (Cramer et al. 2011). Low temperature stress to the plants may be chilling (>0 ∘ C) or freezing (≤0 ∘ C), and frost is considered as another form of such stress. There is an active process called cold acclimation or hardening off, where plants gain freezing tolerance owing to exposure to low nonfreezing temperatures. The freezing-tolerant plants cannot survive at −3 to −5 ∘ C if they are not cold acclimated while they can withstand much colder freezing temperatures if cold-acclimated. With greater duration of cold-acclimation exposure, there is a chance to enhance the freezing tolerance. It is essential to possess freezing tolerance for the temperate crops to survive during the winter months as they have to go through a prolonged vernalization period to reach the reproductive growth phase from the vegetative stage. There lies a significant link between cold acclimation and vernalization (Dhillon et al. 2010). Regading frost tolerance, crops must have the ability to resist desiccation owing to frost during the vegetative period so that they can recover, flower, and produce the desired yield as well (Visioni et al. 2013). Here we represent MAS for low temperature tolerance in barley, pea, oilseed Brassica, and potato. 18.7.3.1

Barley (Hordeum vulgare)

Strong winters or even mild frost may cause severe injury to temperate cereals like barley. Barley is recognized as an excellent model crop among fall-sown cereals regarding molecular genetic analysis of low-temperature tolerance. Cold acclimation-induced freezing tolerance resulting in winter hardiness is needed for assured winter survival and yield in barley. In cold-acclimated winter barley plants, proline accumulation confers frost tolerance (Tantau et al. 2004). Hayes et al. (1993) mapped QTL controlling traits associated with winter hardiness in a barley population of 100 F1 -derived doubled haploid lines on chromosome 7. Cold acclimation also increases recovery ability from freezing injury in barley by improving photosynthetic stability (Dai et al. 2007). Functions of antioxidative enzymes (such as peroxidase, ascorbate peroxidase, glutathione reductase, etc.) were increased in acclimated barley along with enhanced glutathione and ascorbate accumulation throughout freezing and revival phases (Dai et al. 2009). Giorni et al. (1999) found significant accumulation of several COR (cold regulated) genes such as pt59, pao86, and paf93 and proteins (COR14a and COR14b) in winter cultivars of barley and suggested that a high level of COR14 may be associated with the winter hardiness capacity in barley. Rapacz et al. (2008) observed a higher transient induction

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of COR14b expression on the initial days of cold acclimation in freezing-tolerant plants. Dai et al. (2007) indicated some chlorophyll fluorescence parameters in the identification and evaluation of cold tolerance in barley during freezing shock recovery. Francia et al. (2004) identified vernalization response and low temperature tolerance QTL on chromosome 5B. Tóth et al. (2004) developed two STS markers, having strong linkages with the frost tolerance QTL, derived from the RFLP probes WG644 and PSR637 and validated them together with one selected RAPD marker, OPA17, in a double haploid population obtained from a Nure (highly tolerant) × Tremois (susceptible), and mapped them on the long arm of chromosomes 5H and 2H. HvBM5A is a candidate gene for vernalization locus VRN-H1. To characterize barley vernalization genes, von Zitzewitz et al. (2005) reported winter barley genotypes to possess a VRN-H2 locus while this locus is being deleted from the facultative and spring barley genotypes where VRN-H1 is present. Tondelli et al. (2006) found HvCBF, ICE1, and FRY1 candidate genes for the cold tolerance stress in barley by developing STS, SNP, and SSCP (single-strand conformation polymorphism) markers; the HvCBF genes were co-mapped with the cold tolerance QTL FR-H2. Francia et al. (2007) validated both FR-H1 and FR-H2 in the segregating population from Nure × Tremois, and constructed a high-resolution genetic map of FR-H2 QTL, based on a cluster of seven HvCBF candidate genes. Cockram et al. (2007) described the haplotypic divergence of VRN-H1 and VRN-H2 in European barley to detect three original VRN-H1 alleles and 17 VRN-H1/VRN-H2 multilocus haplotypes. Ten VRN-H1 markers including SNPs, intron I InDel and (CGCT)2–5 SSR, employed by them could be utilized in a diagnostic test for a vernalization response in barley germplasms. Chen et al. (2009) identified winter alleles at the VRN-H1 locus on 5H associated with low temperature tolerance and associated with earlier flowering. On the other hand, Flt-2L locus on 2HL was linked to tolerance and late flowering. Fricano et al. (2009) investigated the normal allelic discrepancy in four CBF genes (HvCBF3ee, HvCBF6, HvCBF9, and HvCBF14) positioned at the FR-H2 locus. Two-nucleotide variants of HvCBF14 and a one-nucleotide variant of VRN-H1 were identified to be associated with frost tolerance as revealed by their study. FR-1 has the pleiotropic effects of VRN-1 (Dhillon et al. 2010) while QTL FR-2 has a cluster of C-Repeat Binding Factor (CBF) genes (Knox et al. 2010). Akar et al. (2009) tested FR-H1 and FR-H2 in highly frost-tolerant barley accessions treated at −12 ∘ C using STS markers targeting three HvCBF genes, and one STS marker PSR637 along with a CAPS marker seeking HvBM5A (VRN-H1) and the STS marker for VRN-H2. Dhillon et al. (2010) reported that allelic variation in VRN-1 is appropriate to establish the disparities in freezing tolerance. Owing to the existence of varying copy number in the CBF gene within the Triticeae, Knox et al. (2010) suggested considering both VRN-1 and FR-2 alleles for selection of winter hardiness in barley. Fisk et al. (2013) discovered a new major QTL FR-H3 corresponding to superior low-temperature tolerance on barley chromosome 1H by analysis of two populations derived from a facultative genotype OR71 hybridizing with NB3437f (facultative) and the NB713 (winter). A genome-wide association study of winter hardiness traits employing two Illumina GoldenGate oligonucleotide pool assays for 3072 SNPs was conducted by von Zitzewitz et al. (2011). They predicted maximum low temperature tolerance to be associated with facultative growth habit involving FR-H1, FR-H2, and VRN-H2. Visioni et al. (2013) performed a genome-wide association study with 1536 SNP markers and found two of the substantial SNP links that are firmly associated with the Fr-H2 and HvBmy

18.7 Marker-assisted Selection for Drought Tolerance

(beta amylase barley unigene) loci on chromosomes 5H and 4HL, correspondingly. For the establishment of the transcriptome profiling and genotypic difference during short acclimation followed by mild freezing shock, Wang et al. (2016) performed an Illumina RNA-sequencing in Nure and Tremois, and identified the HvCBFs cluster within FR-H2 locus on 5B chromosome. 18.7.3.2

Pea (Pisum sativum)

Pea is one of the most economically important winter legumes and the birth of the science genetics is associated witho this model crop plant since Mendel’s experiment. On the basis of end use the crop pea is classified as a green or field pea; the green immature seeds of the former one are eaten as vegetable whereas the dry seeds of the latter are utilized. Winter cultivars have a longer life cycle and produce higher biomass than the spring cultivars (Baldwin et al. 2014). Winter peas face major challenge in temperate areas regarding plant protein production (Klein et al. 2014). Frost and chilling stress cause seedling death, limited adaptability and productivity in pea as well abortion of buds, flowers, and pods, and reduction in seed size (Stoddard et al. 2006; Shafiq et al. 2012; Liu et al. 2017). Superior chilling and freezing tolerance depend on a higher inherent photosynthetic capability after cold exposure owing to cold-induced changes at the chloroplast level during cold acclimation (Grimaud et al. 2013). “Cold acclimation also induces an increase in the degree of methylesterification of cell wall pectins indicating a role for esterified pectins in cold tolerance” (Baldwin et al. 2014). Liesenfeld et al. (1986) found black-pigmented hilum to be linked with winter hardiness in pea. Under chilling stress, the levels of sugars like sucrose, glucose-6-phosphate, fructose- 6-phosphate, mannose-6-phosphate were found to be significantly increased in pea leaves (Streb et al. 2003). In an experiment conducted by Bourion et al. (2003) in spring as well as winter peas, cold acclimation was not observed in low light. Early flowering winter peas have increased risk of frost exposure to the flowers; late-flowering pea genotypes might have frost tolerance (Lejeune-Hénaut et al. 2004). Weller et al. (2012) identified QTL HR (HIGH RESPONSE TO PHOTOPERIOD) as an ortholog of circadian clock gene EARLY FLOWERING 3 (ELF3). Klein et al. (2014) mentioned HR to have a pleiotropic effect on frost tolerance in pea. Lejeune-Hénaut et al. (2008) evaluated RILs from Champagne (tolerant) × Terese (susceptible) and detected six QTLs for winter frost tolerance among three, located on pea linkage groups (LG) 3, 5, and 6 respectively, namely WFD 3.1, WFD 5.1 and WFD 6.1, which showed consistency; the microsatellite markers close to those were AA175 and AA475 for the first two QTLs, respectively, and for the third QTL the close markers were AA200, AD159, AD141, and AD59. The dominant allele of the flowering locus HR co-localizes with the highest explanatory WFD 3.1, hence this could be utilized for MAS. QTL analysis of frost damage in pea RILs derived from China (freezing tolerant) × Caméor (highly frost sensitive) by Klein et al. (2014) revealed several QTLs related to frost tolerance on different genomic regions including LG3, LG5, and LG6. Their results suggested selection of winter frost tolerance in pea independently of seed yield and quality. Dumont et al. (2009) detected an association between frost damage resistance QTL with two raffinose QTLs and few protein quantitative loci on LG5 and LG6. Legrand et al. (2013) developed molecular markers from the differentially expressed genes and genotyped them on RILs from Champagne × Terese. They additionally recognized five candidate genes coexisting with three distinctive frost damage-linked QTLs on LG3,

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LG6, and LG7 and a protein quantitative loci-rich region located on LG6 rich region. Freezing tolerance is concomitant with reduced photosynthetic gene expression and the expression of genes permitting the assembly of cryoprotectant agents such as cysteine and methionine. Using highly multiplexed SNP genotyping, Deulvot et al. (2010) identified many SNP markers representing different genes associated with cold acclimation in pea. Mt-FTQTL6 is a vital freezing tolerance QTL detected on chromosome 6 of Medicago truncatula Gaertn. Five Mt-FTQTL6-linked markers—MTIC153, NT6067, Ps92K09T, NT6012, and NT6032—were also mapped on freezing damage QTL on pea LG6, indicting the utility of cross-legume markers in cool-season legumes (Tayeh et al. 2013). Using the F2 population of pea derived from G0003973 (winter hardy) × G0005527 (cold sensitive), Sun et al. (2014) mapped 157 SSR markers in 11 pea LGs with an average interval of 9.7 cM. Using the Illumina HiSeq 2500 System (next generation sequencing) in the same F2 population of pea, Yang et al. (2015) identified 33 polymorphic SSR markers. The pea breeder could utilize those SSRs for MAS regarding low temperature tolerance. Conducting marker-trait association analysis of frost tolerance, Liu et al. (2017) screened the 16 most winter-hardy germplasms from 672 diverse pea accessions and identified the frost tolerance-associated marker EST1109 on LG6; it is associated with a gene involved in the glycoprotein metabolism in response to chilling stress, providing a novel mechanism of frost tolerance. These findings could be helpful in marker-assisted breeding for winter-hardy pea cultivar. 18.7.3.3

Oilseed Brassica

Compared with other vegetable oils, rapeseed/canola (B. napus L.) is considered to be healthy for human consumption owing to appropriate combinations of the essential fatty acids in the seeds. Canola plants can be injured by exposure to frost. Biennial types of canola grow as overwintering crops in cooler climates and vernalization is required for their flowering. They possess winter survival characteristics and for higher expression of such winter survival they should be acclimated with freezing tolerance. B. rapa has better cold resistance than other Brassica species (Li 2011). Fiebelkorn and Rahman (2016) recommended adaptation of two-week old plantlets for 7 days at 4 ∘ C and after that frost treatment at −4 ∘ C for 16 h to assess frost tolerance in B. napus. Teutonico et al. (1993) established a correlation between winter survival and acclimated freezing tolerance. Kole et al. (2002) analyzed RIL populations of oilseed B. rapa and doubled haploid lines of B. napus. For the traits like winter survival, nonacclimated freezing tolerance, acclimated freezing tolerance, and nonvernalized flowering period, the number of identified QTLs was 5, 1, 2, and 4, including FR-2, respectively, in the B. rapa population. In case of B. napus population 16, 1, and 4 QTLs were identified respectively for the traits, namely winter survival, acclimated freezing tolerance, and nonvernalized flowering period; six QTLs for winter survival were found to be important in this case. From the corresponding QTL map significant allelic deviation at homologous loci in B. rapa and B. napus was detected. Findings from both of the aforesaid research groups suggested the existence of polygenic control on winter survival and freezing tolerance as revealed by the report of transgressive segregants for the mentioned traits; in those studies the parents delivered positive and negative effects on the concerned traits. A linkage map assigning nine LGs was constructed from the F2:3 populations of B. napus SLMO46 (winter type and cold resistant) × Quantum (spring type and cold susceptible) by Asghari et al. (2007). They

18.7 Marker-assisted Selection for Drought Tolerance

detected one putative QTL for winter survival explaining only 5% phenotypic variation using RAPD markers and the QTL was positioned on LG6 in between 670 and 650 bp sized RAPD markers with 4 cM distance from the 650 b marker. In another similar population using polymorphic 32 SSR and 47 RAPD markers, a linkage map assigning 14 LGs was prepared and four putative QTLs were detected on LGs 3, 8, 9, and 10, cumulatively explaining 24% variation in the freezing tolerance (Asghari et al. 2008). Pu and Sun (2010) reported some chemical and physiological indexes such as activities of superoxide dimutase, catalase, soluble protein content and malondialdehyde (MDA) content conferring the cold resistance on winter turnip rape (B. campetris) to some degree. The relative conductivity is also considered to be an important indicator, having a significantly negative correlation with cold resistance in B. napus (Huang et al. 2014). Using B. rapa EST and Microarray Database (BrEMD) in analysis of KBGP-24 K oligo chip data, 417 genes were primarily identified as cold responsive genes in B. rapa and one gene conferring cold stress resistance was finally confirmed and named as BrCSR (Yu and Park 2014). Huang et al. (2017) showed the relation of frost damage to the relative conductivity and MDA content in B. rapa. They also correlated 10 SSR markers with relative conductivity and 11 SSR markers with MDA content. Four SSR markers (Na10-C03, BrGMS4511, BrGMS397, and BnGMS164) and five SSR markers (Na10-C03, BrGMS4511, BrGMS397, BRAS011, and BnGMS67) showed highly significant positive association with respect to relative conductivity and MDA. Three SSR markers (Na10-C03, BrGMS4511, and BrGMS397), showing significant positive correlation to both of these two indexes, could be utilized to screen cold-resistant genotypes. 18.7.3.4

Potato (Solanum tuberosum)

Potato is a vital noncereal food crop and it is considered to be central to world food security (The Potato Genome Sequencing Consortium 2011). It is a winter crop and is grown mainly in temperate areas; hence irregular frosts frequently decrease its yield and quality. The threshold temperature for cold acclimation was found to be ∼12 ∘ C. Under 12 ∘ C, tuber hardiness gradually increased; the extreme level of resilience was discovered following 15 days of cold acclimation (Chen and Li 1980). Hijmans et al. (2003) found strong association of frost tolerance with the wild potato species. Chen and Li (1980) classified tuber-bearing Solanum species in terms of cold tolerance and cold acclimation into five sets, viz. “(I) frost resistant and able to cold harden, (II) frost resistant but unable to cold harden, (III) frost sensitive but able to cold harden, (IV) frost sensitive and unable to cold harden, and (V) chilling sensitive.” S. tuberosum falls under the group IV. Furthermore, this potato species showed its inability to be acclimated to cold conditions and suffered damage at −3 ∘ C as revealed from the study. Therefore we can say that potato is highly susceptible to low temperatures among the temperate crops. Vega and Bamberg (1995) screened high levels of frost hardiness in some wild potato germplasms such as S. acaule, S. albicans, S. commersonii, S. demissum, and S. paucissectum which expand the donor options for the potato breeders. Earlier Stone et al. (1993) explained the possibility of incorporating the freezing tolerance and acclimation ability traits from the wild germplasm of potato into the cultivated potato by independent selection, as also supported by Valverde et al. (1999). Vega et al. (2003) constructed a partial genetic linkage map in a backcross population obtained from frost-tolerant S. commersonii × frost-sensitive S. cardiophyllum with the aid of 113 RAPD markers

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and 12 potato SSR markers, and identified two QTLs each for “nonacclimated relative freezing tolerance” and “cold acclimation capacity” at separate genomic regions in a linkage assembly, detected as a portion of chromosome V; these are the two key constituents of tolerance against freezing that display independent genetic control.

18.8 Outlook Conventional methods of crop breeding related to abiotic stress tolerance or stress resistance face hurdles in selecting high yielders owing to the complex nature of the genes involved. Environmental influences play a crucial role in expressing such traits, as in all of the cases where the QTLs are associated. Owing to the introduction of molecular genetics and marker concepts into breeding science in the last few decades, a faster route to crop improvement has been constructed. Naturally, more detailed knowledge of the putative genes and QTLs corresponding to the traits accompanied by their relative positions amongst the linked markers in the relevant genetic map makes the path smoother. Discovery of the DNA markers has enriched the genomic database and obviously genome exploitation and crop breeding have improved. Phenotyping, the bottleneck in crop breeding, is significantly compensated for by the application of MAS in terms of time and cost savings. Without affecting the ‘genotype × environment’ interaction, gene epistasis, or even the plant growth phases, molecular markers easily pick up homozygous lines from the segregating or mapping populations for the desired characteristic. MAS approaches in crop breeding for abiotic stress tolerance not only accelerate the screening of the donor lines, furthermore it also aids in the identification and characterization of the tolerance-specific loci, as well as their introgression into susceptible high yielders. In the previous sections we reviewed some research stories utilizing MAS techniques for the abiotic threats, such as heat, drought, salinity, and low temperature, in some major cereals, pulses, oilseeds, fruit, and vegetables. This might deliver encouragement to the readers in developing ideas because more studies are necessary to generate climate-resilient crops for uninterrupted food supply for our climate-fluctuating Earth; here MAS could be an obvious choice.

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19 Transgenes: The Key to Understanding Abiotic Stress Tolerance in Rice Supratim Basu, Lymperopoulos Panagiotis, Joseph Msanne, and Roel Rabara NMC Biolab, New Mexico Consortium, Los Alamos, NM, USA

19.1 Introduction Environmental stresses like salinity, drought, heavy metals, and temperature extremes, adversely affect crop yield and productivity (Atkinson and Urwin 2012; Singh et al. 2015; Tripathi et al. 2016, 2017; Kumar et al. 2017; Singh et al. 2017). Exposure of plants not only reduces yield, but also affects plant growth and subsequently leads to plant death (Arif et al. 2016a,b; Liu et al. 2018). Salinity stress predominantly leads to abrupt closing of the stomata, thereby creating an imbalance in carbon dioxide/oxygen ratio that consequently leads to the buildup of negatively charged reactive oxygen species (ROS) and hence oxidative stress. This excessive ROS generation under stress conditions cannot be neutralized by the antioxidant defense machinery of the cell and hence is detrimental to the plants (Basu et al. 2010; Arif et al. 2016b). Over the years, damage to agricultural lands owing to drought or salinity has led to a severe threat to the maintenance of a continuous food supply to feed the ever increasing world population. The threat has accelerated tremendously owing to the looming effects of climate change. Considerable advances were made in the last century by the plant breeders in developing drought- and salt-tolerant cultivars using conventional breeding strategies. Marker-assisted breeding (MAB) and quantitative trait loci (QTL) are techniques commonly used by breeders to meet the needs of developing stress-tolerant crops. MAB has facilitated the identification of several operational regions in the genome for a crop under stress and also enhanced the ability of maize to tolerate water logging and hence improved yield and productivity, thereby clearly justifying it as an efficient approach. QTL analysis, on the other hand, refers to the identification of specific genomic regions in a stress-tolerant crop that correlates to the genes expressed under conditions specific to stresses (Dixit et al. 2015; Kumar et al. 2014). Several salt- and drought-tolerant QTLs have been identified for a variety of traits. Compared with MAB, which is less expensive and error prone, QTL analysis is more specific, but it refers to a big region on the chromosome that needs to be delineated eventually. To find a solution to the problems, it is necessary to improve crop varieties so that they can yield under abiotic stress conditions. Under these circumstances, a transgenic approach seems to be the most preferred solution that is more substantial and useful. It has been pursued all over the world to improve quantitative and qualitative traits Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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pertaining to salt and drought stress. Thus, the primary aim is to develop plants using a transgenic approach that can maintain biomass and yield well under stress conditions. Transgenic approaches make use of genes either from the native plants or other plant species or bacteria that are either overexpressed or knocked out using RNAi or gene-editing techniques. These genes mainly include transcription factors (TFs), genes encoding for metabolite production or cofactors involved in the post-translational modification of protein. The transgene expression can be achieved by integrating it stably into the plant genome mediated either by plant vectors or by transforming the nuclear genome. The transgenic plants that have been developed include not only rice, but also other plant species like tomato, maize, and barley. Our chapter focuses on the various aspects of transgenic approaches in rice that have been developed and employed over the years to enhance tolerance to salt and drought stress.

19.2 Drought Effects in Rice Leaves Conventional QTL analysis of drought tolerance in rice, often based on a bi-parental cross between lowland and upland rice varieties, has been studied extensively. Flag leaf is the key contributor to grain filling. To enable successful grain filling under drought, the flag leaf has to remain unaltered or be reprogrammed considerably so that it can continue the synthesis and translocation of photoassimilates. Over the years, several traits of flag leaf have been suggested for selecting plants that can tolerate drought, like area of the leaf, relative dry weight, weight loss of excised leaf, content of chlorophyll, late senescence and carbon isotope discrimination (Biswal and Kohli 2013). Genotypic variation in rolling of the leaf is normally genetic, and several reports on rice have identified QTLs associated with leaf rolling (Subashri et al. 2009; Salunkhe et al. 2011). Leaf rolling is an adaptive response to combat water deficit in rice, while leaf angle is related to developmental plasticity when drought stress occurs (Chutia and Borah 2012). In addition, there are other parameters which are affected by drought, like decrease in leaf number (Cerqueira et al. 2013). Hence, it can be concluded that parameters like leaf number, area, angle of leaf and developmental plasticity contributing to rolling and unrolling of leaf can be election criteria for drought tolerance. In the recent era, statistical analysis of the association between genotypes and phenotypes based on linkage disequilibrium has been the preferred method of analysis for identifying loci contributing to particular traits. The availability of whole genome sequences in rice has released unprecedented information about polymorphisms, notably single nucleotide polymorphisms. This genome-wide association mapping, when compared with QTLs from multiple bi-parental populations, will reveal information on markers of drought tolerance. Co-localization of QTLs/genes will suggest mechanistic links between parameters (i.e. between plant hormones and root architecture or between stomatal conductance and drought tolerance). Almost 1.7 million single nucleotide polymorphisms have been identified in rice, by comparative analysis of the draft genomic sequences of cv. Nipponbare (Kurokawa et al. 2016).

19.3 Molecular Analysis of Drought Stress Response Drought stress is a polygenic trait. Research over the years has identified several genes that are known to contribute to drought stress response and hence tolerance

19.4 Omics Approach to Analysis of Drought Response

both transcriptionally and posttranscriptionally (Shinozaki and Yamaguchi-Shinozaki 2007) A thorough dissection of these complex traits into their genetic components is of utmost importance before moving onto manipulating them. QTLs are regions within the genome comprising genes that are predictably associated with a particular quantitative trait and can be identified by genome mapping using molecular genetic markers (Manickavelu et al. 2006). Although several QTLs contributing to drought tolerance have been identified at the seedling or vegetative stage, recent studies are mainly focused on identifying QTLs contributing to yield under drought.

19.4 Omics Approach to Analysis of Drought Response Omics approaches can be grouped into three different categories: (i) transcriptomics; (ii) metabolomics; and (iii) epigenomics 19.4.1

Transcriptomics

A global picture of drought stress-induced changes in gene expression can be obtained from transcriptome analysis (Roychoudhury and Banerjee 2015). A thorough analysis of the identified QTLs and the drought-responsive rice genes has identified the involvement of several TFs that belong to either abscisic acid (ABA)-dependent or ABA-independent pathway. The ABA-dependent TFs mainly include (i) the bZIP class of TFs and (ii) NAM, ATAF, CUC2 (NAC). The bZIP class of TFs has a conserved basic leucine zipper domain that recognizes the ABA-responsive element (ABRE) in the promoter regions of downstream target genes and hence they are also known as ABA responsive element binding (AREB)/ABRE binding factors (ABFs) (Yang et al. 2010; Banerjee and Roychoudhury 2017). Previous studies have shown that AREB1, AREB2, and ABF3 confer drought tolerance by acting cooperatively in an ABA-dependent manner (Yoshida et al. 2010). Again OsABF2 (Oryza sativa ABA-responsive element binding factor 2) has been identified to contribute to drought tolerance in rice in an ABA-dependent manner by regulating genes inducible by ABA (Hossain et al. 2010; Roychoudhury et al. 2013). Recently, researchers have also identified that overexpression of OsbZIP23, OsbZIP46, and OsbZIP72 not only increased drought tolerance in rice but also improved tolerance to other environmental adversities like salinity and temperature in an ABA-dependent manner (Xiang et al. 2008; Lu et al. 2009; Tang et al. 2012). Zhang et al. (2017) have identified OsABF1 as the master regulator of drought tolerance in rice operating by the ABA-dependent pathway by regulating the manifestation of bZIPs, OsPP48, OsPP108, and COR413-TM1 that encode a membrane protein associated with thylakoid. NACs are another group of ABA-dependent TF families in rice that are responsible for conferring drought tolerance. Previous reports have shown that overexpression of SNAC1 in rice improved tolerance to drought by reducing water loss and increased sensitivity to ABA (Hu et al. 2006). SNAC3, ONAC045, and ONAC022 have also been shown to improve drought tolerance in rice by increasing the accumulation of ABA and level of osmolytes like proline, and also by modulating the expression of genes responsive to ABA (Zheng et al. 2009; Fang et al. 2015; Hong et al. 2016). Although NAC TFs confer tolerance to abiotic stresses, recent studies by Huang et al. (2016) have shown that ONAC095 confers

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tolerance to cold, but promotes drought sensitivity. AP2/ERFs, myeloblastosis (MYB), WRKYs, and zinc fingers are another group of TFs that are known to play a role in drought tolerance via the ABA-independent pathway. Previously, it has been shown that overexpression of HYR (High Yield Rice) and OsAP37 not only increased drought tolerance at the vegetative stage, but also improved yield under drought (Oh et al. 2009; Ambavaram et al. 2014). Overexpression of OsERF48 in rice enhanced drought tolerance by regulating the expression of calmodulin-like protein OsCML16 and improving root growth (Jung et al. 2017). Dehydration-responsive-element-bindings (DREBs), which are ABA-independent dehydration-responsive TFs, bind to the dehydration-responsive element (DRE) or C-repeat (CRT) in the promoters of droughtor cold-responsive genes. DREB/C-repeat binding factors belong to the superfamily of AP2/ERF. When OsDREB1A was overexpressed in Arabidopsis, it resulted in dehydration tolerance (Dubouzet et al. 2003). However, overexpression of OsDREB1F in rice enhanced drought tolerance by inducing the expression of genes belonging to both ABA-independent and ABA-dependent pathways. Thus, OsDREB1F can be regarded as a molecular bridge between the ABA-dependent and ABA-independent pathways (Wang et al. 2008). WRKY TFs are involved in several biological processes and have been predicted to be a key link between responses of plants to abiotic and biotic stresses (Banerjee and Roychoudhury 2015). Research from independent groups has shown that overexpression of OsWRKY11, OsWRKY13, and OsWRKY30 enhanced drought tolerance, while OsWRKY45-2 had a negative effect (Wu et al. 2009; Tao et al. 2011; Shen et al. 2012; Xiao et al. 2013). MYBs are another class of TFs that comprise the MYB domain with 52 amino acids containing one to four imperfect amino acid sequence repeats (R). MYBs are primarily classified into four subfamilies based on the repeats: MYB-related (one single MYB domain); R2R3-MYB (two MYB domains); R1R2R3-MYB (three MYB domains); and 4R-MYB (four MYB domains). Yang et al. (2012) have shown that overexpression of OsMYB2 in rice enhanced drought tolerance by increasing the concentration of antioxidant enzymes like peroxidase, superoxide dismutase, and catalase. In addition, it has also been reported that overexpression of OsMYB48-1 and OsMYBR-1 improved drought tolerance by increasing the endogenous ABA concentration (Xiong et al. 2014; Yin et al. 2017). Besides overexpression of these TFs in rice, when some of them like OsMYB55 and OsMYB3R-2 were overexpressed in maize and Arabidopsis, respectively, they were shown to confer drought tolerance (Dai et al. 2007; Casaretto et al. 2016). C2H2-type ZFPs have been reported to be inducible by abiotic stresses and also confer drought tolerance. Overexpression of OsZFP252 and ZAT10 has been shown to confer drought tolerance by either DREB1 pathway or H2 O2 -mediated closure of stomata (Xu et al. 2008; Xiao et al. 2009). In contrast, it has been observed that overexpression of DST (drought and salt tolerance) (Huang et al. 2009) or OsiSAP8 (Kanneganti and Gupta 2008) resulted in a drought-sensitive phenotype. Over the years, many TFs have therefore been identified to be inducible by drought stress. 19.4.2

Metabolomics

Metabolome refers to the total metabolite content of the cell that includes hormones, signaling molecules, and secondary metabolites and is analyzed by HPLC-MS, GC-MS,

19.4 Omics Approach to Analysis of Drought Response

CE-UV, and HPTLC (chromatographic) and LC-MS-NMR, GC-IT-MS-MS, and LC-MSMDF (combined). A comprehensive analysis of the metabolite profile of stressed and nonstressed rice genotypes will give an idea about the changes in metabolite pool to restore homeostasis and lead to stress tolerance. Some of the stress-associated metabolites include polyols, betaines, amino acids, and sugars like sucrose, glucose, and fructose (Basu et al. 2010). The metabolites play an essential part in steadying the photosystem II complex, integrity of the membrane, scavenging of the ROS, chelators, and signaling molecules (Alcázar et al. 2010). Metabolome analysis can be used periodically to dissect the tolerance mechanism in rice where the drought tolerance mechanism has been characterized as ABA dependent or ABA independent with reference to ABA levels (Shinozaki and Yamaguchi-Shinozaki 2007). A study performed by Shu et al. (2011) has shown that under drought stress there was an enhanced consumption of energy that came from a reserved substance under drought stress. In fact, there was an enhancement of the expression of genes involved in anabolic pathways leading to amino acid biosynthesis as well as enhanced levels of energy transfer from fatty acids and carbohydrates into amino acids. 19.4.3

Epigenomics

A well-known concept in abiotic stress acclimation of plants is retaining the memory of stress, but for a short period of time (Thomashow 1999). Short-term response memory accrual in plants on exposure to stress depended on the half-life of stress-induced transcript accumulation, proteins, and metabolites, and could be prolonged depending on alteration in morphology and phenology. Epigenetic regulation like histone modifications, effects of small RNAs and DNA methylation, can be attributed to long-term stress memories (Chinnusamy and Zhu 2009; Banerjee and Roychoudhury 2018). Previously epigenetic regulation has been proposed to be involved in drought response in rice and Arabidopsis and hence epigenomics that involves the use of techniques like ChiP-sequencing (this method combines ChiP with next-generation sequencing), shotgun bisulfite sequencing, and methylated-DNA immunoprecipitation can be used to derive a bigger picture. Comparative analysis of DNA methylation pattern in diverse rice genotypes in response to drought stress identified that alteration of DNA methylation pattern is genotype, tissue, and stage specific (Wang et al. 2011). In addition, recent reports have shown that adaptation of plants to prolonged drought stress is correlated with several trans-generational epimutations. These epimutations are inherited in the progenies as well and thereby result in the methylation of DNA of several drought-responsive genes (Zheng et al. 2017). At least 18 homologs of histone deacetylases (HDACs) have been identified in rice and proposed to be involved in drought stress. These HDACs have been classified into three groups, reduced potassium dependency3/histone deacetylase 1 (RPD3/HDA1), silent information regulator 2 (SIR2) and HD2. Although several studies have been carried out over the years on the role of epigenetic regulation in drought tolerance, only little progress has been achieved. The observed results have shown the importance of the emerging role of epigenomics in dissecting the complex mechanism of drought tolerance. OsHDAC1 has been reported to epigenetically regulate the expression of OsNAC6 that regulates the expression of genes controlling drought tolerance like nicotinamine biosynthesis

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and glutathione relocation (Lee et al. 2016; Banerjee and Roychoudhury 2018). Other than the HDACs, there are HATs (histone acteyl transferases) that have been reported to be induced during drought stress in rice, like OsHAC703, OsHAG703, OsHAF701, and OsHAM701 (Fang et al. 2014). In addition, Miniature inverted–repeat transposable elements have been also reported to positively influence ABA signaling and drought tolerance in rice by controlling the expression of siRNAs like siR441 and siR446 (Yan et al. 2011).

19.5 Plant Breeding Techniques to Improve Rice Tolerance Recently, gene editing techniques have been developed and rely on meganucleases, transcription-activator like effector nucleases and the CRISPR/Cas system. In addition to this, intragenesis, RNA-dependent DNA methylation, reverse breeding, and agro-infiltration are still considered. OsDERF1, an AP2 domain protein, has been targeted using the CRISPR/Cas9 system, and inferred to play a role in drought stress tolerance (Zhang et al. 2014). For a long time, it has been believed that food security can be obtained by developing drought-tolerant rice crops that can survive the detrimental effects of drought and water shortage (Xiao et al. 2009). Tolerance to drought is a complex phenomenon involving biochemical, physiological, developmental, and cellular adjustments. A number of these changes include elongation of guard cells, root growth, osmotic adjustment, photosynthetic alterations, and the production of antioxidants and defense proteins. Plant breeders have used several strategies for enhancing drought avoidance by reducing the duration of crop growth, and hence decreasing recurrent transpiration (Tuong 1999), while sustaining yields through superior harvest indices, or controlling transpiration through the cuticle either to keep the canopy of the crop cool or reduce nonbeneficial depletions. The length of the roots, root density and surface area, and water uptake rate are some of the essential traits that are also closely associated with water and nutrient acquisition ability and in turn can affect the ability of the crops to suppress weeds (Korres et al. 2016). In spite of the systematic breeding efforts in rice in regulating the properties of the root system, there are only limited numbers of reports where root traits have been targeted. Transgenic rice generated by overexpressing ots A and ots B genes from Escherichia coli have resulted in increased trehalose biosynthesis and consequent tolerance to drought and salinity (Garg et al. 2002).

19.6 Marker-assisted Selection Marker assisted selection (MAS) is an indirect method of selection for genetic determinant or determinants contributing to a particular trait of interest like tolerance to abiotic stresses, resistance to disease, yield/productivity (Prabhu et al. 2009). Selection of the plants carrying the genomic region of interest and contributing to the trait expression is done using molecular markers. MAS has become increasingly common these days for identification of traits associated with key genes and QTLs, owing to the availability of dense molecular maps and an array of molecular markers (Choudhary et al. 2008). The successful application of MAS is dependent on several factors that not only include the target genes to be transferred, but also rely on the distance between the

19.7 Transgenic Approach: Present Status and Future Prospects

markers flanking the target (Perumalsamy et al. 2010). MAS enables genotypic selection of the individual plants during the selection process. Moreover, MAS can be used for the selection of donor parents, thereby increasing the effectiveness of breeding by backcross and sex-limited traits (Zhou et al. 2007). In addition, MAS finds its application for investigating heterosis for hybrid crop production and also allows for the prospective use of DNA markers, as well as phenotypic data for selecting the hybrids (Jordan et al. 2003). Furthermore, MAS selection can be performed at the seedling stage and plant phenotypes that are undesirable can be eliminated (Khan et al. 2015). To conclude, it can be said that the advantages associated with markers include consistency, speed, and biosafety, and allow provision for skewing the odds in our favor when dealing with complex traits.

19.7 Transgenic Approach: Present Status and Future Prospects In an effort to dissect the complexity of the tolerance mechanism in rice, several drought-responsive genes that are related to posttranslational modification, signal transduction, or metabolite biosynthesis have been overexpressed in rice (Yang et al. 2010). These drought-responsive genes have been transformed in rice using constitutive or drought-inducible promoters. Initial research on drought tolerance using a transgenic approach primarily focused on structural or functional genes that encoded late embryogenesis abundants (LEAs), osmolytes like proline or heat shock proteins, or transporters or key genes of the phytohormone biosynthesis pathway (Kishor et al. 1995; Xiao et al. 2007; Sato and Yokoya 2008; Xiao et al. 2009; Hwang et al. 2018). Genes contributing to posttranslational modification and hence drought tolerance have been grouped into two categories based on their function. One group is related to protein farnesylation and includes genes like OsCDPK7 that regulate the expression of LEA genes (Saijo et al. 2000). Another key member of this group are the kinases [Receptor like cytoplasmic kinase (RLCK), mitogen activated protein kinases (MAPKs)] that regulate the expression of downstream genes by phosphorylation. GUDK (Growth Under Drought Kinase) and OsCIPK03 have been reported to regulate its downstream target genes by phosphorylating them either alone or as a complex (Yang et al. 2010; Ramegowda et al. 2014). TFs play a crucial role in improving drought tolerance by binding to the cis-elements in the promoter region of downstream target genes and hence are referred to as a TF regulon. The major TF regulons that have been characterized in rice refer to the DREB, MYB, NAC or bZIP (Nuruzzaman et al. 2013). In spite of the identification of several regulatory elements, the major concern that still remains is the identification of promoters and developing a clear understanding of the physiological, molecular, and metabolic perspectives for a desired trait. Comparative analysis of promoters has been carried out in the past by overexpressing DREB1A under the control of an inducible promoter from rd29A as well as CaMV35S promoter and it was observed that transgenic plants behaved better under the control of the rd29A promoter in drought stress (Kasuga et al. 1999; Yamaguchi-Shinozaki and Shinozaki 2001). Growth and developmental stage-specific expression of genes is also another important aspect that needs to be taken into account for gene expression under drought stress. In addition, the short- and long-term effect of genes affecting

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the cellular function or the plant phenotype is another factor that is to be considered. The primary consideration for evaluation of transgenic plants for drought tolerance is that they are tested under greenhouse conditions and need to be examined for their effectiveness in field conditions as well, but this is restricted by biosafety regulations (Todaka et al. 2015). Hence, this dilemma has two schools of thought—one that still believes that transgenics are still the most effective way to improve drought tolerance, and the other that believes that there are some modifications that need to be made, like (i) targeting multiple genes at a time instead of a single gene, since drought tolerance needs a concerted approach, (ii) assessing the effect of stress on yield and the biological cost of producing the metabolites under the stress condition, and (iii) identifying the physiological responses of plants to drought stress and then choosing the promoters appropriately (Bhatnagar-Mathur et al. 2008; Paul and Roychoudhury 2018). Under the current scenario, it is necessary to develop an integrated strategy combining conventional molecular breeding to identify the QTLs for yield under drought, undertake fine mapping, clone them and then perform transgenic biology to confirm the function of the identified candidate genes.

19.8 Looking into the Future for Developing Drought-tolerant Transgenic Rice Plants Transgenic rice plants have been reported with improved yield under drought in the field, but extensive research needs to be carried out to identify the mechanism governing drought tolerance under field conditions. A close insight into these studies will enable identification of novel candidate genes that will improve drought tolerance without yield penalty. One more step toward this research will be to dissect the mechanism of stress tolerance in stress-adapted or acclimatized extremophiles like halophytes, cold-water fishes, thermophilic bacteria, or desert plants (Mittler and Blumwald 2010). It has been observed that, even for well characterized genomes, the function of 18–38% of the total proteins is still unknown (Gollery et al. 2006). Drought tolerance in rice can be improved by modifying the root architecture, as has been observed by overexpressing DRO1 (Deeper Rooting 1) in rice, which improved root angle and hence yield under drought (Uga et al. 2013). In rice, several studies have been carried out for identifying submergence tolerance mechanisms and have shown that drought-tolerant rice genotypes with a background from submergence-tolerant cultivars are exceptional crop species that can survive under both conditions of low and high water availability. Change in climatic conditions may eventually force the plants to be exposed consecutively to both drought and flooding and hence efforts need to be made toward developing rice cultivars with water usage flexibility that can help meet the crisis.

19.9 Salinity Stress in Rice Another important environmental factor that affects rice production worldwide is soil salinity. It is estimated to affect up to 22% of Earth’s land area and around 77 Mha of irrigated land (Eynard et al. 2005; Guo et al. 2014). Yield loss owing to salt stress can

19.9 Salinity Stress in Rice

be as high as 90%, as in the case of wheat and sugarcane (Eynard et al. 2005). In rice, a glycophyte, soil salinity could result in yield loss as high as 69% as observed in the Indus Basin in Pakistan (Roychoudhury and Chakraborty 2013; Al-Tamimi et al. 2016). Under high-salinity conditions, plants are under two major stresses: osmotic stress and ionic stress (Horie et al. 2012). Under osmotic stress, plants grown in saline soil can show growth reduction (Al-Tamimi et al. 2016). Salinity can also induce ionic stress in plants, which is the result of accumulation of toxic concentrations of Na+ and Cl− in the cells in plant shoots. Owing to its impact on rice production, breeding for salinity tolerance had been one of the major goals in rice research with emphasis on growth, yield, and yield components (Ali et al. 2014; Hoang et al. 2015). Physiological markers have also been used as criteria for screening salt tolerance in rice (Zeng et al. 2003; Hoang et al. 2015). Zeng et al. (2003) proposed the Na–Ca selectivity as one physiological component in rice tolerance to salinity. This group observed that there is highly significant correlation between Na–Ca selectivity and ranking in genotypes for grain yield (Zeng et al. 2003). Tolerance to salt stress in rice occurs by two mechanisms, exclusion of ions and osmotic tolerance (Munns and Tester 2008; Roychoudhury et al. 2008). The exclusion of ions primarily refers to the transport of Na+ and Cl− in roots, thereby preventing the excessive accumulation of ions in the leaves. In addition, ion exclusion involves the efflux of negatively charged radicals into the soil, while recovering the Na+ from the xylem. Long-distance signals are known to regulate osmotic tolerance and to get activated before Na+ accumulation in shoot, and subsequently reduce the growth of the shoot. Hence, it is postulated that osmotic tolerance is the innate ability of the plant to counter osmotic stresses like salt and drought by maintaining the expansion of the leaf and stomatal conductance (Rajendran et al. 2009). Some of the important classes of genes are OsKCO1 (K+ outward-rectifying channel), OsAKT1 (K+ inward-rectifying channel) (Yang et al. 2014), OsSOS1, OsHKT2.1 (Na+ /K+ symporter) (Mishra et al. 2016), OsNHX1 (Na+ /H+ antiporters) (Amin et al. 2016), OsCAX1 (H+ /Ca+ antiporter) (Kumar et al. 2013), OsCLC1 (Cl− channel) (Diedhiou and Golldack 2006), OsTPC1 (Ca2+ permeable channel) (Kurusu et al. 2012), and OsNRT1.2 (nitrate transporter) (Wang et al. 2012). In a recent article, Chunthaburee et al. (2016) evaluated 12 rice cultivars, four white rice and eight black glutinous rice cultivars. They found an increase in hydrogen peroxide activity, proline, and anthocyanin in all cultivars. Salt stress induced physiological changes in all rice cultivars. K+ /Na+ is highly correlated with several physiological parameters, including salt injury scores, proline, anthocyanin, H2 O2 , peroxidase, and catalase activity, which could be used as supplementary or alternative indicators for salt tolerance screening. The black glutinous rice Niewdam was identified as the most salt-tolerant cultivar, as indicated by cluster analysis, which can be used as a target cultivar for selection and breeding programs for salt tolerance improvement in future studies. Currently, there are three major methodologies for improving salt tolerance in plants: (i) conventional breeding; (ii) MAS; and (iii) genetic engineering (Hoang et al. 2015). The utilization of QTL mapping is being widely adopted in genetic dissection of quantitative traits such as salt tolerance (Thomson et al. 2010; Tiwari et al. 2016). Generated QTL maps could be the basis for map-based cloning of genes responsible for tolerance traits as well as for a MAS breeding approach for crop improvement (Tiwari et al. 2016). A major QTL for salt tolerance, Saltol, was identified in chromosome 1 of a recombinant inbred lines (RILs) population derived from crosses between the salt-tolerant rice Pokkali and salt-sensitive IR-29

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(Gregorio 1997; Bonilla et al. 2002). Analyses of series of near-isogenic lines and backcross lines revealed that the Saltol locus mainly acted to control homeostasis of Na+ /K+ in the shoot and that it requires multiple QTLs for achieving the highest level of salt tolerance in rice (Thomson et al. 2010). High-throughput phenotyping has also been employed to characterize a large pool of rice genotypes to identify rice responses to salt stress. Al-Tamimi and colleagues used plant imaging technology to phenotype 553 rice genotypes belonging to indica and aus groups and revealed that the indica group maintains growth and transpiration better than the aus group under 150 mM NaCl condition (Al-Tamimi et al. 2016). Their findings also revealed that indica have a higher early growth response index than aus. They also validated the higher early growth response index value of the salt-tolerant Pokkali rice compared with salt-sensitive rice IR-28. These findings suggested that early rice response to stress is an important component of the overall mechanism of rice tolerance to salt stress (Al-Tamimi et al. 2016). The use of genetic engineering technology is another important approach for developing rice with improved tolerance to salt stress. Research was carried out by overexpressing genes from diverse sources regulating programmed cell death and their findings revealed that these genes were able to maintain metabolic activity in transgenic rice grown at 100 mM NaCl (Hoang et al. 2015). Hoang and colleagues overexpressed programmed cell death-involved genes (AtBAG4 from Arabidopsis, Hsp70 from Citrus tristeza, and p35 from baculovirus) in rice and demonstrated that the transgenic rice performed better both at seedling and at reproductive stages under salt stress (Hoang et al. 2015). Overexpression of TFs had also been employed to develop salt-tolerant rice. Xiong et al. (2014) overexpressed an MYB-related gene, OsMYB48-1 in rice and phenotyping of three-day-old transgenic rice grown under 150 mM NaCl for 10 days showed that the transgenic lines had longer shoot length and higher fresh weight compared with the wild type. The transgenic lines also showed improved tolerance to drought and showed high expression of ABA-related genes like OsNCED4, OsNCED5, OsLEA3, OsPP2C68, RAB16D, OSRK1, RAB16C, and RAB21, under drought stress as compared with the wild type. Overexpression of group 2 LEA gene Rab16A in tobacco and rice led to enhanced salt tolerance by regulating the antioxidative machinery and osmolyte levels (Roychoudhury et al. 2007; Ganguly et al. 2012).

19.10 Candidate Genes for Salt Tolerance in Rice Several studies have identified potential candidate genes that play an important role in salinity tolerance in rice. Al-Tamimi et al. (2016) considered the interaction of treatments with genetic markers to identify novel loci that may have an important role in the response of rice to salinity. Their group was able to identify new candidate genes (Table 19.1) that are involved in the early response of rice to salinity stress. Through the interaction model approach, they further noted that the early response of rice to stress is related to signaling mechanisms as exhibited by the expression of

19.11 QTL Associated with Rice Tolerance to Salinity Stress

Table 19.1 Candidate genes involved in rice response to salinity stress. Candidate gene

Chromosome

Gene annotation

Os03g16130

3

Calcium/calmodulin dependent kinase

Os03g16120

3

Myosin heavy chain-related

Os03g16334

3

Fringe-related protein

Os05g15920

5

Glycosyl hydrolase

Os05g39870

5

CAMK_KIN1 calcium/calmodulin dependent protein kinase

Os05g46320

5

OsFBX173—F-box domain containing protein

Os05g46350

5

IQ calmodulin-binding motif domain containing protein

Os05g39900

5

CBL-interacting serine/threonine-protein kinase 15

Os05g46490

5

Hydrolase, 𝛼/𝛽 fold family domain containing protein

Os05g49120

5

Nuclear LIM (Lin11, Isl-1 & Mec-3) interactor factor-like phosphatase

Os05g47670

5

Zinc finger, C3HC4 type domain containing protein

Os05g46490

5

Hydrolase, 𝛼/𝛽 fold family domain containing protein

Os08g18740

8

Zinc knuckle-family protein

Os11g07240

11

Serine/threonine-protein kinase BRI1-like 2 precursor

Os11g05930

11

Response regulator receiver domain

Os11g05935

11

Mucin

signaling-related genes such as Os05g39870 (encoding OsCIPK28 and CAMK_KIN1, calcium/calmodulin-dependent protein kinase), Os05g39900 (encoding a calcineurin-B like, CBL-interacting serine/threonine-protein kinase 15), Os03g16130 (encoding a calcium/calmodulin-dependent kinase), Os05g47670 (containing a zinc-finger motif, a C3HC4-type domain-containing protein) and Os05g46320 (encoding OsFBX173, an F-box domain-containing protein).

19.11 QTL Associated with Rice Tolerance to Salinity Stress Quantitative traits are complex and may involve multiple genes. Identification of the underlying genetic control mechanisms usually involves mapping of the QTLs. Several reports have been published on QTLs identified to be involved in rice tolerance to salinity. One of the well-characterized QTLs involved in rice tolerance to salt is Saltol (Gregorio et al. 1997; Bonilla et al. 2002), associated with Na+ /K+ ratio and salinity tolerance at seedling stage identified in chromosome 1 of rice (Bonilla et al. 2002). Thomson et al. (2010) extensively mapped QTLs from 140 IR-29/Pokkali RILs and identified 17 QTLs associated with salinity tolerance (Table 19.2).

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Table 19.2 QTL involved in rice response to salinity stress. QTL

Chromosome

Associated trait

Reference

Saltol

1

Maintaining the Na+ /K+ homeostasis

Gregorio et al. 1997

qPH2

2

Seedling height

Thomson et al. 2010

qPH4

4

Seedling height

qSNC1

1

Shoot Na+ concentration

qSKC1

1

Shoot K+ concentration

qSNK1

1

Shoot Na+ /K+ ratio

qSNK9

9

Shoot Na+ /K+ ratio

qRKC1

1

Root K+ concentration

qRKC2

2

Root K+ concentration

qRKC6

6

Root K+ concentration

qRNK1

1

Root Na+ /K+ ratio

qRNK6

6

Root Na+ /K+ ratio

qRNK9

9

Root Na+ /K+ ratio

qSES4

4

Final standard evaluation system (SES) tolerance score

qSES9

9

Final SES tolerance score

qCHL2

2

Leaf chlorophyll content

qCHL3

3

Leaf chlorophyll content

qCHL4

4

Leaf chlorophyll content

19.12 The Saltol QTL Saltol is an important QTL identified in rice that is responsible for salinity tolerance at seedling stage (Bonilla et al. 2002). This QTL was mapped from a population of RILs developed at the International Rice Research Institute from indica varieties IR-29 and Pokkali (Gregorio 1997). Further analysis by Bonilla et al. (2002) on 54 RILs screened under a hydroponic system showed that this QTL explained 43% of the variation for seedling shoot Na+ /K+ ratio in this population. Screening of these RIL populations could identify one highly salt-tolerant line, FL478 (IR 66946-3R-178-1-1), that was promoted as an improved donor for breeding programs, because of its high level of seedling stage salinity tolerance and being photoperiod insensitive, shorter with earlier flowering than the original Pokkali landrace. Transcriptomic analyses between IR-29 and FL478 revealed upregulation of ion transport and cell wall-related genes, while differential expression was observed in roots for cation transport proteins (Walia et al. 2005, 2009; Senadheera et al. 2009).

References

19.13 Conclusion Environmental stresses, primarily salinity and drought, cause immense crop losses worldwide. To maintain sustainable agriculture in the face of global climate change and increasing world population, it is of utmost importance for plant breeders to enhance the abiotic stress tolerance of crops. All of the studies carried out in the recent past for abiotic stress tolerant rice focused on a single gene either from a native source or from other plants, including genes like ion transporters, TFs, osmolytes, and signaling molecules. Hence, it is necessary to focus on a multigenic approach, keeping in mind the fact that drought stress in the field is multidimensional and hence plants need to modify the stress response accordingly. Owing to the research carried out over the years, we have been able to identify genes contributing to salt and drought tolerance, and partially understand the mechanism governing abiotic stress tolerance; however, we are still far from identifying the factor that is responsible for activating a gene under a specific stress condition. It is essential that an ideal genetically modified crop needs to have a well-orchestrated stress regulatory mechanism and should not affect the productivity of the plant under well-watered conditions. To conclude, it can be said that acquiring the knowledge about the molecular and physiological mechanism involved in the signaling pathways and hormonal crosstalk in response to salinity and drought stress will help in manipulating the crops easily and enhance the yield in the future.

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20 Impact of Next-generation Sequencing in Elucidating the Role of microRNA Related to Multiple Abiotic Stresses Kavita Goswami 1,2 , Anita Tripathi 1 , Budhayash Gautam 2 , and Neeti Sanan-Mishra 1 1 2

Plant RNAi Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, India

20.1 Introduction Decoding the genetic information packaged into the tiny chromosomes has always been a scientific fantasy. The ability to sequence the DNA of an organism was a significant development that radically influenced several aspects of biology. The process was initiated by a preliminary method based on extension of location-specific primers. Later, Maxam and Gilbert developed the method of chemical sequencing (Gilbert 1981). This method required radioactive labeling at the 5′ end of the DNA fragment to be sequenced and purification of the DNA. Chemical treatment was performed to generate breaks at a small proportion of one or two of the four nucleotide bases in four separate reactions (G, A+G, C, C+T). The technical complexity of using radioactivity in this technique deterred its general application. Around the same time, Fredric Sanger and his team established another strategy for “sequence-by-synthesis” using chain-terminating inhibitors (Jay et al. 1974). This developed it into an efficient and dependable technique of DNA sequencing, which soon became a method of choice both commercially and in laboratories. The ease and consistency of Sanger sequencing led to the development of its automated version, which laid the foundation for a first-generation DNA sequencer and Applied Biosystems developed the first automatic sequencing machine, AB370, in 1987. It adapted capillary electrophoresis to enhance sequencing speed and accuracy. Automated Sanger sequencing equipment and software were utilized for deciphering the first human genome in 2001 (Collins et al. 2003). The first plant genome to be sequenced was that of Arabidopsis thaliana (Kaul et al. 2000), by rice (Sasaki 2005) and maize (Pennisi 2008) genomes. The next revolutionary development in the sequencing technology was the discovery of a luminescent method for measuring pyrophosphate production during DNA synthesis (Nyrén and Lundin 1985; Hyman 1988). The method consisted of a two-step enzymatic process in which adenosine triphosphate (ATP) sulfurylase converted pyrophosphate into ATP, which served as the substrate for luciferase, thus producing light in proportion to the amount of pyrophosphate. This pyrosequencing technique * Kavita Goswami and Anita Tripathi contributed equally. Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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proved to be beneficial as it could be performed using natural nucleotides instead of the heavily modified deoxy nucleotide diphosphate (dNTPs) used in the chain-termination protocols, and the results could be obtained in real time instead of requiring lengthy electrophoresis (Ronaghi et al. 1998). Pyrosequencing was licensed to 454 Life Sciences (later purchased by Roche), where it evolved into the first major successful commercial Next Generation Sequencing (NGS) technology. The various NGS machines produced a paradigm shift by performing huge numbers of parallel sequencing reactions on a micrometer scale, greatly increasing the amount of DNA that can be sequenced in any one run (Margulies et al. 2005). The large scale facilitated the sequencing of long DNA fragments as well as larger pools of small DNA sequences. This method allowed the sequencing and assembly of unknown genomes and was thus termed as de novo sequencing, whole genome shotgun sequencing (WGS) or high-throughput sequencing. It was used to sequence several other plant genomes like Populus trichocarpa (Tuskan et al. 2006), grapevine (Jaillon et al. 2007), and sorghum (Paterson et al. 2009).

20.2 NGS Platforms and their Applications In the last couple of decades, sequencing technologies have undergone tremendous advances as a result of improvements in microfabrication and high-resolution imaging. It is now possible to perform single molecule sequencing and real-time sequencing. The NGS technology is becoming economical, fast, and easy to use (Figure 20.1). When coupled with bioinformatics and experimental methods, NGS can efficiently and quickly solve various complicated biological problems. This has opened up broad aspects for researchers and broadened its applications from viewing the complete genome or transcriptome to differentiating between strand-specific expression patterns, identifying sodium nitroprussides (SNPs) at a low coverage, assessing DNA–protein interactions, characterizing structural rearrangements, and identifying new transcripts or splice variants (Grada and Weinbrecht 2013). 20.2.1

NGS Platforms

Various NGS platforms are being used these days and a comparative description of those commonly used is provided in Table 20.1. 20.2.1.1

Roche 454

The earliest NGS methodology was developed in 2005 and promoted by 454 Life Sciences. It utilizes a method of base calling for determination of the sequence from captured signals. First, a library of DNA molecules is attached to beads via a fournucleotide known adapter sequence. Then, a water-in-oil emulsion polymerase chain reaction (PCR) (Tawfik and Griffiths 1998) is performed to coat each bead such that, on average, one DNA molecule ends up on one bead and amplifies in its own droplet in the emulsion. These DNA-coated beads are then washed over a picoliter reaction plate that fits one bead per well. The smaller bead-linked enzymes and dNTPs are washed over the plate and sequencing occurs by measuring the pyrophosphate release using a charged couple device sensor beneath the wells. The adapter sequence, present

20.2 NGS Platforms and their Applications

Selection and isolation of target DNA

Sample preparation

Clonal amplification of sequencing template

Amplification

Chemistry

Bridge amplification PCR (cluster generation of solid phase)

Emulsion PCR

Sequence by synthesis

Illumina/solexa sequencing

Pyrosequencing

Ion torrent squencing

Roche 454

Sequencing by ligation

ABi SOLiD

Data analysis

Figure 20.1 Schematic representation of the various next generation sequencing platforms.

at the start of the flowgram, is used to determine the sequence of the fragment. Peak intensity in the flowgram is directly proportional to the number of nucleotides in the sequence (Margulies et al. 2005). This technique is fast, suitable for sequencing a read length of 450–900 bases and has low capital cost and cost per experiment. The major disadvantages are low throughput, high cost per Mb of data and error-prone (polybase >6) sequencing. 20.2.1.2

ABI SoLid

This involves sequencing by Olignucleotide Ligation and Detection (SOLiD) and was developed by Life Technologies in 2006. In this method, a two-base fluorescent probe is used and sequences are obtained by ligation, building on principles previously developed in George Church’s group (Shendure et al. 2008). It utilizes the mismatch sensitivity of DNA ligase for identification of the nucleotide at a specified position in sequence. Emulsion PCR is performed using a library of DNA fragments (prepared from the sample to be sequenced) to generate clonal bead populations so that only one species of fragment will be present on the surface of each magnetic bead. The fragments attached to the magnetic beads carry a universal adapter sequence so that the starting sequence of every fragment is both known and identical. A set of four fluorescently labeled di-base probes compete for ligation to the sequencing primer. Specificity of the

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20 Impact of NGS in Elucidating the Role of microRNA Related to Multiple Abiotic Stresses

Table 20.1 Comparison of some commonly used sequencing platforms. Illumina/HiSeq

Ion torrent

SoliD ABI

Roche 454

Sequencing principle

Sequencing by synthesis

Sequencing by synthesis

Ligation and two base coding

Pyrosequencing

Instrument cost

Instrument $690 000, $6000/(30×) human genome

$80 000

Instrument $495 000, $15 000/100 Gb

Instrument $500 000, $7000 per run

Sequencing $0.07 cost per million base

$5

$0.13

$10

Technology

emPCR, H+ detection

emPCR ligation with cleavable dye terminators

emPCRpyrosequencing

Run time (days 5–6 per run)

2–4

7

0.95

Accuracy

99%

99.94%

99%

Polonies cleavable dye terminators

99.9%

Memory

48 Gb

10 Gb

16 Gb

48 Gb

Read length

100–150

150–200

35–75

450–900

Output range

600 Gb

1–10 Gb

120 Gb

0.7 Gb

Primary errors

Substitution

Indel

AT bias

Indel

Error rate

0.26

1.71

0.01

0.1

Capacity for paired end

Yes

No

Yes

Yes

Advantage

High throughput

Low cost, fast, optical detection not needed

Accuracy

Read length, fast

Disadvantage

Short read assembly

Higher error rate in Short read case of long reads assembly

Error rate with polybase >6, high cost, low throughput

di-base probe is achieved by interrogating every first and second base in each ligation reaction. Multiple cycles of ligation, detection, and cleavage are performed with the number of cycles determining the eventual read length. Several rounds and cycles permit the individual examination of every base in two different ligation reactions and two different probes, hence they are effective in increasing the specificity and accuracy of template strand (Margulies et al. 2005; Massingham and Goldman 2012). The method is limited in sequencing 35–75 bases per read, the presence of a high number of gaps in the assemblies and high capital cost. 20.2.1.3

ION Torrent

Like others, this also requires the amplification of sequence; however, instead of luminescence-based camera scanning or fluorescence detection, it uses electrochemical detection. In a manner analogous to that described earlier, emulsion PCR is performed to produce beads bearing clonal populations of DNA fragments. These are washed over a picowell plate and addition of each nucleotide is measured by the

20.2 NGS Platforms and their Applications

393

difference in pH caused by the release of protons (H+ ions) during polymerization. This is made possible by the complementary metal-oxide-semiconductor technology used in the manufacture of microprocessor chips (Rothberg et al. 2011). This technology can sequence 150–200 bases per read, allowing for very rapid sequencing during the actual detection phase, but is less sensitive to interpret homopolymer sequences. 20.2.1.4

Illumina

This is amongst the most successful and widely used NGS technologies, having a huge range of applications related to genome, transcriptome, and regulome. The sequencing approach is constructive for both single-read and paired-end (both 3′ and 5′ ends) reads of long or short inserts. The Illumina sequencing includes bridge amplification to clonally build the fragments, which can be then sequenced by synthesis. It is capable of sequencing 100–150 read lengths with a maximum daily throughput of 360–500 Gb, which is the highest among all four majorly used sequencing techniques. A run may take around five or six days with a memory of 48 Gb (Liu et al. 2012). Illumina uses different sequencing platforms such as Hiseq 2000/HiSeq 2500, GAIIx (Genome analyzer), MiSeq, HiSeq X Ten, NextSeq 500, etc. The various platforms have been compared in Table 20.2. This technology has been most useful for small RNA sequencing. Compatible applications include identification of new miRNAs, characterizing isomers with single-base resolution and differential expression analysis. The process requires library Table 20.2 Comparison of some commonly used Illumina sequencing platforms. Plastform

MiSeq

NextSeq

HiSeq X Ten

GAIIx

HiSeq 2000

Instrument cost

$128 000

$80 000

$695 000

$256 000

$654 000

Sequence yield per run

1.5–2 Gb

20–50 Mb on 314 chip, 100–200 Mb on 316 chip, 1 Gb on 318 chip

100 Mb

30 Gb

600 Gb

Sequencing cost per Gb

$502

$1000 (318 chip)

$2000

$148

$41

Run time

27 h

2h

2h

10 days

11 days

Reported accuracy

Mostly >Q30

Mostly Q20

Q30

Mostly >Q30

Observed raw error rate

0.8%

1.71%

12.86%

0.76%

0.26%

Read length

2 × 300 bp max read length

∼200 bases

Average 1500 bases (C1 chemistry)

2 × 150 bp

2 × 100 bp max read length

Output range

15 Gb

129 Gb

1.8 Tb

95 Gb

600 Gb

Typical DNA requirements

50–1000 ng

50–1000 ng

50–1000 ng

Run types

Single and paired-end

Paired-end

Single and paired-end

Single and paired-end

Single and paired-end

Reads per run

25 million

420 million

1.8 Tb

80–100 million

3 billion

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20 Impact of NGS in Elucidating the Role of microRNA Related to Multiple Abiotic Stresses

3′ Ligation 3′ RNA Adapter 5′ Ligation 5′ RNA Adapter

3′ RNA Adapter

cDNA Synthesis RNA RT primer RNA PCR primer

Index Sequence

PCR Amplification

Sequencing

RNA PCR primer

1st Read sRNA Sequencing primer

2nd Read Multiplexing index read primer

Figure 20.2 Schematic representation of small RNA sample preparation.

preparation, cluster generation, and sequencing. Specialized kits are available for generating adapter-ligated small RNA libraries from total RNA. The adapter-ligated small RNAs are attached to the flow cell and solid-phase bridge amplification generates a small cluster of RNA molecules with the same sequence (Figure 20.2). The initial sequencing cycle starts by addition of reversible terminator nucleotides labeled by fluorescent dye, primers, and polymerase to the flow cell (Ju et al. 2006). The primer attaches to the fragment which is being sequenced and the polymerase adds the labeled terminator nucleotide (chemically cleavable moiety) at the 3′ OH position of the new strand. Each nucleotide contains a different fluorescently labeled terminator group for easy detection and recognition. The instrument uses dual surface imaging technology, which increases the accuracy of sequencing by reducing signal interference. The fluorescent label is detached from the incorporated base at the end of each sequencing cycle and the process repeats to decode the sequence, one base at a time (Ju et al. 2006). 20.2.2

Applications of NGS

NGS technologies have a tremendous range of applications, which have allowed rapid progress in many biological fields. NGS proved to be an asset for comparative biological studies of wide range of organisms by utilizing whole genome sequencing. It is also widely being used in gene expression studies with the help of RNA-Seq. This gives researchers the ability to visualize RNA expression in sequence form and is fast replacing microarray analysis (Grada and Weinbrecht 2013). The various other applications include exome or transcriptome sequencing, whole genome re-sequencing, target region capture sequencing, small RNA sequencing, degradome sequencing and methylation (reduced representation bisulfite sequencing, methylated DNA immunoprecipitation sequencing and chromatin immunoprecipitation (ChIP) sequencing) sequencing. The use of various NGS platforms over the last decade is plotted in Figure 20.3. As NGS continues to grow rapidly, it is imperative to discuss some widespread applications.

ION_TORRENT

454 GS FLX Titanium

ABI_SoLiD

20 14

30000

20 11

20.2 NGS Platforms and their Applications Illumina

25000 20000 15000 10000 5000

) r il

20 16

20 17 (A p

20 15

20 13

20 12

20 10

09 20

20

08

0

Figure 20.3 Graphical representation of the usage of various sequencing platforms over time.

20.2.2.1

Genomics

This includes systematic study on a whole-genome scale to gain understanding of gene functions and genomic regions (Novelli et al. 2010). Earlier, candidate-gene approaches were used with a focus on the genes involved in well-defined molecular pathways. Recent advances in high-throughput genomic technologies have enhanced our knowledge of genetic linkage, association studies, DNA copy number, and gene expression analysis (Roychoudhury et al. 2011). NGS has emerged as an effective tool for genome mapping, assessing allelic transmission and identifying quantitative trait loci. The various applications in this context NGS are detailed below: (1) De novo sequencing refers to generation of the first genome sequence draft of a new species without any reference genome. This involves the sequencing and assembly of small DNA fragments into longer contig sequences and arranging the contigs in a series to get a complete genome sequence. The de novo assembly of plant genomes is a challenge owing to their complex organization, large size (Pellicer et al. 2010), high ploidy levels (Meyers and Levin 2006), large gene families, heterozygosity (Gore et al. 2009), and the presence of numerous transposons, repeats, and pseudogenes with nearly similar sequences. In 2007, the grape genome was sequenced using both Sanger sequencing and pyro-sequencing to generate a consensus sequence (Velasco et al. 2007). Woodland strawberry was the first plant genome sequenced using NGS alone by combining the 454, Illumina, and SOLiD platforms (Edwards and Batley 2010; Shulaev et al. 2011). Illumina has emerged as the preferred NGS platform and has provided the genomic sequence of banana (D’Hont et al. 2012), potato (Xu et al. 2011), chickpea (Edwards et al. 2013; Varshney et al. 2013), pigeon pea (Varshney et al. 2012), and many more plants. (2) Whole genome sequencing is a procedure to determine the complete DNA sequence of an organism’s genome. In plants, it involves sequencing all of the chromosomes and DNA of the mitochondria and chloroplast. This is useful for re-sequencing of different species for which the genome sequence is already available. Comparative analysis using the available sequence as a reference is useful for identification of mutations, polymorphism, and structural variations between organisms. The 1001 Genomes Project was launched at the beginning of 2008 to discover detailed WGS

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variation in at least 1001 strains (accessions) of A. thaliana. These accessions are naturally inbred lines that are products of natural selection under diverse ecological conditions, enabling a research program that links genotypes and phenotypes to fitness effects in the laboratory and the field. (3) The targeted sequencing approach is used for the sequencing and analysis of any part or region of a gene or genome. This facilitates researchers to invest their precious time, effort, and expense in specific areas of interest. In this case, NGS helps to achieve higher sequencing coverage, while generating smaller and manageable amounts of data. This has proved to be beneficial in the identification of rare variants and SNPs. It is facilitated by either pre-made and pre-selected or custom-designed sequencing panels for specific areas of research interests. Custom-targeted sequencing is suitable for investigating genes involved in specific pathways or in follow-up studies of WGS. (4) Exome sequencing is a broadly used targeted sequencing method that involves the sequencing of exonic regions of the genome. It is beneficial as several disease-causing variants lie within exonic regions or protein-coding regions of the genome. It allows affordable sequencing of a larger number of species, with higher sequence depth in less time and cost, as compared with WGS (Van Dijk et al. 2014). It can also be utilized to achieve sequencing of agriculturally important plants with complex, repetitive, and mostly polyploid genomes which are not much suited to WGS, such as Triticum aestivum. 20.2.2.2

Metagenomics

This includes a direct study of genetic material recovered from microbial communities present in environmental samples (Buermans and Den Dunnen 2014). This has aided the discovery of known pathogens and novel microbes (Prabha et al. 2013), making it useful in the development of effective techniques that can pre-detect the threats to crop production and harmful microbial contaminants to achieve food safety. This also enables the strategizing of beneficial attributes of a microbial community in both plants and animals (National Research Council 2007). Metatranscriptomics of the microbiome is emerging as a promising new tool for sequencing and studying the microbial activities regulated by gene expression (Carvalhais et al. 2013; Pereira de Castro et al. 2013; Bashiardes et al. 2016; Meena et al. 2017). Adams and his team have applied this for investigating the total RNA from tomatoes infected with the Pepino mosaic virus (Adams et al. 2009; Van der Vlugt 2011). Specific applications of metatranscriptomics involve isolation of total RNA and its enrichment based on the type of RNA to be sequenced (i.e. mRNA, lincRNA, and microRNA) (Bikel et al. 2015). For instance it is known that virus-derived small interfering RNAs (siRNA) constitute a substantial amount of host cell small RNAs in plants infected with viruses. This type of study was performed on soyabean to identify various pathogenic and symbiotic microorganisms (Molina et al. 2012). 20.2.2.3

Epigenomics

Epigenomics is associated with the investigation of heritable or acquired alterations in DNA sequences caused by processes that may exert an indirect influence on DNA sequence or structure. It involves DNA methylation, DNA–protein interactions, histone modification, small RNA-mediated regulation, etc. (Banerjee and Roychoudhury 2017, 2018; Banerjee et al. 2017). The miRNAs and other noncoding small RNAs

20.2 NGS Platforms and their Applications

are the key regulators of the epigenetic processes and gene networks operative during plant growth and response to the environment (Banerjee et al. 2016). They activate silencing of transcription by directing chromatin alterations through recruitment of histone and DNA methyltransferases (National Research Council 2007; Schnable et al. 2009; Prabha et al. 2013; Holoch and Moazed 2015; Deschamps and Llaca 2016; Soto et al. 2016). The first evidence of the role of RNA-directed DNA methylation was demonstrated in Arabidopsis (Bao et al. 2004; Wu et al. 2010) and now several reports have described the role of siRNA in DNA methylation (Onodera et al. 2005). The role of miRNA in this process was described by the miR165/166 directed DNA methylation of phabulosa and phavoluta (Bao et al. 2004; Wu et al. 2010). The NGS-based applications combining chromatin immunoprecipitation (ChIP) assays with sequencing are used to study protein binding sites on genomic DNA to obtaining an extensive view of the epigenetic changes. The ChIP-seq has enabled the identification of genome-wide DNA binding sites for transcription factors (TFs), histone modifications, and other proteins. This application has revealed insights into gene regulation events that play a role in various diseases and biological pathways. It also enables thorough examination of the interactions between proteins and nucleic acids on a genome-wide scale. 20.2.2.4

Transcriptomics

NGS has enhanced studies on the transcriptome by providing accurate measures of total cellular gene expression. RNA-seq enables researchers to identify both annotated and unannotated features of genes in a single assay. It further supports the identification of gene fusion, single nucleotide variants, transcript isoforms, and gene expression specific to alleles. Total RNA and mRNA sequencing was used to obtain a complete view of the whole transcriptome in a selected biological condition and thus increase the power of RNA discovery techniques. It allows the identification and profiling of both common and rare transcripts. In addition, it can be used in aligning sequencing reads across splice junctions, detections of isoforms, gene fusions, and new transcripts (Roychoudhury and Banerjee 2015). (1) Targeted RNA sequencing is used for the identification of transcripts of specific interest. This enables the tapping of their differential expressions profiles, identification of isoforms, determination of the expression of specific alleles, detection of gene fusion, pinpointing of SNPs and the finding of splice junctions. It is also a powerful method for confirming the results obtained from whole-transcriptome sequencing or gene expression microarray. (2) Ribosome profiling is a technique based on sequencing of mRNA fragments protected by ribosomes to obtain a global view of the functional transcripts at a specific time point in a cell. It provides systematic information for predicting the protein abundance and defining the proteome in a cell. This method is also useful for quantitating gene expression and finding the rate of protein synthesis. The approach has been used for the identification of small RNA targets and understanding their regulatory effects at translation level. Wang and his team have used this technique to experimentally identify known and novel regulatory targets of Escherichia coli small RNA RyhB (Wang et al. 2015). Ribosome profiling was also used to study MicL small RNA, which represses Lpp outer membrane protein and links copper-sensitive phenotype to the loss of MicL (Guo et al. 2014a).

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(3) Small RNA sequencing is a method to isolate and sequence small noncoding RNA species such as siRNAs and miRNAs. This has played an important role in the discovery of novel miRNAs (Tripathi et al. 2018b; Sunkar and Zhu 2004; Sunkar et al. 2008; Lan et al. 2012) and other small noncoding RNAs. This is being widely utilized for identifying and analyzing the differential expression patterns of all small RNAs in any sample (Mittal et al. 2013; Sharma et al. 2015). The small RNA repertoire of Arabidopsis was initially visualized using the NGS approach (Fahlgren et al. 2007; Tripathi et al. 2015) and in 2008 Sunkar and his team used NGS for rice miRNA expression profiling (Sunkar et al. 2008). Since then, it has been used to identify several conserved and novel miRNAs and capture their profiles under a variety of stress conditions, e.g. water deficit and rust infections in soybean (Kulcheski et al. 2011), cold stress in orange (Zhang et al. 2014b), drought and salinity stress in Gossypium hirsutum (Xie et al. 2014), and tomato leaf curl virus (ToLCV) infection in tomato (Tripathi et al. 2018b). The NGS data has also helped to explore and identify end modifications in miRNAs (Ebhardt et al. 2009; Kim et al. 2010; Kaushik et al. 2015; Tripathi et al. 2018b). (4) Degradome sequencing, also referred to as parallel analysis of RNA ends (PARE), is useful in identification of miRNA cleaved targets. It is known that miRNA guides argonaute (AGO) protein to cleave target mRNA between the ninth and eleventh nucleotide from the 5′ end. The cleaved products are employed for the validation of the miRNA–mRNA target pair by degradome sequencing. It also gives information on the cleavage site and has helped in the identification of target transcripts of many known and novel plant miRNA and trans-acting siRNAs (ta-siRNAs) (Tripathi et al. 2018a; Addo-Quaye et al. 2008a; Dutta et al. 2017; Song et al. 2017).

20.3 Understanding the Small RNA Family Small RNAs are usually noncoding in nature, but play significant roles in gene regulation. They are a large family consisting of siRNA, microRNA (miRNA), piwi RNA (piRNA), tRNA, rRNA, snRNA, snoRNAs, etc. Among these, the housekeeping noncoding RNAs like tRNA, rRNA, snRNA, and snoRNA, perform structural and catalytic roles, while the regulatory RNAs like miRNAs and siRNAs control gene expression by silencing transcription or translation (Guleria et al. 2011). The piRNAs are 23–29 nt in size and are mostly found in animals. They are derived from distinct transposons as well as from repetitive genomic elements and are involved in providing genome stability by silencing the transposons (Le Thomas et al. 2014; Weick and Miska 2014). The characteristic features of the miRNA and siRNA biogenesis and function are described in Figure 20.4 and a comparison is provided in Table 20.3. 20.3.1

Small Interfering RNAs

Endogenous siRNAs are 21–24 nt long double-stranded molecules with 2 nt overhang at the 3′ end. They act as mediators of RNA interference (RNAi) pathways in transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS) (Chau and Lee 2007; Yu et al. 2014). In animal as well as plant cells, they originate from double-stranded RNA (dsRNA) or short hairpin RNA (shRNA) by the action

miRNA precursor gene

5′ cap

DCL1

AAAAA

DDL SE HYL1

l Po

TAS or PHAS precursor gene

NAT precursor gene

RAS precursor gene

Stress induced transcript

II

l Po

II l Po

I lV Po

VI

miRNA precursor DCL 1

DCL2

DDL SE HYL1

TGH DRB

TGH

DCL3 HEN1

23–24nt ra-siRNA

Nat-siRNA duplex

CH3

CH3

PollVa

RDR2

HEN1

DRB ?

AGO?

24-nt Nat-siRNA HEN1

Nucleus

Nat-siRNA cleaved product RDR6

CH3 AGO

O7 AG

SGS3

One Hit: 22-nt miRNA

Two Hit: 21-nt miRNA O7 AG

DCL2 HEN1 DRB

O7 AG

Nat-siRNA RDR6

Phasing

SGS3 DCL4 DRB4

RDR6 SGS3 DCL4/5 DRB4

O7 AG

Phasing DCL5

Nat-siRNA precusror

l Po

CH

BRB4

O7 AG

I

DRM1/2

DRD 1

CH3

3

CH

3

CH

3

CH

21-nt Nat-siRNA 3

Figure 20.4 Schematic representation to show the steps in biogenesis of different classes of small RNAs.

CH

Cytoplasm

3

O7 AG

CH

O7 AG

3

O7 AG

CH

21-,22-and 24-nt Ta-siRNA, PhasiRNA, CasiRNA

3

Table 20.3 Comparative account of biogenesis and function of different small RNA. microRNA

Small interfering RNA (siRNA)

Small RNA

miRNA

Ta-siRNA (PhasiRNA)

Nat-siRNA (cis-NATs)

hc-siRNA

ra-siRNA

Origin

Distinct genomic loci

Transposable elements, noncoding Tas-si or Pha-si transcript

Overlapping transcripts of two adjoining genes

Genomic repeats such as transposons and retroelements

Genomic repeats and retrotransposons

Configuration of precursor

Single-stranded, hairpin-structured

Double-stranded with overhangs

Double-stranded

Double-stranded

Double-stranded

Proteins involved in biogenesis

RNA PolII, DDL, HEN1, SE, DCL1, TGH, HEN1, HST1, AGO

RdRp/RDR6, SGS2/SGS3, SDE1, DCL4, DCL2, AGO1, AGO7, DSRBF4

Pol IV, RDR2, NRPD1a, SGS3, HYL1, DCL1/DCL2/DCL3, HEN1, AGO

Pol IV, RDR2, HEN1, DCL3, AGO4, RdDM

Pol IV, RdRP/RDR2, HEN1, DCL3, AGO4/AGO6

Length

21–24 nt

21 and 22 nt

21–24 nt

24 nt

24–26 nt

Sequence conservation

Conserved in related organism

Conserved in related organism

Not confirmed

Not conserved

Conserved

Mode of action (level of regulation)

Transcript cleavage (PTGS); DNA methylation (TGS)

Transcript cleavage (PTGS); DNA methylation (TGS)

Translation repression and transcript cleavage (PTGS)

DNA and histone methylation (TGS)

Modification of histone and/or DNA (TGS)

Complementarity with target gene

Imperfect with mismatch and gaps

Partially identical sequences

Perfect base-pairing

Perfect base-pairing

Perfect base-pairing

Function

Regulating growth, development, and stress response

Regulating growth and development

Regulating stress response

Genome stability by regulating transposons and host defense

Genome stability by regulating transposons and host defense

20.3 Understanding the Small RNA Family

of Dicer/Dicer-like (DCL) enzyme (Chellappan et al. 2010). The endogenous siRNAs can be categorized into various classes including miRNA-directed ta-siRNAs, natural antisense siRNAs (nat-siRNAs), repeat-associated small interfering RNAs (ra-siRNAs) and heterochromatic siRNAs (hc-siRNA). (1) ra-siRNAs originate from dsRNA generated from genomic repeats (5S rDNA repeats) and retro transposons (AtSN1) by the action of putative RNA-dependent RNA polymerase, RdRP/RDR2 proteins (Figure 20.4). The dsRNA is processed by DCL3 into 24 nt-long siRNAs, that are subsequently O-methylated by HUA Enhancer 1 (HEN1) (Xie et al. 2004; Vazquez 2006). The ra-siRNAs play a central role in chromatin modifications and maintenance of DNA and histone methylation on certain retro-elements and repetitive DNA. They associate with AGO4/AGO6 (Hamilton et al. 2002) and recruit de novo methyltransferases, DRM1/DRM2, for methylation of target DNA (Cao et al. 2003; Zilberman et al. 2003; Vazquez 2006). (2) hc-siRNAs comprise the 23–24 nt class of siRNAs. They are produced from intergenic regions or genomic repeats such as transposons by the activity of RNA Polymerase IV (Pol IV). The RDR2 processes them into a perfectly complemented duplex which is further acted upon by DCL3 (Chapman and Carrington 2007; Matzke et al. 2009). The hc-siRNAs help to sustain genome integrity by AGO4-mediated chromatin modifications (heterochromatin formation) by enhancing DNA and histone methylation (Chellappan et al. 2010; Fei et al. 2013). (3) nat-siRNAs are formed by the stimulation of partially overlapping transcripts of two neighboring genes under various biotic and abiotic stresses (Figure 20.4). They might arise from sense and antisense transcripts so they can be categorized as trans-nats and cis-nats, respectively. The complementary dsRNA are recognized by DCL1/DCL2/DCL3to produce 21–24 nt-long nat-siRNAs (Phillips et al. 2007). Their biogenesis involves the interplay of several other proteins like suppressor of gene silencing (SGS3), RDR2, Pol IV, HEN1, and hyponastic leaf-1 (HYL1). Studies on Arabidopsis and rice report that the induction of specific biotic and abiotic stresses (salt stress) increases the intensity or number of nat-siRNAs. They mainly act at the posttranscriptional level by either cleavage or translational suppression of target transcripts, although a few instances have been described in which they can also direct DNA methylation (Wu et al. 2012; Fei et al. 2013). Their function is associated with enhancing stress tolerance in plants (Naqvi et al. 2011; Zhang et al. 2012b). (4) ta-siRNAs are a class of plant specific endogenous secondary siRNAs that regulate mRNAs in trans at the transcriptional level by increasing AGO4 mediated DNA methylation (Chellappan et al. 2010). Their origin depends on miRNA directed cleavage of trans-acting siRNA (TAS) transcript. The TAS locus is transcribed by RNA Polymerase II (Pol II) (Vazquez et al. 2004; Felippes and Weigel 2009) and is cleaved by the 22 nt-long miRNAs. The cleaved transcripts are converted into dsRNA by RDR6 and then processed into 23–27 nt siRNAs by DCL4 protein (Vazquez et al. 2004; Felippes and Weigel 2009). The dsRNA can also be processed by DCL5 to produce 24 nt phased small interfering RNA (phasi-RNAs). They are involved in regulating plant development and response to various biotic and abiotic stresses (Felippes and Weigel 2009; Yoshikawa et al. 2005).

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20.3.2

microRNA

The miRNAs are 21–24 nt noncoding single-stranded RNA molecules, produced endogenously from RNA transcripts (Bartel 2004; Xie et al. 2015; Shriram et al. 2016). They negatively regulate gene expression at transcriptional and posttranscriptional levels (Guleria et al. 2011). This regulation results in the maintenance of chromatin structure, chromosome assortment, RNA editing, RNA stability, and protein synthesis (Carthew and Sontheimer 2009; Banerjee et al. 2016). The miRNAs regulate several developmental processes in plants such as seed germination (Wang et al. 2011), root and shoot growth (Guo et al. 2005; Wu and Poethig 2006), vascular development (Kim et al. 2005), flowering (Chen 2004), panicle development (Zhang et al. 2013d), leaf senescence (Li et al. 2013b), and floral differentiation (Aukerman and Sakai 2003). During biogenesis they are transcribed by Pol II as large primary transcripts that are processed by a protein complex, containing DCL1, Tough (TGH), HYL1, HEN1, and Dwaddle (DDL) into miRNA precursor (pre-miRNA) (Tripathi et al. 2015). The premiRNA is subsequently processed into mature miRNA/miRNA* (Figure 20.4). The mature duplex is methylated by the action of HEN1 protein and transported to the cytoplasm, where one strand is associated with AGO containing RNA-induced silencing complex (RISC) to guide the cleavage or translation inhibition of its cognate mRNA in a sequence-specific manner. The other strand known as miRNA* is normally degraded. The miRNAs regulate various aspects of plant development and many computational approaches have been adopted to predict their targets and hence the function of miRNAs (Mallory and Vaucheret 2006). Sometimes, different length variants of miRNAs are produced from the pre-miRNAs and these are termed isomiRs. These can contain modifications in the form of templatebased or nontemplate-based additions or deletions at the 5′ end, 3′ end or both (Neilsen et al. 2012). Both miRNAs and isomiRs may have functions in the same biological pathway or have totally different functions owing to the sorting into RISC with different AGO proteins (Cloonan et al. 2011; Tan and Dibb 2015; Goswami et al. 2017; Khan et al. 2018; Tripathi et al. 2018b). The generation and function of isomiRs are still questioned because of their inconsistent presence in the NGS datasets. This raises the possibility that they may arise as a result of sequencing artifact or low-quality RNA (Khan et al. 2018; Goswami et al. 2017; Tripathi et al. 2018b)

20.4 Criteria and Tools for Computational Classification of Small RNAs Development of NGS technologys has brought a revolution in the discovery of small RNA molecules. When used along with computational tools it helps to identify and differentiate between small RNAs species and also provides understanding of their biological function. Various tools or software and databases are available to process and analyze the NGS data. The analysis of raw sequencing data begins with pre-processing for adapter trimming and quality filtering followed by prediction and identification of known and novel small RNAs.

20.4 Criteria and Tools for Computational Classification of Small RNAs

403

Table 20.4 List of available tools or software for pre-processing the NGS data. Tool/software

Description

References

cutadapt

A command line tool for adapter removal (http://code.google.com/ p/cutadapt)

Martin (2014)

FASTX toolkit

A collection of command line tools for preprocessing short reads FASTA/FASTQ files (http://hannonlab.cshl.edu/fastx_toolkit)

Gordon and Hannon (2015)

UrQt

Unsupervised quality trimming of next-generation sequencing reads (https://lbbe.univ-lyon1.fr/-UrQt-.html)

Modolo and Lerat (2015)

Fastq_clean

An optimized pipeline to clean the Illumina sequencing data with quality control (https://github.com/gaoshanT/Fastq_clean)

Zhang et al. (2014a)

NGS toolkit

A toolkit for the quality control (QC) of NGS data (http://www .nipgr.res.in/ngsqctoolkit.html)

Patel and Jain (2015)

AdapterRemoval

A comprehensive tool for pre-processing of both single and paired-end NGS data (http://code.google.com/p/adapterremoval)

Schubert et al. (2016)

Bowtie/Bowtie2

Shortread alignment to the reference genome, allowing up to three mismatches (http://bowtie-bio.sourceforge.net/index.shtml)

Langmead and Salzberg (2012)

Burrows-Wheeler Aligner (BWA)

A software package for mapping low-divergent sequences against a large reference genome (https://github.com/lh3/bwa)

Li and Durbin (2009)

Short Oligonucleotide Analysis Package

Alignment tool that provides various tools in a single package. SOAPaligner/soap2, SOAPsnp, SOAPindel, SOAPsv, SOAPdenovo,SOAP3/GPU (http://soap.genomics.org.cn)

Li et al. (2008a, 2009)

SeqMap

Alignment tool that allows up to 5 nt mismatch (insertion/ deletion) with many options for modification and input/output format (http://www-personal.umich.edu/∼jianghui/seqmap/)

Jiang and Wong (2008)

20.4.1

Pre-processing (Quality Filtering and Sequence Alignment)

The sequencing data is obtained in FASTQ format and is contaminated by the sequence of the ligated adapters at both 5′ and 3′ ends. So as an initial step, quality filtering and adapter trimming are performed to remove low-quality reads and adapter sequences, respectively. The NGS QC Toolkit is a useful tool which provides stringent quality control platform for Illumina (IlluQC) and Roche454 (454QC) separately (Patel and Jain 2015). Additionally, it also facilitates adapter trimming, format conversion (FASTQ to FASTA) and some other statistical analysis of sequencing data. The second step in preprocessing involves aligning or mapping the reads to the reference genome and annotating to exons to filter off the overlapping noncoding RNAs like rRNA, tRNA, snRNA, snoRNA, etc. Various tools are freely available for quality filtering (Table 20.4). 20.4.2

Identification and Prediction of miRNAs and siRNAs

As a first step the mapped reads are used for identification of known miRNAs by searching through the authentic repositories for mature miRNAs and their precursors such asmiRBase database (www.mirbase.org) (Kozomara and Griffiths-Jones 2013). The remaining sequences are used for predicting novel miRNAs, isomiRs, and siRNAs

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20 Impact of NGS in Elucidating the Role of microRNA Related to Multiple Abiotic Stresses

(ta-siRNA, pha-siRNA, nat-siRNA, hc-siRNA, and ra-siRNA) using a variety of different computational tools. Various valuable computational approaches are available online, including web servers, tools, and standalone programs for small RNA identification, and more are being routinely developed (Tables 20.5 and 20.6). The knowledge of target transcripts of the miRNAs and other small RNAs is equally important to gain insight into their function as well as their targets. Several integrated softwares are available for prediction of target genes. A few well-known databases/toolkits which provide comprehensive platform for NGS data analysis are shown below. (1) miRBase is a miRNA repository that provides complete information on miRNA mature sequences, precursor sequences and annotation, genome coordinates, etc. (Kozomara and Griffiths-Jones 2013). (2) ARMOUR is a rice-specific miRNA database that contains information on predicted and known miRNAs, precursors and the target transcripts including Gene Ontology (GO) or KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology (KO) annotation (http://armour.icgeb.trieste.it). It includes experimentally validated expression profiles of miRNAs under different developmental and abiotic stress conditions across seven Indian rice cultivars. (3) PMRD is a publicly available database of plant miRNAs that provides complete information on secondary structure, target genes, expression profiles, etc. In PMRD, there are 8433 miRNA identified in 121 plant species including Arabidopsis, wheat, rice, sorghum, soybean, and maize integrated with their respective databases for the putative targets (Zhang et al. 2010). PMRD also facilitates microarray based expression profiles of miRNA related to the oxidative stress. (4) UEA sRNA workbench is a collection of tools that provide a comprehensive platform with many tools to analyze NGS data (Mohorianu et al. 2017) including adapter removal (to remove the adapter from both ends), miRCat (the package for novel miRNA prediction) (Paicu et al. 2017), TA-SI Prediction (tool for prediction of ta-siRNA and phased reads), and PAREsnip (for target prediction from degradome data). Along with miRNA prediction, other tasks such as expression profiling of small RNAs can be performed using CoLide tool (http://srna-workbench.cmp.uea .ac.uk) (Mohorianu et al. 2017). (5) sRNAtoolbox is a collection of tools that can be used independently to analyze the small RNA sequencing data. It includes sRNAbench, for prediction of novel miRNAs and their length variants (isomiRs) as well as expression profiling. miRNAconsTarget provides an integrated platform for target prediction from both animals and plants (http://bioinfo5.ugr.es/srnatoolbox) (Rueda et al. 2015). (6) Massively parallel signature sequencing ( MPSS) database is a unique signaturebased transcription resource for analysis of mRNA and small RNA. It facilitates searching for gene expression data in model plants (Arabidopsis, rice, etc.) (https:// mpss.danforthcenter.org/dbs/index.php?SITE=at_sRNA) (Nakano et al. 2006). The MPSS database provides various sequencing datasets such as small RNAs, PARE data, mRNA differential expression, DNA methylated data, and ChIP sequencing data. High-throughput sequencing data can also be accessed from the the Gene Expression Omnibus (GEO) short-read archive (https://www.ncbi.nlm .nih.gov/geo) and many tools are available for the analysis of plant small RNAs and

20.4 Criteria and Tools for Computational Classification of Small RNAs

405

Table 20.5 A list of major softwares or resources and repositories available for identification and prediction of miRNAs and their targets from NGS data. Tools/software

Description

References

miRU

Web server for plant miRNA target prediction

Zhang (2005)

CleaveLand4

A pipeline detection of targets using degradome data (https:// github.com/MikeAxtell/CleaveLand4/blob/master/ CleaveLand4.pl)

Addo-Quaye et al. (2008b)

miRexpress

Database-supported tool for comparing miRNA expression from NGS (http://mirexpress.mbc.nctu.edu.tw/index.php)

Wang et al. (2009)

TAPIR

Target prediction for Plant miRs (http://bioinformatics.psb .ugent.be/webtools/tapir)

Bonnet et al. (2010)

PMRD

Integrated database of miRNAs expression profiling and target genes (http://bioinformatics.cau.edu.cn/PMRD)

Zhang et al. (2010)

psRNAtarget tool

Web server for plant small RNA target prediction, designed for NGS analysis data (http://plantgrn.noble.org/psRNATarget)

Dai and Zhao (2011)

miRanalyzer

A web server for the identification of mature miRNA, isomiRs, and prediction of novel miRNAs (http://web.bioinformatics .cicbiogune.es/GAP/group/index.html)

Hackenberg et al. (2009, 2011)

miRTour

Homology-based plant miRNA discovery and target prediction tool (https://omictools.com/mirtour-tool)

Milev et al. (2011)

miRNEST

An integrative resource of animal-, plant- and virus-associated miRNA, targets, mirtrons, miRNA gene structures (http:// rhesus.amu.edu.pl/mirnest/copy)

Szcze´sniak et al. (2011) and Szcze´sniak and Makałowska (2014)

Semirna

Searching for plant miRNAs in the genome using putative target gene (http://www.bioinfocabd.upo.es/node/5)

Muñoz-Mérida et al. (2012)

miRDeepFinder

A software package to identify the miRNAs and their targets (http://www.leonxie.com/DeepFinder.php)

Xie et al. (2012)

mirTools

A tool for discovery and profiling of noncoding RNAs and target prediction (http://centre.bioinformatics.zj.cn/mirtools)

Wu et al. (2013)

PASmiR

A literature-curated database for Abiotic stress responsive miRNAs (http://pcsb.ahau.edu.cn:8080/PASmiR)

Zhang et al. (2013c)

miRPlant

An integrated tool for identification novel miRNAs (http:// sourceforge.net/projects/mirplant)

An et al. (2014)

miRBase

A searchable database of published miRNA, precursor; it is a central repository for miRNA sequence information (http:// www.mirbase.org/index.shtml)

Kozomara and Griffiths-Jones (2013)

PNRD

PNRD is a comprehensive analysis platform for plant ncRNA research, updated version of PMRD (http://structuralbiology .cau.edu.cn/PNRD)

Yi et al. (2014)

NGSmirPlant

A specialized web tool for comprehensive characterization of the small RNA transcriptome of plant (http://122.228.158.106/ NGSmirPlant/)

Bai et al. (2015)

mirPRo

A standalone tool for detection of known and novel miRNAs and miRNAs variants (https://sourceforge.net/projects/mirpro)

Shi et al. (2015)

PmiRExAt

Online web resource that provides plant miRNA expression profiles in various tissues and developmental stages (http:// pmirexat.nabi.res.in.)

Gurjar et al. (2016)

miRCat2

A new entropy-based approach to detect miRNA loci (http:// srna-workbench.cmp.uea.ac.uk)

Paicu et al. (2017)

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20 Impact of NGS in Elucidating the Role of microRNA Related to Multiple Abiotic Stresses

Table 20.6 List of tools used for prediction of isomiRs and siRNAs. Tools/software

Small RNA

Description

References

miRMOD

isomiRs

A tool for the identification of both template and nontemplate based isomiRs (http://bioinfo.icgeb.res .in/miRMOD)

Kaushik et al. (2015)

DeAnnIso

isomiRs

A web-based tool for detection of isoform of miRNAs (isomiRs) from small RNA sequencing data (http:// mcg.ustc.edu.cn/bsc/deanniso)

Zhang et al. (2016b)

isomiRex

isomiRs

A web-based platform for identification of isomiRs and graphical visualization of the differentially expressed miRNA (http://bioinfo1.uni-plovdiv.bg/ isomiRex)

Sablok et al. (2013)

IsomiRage

isomiRs

A stand-alone application for the profiling of miRNA/isomiRs from NGS data (http://cru.genomics .iit.it/Isomirage)

Muller et al. (2014)

IsomiR Bank

isomiRs

A database, containing the predicted isomiRs from various samples from plant and animal NGS dataset (http://mcg.ustc.edu.cn/bsc/isomir)

Zhang et al. (2016a)

phasetank

siRNA

A open source tool for the prediction of phased siRNA from small RNAs and their target (http://phasetank .sourceforge.net)

Guo et al. (2014b)

TasiAyser

siRNA

A web tool for ta-siRNA prediction from NGS data (http://mcube.nju.edu.cn/jwang/lab/soft/TasiAyser)

Zhang et al. (2012a)

NATpipe

siRNA

A tool for predicting natural antisense transcripts, including the detection and functional analysis of nat-siRNA and phase-distributed nat-siRNAs (http:// www.bioinfolab.cn/NATpipe/NATpipe.zip)

Yu et al. (2016)

PlantNATsDB

siRNA

An integrated platform for discovery of functional NAT genes (http://bis.zju.edu.cn/pnatdb)

Chen et al. (2011a)

pssRNAMiner

siRNA

A web server for identification of ta-siRNA and phased small RNA (http://bioinfo3.noble.org/ pssRNAMiner)

Dai and Zhao (2008)

plantDARIO

siRNA

A web server for the analysis and expression of plant ncRNA from small RNA-seq data (http://plantdario .bioinf.uni-leipzig.de)

Patra et al. (2014)

Ta-siRNAdb

siRNA

A database for identification of miRNAs associated with ta-siRNA regulatory pathway, including TASs, ta-siRNAs, and ta-siRNA targets (http://bioinfo.jit .edu.cn/tasiRNADatabase)

Zhang et al. (2013a)

target prediction: comPARE, a web resource to sort and examine miRNA–target interaction, can be validated using PARE data (https://mpss.danforthcenter.org/ tools/mirna_apps/comPARE.php); sPARTA, small RNA–PARE Targets Analyzer, a software for the validation of plant miRNAs or sRNA targets, can be used for whole genome analysis; miTRATA (MicroRNA Truncation and Tailing Analysis) is a web-based tool to find differential of 3′ nucleotides modifications of miRNA (3′ isomiRs) in respect to the canonical sequences (https://wasabi.ddpsc.org/

20.5 Role of NGS in Identification of Stress-regulated miRNA and their Targets

~apps/ta). The MPSS database also facilitates the identification of known and novel ta-siRNA loci in TAS transcripts with respect to many plant species (Nakano et al. 2006).

20.5 Role of NGS in Identification of Stress-regulated miRNA and their Targets Plants have evolved intricate molecular mechanisms for sensing and responding to different environmental cues (Wani et al. 2016). This is mediated by a complex network of genes that are under tight transcriptional and/or translational control (Ku et al. 2015). The RNAi pathway with its repository of small regulatory RNAs plays an important role in modulating the expression of various stress-related genes and transcription factors (Barciszewska-Pacak et al. 2015; Mirlohi and He 2016). The advances in NGS technologies have proved to be useful in capturing the expression profiles of the small RNAs and their targets under various stresses, to yield useful insights into the molecular mechanisms of gene regulation (Mirlohi and He 2016). The role of miRNAs in regulating the abiotic stress response is well studied in a number of economical important crops such as rice, wheat, maize, barley, legumes, potato, tomato, and sugarcane (Deschamps and Campbell 2010; Schreiber et al. 2011; Karlova et al. 2013; Zhang et al. 2013b; Sharma et al. 2015; Hamza et al. 2016) The involvement of miRNA regulations in abiotic stress responses in plants was revealed by the study of Arabidopsis mutants like hyl1, hen1, and dcl1, which represent important components of the miRNA biogenesis module. The mutants were not only defective in miRNA production and function but were also hypersensitive to different abiotic stresses and the stress hormone abscisic acid (ABA) (Lu and Fedoroff 2000; Zhang et al. 2008). In addition, correlation of miRNA expression profiles with abiotic stress responses has made it apparent that several miRNAs from diverse species are responsive to abiotic stress (Sunkar and Zhu 2004; Jeong et al. 2011). Stress signals can be transduced in ABA-independent or ABA-dependent pathways (Sahoo et al. 2013). It has been reported that transcription factors like NFYA, NAC, MYC MYB, ABF, zinc finger protein, bZIP, and ARF/ERF share an association with both the pathways and play important roles in gene expression (Bartels and Sunkar 2005; Sahoo et al. 2013; Pradhan et al. 2015). Among stress-responsive genes, nuclear factor YA (NF-YA), a plant-specific transcription factor family, acts as a positive regulator by providing tolerance to drought stress in response to ABA induction in Arabidopsis (NFYA5) and soybean (GmNFYA3). Many of these also influence the expression of miRNA genes. The changes in miRNA expression under stress do not confirm that they are involved in plant adaptation to stress conditions. However, this shows that in response to stress, the miRNAs can directly modulate the expression of stress-inducible genes by targeting the mRNAs (Jones-Rhoades et al. 2006; Alptekin and Budak 2017). Typically stress-upregulated miRNAs are expected to downregulate the expression of their cognate targets, while their stress downregulated miRNAs should direct the accumulation of their target transcripts (Chinnusamy et al. 2007). miRNA-mediated regulation can indirectly affect the gene expression by targeting the transcripts encoding transcription factors. Some miRNA-regulated TF families like ARF (Xu et al. 2016), HD-ZIP

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20 Impact of NGS in Elucidating the Role of microRNA Related to Multiple Abiotic Stresses

(Hivrale et al. 2016), DREB (Kudo et al. 2017), TIR/AFBs (Naser and Shani 2016), GRF (Debernardi et al. 2014), MYC (Noman et al. 2017), bZIP (Hu et al. 2016), HSF (Ohama et al. 2017), zinc-finger (Muthamilarasan et al. 2014), NAC (Hernandez and Sanan-Mishra 2017), MYB/TCP (Shriram et al. 2016), WRKY (Karanja et al. 2017), Scarecrow-like/GRAS (Kasschau et al. 2003), AP2/ERF (Shu et al. 2016), MADS-Box (Zhang and Forde 1998), bHLH (Abe et al. 2003), F-box (Yan et al. 2011), HAP12 –CCAAT-Box TF Complex (Bottino et al. 2013), NF-YA or HAP2 (Sorin et al. 2014) and SPL (SBP) (Cui et al. 2014) are known to play a crucial role in influencing the plant stress responses. Various plant miRNAs have been documented to be regulated by soil salinity (Sunkar et al. 2008; Bottino et al. 2013; Dong et al. 2013), UV-B radiation (Eldem et al. 2012), metal stress (Barciszewska-Pacak et al. 2015), heat stress (Chen et al. 2012; Goswami et al. 2014), and nutrient deficiency (Lu et al. 2014). NGS-based studies have revealed that several conserved miRNAs like miR156, miR159, miR160, miR164, miR166, miR167, miR168, miR167, miR170, miR169, miR171, miR172, miR319, miR395 miR398, miR390, miR393, and miR820 are responsive to multiple stresses (Jeong et al. 2011; Budak et al. 2014, 2015; Goswami et al. 2014; Barciszewska-Pacak et al. 2015; Sharma et al. 2015); however, the extent of their deregulations vary in diverse plant species depending on their adaptability to stresses. The NGS profiles indicate that specific members of families like miR156, miR158, miR159, miR164, miR165, miR168, miR169, miR171, miR319, miR393, miR394, miR396, miR397, miR398, and miR1029 are differentially deregulated in stress in diverse plants (Liu et al. 2008; Frazier et al. 2011). For example, miR396 is downregulated under salt stress in rice but is upregulated in Arabidopsis (Liu et al. 2008; Zhou et al. 2010; Pagliarani et al. 2017) and maize (Ding et al. 2009). Similarly, expression of miR408 was induced in Arabidopsis but inhibited in rice under drought stress conditions (Liu et al. 2008; Zhou et al. 2010; Zhang 2015). The information obtained by NGS-based studies on key miRNA families is summarized below. 20.5.1

miR156

miR156 is a conserved miRNA family that was shown to be upregulated by salt, heat, and drought stress in Arabidopsis (Liu et al. 2008; Kim et al. 2012; Stief et al. 2014; Pagliarani et al. 2017). It was downregulated under salt stress in soybean, cotton, rice, and maize (Ding et al. 2009; Zhou et al. 2010; Kohli et al. 2014; Zhang 2015; Sun et al. 2016). Guray Akdogan (2016) reported its downregulation in leaf and root tissues of rice under water-deficit conditions. miR156 targets SPL transcription factors, which regulate the vegetative phase transition and flowering (Wu et al. 2009; Akdogan et al. 2016). The heat induction of miR156 downregulates SPL, which in turn suppresses the FT and FUL gene expression to influence flowering time (Kim et al. 2012; Stief et al. 2014). The isoforms of miR156 are hypothesized to play a significant role in heat stress memory (Cui et al. 2014; Stief et al. 2014). 20.5.2

miR159

miR159 is induced under osmotic, ABA, and salt stress in wheat and Arabidopsis but downregulated under drought stress (Chinnusamy et al. 2007; Fang et al. 2014; Gupta

20.5 Role of NGS in Identification of Stress-regulated miRNA and their Targets

et al. 2014; Zhang 2015; Pagliarani et al. 2017). This has been shown to influence plant response to stress by regulating the ABA-dependent transcripts (Fang et al. 2014). 20.5.3

miR160

miR160 seems to play an important role in response to salt, heat, and drought stress. Its expression was upregulated under salt stress in the salt-sensitive variety of Vigna unguiculata (Paul et al. 2011; Feng et al. 2017) but was downregulated under salt stress in salt-tolerant Populus euphratica (Li et al. 2013a). miR160 targets auxin response factors, ARF10, ARF16, and ARF17. The miR160 also triggers the production of TAS3 (ARF family proteins) (Montgomery et al. 2008a,2008b; Khraiwesh et al. 2012; Vazquez and Hohn 2013; Yoshikawa 2013). Its role in root development was inferred from observations made on seeds expressing an miR160-insensitive-ARF17. These seeds developed into plants with reduced root and hypocotyl lengths and decreased root branching apart from shoot and floral organ defects (Mallory et al. 2005). The overexpression of miR160 variants in Medicago truncatula also resulted in distinct defects in root growth (Bustos-Sanmamed et al. 2013). 20.5.4

miR164

miR164 is upregulated in response to salt stress (Fu et al. 2017; Goswami et al. 2017) and downregulated by drought stress (Bakhshi et al. 2014). It targets the NAC transcription factor gene family, which is involved in biotic and abiotic stress responses as well as plant growth and development. Many genes such as SNAC2 (Nakashima et al. 2007; Hu et al. 2008), OsNAC5 (Sperotto et al. 2009), ONAC045 (Zheng et al. 2009), OsNAC10 (Jeong et al. 2010), ONAC022 (Hong et al. 2016), OsNAC6 (Nakashima et al. 2007), OsNAC2 (Shen et al. 2017), ANAC 019, ANAC 055, ANAC 072 (Nuruzzaman et al. 2013; Fang et al. 2014), ANAC092/AtNAC2 (Balazadeh et al. 2010), and CmNAC1 (Cao et al. 2017) have been reported to be involved in various abiotic stress responses such salt, drought, and ABA. The SNAC1 gene in rice provides tolerance to drought by stimulating stomatal closure and inhibiting auxin signals (Hu et al. 2006; Fang et al. 2014). 20.5.5

miR166

miR166 is downregulated under salt stress in salt-tolerant maize line “NC286” (Ding et al. 2009). It regulates the expression of the class III homeodomain-leucine zipper (HD-ZIP III) family of transcription factors that are involved in plant development (Carlsbecker et al. 2010; Goswami et al. 2017). Mutants of HD-ZIP III genes, such as Phabulosa, Phavoluta, and Revoluta show reduced lateral root number along with defects in primary root formation in some cases (Hawker and Bowman 2004). Overexpression of miR166 results in downregulation of HD-ZIP III genes, which in turn affects root development (Singh et al. 2014). In wheat and barley, miR166 shows upregulation in response to high temperature (Xin et al. 2010; Kruszka et al. 2014). 20.5.6

miR167

miR167 showed drought-induced expression in both Arabidopsis and P. euphratica (Sunkar et al. 2012), but in sorghum it was downregulated (Hamza et al. 2016). In wheat

409

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20 Impact of NGS in Elucidating the Role of microRNA Related to Multiple Abiotic Stresses

and barley, it showed differential regulation in response to high temperature (Xin et al. 2010; Kruszka et al. 2014). ARFs (ARF8, ARF10, ARF16, and ARF17), are targeted by miR167 in rice and switch grass in response to drought stress in an ABA-dependent manner (Yang and Zeevaart 2006; Liu et al. 2007; Liu and Chen 2009; Sanan-Mishra et al. 2013). miR167 may inhibit plant development under stress by regulating the production of TAS3 (ARF family proteins) along with miR160 and miR390 (Montgomery et al. 2008a,2008b; Khraiwesh et al. 2012; Vazquez and Hohn 2013; Yoshikawa 2013). 20.5.7

miR168

miR168 showed induced expression in response to water-deficit conditions in Arabidopsis while in rice and P. euphratica, it showed downregulation. In response to cold stress, miR168 expression is upregulated in Arabidopsis but downregulated in rice (Liu et al. 2008; Lv et al. 2010; Zhou et al. 2010; Sunkar et al. 2012; Feng et al. 2017). 20.5.8

miR169

miR169 is downregulated under drought and salt stress (Li et al. 2008b; Fang et al. 2014; Si et al. 2014; Deng et al. 2015), leading to accumulation of NFYA gene (Sunkar 2010). In rice, some members of the miR169 family were induced by drought (Zhao et al. 2007) and they also played an important role in salt stress (Kantar et al. 2011). 20.5.9

miR172

miR172 showed upregulation in response to heat in Arabidopsis, wheat, and Helianthus annuus (May et al. 2013; Li et al. 2014). It targets AP2-like genes, viz., TARGET OF EAT1 (TOE1), TOE2, and SCHLAFMUTZE (SMZ), which are downregulated in response to heat stress (May et al. 2013; Li et al. 2014). 20.5.10

miR393

miR393 showed upregulation in response to salt, cold, drought, and ABA treatment in various plant species such as Arabidopsis, rice, P. euphratica, M. truncatula, etc. (Chinnusamy et al. 2007; Khraiwesh et al. 2012; Feng et al. 2017). It was downregulated in cotton and cowpea (Barrera-Figueroa et al. 2011; Sunkar et al. 2012; Xie et al. 2015). It regulates the expression of Auxin Signaling F-box Protein (AFB)/TAAR, auxin receptor transport inhibitor response1 (TIR), E3 ubiquitin ligase, and SCF (Skp, Cullin, F-box containing complex) complex. AFB is involved in adaptation to oxidative and salt stress during plant development (Chen et al. 2011b; Si-Ammour et al. 2011; Iglesias et al. 2014; Windels et al. 2014). Later 5′ RACE (rapid amplification of cDNA ends) experiments confirmed the involvement of miR393:F-box protein (TIR/AFB) in response to Al stress in barley (Bai et al. 2017). The miRNA regulatory module affected MYB transcription factor to influence root development under Al stress (Deng et al. 2015). miR393 potentially triggers the production of siRNAs in Arabidopsis from its target gene TIR/AFB2 auxin receptor (TAAR) (Si-Ammour et al. 2011). In Arabidopsis, miR173 produced ta-siRNA targets HTT1 (HEAT-INDUCED TAS1 TARGET1) and HTT2, which were observed to be highly expressed and to provide thermo-tolerance

20.6 Conclusion

in heat stress accumulation (Khraiwesh et al. 2012; Li et al. 2014). The expression levels of the HTT genes were reduced with higher accumulation of TAS1a gene expression, which negatively regulates the expression of HTT gene and causes higher sensitivity to heat stress (Zhao et al. 2016). 20.5.11

miR396

miR396 was downregulated in response to drought stress in rice (Pagliarani et al. 2017), but its expression was induced in drought-stressed Arabidopsis, wheat, and cotton (Liu et al. 2008; Zhou et al. 2010; Sunkar et al. 2012; Budak et al. 2014; Xie et al. 2015). 20.5.12

miR398

miR398 is reported to be heat inducible and it plays a crucial role in thermo-tolerance in Arabidopsis (Guan et al. 2013; Lu et al. 2013). It targets Cox5b-1 (a subunit of the mitochondrial cytochrome c oxidase), CCS1 (a copper chaperone for superoxide dismutase), CSD1 and CSD2 (closely related copper/zinc superoxide dismutases) (Sunkar and Zhu 2004; Zhu et al. 2011), which are allied with biosynthesis of heat shock factors and heat shock protein (Guan et al. 2013; Lu et al. 2013). The CSDs are the key reactive oxygen species scavengers, and CSD/CCS negatively regulate the reactive oxygen species pathway (Mittler 2002; Sunkar et al. 2006). Expression of miR398 reduces during oxidative stress, while the expression of its target genes CSD1 and CSD2 is elevated. There are many other miRNA such as miR171, miR172, miR319, miR394a, miR395, miR397, miR402, miR5655, and miR2933 that were observed to be upregulated in drought and related stresses, whereas miR161 and miR389 were downregulated under drought stress (Sunkar and Zhu 2004; Liu et al. 2008). The differential regulation by stresses was captured for miR319c, which is to be upregulated by cold, but not dehydration, NaCl or ABA (Sunkar and Zhu 2004). The action of miRNA also influences other pathways by deregulating the ta-siRNA production. In Arabidopsis, miR173 and miR161 play a crucial role in recognition of ta-siRNAs from TAS1a,b,c (flavin adenine dinucleotide-binding domain-containing protein) and TAS2 (pentatricopeptide subfamily proteins), respectively (Vazquez et al. 2004; Allen et al. 2005; Yoshikawa et al. 2005; Li et al. 2014). In hypoxia (low oxygen condition), tasi289 generated from TAS1 downregulates the expression of pentatricopeptide repeat-containing proteins to provide protection in response to hypoxia (Moldovan et al. 2009). The list is never-ending and functional analysis is required to understand the distinct behavior of each miRNA in response to stress in crop plants.

20.6 Conclusion Changes in the various environmental factors exert stress on plants, thereby affecting their growth and development. Scientific research has unraveled the molecular mechanisms controlling the stress responses. The rapid advances in NGS, during the last few decades have significantly enhanced our knowledge on the stress-induced transcriptome and the genetic machinery involved in its regulation. The discovery of the regulatory small RNAs including the miRNAs has enabled identification of the genetic

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modules involved in reprogramming plant responses that are influenced by stress. NGS is playing an important role in the discovery, expression profiling, and target identification of the miRNAs. It is envisioned that global information on the genomics, transcriptomics, and regulomics through NGS approaches will provide in-depth understanding of the miRNA-mediated regulation of genetic networks. This will help in gaining molecular insights into the stress responses which will be useful in developing plants with greater adaptability to variations in the climate.

Acknowledgments We apologize to colleagues whose work could not be included owing to space constraints. The study on miRNAs in our laboratory was supported by financial grants from the Department of Biotechnology.

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21 Understanding the Interaction of Molecular Factors During the Crosstalk Between Drought and Biotic Stresses in Plants Arnab Purohit 1 , Shreeparna Ganguly 1 , Rituparna Kundu Chaudhuri 2 , and Dipankar Chakraborti 1 1 Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata, 700016, West Bengal, India 2 Department of Botany, Krishnagar Government College, Krishnagar, 741101, West Bengal, India

21.1 Introduction Plants often encounter multiple stress factors continuously and simultaneously during their growth in natural habitats. Range of biotic (e.g. viruses, bacteria, fungi, and insects) and abiotic (e.g. heat, cold, drought, and salinity) factors has a huge impact on plant growth and fitness, crop productivity and global food security (Suzuki et al. 2014; Singh et al. 2015; Zhu 2016; Tripathi et al. 2016, 2017a,b; Singh et al. 2017; Liu et al. 2018). Plants have developed complex mechanisms to detect and defend against individual as well as combined stresses. Various immune response pathways triggered by biotic and/or abiotic stress interactions minimize damages and protect the plant to safeguard valuable resources. After successful stress recognition, defense mechanisms are activated in a rapid and efficient way and transcriptional reprogramming is orchestrated in a two-layered manner. Pathogen-associated molecular patterns (PAMPs) triggered immunity is the general and nonspecific first line of defense response which provides basal resistance against abiotic and biotic stresses. PAMP-triggered immunity triggers a cascade of defense responses including production of reactive oxygen species (ROS), activation of specific ion channels and kinase cascades, and accumulation of defense-related hormones like abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). Across similar types of biotic and abiotic stresses, PAMP- triggered immunity has comparable molecular patterns. Effector-triggered immunity is the second line of plant defense response activated by resistance (R) genes when effectors (pathogen virulence factors) are released into plant cells. Effector-triggered immunity is specific to the pathogen infection and produces defense proteins that cause programmed cell death. Most past research activities are focused on plant responses to biotic or abiotic stresses in isolation. However, when subjected to combined stresses simultaneously, plants respond in a unique and specific way that is completely different from their response to either of the stresses alone (Basu and Roychoudhury 2014). Plants are supposed to be exposed to a greater number of environmental stresses with constantly Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

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changing climatic conditions. Reports suggest that altering environmental conditions will also increase the host range of pathogens and their virulence. As a result, the effects of combined stress are expected to be augmented in future. Therefore, understanding major factors affecting signaling pathways to single and combined stresses, their common and overlapping responses as well as crosstalks are crucial for the development of plants that are tolerant to multiple stresses.

21.2 Combined Stress Responses in Plants Previous research activities on simultaneously occurring multiple stress factors revealed that plant responses to combined stresses can interact in an either antagonistic or synergistic manner. Plant responses to multiple stress factors depend on the field conditions, developmental stages of the plant, intensity of the stress factors, combination of stress factors, degree of simultaneity, tissues, geographical origin, and the plant species (Fujita et al. 2006; Mittler and Blumwald 2010; Fraire-Velázquez et al. 2011; Ramegowda et al. 2013; Ramegowda and Senthil-Kumar 2015). Reports on simultaneous abiotic stress and pathogenesis in plants suggest that abiotic factors can lead to enhanced disease susceptibility in plants. Salinity stress significantly increased susceptibility to powdery mildew in tomato plants (Kissoudis et al. 2015). Both basal and R gene-mediated responses to Pseudomonas syringae were shown to be inhibited by high-temperature treatment in Arabidopsis thaliana and Nicotiana benthamiana (Wang et al. 2009). In contrast, resistance responses against pathogen attack owing to abiotic stresses have also been documented in several reports. Salt stress significantly reduced infection by the biotrophic fungus Odium neolycopersici (Achuo et al. 2006). There is evidence that high temperature provides durable protection against stripe rust caused by Puccinia striiformis in spring wheat (Carter et al. 2009).

21.3 Combined Drought–Biotic Stresses in Plants Drought, the most important and frequent abiotic factor, occurs simultaneously with several biotic stress factors like fungi, bacteria, virus, and pests (Pandey et al. 2015). The combination of simultaneous drought and biotic stress is well demonstrated among different biotic–abiotic stress combinations (Mayek-Perez et al. 2002; McElrone et al. 2003; Xu et al. 2008; Ramegowda et al. 2013). Since there is always gradual development of drought stress in plants, the emergence of this type of combination stress is not rapid. Pathogens can infect drought-stressed plants or pathogen-infected plants can develop drought stress in field conditions (Pandey et al. 2015; Ramegowda and Senthil-Kumar 2015). The duration and intensity of combinatorial stresses modulate the response of plants against concurrent drought–biotic interaction. Depending on these factors, simultaneous drought and biotic stresses can have two types of consequences: (i) they can hamper plant growth and development; or (ii) drought-stressed plants show enhanced tolerance against pathogen or pathogens enhance plant resistance to drought stress (Figure 21.1). Plants can proficiently modulate the existing defense mechanisms in a unique manner, which is the probable reason for their tolerance of combined stresses (Atkinson and Urwin 2012; Ramegowda and Senthil-Kumar 2015; Nejat and Mantri

21.3 Combined Drought–Biotic Stresses in Plants Plant

Fungus/bacteria/ virus/pest

Drought

(+) Fungus/ bacteria/virus/pest

(+) Drought

Susceptibility

Tolerance

Susceptibility

1. Drought stressed common bean charcoal rot fungus Macrophomina phaseolina (Mayek-Perez et al., 2002) 2. Parthenocissus quinquefolia grown on low soil moisture bacterial leaf scorch agent Xylella fastidiosa (McElrone et al., 2003) 3. Drought stress of bean plants to tobacco mosaic virus (TMV) (Yarwood, 1955).

1. Drought stressed tomato - gray mold Botrytis cinerea (Achuo et al., 2006) 2. Drought stressed Nicotiana benthamia na - bacterial speck agent Pseudomonas syringae pv. tomato (Ramegowda et al., 2013) 3. Drought stressed tomato - tomato spotted wilt virus (Cordoba et al., 1991) 4. Drought stressed tomato - herbivore Spodoptera exigua (English-Loeb et al., 1997)

1. Maize dwarf mosaic virus infected Zea mays var. saccharata - drought stress. (Olson et al., 1990).

Tolerance 1. Brome mosaic virus (BMV), Cucumber mosaic virus (CMV) or TMV infected N. benthamiana drought stress (Xu et al., 2008) 2. Verticillium longisporum infected Arabidopsis drought stress (Reusche et al., 2012).

Figure 21.1 Potential responses of plant exposed to drought–biotic stress combinations.

2017). On the contrary, failing to modulate resistance mechanisms to one stress results in susceptibility to the other. 21.3.1

Plant Responses Against Biotic Stress during Drought Stress

During drought stress, plants can become susceptible to biotic stress factors. Drought-stressed common bean was more susceptible to charcoal rot causing fungus Macrophomina phaseolina (Mayek-Perez et al. 2002). Drought stress, in combination with bacterial infection, was found to increase the disease susceptibility of plants. Infection of xylem-limited bacterial leaf scorch pathogen, Xylella fastidiosa, on an ornamental plant, Parthenocissus quinquefolia, resulted in intense scorch symptoms when grown under limited soil moisture levels, in comparison with infected plants generated under normal soil moisture conditions (McElrone et al. 2003). There are reports on the susceptibility of drought-stressed plants to viral infection. Fifteen percent drought induced by watering regulation made bean plants more susceptible to tobacco mosaic virus (Yarwood 1955). Susceptibility of Arabidopsis to Turnip mosaic virus was increased during simultaneous heat and drought stress, as abiotic stress conditions suppressed defense responses to biotic stress (Prasch and Sonnewald 2013). Drought stress conditions affect the interactions between plant and insect pests.

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This influences the occurrence of pests and contributes to the transformation of a minor pest to a major one. In contrast, drought-stressed plants can resist those pathogens which need wet or humid climatic conditions for their growth. Drought stress was reported to reduce gray mold Botrytis cinerea infection in tomato by 50% (Achuo et al. 2006). N. benthamiana subjected to drought exhibited limited symptoms of bacterial speck disease caused by P. syringae pv. tomato compared with healthy plants (Ramegowda et al. 2013). Cordoba et al. (1991) reported that drought slowed down the multiplication of Tomato spotted wilt virus. Feeding on tomato leaf tissue by the herbivore Spodoptera exigua was reduced during drought-stressed conditions. Drought stress elevated the levels of defense compounds in tomato leaves (English-Loeb et al. 1997). 21.3.2

Plant Responses Against Drought Stress during Biotic Stress

Pathogen infection reduced the basal defense potential in plants which in turn increased the susceptibility to drought stress. Simultaneous exposure of Zea mays to Maize dwarf mosaic virus and drought stress exhibited contraction of a number of productivity-oriented characteristics (Olson et al. 1990). Conversely, combined stress also provided pathogen-induced drought tolerance. Brome mosaic virus, Cucumber mosaic virus, or tobacco mosaic virus-infected plants of N. benthamiana exhibited late disease responses under combined virus and drought stress. This combined stress also resulted in the formation of several osmoprotectants in plants (Xu et al. 2008). Additionally, the infected N. Benthamiana leaves exhibited low transpiration rate owing to partial stomatal closure, which increased the water-holding potential. The mentioned study demonstrated that accumulation of certain metabolites during virus infection led to changes in the physiology of the host and effective resistance to drought stress, resulting in combined stress tolerance. Verticillium longisporum-infected Arabidopsis was found to induce the synthesis of Vascular-Related NAC domain 7 transcription factor, which prompted de novo xylem development balancing the water storage, which in turn improved drought tolerance (Reusche et al. 2012).

21.4 Varietal Failure Against Multiple Stresses Improved stress tolerance varieties of crop plants have been developed through conventional breeding or transgenic strategies routinely all over the world. Success has been achieved in almost all areas of crop improvement, including drought and other biotic stress tolerance individually. However, crop improvement programs aiming to develop tolerance to individual stress did not effectively exhibit tolerance to concurrent biotic and abiotic stresses. These improved varieties, when grown in field conditions, may respond unpredictably and can have unforeseen consequences. It has been reported that drought-tolerant rice cultivars were prone to nematode attack owing to their longer roots (Kreye et al. 2009). Similarly, Bacillus thuringiensis insecticidal Cry protein-expressing transgenic cotton plants exhibited reduced toxin levels during elevated temperature or drought stress, which led to the breakdown of insect resistance (Dong and Li 2007). Therefore, a comprehensive approach should be undertaken where tolerant or resistant traits will be tested in the presence of a range of

21.5 Transcriptome Studies of Multiple Stress Responses

concurrent biotic or abiotic stress factors (Mittler and Blumwald 2010). It was proposed that true characterization of plant responses to multiple stresses can be achieved by imposing those factors in combination and studying each set of stress factors as a novel one (Mittler 2006).

21.5 Transcriptome Studies of Multiple Stress Responses Complex signaling pathways were demonstrated during molecular responses to biotic and abiotic stress conditions. Overlapping transcriptional patterns were found to be an important phenomenon during molecular interactions in plants against multiple stresses (Roychoudhury and Banerjee 2015). These overlapping sets of genes were then identified and proposed to represent points of crosstalk among signaling pathways (Mantri et al. 2010; Narsai et al. 2013; Shaik and Ramakrishna 2013; Sham et al. 2014; Zhang et al. 2016). It may be assumed that these common or overlapping genes can be the targets for stress-tolerant crop development (Atkinson and Urwin 2012; Rejeb et al. 2014; Nejat and Mantri 2017). Microarray was used to analyze the chickpea transcriptome in response to drought, cold, and high salinity in the presence of the necrotrophic fungal pathogen Ascochyta rabiei (Mantri et al. 2010). In the presence of A. rabiei and high salinity, chickpea seedlings shared the highest number of differentially expressed transcripts in comparison with A. rabiei and cold, and A. rabiei and drought. It was also demonstrated that out of 51 differentially expressed A. rabiei-responsive transcripts in shoot tissues, 21 were commonly expressed in the presence of one or more abiotic stresses. No transcript was found to be common to the four stresses involved. However, this kind of study is unable to provide adequate information to understand simultaneous stress response, as combined stress induces some unique genes that are not activated during plant response to either of the stresses independently. In a recent study, transcriptome dynamics was examined in chickpea under a combination of drought and wilt-causing Ralstonia solanacearum (Sinha et al. 2017). In this study, drought-stressed chickpea plants were subjected to pathogenic infection. Under this combined stress condition, chickpea plants were allowed to grow for two and four days, termed short-duration (SD) stress and long-duration (LD) stress, respectively. This report demonstrated decreased pathogenesis during SD combined stress in comparison with SD pathogen, whereas insignificant alteration in multiplication of pathogens was observed in LD combined stress with respect to LD pathogen. Through microarray analysis, it was found that there were 821 and 1039 uniquely expressed differential genes during SD and LD combined stresses, respectively. Three and 15 differentially expressed genes were found to be common components in all SD and LD stress situations, respectively. Lignin and cellulose biosynthetic genes were upregulated in three different conditions, viz., SD combined, LD combined, and LD pathogen stress. Transcriptomic profiling of different species demonstrated a number of signal transduction pathways to be crucial components for determining plant responses to multiple stresses (Zhang et al. 2016). These can be targeted for the improvement of crops against simultaneous stresses. In spite of developing research interest in simultaneous biotic and abiotic stresses in plants, there have been few studies on global transcriptomic changes during combined stresses.

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21.6 Signaling Pathways Induced by Drought–Biotic Stress Responses Complex arrangement of interacting factors results in biotic and abiotic stress signal transduction (Fujita et al. 2006). Multiple stresses are controlled by certain crucial gene products and thereby specific responses were obtained during both biotic and abiotic stress signaling (Mauch-Mani and Mauch 2005). Insights into those signal transduction pathways and their analysis enhanced our understanding of multiple stress tolerance (Figure 21.2). 21.6.1

Reactive Oxygen Species

It has been established that ROS perform crucial functions during biotic and abiotic stress (Fujita et al. 2006; Ton et al. 2009). The concentration of ROS increases during abiotic stresses such as drought, osmotic stress, salinity, and high light. The antioxidants and ROS-scavenging enzymes are produced by plants to reduce damage caused by these potentially harmful molecules (Apel and Hirt 2004; Roychoudhury and Basu 2012). Conversely, plants also generate ROS upon pathogen encounter through oxidative burst, which restricts the growth of the pathogen by initiating a hypersensitive response and Pathogen/ pest

Drought

Plant

ROS

HR/PCD

Hormones

MAPK cascades

Callose

TFs

Downstream response genes

Stress response

Figure 21.2 Key events in the signaling pathway activated during plant’s response to concurrent drought–biotic stresses.

21.6 Signaling Pathways Induced by Drought–Biotic Stress Responses

ultimately cell death. This phenomenon requires downregulation of ROS-scavenging pathways (Apel and Hirt 2004; Torres 2010). Plants have evolved mechanisms to utilize ROS as molecules for stress signaling. The identifiable impact of ROS-responsive genes was reported during biotic and abiotic stresses (Gadjev et al. 2006). ROS were reported to be a probable control mechanism conferring crosstolerance to abiotic and biotic stress response pathways (Atkinson and Urwin 2012). It was found that ROS-sensitive transcription factors can sense the ROS production in Arabidopsis (Miller et al. 2008). These transcription factors were found to induce genes participating in stress responses. ROS were reported to be inducers of tolerance or resistance, and to activate stress responsive transcription factors (TFs), mitogen-activated protein kinases (MAPKs), antioxidant scavenger enzymes, heat shock proteins (HSPs), and pathogenesis-related (PR) proteins (Gechev et al. 2006). Immediately after pathogen infection or wounding, H2 O2 is generated by membrane-bound NADPH oxidases. H2 O2 then diffuses into adjacent as well as distal plant tissues and activates various defense mechanisms like HR, phytoalexins, lignin, SA, and hydrolytic enzyme biosynthesis (Apel and Hirt 2004). The positive effect of ROS development was reflected by ABA-driven stomatal closure. ABA-induced NADPH oxidases, Arabidopsis Respiratory Burst Oxidase Homologue D and Respiratory Burst Oxidase Homologue F generated ROS in guard cells as signaling intermediates (Torres 2010; Atkinson and Urwin 2012). In contrast, ABA can also negatively affect the ROS-mediated pathogen defense mechanism. This was demonstrated by an ABA-deficient tomato mutant, sitiens, which was found to accumulate H2 O2 immediately after infection with B. cinerea. These led to improved defense in comparison with wild type (Asselbergh et al. 2007). It was proposed that the mentioned defense response was due to accumulation of ABA precursors in sitiens plants which disrupted redox homeostasis and caused elevated ROS generation (Ton et al. 2009). Coordinated ROS generation as well as scavenging is a necessary step to combat combined stresses. In Arabidopsis, a zinc-finger transcription factor ZAT12 was induced in response to elevated H2 O2 during abiotic and biotic stress. ZAT12, in turn, upregulated genes like ROS scavenging ascorbate peroxidase (APX1) (Rizhsky et al. 2004; Fujita et al. 2006). APX1, a crucial multiple-stress responsive enzyme, was accumulated in Arabidopsis during combined stresses but not individual stress (Koussevitzky et al. 2008). APX1 knockout mutants had a significantly lower survival rate under combination stress in comparison with wild-type plants, whereas, they survived under single stress. From the above studies, it may be inferred that there is complexity in ROS signaling during different stress responses. Identification of master regulators of ROS signaling is likely to provide probable candidates for development of multiple stress-tolerant plants. 21.6.2

Mitogen-activated Protein Kinase Cascades

The important component for perception of environmental stimuli and transducing the signal into internal regulatory pathways is mediated by MAPK cascades (Rodriguez et al. 2010; Roychoudhury and Banerjee 2017). Several MAPK components are participants in such signal transduction networks during abiotic and biotic stress responses and some of the kinases are common to both kinds of stresses (Teige et al. 2004; Fujita et al. 2006; Brader et al. 2007). Transmembrane receptors from plants, viz., flagellin sensitive 2 (FLS2) and elongation factor Tu receptor (EFR), corresponding to PAMPs, flagellin, and

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elongation factor Tu, respectively triggered MAPK cascades to initiate defense signaling (Nakagami et al. 2004; Chinchilla et al. 2007; Pitzschke et al. 2009). On the contrary, abiotic stress signals perceived by the transmembrane osmoreceptor, histidine kinase ATHK1, induced MAPK cascades such as the MEKK1/MKK2/MPK4/MPK6 pathway in Arabidopsis (Teige et al. 2004; Rodriguez et al. 2010). After activation of MAPK cascades, several TFs, viz., WRKY family TFs, and ETHYLENE INSENSITIVE3 (EIN3) were induced (Andreasson and Ellis 2010). MAPKs were responsible for controlling crosstalk between multiple stress responses and stress-induced hormones (Rohila and Yang 2007; Andreasson and Ellis 2010). Pathogen-induced MAPK cascades mediated by SA results in PR gene expression. Protein virE2 interacting protein 1 (VIP1) was found to be translocated into the nucleus following phosphorylation by MPK3 in Arabidopsis. Phosphorylated VIP1 was reported as an indirect inducer of PR1 (Pitzschke et al. 2009). Arabidopsis MPK6 functioned during cold and salt stress, stomatal control, ethylene synthesis, and pathogen signaling (Rodriguez et al. 2010). MPK3 and MPK6 responded to various biotic and abiotic stresses (Gudesblat et al. 2007; Beckers et al. 2009) and could be crucial to demonstrate tolerance to multiple stresses. Five MAPK genes were induced during biotic and abiotic stresses in rice (Rohila and Yang 2007). Among these, some MAPKs influenced both types of stress responses. Rice MAPK5 regulated cold-, drought-, and salt stress-responsive pathways positively and at the same time suppressed the expression of PR genes (Xiong and Yang 2003). Downregulation of rice MAPK5 by RNA interference (RNAi) lines exhibited resistance to pathogenic infections, whereas overexpressor mutants conferred abiotic stress tolerance. MPK16 identified from cotton, upon overexpression, demonstrated resistance to pathogens and sensitivity to drought in Arabidopsis (Shi et al. 2011). MPK6a, also identified from cotton, negatively regulated both biotic and abiotic stress (Li et al. 2013). MAPK cascades are induced by ROS, and also regulate ROS generation (Apel and Hirt 2004; Fujita et al. 2006; Takahashi et al. 2011). The function of ROS in drought-mediated multiple stress tolerance and signal crosstalk has been discussed in a previous section. Additionally, there are reports on the interaction of MAPK and ABA signaling, which led to improved plant defense and induced crosstolerance to both kinds of stresses (Lu et al. 2002; Miura and Tada 2014; Zhou et al. 2014). 21.6.3

Transcription Factors

Transcription factors regulate the reprogramming of molecular machinery after detection of a given stress. They are the key players to generate specificity in stress responses. These include members of the APETALA2/ETHYLENE-RESPONSE ELEMENT BINDING FACTOR (AP2/ERF), basic–helix–loop–helix (bHLH), basic domain leucine zipper (bZIP), MYB, NAC, and WRKY families (Ford et al. 2015; Banerjee and Roychoudhury 2017). TFs are multifunctional proteins in plants, which mediate diverse aspects of developmental pathways, respond to various biotic–abiotic stresses, and precisely modulate the transcription rate through either repression or activation of gene expression (Nejat and Mantri 2017). TFs control a wide range of downstream pathways and are ideal candidates for gene manipulation and transgenic development, which may confer multiple stress tolerance (Xu et al. 2011).

21.6 Signaling Pathways Induced by Drought–Biotic Stress Responses

Biotic stress/ wounding

Drought

Plant

ET

ABA

JA MYC2 NAC TFs ANAC019 ANAC055 ANAC072

MYB96 SA synthesis PR gene expression

RD22 Wax biosynthesis

Stomatal closure

Pathogen defense

PDF1.2 VSP1

Drought tolerance

Pathogen defense

Insect defense/ wounding

Figure 21.3 The schematic representation of crosstalk between hormones, transcription factors, and other regulatory components during concurrent drought and biotic stresses. ROS, Reactive oxygen species; ABA, abscisic acid; JA, jasmonic acid; SA, salicylic acid; PR, pathogenesis-related. Arrow head indicates activation and T-head indicates suppression.

The functions of defense-responsive TFs are mostly mediated by hormones like ABA, SA, JA, and ET. A JA-mediated signaling pathway, controlled by BOTRYTIS SUSCEPTIBLE 1 (BOS1), expressing an R2R3MYB transcription factor, demonstrated resistance to a range of pathogens (necrotrophic fungi B. cinerea and Alternaria brassicicola and biotrophic bacterial pathogen P. syringae) and abiotic stresses including drought in Arabidopsis. This was further verified by analyzing a knockout mutant of BOS1 which exhibited susceptibility to the mentioned pathogens and abiotic stresses (Mengiste et al. 2003). Another MYB transcription factor, MYB96, was induced by ABA-mediated drought signals and triggered the biosynthesis of cuticular wax, which contributed to drought tolerance in Arabidopsis (Seo et al. 2011) (Figure 21.3). Additionally, MYB96 is also involved in ABA-dependent SA biosynthesis, which caused resistance against pathogens owing to enhanced PR gene expression (Seo and Park 2010). bHLH TF MYC2 or JIN1 was reported as a central component between pathogen-induced and drought signaling pathways (Anderson et al. 2004). MYC2 regulated JA-induced insect/wounding responsive defense gene VSP1 through NAC TFs ANAC019 and ANAC055, but repressed JA/ET-induced pathogen defense gene PDF1.2 (Anderson et al. 2004; Pieterse et al. 2009; Kazan and Manners 2013). MYC2 also activates NAC TF ANAC072, in addition to ANAC019 and ANAC055. These TFs suppressed SA levels by inducing SA catabolism and suppressing SA synthesis (Laurie-Berry et al. 2006; Kazan and Manners 2013). MYC2 was found to activate ABA responsive gene, RESPONSIVE TO DESICCATION22 (RD22), which was responsible

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for stomatal closure and subsequent drought tolerance. MYC2 knockout Arabidopsis mutants were devoid of ABA-induced gene expression (Abe et al. 2003). This partial synergy in ABA-JA signaling may play a role in drought-mediated enhanced pathogen resistance (Atkinson and Urwin 2012; Kazan and Manners 2013). Mutant myc2 and the anac019-anac055 double mutant in Arabidopsis showed enhanced resistance to B. cinerea. Expression of either ANAC019 or ANAC055 exhibited more susceptibility to B. cinerea and increased tolerance to drought in mutant Arabidopsis plants (Kazan and Manners 2013). A NAC family TF Arabidopsis thaliana activating factor 1 (ATAF1) was induced in response to multiple stresses. ATAF1 was found to induce drought tolerance by stomatal closure in both an ABA-dependent and an ABA-independent manner (Lu et al. 2007; Wu et al. 2009). Additionally, in ATAF1 knockout mutant, ABA biosynthesis aldehyde oxidase gene (AAO3) was induced upon powdery mildew (Blumeria graminis f.sp. hordei [Bgh]) infection and the plants became susceptible, whereas AAO3 knockout mutant showed resistance to Bgh penetration (Jensen et al. 2008). Moreover, in the mentioned ATAF1 knockout mutant JA/ET-activated transcription of plant defensin genes was abolished as compared with wild-type plants. Another NAC TF isolated from rice, OsNAC6, having high similarity to genes of the ATAF subfamily, was reported as a regulator for enhanced tolerance to biotic and abiotic stresses (Nakashima et al. 2007). It was induced by blast disease and wounding as well as drought, cold, and high salinity (Ohnishi et al. 2005; Nakashima et al. 2007). Overexpression of OsNAC6 in rice showed increased tolerance to abiotic factors like drought and salinity, in addition to moderate tolerance against Magnaporthe oryzae. Pathogens and JA, as well as drought and ABA, induced the NAC transcription factor RD26 in Arabidopsis (Fujita et al. 2004). A bZIP TF, AREB1, obtained from tomato, conferred tolerance to salt and drought stress and also activated defense-responsive genes (Orellana et al. 2010). 21.6.4

Heat Shock Proteins and Heat Shock Factors

HSPs are molecular chaperones, regulating protein synthesis, folding, assembly, translocation, and degradation. These are expressed constitutively in both prokaryotes and eukaryotes (Wang et al. 2004; Banerjee and Roychoudhury 2018). However, HSPs are upregulated in response to adverse environmental factors and protect cells by preventing protein aggregation, stabilizing misfolded proteins and maintaining cellular homeostasis (Wang et al. 2004; Sham et al. 2014). The expression of HSPs is regulated by heat shock transcription factors (HSFs) which have an integral role in response to biotic and abiotic factors (Chung et al. 2013; Xue et al. 2015). HSF genes were upregulated upon induction of heat, drought, and other abiotic stresses as well as powdery mildew and Podosphaera aphanis infection in strawberry (Hu et al. 2015). Overexpression of Arabidopsis HSFA1b in oilseed rape demonstrated multiple stress tolerance, maintaining the productivity. HSFA1b was involved in regulation of more than 500 genes controlling stress tolerance to biotic and abiotic factors (Bechtold et al. 2013). The ABA-dependent signaling pathway was found to be involved in regulation of HSF expression. This was demonstrated by overexpression of HSFs upon exogenous ABA application (Hwang et al. 2014; Hu et al. 2015). Additionally, HSFs were reported to be sensors to detect ROS generation and activate corresponding downstream genes (Miller and Mittler 2006; Hu

21.6 Signaling Pathways Induced by Drought–Biotic Stress Responses

et al. 2010). The redox sensor HSFA4a was found to be induced in response to H2 O2 in Arabidopsis. Downstream, it can control the synthesis of ROS-scavenging enzymes (Miller and Mittler 2006). Moreover, HSPs were also reported to be regulators for the function of R genes in response to biotic and abiotic stresses, which was demonstrated by analyzing the function of an Arabidopsis mutant carrying a point mutation in HSP90-2 (Belkhadir et al. 2004). This might be the explanation for the stress responses in a study on transcriptomic and metabolomic analyses in Arabidopsis during heat, drought, and Turnip mosaic virus together or in isolation. The results revealed that the highest number of R genes was induced under heat as well as combined heat and drought stress, whereas only a few R genes were expressed under all three stresses in combination (Prasch and Sonnewald 2013). From the above observations, it was inferred that HSPs regulate the activities of R genes in response to biotic and abiotic stresses. 21.6.5

Role of ABA Signaling during Crosstalk

The responses of plants to environmental stress factors are largely controlled by specific hormones. Abiotic stresses are mostly controlled by ABA (Roychoudhury et al. 2009), whereas resistance or tolerance responses against biotic factors are mediated by SA and/or JA/ET signaling pathways. However, both negative and positive roles of ABA were demonstrated in response to pathogenic infection. P. syringae pv. tomato DC3000 (PstDC 3000) infection in tomato exhibited higher levels of ABA accumulation which inhibited other defense responses (Truman et al. 2006; de Torres-Zabala et al. 2007). Additionally, ABA treatment increased the susceptibility of plants to pathogen infections, e.g. susceptibility of Arabidopsis to an avirulent strain of P. syringae (Mohr and Cahill 2003). Higher levels of ABA accumulation can suppress the defense responses mediated by SA, JA, or ET under combined abiotic and biotic stresses (Audenaert et al. 2002; Mauch-Mani and Mauch 2005; Asselbergh et al. 2008a; Yasuda et al. 2008). The systemic acquired resistance pathway upstream and downstream to SA was repressed by ABA treatment in Arabidopsis and tobacco (Mohr and Cahill 2007; Yasuda et al. 2008; Kusajima et al. 2010). Additionally, ABA-deficient tomato sitiens mutant exhibited increased accumulation of SA-dependent PR1, with improved tolerance to B. cinerea (Audenaert et al. 2002; Asselbergh et al. 2007). Furthermore, ABA application suppressed the expression of JA-mediated PR genes like PDF1.2 ( plant defensin 1.2), CHI (basic chitinase), and HEL (hevein-like protein) in Fusarium oxysporum- and Erwinia chrysanthemi-infected Arabidopsis plants (Fujita et al. 2006; Asselbergh et al. 2008a). A positive role of ABA in biotic stress tolerance was also demonstrated in plants, particularly during pre-invasive and early post-invasive defense against pathogens (Asselbergh et al. 2008b; Ton et al. 2009; Garcia-Andrade et al. 2011; Luna et al. 2011). Pre-invasive defense induced by ABA was reported in Arabidopsis. This kind of defense was mediated by ABA-induced stomatal closure to resist microbial invasion in a mechanism supported by SA signaling. It was demonstrated that, in ABA-deficient Arabidopsis mutant aba3-1, stomatal closure was hampered after applying pathogen-derived elicitors (Melotto et al. 2006). Additionally, callose accumulation in pathogen-infected plants was regulated by the ABA signaling pathway (Ton and Mauch-Mani 2004; Ton et al. 2005). Improved resistance was obtained by ABA-induced callose biosynthesis (e.g. resistance of Arabidopsis against Pythium irregulare [Adie

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et al. 2007]) or inhibition of callose degradation (Rezzonico et al. 1998; Jacobs et al. 2003). A model was proposed by Ton et al. (2009) that described the intricate role of ABA in pathogenic infection. The infection time-span and nutritional requirement of the pathogen influenced this ABA-mediated response (Ton et al. 2009). Three phases of pathogen infection were mentioned in this model. In the first phase, ABA-mediated stomatal closure (Kim et al. 2011) allowed a reduction in water loss and water potential was maintained in plant tissue. Additionally, stomatal closure increased resistance to penetration by pathogens. At this phase, there might not be any SA, JA, and ET activation and ABA can antagonize their induction. In the second phase, ABA-dependent callose accumulation strengthens cell walls to regulate early post-invasive defenses during fungal infection. In contrast, ABA-dependent callose accumulation was repressed during bacterial infection. Environmental conditions can control ABA-mediated induction or repression of additional callose accumulation. In the third phase, PAMP-stimulated SA, JA, and ET accumulation was reported to regulate the defense reaction (Zhou et al. 2014). However, during the third phase of disease development, abiotic stress-driven elevated ABA levels could suppress the SA, JA, and ET responsive pathways.

21.7 Conclusion Plants are exposed to multiple environmental stress factors during their growth. They have an inherent potential to combat the combined effects of multiple stress factors, overcoming damage and saving their resources for growth and reproduction. The prevention of yield loss owing to combined stresses was of utmost importance for researchers during the last century. In recent years, global warming has influenced the emergence of new stress combinations that have aggravated the adverse effects of combined stresses. There is crosstalk between the stress-inducible pathways against multiple stresses, with or without affecting each other’s outcome. Such crosstalks have a positive or negative influence on plants; a positive influence may result in crosstolerance, while a negative effect can introduce susceptibility to a previously tolerant plant. Concurrent drought and biotic stress interaction has gained in importance as these stresses are responsible for major crop losses individually. Although a number of resistant cultivars against such individual stresses have been established through conventional breeding programs or transgenic strategies, there is evidence that plants that are tolerant to single stresses become susceptible to a novel stress in field conditions. Thus, there are possibilities for breeding/transgenic failure with the increasing occurrence of combined stresses. With the growing concern about severe yield loss, understanding of molecular factors responsible for combined stress tolerance is highly solicited. Unfortunately, limited research findings are available on whole-genome transcriptomic changes resulting from such combined stresses, in spite of remarkable advancement in high-throughput sequencing technologies and a wide range of bioinformatics tools for genomic and transcriptome analyses. The candidate genes differentially regulated during combined stresses may be the potential targets for genetic modification to develop resistant plants under combined drought–biotic stress conditions. Such genes can also be important components in marker-assisted

References

breeding programs for combined stress-resistant crops. Recent studies based on the identification of common and overlapping components of the plant signaling crosstalk obtained from individual and concurrent stresses will also allow the development of novel tools to combat the combined drought–biotic stresses. Hence, identification of such multifunctional genes responsible for combined drought–biotic stress resistance would accelerate the development of tolerant crop plants. In this direction, the production of ROS, MAPK cascades, TFs, and hormone signaling triggered by both biotic and drought stresses in a specific or nonspecific manner and their roles as common components of the immune system have been studied. The most challenging aspect of the combined drought–pathogen stress tolerance studies in plants is to determine an exact methodology to impose accurate and concurrent stresses under controlled conditions. In most of the studies, the growth stage of plants, duration, and severity of imposed stresses are not well established. Additionally, it is also not clear from most of the reports whether the applied combined stresses were simultaneous or sequential. Owing to these experimental difficulties, specific morphological, physiological, biochemical, and molecular changes in plants exposed to simultaneous or concurrent stress conditions are yet to be elucidated. Taking all of the mentioned difficulties into consideration for studying concurrent stress biology in plants, the proper experimental design and identification of exact molecular tools to find responsible tolerant factors will be decisive steps for the development of improved plants.

Acknowledgments The authors acknowledge St. Xavier’s College (Autonomous), Kolkata for providing infrastructure to support the research activities.

Conflict of Interest There is no conflict of interest.

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447

Index a AAA+ ATPase 229 ABA signalling 94 Abiotic stress 31, 37, 65, 69, 73, 75, 78, 79, 81, 335, 336, 356, 427–438 ABI SoLid 391–392 ABRE-binding factor (ABF) 139, 371 Abscisic acid (ABA) 4, 73, 74, 343, 427, 433, 435–438 deficient mutant 433, 437 dependent 436, 438 dependent SA biosynthesis 435 independent 436 responsive gene 435 signalling 434, 437 Abscisic acid responsive element binding proteins 4 Abscisic acid-responsive elements (ABREs) 139, 210–211, 371 Accessory genes 309 Acclimatization 337 Activator motif (AHA motif ) 230 Additive effect 242 AHKs 213 α-crystallin domain 223 Aluminum-activated malate transporter 249, 251 1 aminocyclopropane-1-carboxylic acid (ACC) 138 Anion channels 242 Anion conductance 250 Anthesis-silking interval (ASI) 345 Anthracene-9-carboxylic acid 258 Antioxidant enzymes 126, 136

ascorbate peroxidase 66, 69, 75, 76, 78, 81, 126, 129 catalase 66, 77, 113, 126, 195, 316, 326 glutathione peroxidase 126 glutathione reductase 79 glutathione-S-transferase 106, 126 guaiacol peroxidase 74, 75, 126, 127 superoxide dismutase 113, 126, 129 Antioxidant system 74, 75, 76, 80, 91, 93 ATP-binding cassette (ABC) transporter 249 ATP hydrolysis 247 Autoinhibition of nodulation 306 Autoregulation of nodulation (AON) 306 Auxin 36, 37, 306

b Bacillus thuringiensis 430 Bacteroids 304 Basic-helix–loop–helix (bHLH) family 158, 434, 435 Basic leucine zipper (bZIP) 11, 155, 375, 434, 436 β-1, 3-glucanase 106 Biological nitrogen fixation 301 Biomodulation 272 Bio-priming 283 Biosynthesized nanomaterials 317 Biotic stress 335, 427–434, 436–439 Blumeria graminis f.sp. hordei (Bgh) 436 Botrytis cinerea 430, 433, 435–437 BOTRYTIS SUSCEPTIBLE 1 (BOS1) 435 Brassinolides 106 Brassinosteroids 186, 272, 306 Breeding 374, 430, 438, 439

Molecular Plant Abiotic Stress: Biology and Biotechnology, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

448

Index

Bridging ligand 243 Brome mosaic virus 430 BY-2 cells 251

c Calcineurin 228 Calcineurin-B like 379 Calcium-activated chloride channel 258 Calcium calmodulin dependent protein kinase (CCaMK) 305 Calcium-dependent protein kinases 250 Calcium-independent SnRK2.6 kinase 250 Calcium signalling 92 Calcium spiking 305 Callose accumulation 437, 438 biosynthesis 437 degradation 438 Calnexin 222 Calreticulin 222 Carbon nanotubes 317, 322 Carbon starvation 150 Carotenoids 66, 76 CBF3 11, 12 CBF genes 352 CBL-interacting serine/threonine-protein kinase 15, 378 Cell membrane receptors 153 Cell signalling 322, 323, 326 Chaperone and Chaperonins 222, 224, 225, 436 ChiP-sequencing 373 Chitinase 289, 437 Chitosan 106 Chloride conductance regulatory protein 249 Chloride co-transporters 249 Chlorofluorocarbon 31 Choline oxidase 96 Cl– efflux 245 Cl– influx 245 CIPK 4 CLCi gene families 249 Climate change 1, 341 Clp 229, 230 Cold acclimation 351, 353 Cold stress 29, 30, 31, 33, 34, 188, 192, 335, 356

Cold tolerance 10 Combined drought–biotic stress 428, 438, 439 drought–pathogen stress 439 stress 427, 428, 430, 431, 433, 438, 439 comPARE 406 Compatible solutes 135 Concurrent Stress 439 COR gene 351, 352 C-Repeat Binding Factor 11 CRISPR/Cas 374 Crosstolerance 433, 434, 438 Cry protein 430 Cucumber mosaic virus 430 CuO-NPs 319, 321 Cyclophilin 274 CYP5 11 Cysteine 195 Cytokinins 209, 306 Cytoprotectants 91

d Dark reaction 189 Dark septate endophytic fungi 288 DEAD-box helicase 16 Deeper rooting 1 (DRO1) 376 Defense phytohormones 210 Defense readiness 278 Degradome sequencing 398 Dehydration-responsive element (DRE) or C-repeat (CRT) 372 Dehydroascorbate reductase 79 DELLA proteins 210 De-novo sequencing 395 de novo xylem development 430 Depolarization 248 Desiccation 336 2, 6-dichloro-isonicotinic acid 106 Differentially expressed gene family 164 Dimethyldiselenide 125 2, 2-diphenyl-1-picrylhydrazyl (DPPH) 327 DNA-binding domain (DBD) 230 DNA helicase 45, 16 DnaJ 227, 228 DnaK 228 DNA methylation 167–168, 373

Index

Downstream transcription factors 146–154 DREB1A 9 DREB genes 161, 372, 375 Drought 29, 30, 32, 33, 335, 345, 346, 370–374, 427–432, 434–439 DroughtGardTM 10 Drought-resistant and drought-sensitive cultivar 190 Drought Stress Sensor 48 Drought Stress Signaling of Stomata 48 Dwarfism 162

e Early nodulation genes (ENOD) 305 Econanotoxicity 319, 321 Econanotoxicology 317 Economic yield 337 Ecotoxicity 320 Effector triggered immunity 427 Electrochemical gradient 247 Electrolytic leakage 113 Elicitors 105 Elongation factor Tu 434 receptor (EFR) 433 Endomutualist 269 Endosymbiosis 306 Engineered nanoparticles 315, 316, 317, 318, 319, 320 Environmental stress 337 Enzymatic defense system 316, 326 Epigenomics and epigenetics 167, 373, 396–397 ERF genes 161 ER stress signalling 288 Erwinia chrysanthemi 437 Ethylene 37, 112, 186, 187, 211, 306, 427, 434–438 ETHYLENE INSENSITIVE3 (EIN3) 434 Evasion 336, 337 Excitation energy capture 244 Exome Sequencing 396 Extremophile plant 150

f Farnesyl transferase 9 Fenton reaction 325 Flagellin 433

Flavonoid 66, 76, 287 Floral induction 109 FLS2 433 Flux localization 245 Food security 1, 337 Free radicals 320, 323, 324, 325, 327 Freezing 30 Functional genes 337 Fusarium oxysporum 437

g GA INSENSITIVE 211 γ-amino butyric acid 137, 251 GA signalling 211 Genetic diversity 337 Genetic engineering 232 Genome-wide association mapping 370 Genotoxicity 322, 326 Genotypic variation 370 Germplasms 335, 355 Gibberellins 209 Global economy 337 Glutathione 74, 75, 126, 127, 195 Glutathione–ascorbate cycle 78, 270 Glutathione synthase 16 Glycinebetaine 92 Grain filling 370 Gravitropism induced systemic tolerance 109 Greener nanoparticles 317 GroL 224 Growth-regulating phytohormones 210 Grp94 228 GrpE 226 GUDK (Growth under drought kinase) 375

h Halophilic crops 347 Halophytes 191 HATs (histone acetyl transferases) 374 hc-siRNAs 401 Heat shock factor (HSF) 436 Heat shock protein (HSP) 192, 433, 436, 437 Heat stress 30, 31, 34, 35, 37, 192 Heat Tolerance 342

449

450

Index

Heavy metal stress 29, 30, 31, 35, 92, 149–154, 337 HEL (hevein-like protein) 437 Heritability 338 Hill reaction 243 Histidine kinase 434 Histone deacetylases (HDACs) 373 Histone deacetylation 167 H2 O2 433, 437 HOG pathway 94 Horizontal resistance 338, 339 Hormonal Regulation of Stomata 49 HOS1 11 HscA 225 HscC 225 HSE 230, 231 HSF 230, 231 HSP 100, 229, 230 HSP40 227, 228 HSP60 224, 225 HSP70 225, 226 HSP90 228, 229 HtpG 228 Hybridization 341, 342 Hydraulic Signaling of Stomata 49 Hydrogen peroxide 69, 244 Hydroxyl radical l69, 244, 316, 323 Hypersensitive response 432 HYR (High Yield Rice) 372

i Illumina 393–394 Incidental nanoparticles 315 Inducer of CBF expression 11 Infection thread 303 In silico analysis 162 Interstitial fibrosis 325 Intracellular compartmentation Intracellular responses 153 Ionic imbalance 241 Ionomics 271 ION Torrent 392–393 Ion toxicity 244 Isoflavanoid 302 isomiRS 406

245

j Jasmonic acid (JA) 427, 434–438 -mediated signaling pathway 435 J-domain 227, 228 JIN1 435

k K+ /Na+ 377 Knockout mutant 435, 436

l Late embryogenesis abundant 4, 9, 154, 211, 375 Leaf rolling 370 Leghaemoglobin 304 Linkage disequilibrium 344, 370 Lipid peroxidation 71, 72, 91, 113, 126–129, 244, 271 Lipochitooligosaccharide (LCO) 302 Lipoxygenase pathway 308

m Macrophomina phaseolina 429 Magnaporthe oryzae 436 Maize dwarf mosaic virus 430 Malondialdehyde 126 Marker-assisted selection (MAS) 335, 341, 374 Mechanosensitive channels of the small conductance-like transporter 259 Membrane spanning helices 250 Metabolomics 372 Metagenomics 396 Metallic nanoparticles 317 Methylamines 91 Methyl jasmonate 106 Methylsulfonium compounds 93 Microarray 431 MicroRNA 9, 402 miRBase 404 Mitogen-activated protein kinase 324, 325, 375, 433, 434, 439 Molecular breeding 336 Molecular markers 339, 340 Monodehydroascorbate reductase 79

Index

MPSS 404 Multidrug-resistance protein 4, 258 Multiple alleles 338 Multiple stresses 427, 428, 431, 432, 434, 436, 438 responses 434 responsive enzyme 433 tolerance 432–434, 436 Multiwalled carbon nanotubes 317, 321, 326 MYB family 96, 372, 375, 434, 435 MYB3R 11 MYC 2, 435 MYC/MYB 158 Mycorrhiza 283

Nodule carbon metabolism 308 Nodule inception (NIN) 305 Nodule organogenesis 305 Nodule primordial 303 Non enzymatic defense system 316, 326 Non-hormonal regulation of stomata 52 NO scavengers 109 NO-synthesizing enzymes 109 Nuclear export signal 230 Nuclear factor Y-B subunits 4 Nuclear Localization Signal 230 Nucleotide binding domain 229 Nucleotide exchange factors 226 Nutrient Imbalance 244

o n NAC family 159, 371, 372, 375, 434 NADPH oxidase 05 Na+ /H+ antiporter 12 Nanoclays 315 Nanoparticle mediated oxidative stress 324 Nanoparticles transformation 318 Nanotechnology 315 Nanotoxicity 319, 322, 328 NARK signal transduction 307 nat-siRNAs 401 Natural nanoparticles 315 Natural selection 338 Net transpiration 242 Next generation sequencing (NGS) 390–393 9-cis-epoxycarotenoid dioxygenase gene (NCED) 138 Nitrate assimilation 308 Nitrate excretion transporter 259 Nitrate transporter 1/peptide transporter 249 Nitric oxide 109 Nitric oxide synthase inhibitors 109 Nitrogenase 305 Nitrogen fertilizers 301 Nod Factors 302 Nodule 302

Odium neolycopersici 428 Oligomerization domain 230 Organic osmolytes 93 OsAKT1 377 OsCAX1 377 OsCDPK7 375 OsCIPK28 378 OsCLC1 377 OsDERF1 374 OsHKT2.1 377 OsKCO1 377 OsLEA3 377 Osmolytes 375 Osmoprotectant 4, 10, 93, 430 Osmotic potential 244 Osmotic pressure 147–149 Osmotic shock 91 Osmoticum 242 Osmotin 271 OsMYB48-1 377 OsNCED4 377 OsNCED5 377 OsNHX1 377 OsPP2C68 377 OsSOS1 377 OsTPC1 377 Outward rectifying depolarization-activated anion channels 248

451

452

Index

Oxidative burst 432 Oxidative damage 73, 74, 75, 76, 290, 316, 319–322, 324–326 Oxidative stress 195, 316, 320, 321, 322, 324, 325, 326 Oxidizing atmosphere 147

p PAL 113 Pangenome 309 PARP 9 Patch-clamped protoplasts 250 Pathogen-associated molecular pattern (PAMP) 433, 438 triggered immunity 427 Pathogenesis-related (PR) 106, 433–435, 437 Peribacteroid membrane 304 Peroxidase 77, 195, 316 Peroxidation of lipids 191 Peroxisomes 70 PGPB 285 PGPR 284 Phenolics 77 Phenotyping 335, 356 Phosphate/nitrogen starvation 161 Phosphorylation 145, 147, 149, 152, 154, 155, 157 Photochemical quenching 244 Photosynthesis 188, 337, 343, 344, 345 Photosynthetic efficiency 189 Photosynthetic electron transport 194 Photosystems 194 Phyllosphere 91 Phytoalexin 106, 433 Phytochrome-Interacting Factor-Like 1 9 Phytohormones 92, 107, 185, 186, 194 Phytoremediation 150 Phytotoxicity 245, 320, 322, 328 Piriformospora indica 269 Pisatin pathway 309 Plant defensin 1.2 (PDF1.2) 435, 437 Plant growth-promoting rhizobacteria (PGPR) 106 Plant senescence 345 PMRD 404 Pod abortion 343

Pollen sterility 343 Poly(ADP-ribosyl)ation 9 Polyamines 9, 91 amelioration 136 arginine decarboxylase 135, 138, 139 cellular antioxidants 136 interaction with abscisic acid 138 interaction with brassinosteroids 137 interaction with ethylene 137 interaction with proline 137 interaction with salicylic acid 138 nitric oxide 137 photosystem II 136 polyamine oxidase (PAO) 137, 138 priming 136, 138 putrescine 135 S-adenosylmethionine decarboxylase 135 spermidine 135 spermidine synthase 135, 138, 139 spermine 135 spermine synthase 135, 138, 139 synthesis 135 Polygenic character 338 Polyphenol oxidase 106 Post translational modification 165 Powdery mildew 428, 436 POX 113 Programmed cell death 427 Proline 113, 126, 127, 136, 375 Protein disulfide isomerase 222 Protein kinases 73 Protein metabolism 193 Protein phosphatase 2C 4 Proteolytic destruction 165 Pseudomonas syringae 428, 435, 437 Puccinia striiformis 428

q Q molecule 307 qRT-PCR 344 QTL 338, 342, 344, 346, 347, 348, 350, 351, 353, 354, 355, 356, 369, 370, 374, 376, 379 QTL-hotspot 345 Quality filtering 403

Index

r RAB 377 Ralstonia solanacearum 431 ra-siRNAs 401 RAV genes 161 RD26 436 Reactive oxygen species (ROS) 2, 12, 16, 37, 66, 91, 112, 123, 125, 135, 136, 145, 196, 305, 316, 319, 320, 321, 322, 323, 324, 342, 369, 427, 432, 433, 434, 439 generation 433, 436 responsive genes 433 sensitive transcription factors 433 signalling 433 Receptor genes 187 Receptor like cytoplasmic kinase (RLCK) 375 Receptor like kinase (RLK) 304 Recombinant inbred lines 343 Redox homeostasis 80, 433 Redox reactions 189, 320 Reduced potassium dependency 3/histone deacetylase 1 (RPD3/HDA1) 373 Reducing atmosphere 147 Relative water content 113 Repressor domain 230 Resilience 336 Resistance (R) genes 427, 428, 437 Respiratory Burst Oxidase Homologue 433 Responsive To Dehydration 29A 4, 375 RESPONSIVE TO DESICCATION22 (RD22) 435 Resurrection plants 336 Rhizobium 301 Rhizosphere 91, 302 Ribosome Profiling 397 RNA-binding proteins 164 RNA interference (RNAi) 370, 434 Roche 454 390–391 Root curling 305 Root symbiosis 283 ROS-MAP kinase networks 95 ROS-scavenging 433 enzyme 432, 437 pathway 433

R2R3MYB 435 R-type channel 250

s Salicylic acid (SA) 191, 344, 427, 433–435, 437, 438 biosynthesis 435 catabolism 435 level 435 signalling 437 Salinity 12, 31, 32, 188, 335, 347, 356 Saltol 347, 380 Salt treatment 159–163 Sebacinales 288 Secondary metabolite 114, 317, 328 Seed dormancy 109, 210 Seed priming 114 Selenium ameliorating property 126–129 antioxidant properties 125 bioaccumulation and metabolism 124, 125 cadmium and lead toxicity 129 drought stress 127, 128 exogenous application 126–129 heavy metal stress 129 low temperature stress 128 plant growth promoters 125 salt stress 126, 127 UV-B stress 128, 129 Selenocysteine and selenomethionine 124, 125 Senescence 109 sHSP 223, 224 Signalling molecules of drought sensing 53 Silent information regulator 2 (SIR2) 373 Simultaneous drought and biotic stress 428 Single nucleotide polymorphisms 370 Singlet oxygen 68, 244 Single-walled carbon nanotubes 317, 322 siRNAs 374 Sitiens 433, 437 Slow anion channel and associated homologs 249 Small interfering RNAs 398–400

453

454

Index

Small RNA Sequencing 398 Sodium nitroprusside 112 Solexa Illumina array 346 SOS pathway 92 Spodoptera exigua 430 sRNA toolbox 404 Stele-specific anion channel 259 Stomatal closure 430, 433, 436–438 Stomatal conductance 47, 151, 242 Stomatal density regulation 57 Stomatal morphology 46 Stomatal movement 47, 112 Stomatal regulation 55, 189, 242 Stress induced hormones 434 inducible promoters 4 responsive genes 93 signaling 432, 433 S-type channel 248 Submergence 91 Sucrose nonfermenting 1-related protein kinase 2, 4 Sulfate transporters, SULTR1; 2 and SULTR1 124 Super crops 291 Superoxide radical 67, 244 Symbiosome 304 Symbiotic interaction 304 Symplastic pathway 245 Systemic acquired resistance 437

Transgenic plants 188, 369–381, 430, 434, 438 Trans-Golgi network 255 Transient stress 336 Translationally controlled tumour protein 154 Transmembrane osmosensors 54 Transpiration rate 430 TRAP1 228 Trehalose 272 Trichoderma harzianum 287 Turgor pressure 242

u Ubiquitination process 165 UEA sRNA workbench 404 UV irradiation 31, 35, 149–154

v Vacuolar H+ pyrophosphatase 12 VaERF057 11 Vascular-related NAC domain 7, 430 Vertical resistance 338, 339 Verticillium longisporum 430 Vigor index 111 Violaxanthin 152 Vir E2 interacting protein 1 (VIP1) 434 Voltage-dependent channels 248 VSP1 435

w t Targeted RNA Sequencing 396, 397 ta-siRNAs 401 Temperature fluctuations 191 Thaumatin 272 TiO2 -NPs 315, 322, 328 Tobacco mosaic virus 429, 430 Tocopherol 75, 194 Tomato spotted wilt virus 430 Total phenolic concentration 113 Transcription factor (TF) 187, 433–436, 439 Transcription regulators of stomatal closure 55 Transcriptome analysis 196 Transcriptomic profiling 431 Transcriptomics 371 Trans-generational epimutations 373

Water deficit 245 Waterflow 150 Water-holding potential 430 Waterlogging 337 Water splitting system 243 Water use efficiency 244 Whole genome sequencing 395–396 WRKY 4, 372, 434

x Xylella fastidiosa

429

z ZAT12 433 Zeatin 106 Zeaxanthin 152 Zinc finger proteins 162 ZnO-NPs 321, 322, 323, 328

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