Protective Chemical Agents in the Amelioration of Plant Abiotic Stress 1119551633, 9781119551638

Table of contents :
Cover
Protective
Chemical Agents in the Amelioration
of Plant Abiotic Stress:
Biochemical and Molecular Perspectives
Copyright
Contents
List of Contributors
1 Role of Proline and Glycine Betaine in Overcoming
Abiotic Stresses
2 Glycine Betaine and Crop Abiotic Stress Tolerance:
An Update
3 Osmoprotective Role of Sugar in Mitigating Abiotic Stress
in Plants
4 Sugars and Sugar Polyols in Overcoming Environmental
Stresses
5 Ascorbate and Tocopherols in Mitigating Oxidative Stress
6 Role of Glutathione Application in Overcoming
Environmental Stress
7 Modulation of Abiotic Stress Tolerance Through
Hydrogen Peroxide
8 Exogenous Nitric Oxide- and Hydrogen Sulfide-induced
Abiotic Stress Tolerance in Plants
9 Role of Nitric Oxide in Overcoming Heavy Metal Stress
10 Protective Role of Sodium Nitroprusside in Overcoming
Diverse Environmental Stresses in Plants
11 Role of Growth Regulators and Phytohormones
in Overcoming Environmental Stress
12 Abscisic Acid Application and Abiotic Stress Amelioration
13 Role of Polyamines in Mitigating Abiotic Stress
14 Role of Melatonin in Amelioration of Abiotic
Stress-induced Damages
15 Brassinosteroids in Lowering Abiotic Stress-mediated
Damages
16 Strigolactones in Overcoming Environmental Stresses
17 Emerging Roles of Salicylic Acid and Jasmonates in Plant
Abiotic Stress Responses
18 Multifaceted Roles of Salicylic Acid and Jasmonic Acid
in Plants Against Abiotic Stresses
19 Brassinosteroids and Salicylic Acid as Chemical Agents
to Ameliorate Diverse Environmental Stresses in Plants
20 Role of γ-Aminobutyric Acid in the Mitigation of Abiotic
Stress in Plants
21 Isoprenoids in Plant Protection Against Abiotic Stress
22 Involvement of Sulfur in the Regulation of Abiotic Stress
Tolerance in Plants
23 Role of Thiourea in Mitigating Different Environmental
Stresses in Plants
24 Oxylipins and Strobilurins as Protective Chemical Agents
to Generate Abiotic Stress Tolerance in Plants
25 Role of Triacontanol in Overcoming Environmental
Stresses
26 Penconazole, Paclobutrazol, and Triacontanol
in Overcoming Environmental Stress in Plants
27 Role of Calcium and Potassium in Amelioration
of Environmental Stress in Plants
28 Role of Nitric Oxide and Calcium Signaling in Abiotic
Stress Tolerance in Plants
29 Iron, Zinc, and Copper Application in Overcoming
Environmental Stress
30 Role of Selenium and Manganese in Mitigating Oxidative
Damages
31 Role of Silicon Transportation Through Aquaporin Genes
for Abiotic Stress Tolerance in Plants
32 Application of Nanoparticles in Overcoming Different
Environmental Stresses
Index

Citation preview

Protective Chemical Agents in the Amelioration of Plant Abiotic Stress

­ rotective Chemical Agents in the Amelioration P of Plant Abiotic Stress Biochemical and Molecular Perspectives

Edited by Aryadeep Roychoudhury

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

Durgesh Kumar Tripathi

Amity Institute of Organic Agriculture Amity University Uttar Pradesh Noida, Uttar Pradesh, India

   

This edition first published 2020 © 2020 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 Office(s) 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: Protective chemical agents in the amelioration of plant abiotic stress: biochemical and molecular perspectives / edited by Aryadeep Roychoudhury, Durgesh Kumar Tripathi. Description: Hoboken : Wiley-Blackwell, 2020. | Includes bibliographical references and index. Identifiers: LCCN 2019051508 (print) | LCCN 2019051509 (ebook) | ISBN 9781119551638 (hardback) | ISBN 9781119551645 (adobe pdf) | ISBN 9781119551652 (epub) Subjects: LCSH: Plants–Effect of chemicals on–Molecular aspects. | Plants–Effect of stress on. | Plant molecular biology. Classification: LCC QK746 .P76 2020 (print) | LCC QK746 (ebook) | DDC 581.3–dc23 LC record available at https://lccn.loc.gov/2019051508 LC ebook record available at https://lccn.loc.gov/2019051509 Cover Design: Wiley Cover Image: © The natures/Shutterstock Set in 9.5/12.5pt STIXTwoText by SPi Global, Chennai, India Printed and bound by CPI Group (UK) Ltd, Croydon, CR0 4YY 10  9  8  7  6  5  4  3  2  1

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Contents

List of Contributors  xix

1

Role of Proline and Glycine Betaine in Overcoming Abiotic Stresses  1 Murat Dikilitas, Eray Simsek, and Aryadeep Roychoudhury Introduction  1 Responses of Crop Plants Under Abiotic Stresses  2 Mechanisms of Osmoprotectant Functions in Overcoming Stress  3 Proline Biosynthesis and Mechanism of Action in Plants  4 Glycine Betaine (GB) Biosynthesis and Mechanism of Action in Plants  7 Application of Osmoprotectants in Stress Conditions  7 Application of Proline  7 Application of GB  10 Transgenic Approaches  11 Negative Effects of Proline Application  12 Conclusion and Future Perspectives  14 Acknowledgment  14 References  15

1.1 1.2 1.3 1.3.1 1.3.2 1.4 1.4.1 1.4.2 1.4.3 1.4.4 1.5 2 2.1 2.2 2.3 2.4 2.4.1 2.4.2 2.4.3 2.4.4 2.5 2.5.1 2.5.2 2.5.3

Glycine Betaine and Crop Abiotic Stress Tolerance: An Update  24 Giridara-Kumar Surabhi and Arpita Rout Introduction  24 Biosynthesis of GB  25 Accumulation of GB Under Abiotic Stress in Crop Plants  26 Exogenous Application of GB in Crop Plants Under Abiotic Stress  27 Drought  27 Salt Stress  28 Temperature Stress  29 Heavy Metal Stress  29 Transgenic Approach to Enhance GB Accumulation in Crop Plants Under Abiotic Stress  33 Drought  34 Salt Stress  34 Temperature Stress  35

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2.6 2.6.1 2.6.2 2.7 2.8 3 3.1 3.2 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.6.1 3.3.6.2 3.3.6.3 3.3.6.4 3.3.7

Effect of GB on Reproductive Stage in Different Crops  35 Role of GB on Flower Initiation  35 GB on Seed Set and Yield Stability  41 Pyramiding GB Synthesizing Genes for Enhancing Abiotic Stress Tolerance in Plants  41 Conclusion and Future Prospective  43 Acknowledgment  43 Reference  44 Osmoprotective Role of Sugar in Mitigating Abiotic Stress in Plants  53 Farhan Ahmad, Ananya Singh, and Aisha Kamal Introduction  53 Involvement of Sugar in Plant Developmental Process  54 Multidimensional Role of Sugar Under Optimal and Stressed Conditions  55 Sugar as Sensing and Signaling Molecules  55 Sucrose and Trehalose Sensing  56 Sugar Alcohol (Polyol) Sensing  57 Sugar and Redox Homeostasis  57 Sugars as Osmoprotectants  58 Sugars and Abiotic Stress Tolerance in Plants  59 Salinity Stress  59 Drought Stress  59 Heat/Cold Stress  61 Mineral Nutrient Deficiency  61 Limitations and Future Prospects  62 References  62

Sugars and Sugar Polyols in Overcoming Environmental Stresses  71 Saswati Bhattacharya and Anirban Kundu 4.1 Introduction  71 4.2 Types of Sugars and Sugar Alcohols  72 4.2.1 Trehalose  72 4.2.2 Sucrose  73 4.2.3 Fructans  74 4.2.4 Raffinose Family Oligosaccharides (RFOs)  75 4.2.5 Sugar Alcohols  75 4.2.5.1 Mannitol  75 4.2.5.2 Sorbitol  76 4.2.5.3 Inositols  77 4.3 Mechanism of Action of Sugars and Polyols  77 4.3.1 As Osmolytes  77 4.3.2 As Antioxidants  79 4.3.3 As Signaling Molecule  80 4.4 Involvement of Sugars and Polyols in Abiotic Stress Tolerance  82 4.4.1 Cold Acclimation  82 4

Contents

4.4.2 4.4.3 4.4.4 4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.5.6 4.6

Tolerance to Drought  83 Salinity Tolerance  84 High Temperature Tolerance  86 Engineering Abiotic Stress Tolerance Using Sugars and Sugar Alcohols  87 Trehalose  87 Fructans  89 RFOs  90 Mannitol  90 Sorbitol  90 Inositol and Its Derivatives  91 Conclusions and Future Perspectives  91 References  92

5

Ascorbate and Tocopherols in Mitigating Oxidative Stress  102 Kingsuk Das Introduction  102 Role of Ascorbic Acid in Plant Physiological Processes  103 Ascorbic Acid—Its Role as Alleviator in Abiotic Stresses  104 Transgenic Approaches for Overproduction of Ascorbate Content for Fight Against Abiotic Stress  104 Ascorbic Acid—Alleviates Temperature Stress  105 Ascorbic Acid—It Confers Photoprotection  107 Ascorbic Acid Can Mitigate Ozone Stress  107 Ascorbic Acid—Fights Against Foliar Injury  108 Tocopherol—Its Occurrence in Plants  108 Tocopherol—Acts as Effective Nonenzymatic Antioxidant  109 Tocopherol and Its Correlation with Other Plant Hormones  109 Tocopherol Content Under Stressed Condition  111 Experiments with Tocopherol-deficient Mutants  111 The Tocopherol–Ascorbate–Glutathione Triad—Capable to Scavenge ROS in Conjugated Manner  111 Tocopherol—Alleviator in Salt Stress  112 Conclusion  113 References  114

5.1 5.2 5.2.1 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.3.6 5.3.7 5.3.8 5.3.9 5.3.10 5.3.11 5.4 6 6.1 6.2 6.3 6.3.1 6.3.2 6.4 6.5 6.5.1

Role of Glutathione Application in Overcoming Environmental Stress  122 Nimisha Amist and N. B. Singh Introduction  122 Glutathione Molecular Structure  123 Glutathione Biosynthesis and Distribution  124 Regulation of Glutathione Biosynthesis  124 Glutathione Distribution and Abundance in Plant Cells  126 Glutathione-induced Oxidative Stress Tolerance  127 Impact of Abiotic Stress on Glutathione Content in Various Plants  129 Glutathione Content Under Heavy Metal Stress  129

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6.5.2 6.5.3 6.5.4 6.5.5 6.6 6.7 6.8

Glutathione Content of Plants Treated with Herbicides  129 Glutathione Content Under Drought  130 Glutathione Content and Heat Stress  130 Glutathione Content Under Salinity  130 Exogenous Application of GSH in Plants  131 Cross Talk on Glutathione Signaling Under Abiotic Stress  131 Conclusion  137 References  137

7

Modulation of Abiotic Stress Tolerance Through Hydrogen Peroxide  147 Murat Dikilitas, Eray Simsek, and Aryadeep Roychoudhury Introduction  147 Abiotic Stress in Crop Plants  149 Mechanisms of Hydrogen Peroxide in Cells  149 Role of Hydrogen Peroxide in Overcoming Stress  154 Conclusion and Future Perspectives  163 Acknowledgment  163 References  163

7.1 7.2 7.3 7.4 7.5 8

8.1 8.2 8.3 8.4 8.5 8.5.1 8.5.2 8.5.3 8.5.4 8.5.5 8.5.6 8.5.6.1 8.5.6.2 8.5.7 8.5.8 8.5.8.1 8.5.8.2 8.5.9 8.5.10 8.5.11 8.5.12

Exogenous Nitric Oxide- and Hydrogen Sulfide-induced Abiotic Stress Tolerance in Plants  174 Mirza Hasanuzzaman, M. H. M. Borhannuddin Bhuyan, Kamrun Nahar, Sayed Mohammad Mohsin, Jubayer Al Mahmud, Khursheda Parvin, and Masayuki Fujita Introduction  174 Nitric Oxide Biosynthesis in Plants  175 Hydrogen Sulfide Biosynthesis in Plants  177 Application Methods of NO and H2S Donors in Plants  178 Exogenous NO-induced Abiotic Stress Tolerance  178 Exogenous NO-induced Salt Stress Tolerance  178 Exogenous NO-induced Drought Tolerance  189 Exogenous NO-induced Metal/Metalloid Toxicity Tolerance  190 Exogenous NO-induced Extreme Temperatures Stress Tolerance  191 Exogenous NO-induced Flooding Stress Tolerance  192 Exogenous NO-induced Atmospheric Pollutant-mediated Tolerance  192 Ozone  192 Herbicides  192 Exogenous NO-induced UV Radiation Tolerance  193 Exogenous NO-induced Light Stress Tolerance  194 High Light  194 Low Light  194 Exogenous H2S-induced Abiotic Stress Tolerance  195 Exogenous H2S-induced Salt Stress Tolerance  195 Exogenous H2S-induced Drought and Hyperosmotic Stress Tolerance  199 Exogenous H2S-induced Metal/Metalloid Stress Tolerance  200

Contents

8.5.13 8.5.14 8.5.15 8.5.16 8.6

Exogenous H2S-induced Heat Stress Tolerance  200 Exogenous H2S-induced Cold Stress Tolerance  201 Exogenous H2S-induced Flood Stress Tolerance  201 Interaction of NO/H2S with ROS and Antioxidant Defense Systems  202 Conclusions and Outlook  202 References  203

9

Role of Nitric Oxide in Overcoming Heavy Metal Stress  214 Pradyumna Kumar Singh, Madhu Tiwari, Maria Kidwai, Dipali Srivastava, Rudra Deo Tripathi, and Debasis Chakrabarty Introduction  214 Nitric Oxide and Osmolyte Synthesis During Heavy Metal Stress  216 Relation of Nitric Oxide and Secondary Metabolite Modulation in Heavy Metal Stress  217 Regulation of Redox Regulatory Mechanism by Nitric Oxide  218 Nitric Oxide-Mediated ROS Regulation During Heavy Metal Stress  219 Nitric Oxide Regulation of Antioxidant Enzyme Activity and Heavy Metal Detoxification  220 Nitric Oxide and Hormonal Cross Talk During Heavy Metal Stress  222 Conclusion  227 References  227

9.1 9.2 9.3 9.4 9.4.1 9.4.2 9.5 9.6 10

Protective Role of Sodium Nitroprusside in Overcoming Diverse Environmental Stresses in Plants  238 Satabdi Ghosh 10.1 Introduction  238 10.2 Role of SNP in Alleviating Abiotic Stress  239 10.2.1 Sodium Nitroprusside Ameliorates Polyethylene Glycol-induced Osmotic Stress  239 10.2.2 Sodium Nitroprusside Ameliorates Nanosilver (AgNP) and Silver Nitrate (AgNO3) Stresses  239 10.2.3 Sodium Nitroprusside Ameliorates Salt Stress  240 10.2.4 Sodium Nitroprusside Ameliorates NaHCO3 Stress  240 10.2.5 Sodium Nitroprusside Ameliorates Arsenic-induced Oxidative Stress  241 10.2.6 Sodium Nitroprusside Ameliorates Heat Stress  241 10.2.7 Sodium Nitroprusside Ameliorates Ultraviolet-B Radiation  242 10.2.8 Sodium Nitroprusside Ameliorates Water Stress  242 10.2.9 Sodium Nitroprusside Ameliorates Metal Toxicity  243 10.2.9.1 Aluminum Toxicity  243 10.2.9.2 Cadmium Toxicity  243 10.2.9.3 Copper Toxicity  244 10.2.9.4 Lead Toxicity  244 10.2.10 Sodium Nitroprusside Ameliorates Chilling Stress  244

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10.3

Conclusion and Future Prospect  245 Acknowledgments  245 References  245

11

Role of Growth Regulators and Phytohormones in Overcoming Environmental Stress  254 Deepesh Bhatt, Manoj Nath, Mayank Sharma, Megha D. Bhatt, Deepak Singh Bisht, and Naresh V. Butani Introduction  254 Function of Classical Plant Hormones in Stress Mitigation  256 Auxins  256 Cytokinins  257 Gibberellins  258 Ethylene  259 Role of Specialized Stress-responsive Hormones  260 Abscisic Acid  260 Brassinosteroids  261 Jasmonic Acid  262 Salicylic Acid  263 Strigolactones  264 Hormone Cross Talk and Stress Alleviation  265 ABA-mediated Signaling with Auxin and Cytokinin  266 ABA-mediated Signaling with GA and MeJA  267 ABA-mediated Signaling with Strigolactone  267 ABA-mediated Signaling with Brassinosteroids  268 Conclusions and Future Perspective  268 References  268

11.1 11.2 11.2.1 11.2.2 11.2.3 11.2.4 11.3 11.3.1 11.3.2 11.3.3 11.3.4 11.3.5 11.4 11.4.1 11.4.2 11.4.3 11.4.4 11.5 12

12.1 12.2 12.3 12.4 12.5 12.6 12.7 12.8 12.9 12.10

Abscisic Acid Application and Abiotic Stress Amelioration  280 Nasreena Sajjad , Eijaz Ahmed Bhat, Durdana Shah, Abubakar Wani, Nazish Nazir, Rohaya Ali, and Sumaya Hassan Introduction  280 Abscisic Acid Biosynthesis  281 Role of Abscisic Acid in Plant Stress Tolerance  282 Regulation of ABA Biosynthesis Through Abiotic Stress  282 ABA and Abiotic Stress Signaling  283 Drought Stress  284 UV-B Stress  284 Water Stress  285 ABA and Transcription Factors in Stress Tolerance  285 Conclusion  286 References  286

Contents

13 13.1 13.2 13.3 13.4 13.5 14

14.1 14.2 14.3 14.4 14.5 14.6 15 15.1 15.2 15.3 16 16.1 16.1.1 16.1.2 16.2 16.2.1 16.2.2 16.2.3 16.2.4 16.2.5 16.3 16.4

Role of Polyamines in Mitigating Abiotic Stress  291 Rohaya Ali, Sumaya Hassan, Durdana Shah, Nasreena Sajjad, and Eijaz Ahmed Bhat Introduction  291 Distribution and Function of Polyamines  293 Synthesis, Catabolism, and Role of Polyamines  293 Polyamines and Abiotic Stress  295 Conclusion  299 References  300 Role of Melatonin in Amelioration of Abiotic Stress-induced Damages  306 Nasreena Sajjad, Eijaz Ahmed Bhat, Sumaya Hassan, Rohaya Ali , and Durdana Shah Introduction  306 Melatonin Biosynthesis in Plants  306 Modulation of Melatonin Levels in Plants Under Stress Conditions  307 Role of Melatonin in Amelioration of Stress-induced Damages  309 Mechanisms of Melatonin-mediated Stress Tolerance  311 Conclusion  313 References  313 Brassinosteroids in Lowering Abiotic Stress-mediated Damages  318 Gunjan Sirohi and Meenu Kapoor Introduction  318 BR-induced Stress Tolerance in Plants  319 Conclusions and Future Perspectives  323 References  323 Strigolactones in Overcoming Environmental Stresses  327 Megha D. Bhatt, and Deepesh Bhatt Introduction  327 Importance of Strigolactones  328 Strigolactone Biosynthesis  328 Various Roles of SLs in Plants  331 In Mitigating Drought and Salinity Stresses  332 In Harmonizing Reactive Oxygen Species  333 In Seed Germination Under High Temperature  333 In Karrikin-induced Signaling and Photomorphogenesis  333 In Augmenting Plant Defense Under Biotic Stress  334 Cross Talk Between Other Phytohormones and SLs  335 Conclusion  336 References  336

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17

17.1 17.2 17.3 17.4 17.4.1 17.4.2 17.4.3 17.4.4 17.4.5 17.4.6 17.5 17.6 17.7 17.8 17.9 17.10 17.11 17.12 17.13 17.14 17.15 17.16 18

18.1 18.2 18.3 18.4 19

19.1 19.2 19.2.1 19.2.2 19.3

Emerging Roles of Salicylic Acid and Jasmonates in Plant Abiotic Stress Responses  342 Parankusam Santisree, Lakshmi Chandra Lekha Jalli, Pooja Bhatnagar-Mathur, and Kiran K. Sharma Introduction  342 Salicylic Acid  343 Biosynthesis and Metabolism of SA  343 SA in Abiotic Stress Tolerance  346 SA and Drought  346 SA and Temperature Stress  347 SA and Salinity Stress  348 SA and Heavy Metals Stress  349 SA and UV-radiation  350 SA and O3 Stress  351 Signaling of SA Under Abiotic Stress  351 Jasmonic Acid  352 Physiological Function of Jasmonates  353 Biosynthesis of Jasmonic Acid  354 JA Signaling in Plants  355 JA and Abiotic Stress  356 Role of Jasmonates in Temperature Stress  357 Metal Stress and Role of Jasmonates  358 Jasmonates and Salt Stress  359 Jasmonates and Water Stress  360 Cross Talk Between JA and SA Under Abiotic Stress  361 Concluding Remarks  362 Acknowledgments  363 References  363 Multifaceted Roles of Salicylic Acid and Jasmonic Acid in Plants Against Abiotic Stresses  374 Nilanjan Chakraborty , Anik Sarkar, and Krishnendu Acharya Introduction  374 Biosynthesis of SA and JA  374 Exogenous Application of SA and JA in Abiotic Stress Responses  377 Future Goal and Concluding Remarks  378 References  383 Brassinosteroids and Salicylic Acid as Chemical Agents to Ameliorate Diverse Environmental Stresses in Plants  389 B. Vidya Vardhini Introduction  389 Overview of PGRs  389 Overview of Brassinosteroids  390 Overview of Salicylic Acid  390 BRs and SA in Ameliorating Abiotic Stresses  390

Contents

19.3.1 19.3.2 19.3.3 19.3.4 19.3.5 19.3.6 19.4

BRs and SA in Ameliorating Heavy Metal Stresses  391 BRs and SA in Ameliorating High Temperature Stress  394 BRs and SA in Ameliorating Low Temperature Stress  395 BRs and SA in Ameliorating Water Stress  396 BRs and SA in Ameliorating Salinity Stress  397 BRs and SA in Ameliorating Radiation Stress  400 Conclusion  400 References  400

20

Role of γ-Aminobutyric Acid in the Mitigation of Abiotic Stress in Plants  413 Ankur Singh and Aryadeep Roychoudhury Introduction  413 GABA Metabolism  414 Protective Role of GABA Under Different Stresses  415 Heat Stress  415 Drought Stress  416 Hypoxia  417 Salinity Stress  418 Arsenic Pollution  418 Conclusion and Future Perspective  419 Acknowledgments  419 Reference  420

20.1 20.2 20.3 20.3.1 20.3.2 20.3.3 20.3.4 20.3.5 20.4 21 21.1 21.2 21.3 21.4 21.4.1 21.4.2 21.4.3 21.5 22 22.1 22.2 22.3 22.3.1 22.3.2 22.3.3 22.3.4

Isoprenoids in Plant Protection Against Abiotic Stress  424 Syed Uzma Jalil and Mohammad Israil Ansari Introduction  424 Synthesis of Free Radicals During Abiotic Stress Conditions  426 Biosynthesis of Isoprenoids in Plants  427 Functions and Mechanisms of Isoprenoids During Abiotic Stresses  428 Stabilization of Membrane and Structure  428 Regulation of ROS  429 Modifications of ROS Signaling Promote Defensive Effects Against Abiotic Stress  430 Conclusion  430 Acknowledgments  431 References  431 Involvement of Sulfur in the Regulation of Abiotic Stress Tolerance in Plants  437 Santanu Samanta, Ankur Singh, and Aryadeep Roychoudhury Introduction  437 Sulfur Metabolism  438 Sulfur Compounds Having Potential to Ameliorate Abiotic Stress  438 Cysteine  439 Glutathione  440 Thioredoxin Systems  440 Vitamins  441

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22.3.5 22.4 22.5 22.6 22.7 22.8 22.8.1 22.8.2 22.9

Other Compounds  441 Role of Sulfur Compounds During Salinity Stress  441 Role of Sulfur Compounds During Drought Stress  443 Role of Sulfur Compounds During Temperature Stress  444 Role of Sulfur Compounds During Light Stress  446 Role of Sulfur Compounds in Heavy Metal Stress  447 Toxic Effects of Heavy Metals in Plants  447 Sulfur Metabolites in Heavy Metal Tolerance  450 Conclusion and Future Perspectives  452 Acknowledgments  452 References  453

23

Role of Thiourea in Mitigating Different Environmental Stresses in Plants  467 Vikas Yadav Patade, Ganesh C. Nikalje, and Sudhakar Srivastava Introduction  467 Modes of TU Application  468 Seed Pretreatment  468 Medium Supplementation  468 Foliar Spray  469 Biological Roles of TU Under Normal Conditions  469 Role of Exogenous Application of TU in Mitigation of Environmental Stresses  470 Salinity Stress  470 Heavy Metal Stress  472 Drought Stress  472 Heat Stress  473 UV Stress  473 Mechanisms of TU-mediated Enhanced Stress Tolerance  474 Success Stories of TU Application at Field Level  476 Conclusion  477 References  478

23.1 23.2 23.2.1 23.2.2 23.2.3 23.3 23.4 23.4.1 23.4.2 23.4.3 23.4.4 23.4.5 23.5 23.6 23.7 24

24.1 24.2 24.3 24.3.1 24.3.2 24.3.3 24.4 24.5 24.6

Oxylipins and Strobilurins as Protective Chemical Agents to Generate Abiotic Stress Tolerance in Plants  483 Aditya Banerjee and Aryadeep Roychoudhury Introduction  483 Signaling Mediated by Oxylipins  484 Roles of Oxylipins in Abiotic Stress Tolerance  484 Oxylipins Regulating Osmotic Stress Tolerance  484 Oxylipins Regulating Temperature Stress Tolerance  485 Oxylipins Regulating Light Stress  485 Role of Strobilurins in Abiotic Stress Tolerance  486 Conclusion  487 Future Perspectives  487 Acknowledgments  487 References  487

Contents

25 25.1 25.2 25.2.1 25.2.2 25.2.3 25.2.4 25.3 25.4 26

Role of Triacontanol in Overcoming Environmental Stresses  491 Abbu Zaid, Mohd. Asgher, Ishfaq Ahmad Wani, and Shabir H. Wani Introduction  491 Environmental Stresses and Tria as a Principal Stress-Alleviating Component in Diverse Crop Plants  493 Metal/Metalloid Stress  493 Salinity Stress  494 Drought Stress  496 Transplantation Shock  496 Assessment of Foliar and Seed Priming Tria Application in Regulating Diverse Physio-biochemical Traits in Plants  497 Conclusion and Future Prospects  499 Acknowledgments  502 References  502

Penconazole, Paclobutrazol, and Triacontanol in Overcoming Environmental Stress in Plants  510 Saket Chandra and Aryadeep Roychoudhury 26.1 Introduction  510 26.2 Nature of Damages by Different Abiotic Stresses  512 26.2.1 Salt Stress  512 26.2.2 Heat Stress  513 26.2.3 Drought Stress  513 26.2.4 Chilling Stress  514 26.2.5 Flooding Stress  514 26.2.6 Freezing Stress  515 26.3 Synthesis of Chemicals  515 26.3.1 Penconazole Synthesis  515 26.3.2 Paclobutrazol Synthesis  516 26.3.3 Triacontanol Synthesis  516 26.4 Role of Exogenously Added Penconazole, Paclobutrazol, and Triacontanol During Stress  516 26.4.1 Penconazole  517 26.4.1.1 Drought Stress  517 26.4.1.2 Salt Stress  518 26.4.1.3 Other Stresses  518 26.4.2 Paclobutrazol  518 26.4.2.1 Morphological Effect  519 26.4.2.2 Yield  519 26.4.2.3 Physiological Response  520 26.4.3 Triacontanol  521 26.4.3.1 Plant Growth  521 26.4.3.2 Physiological and Biochemical Aspects of Plants  521 26.4.3.3 Quality and Production of Crops  522 26.4.3.4 Active Constituents of Plants  522 26.4.3.5 Abiotic Stress Management  522

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26.5

Conclusion  523 Acknowledgment  524 References  524

27

Role of Calcium and Potassium in Amelioration of Environmental Stress in Plants  535 Jainendra Pathak, Haseen Ahmed, Neha Kumari, Abha Pandey, Rajneesh, and Rajeshwar P. Sinha Introduction  535 Biological Functions of Calcium and Potassium in Plants  537 Calcium and Potassium Uptake, Transport, and Assimilation in Plants  538 Calcium- and Potassium-induced Abiotic Stress Signaling  540 Role of Calcium and Potassium in Abiotic Stress Tolerance  542 Drought Conditions  542 Salinity Stress  545 Extreme Temperature (Heat) Stress  546 Low Temperature (Cold) Stress  548 Heavy Metal Stress  549 Waterlogging Conditions  550 High Light Intensity  550 Conclusion  551 Acknowledgments  551 References  552

27.1 27.2 27.3 27.4 27.5 27.5.1 27.5.2 27.5.3 27.5.4 27.5.5 27.6 27.7 27.8 28

Role of Nitric Oxide and Calcium Signaling in Abiotic Stress Tolerance in Plants  563 Zaffar Malik, Sobia Afzal, Muhammad Danish, Ghulam Hassan Abbasi, Syed Asad Hussain Bukhari, Muhammad Imran Khan, Muhammad Dawood, Muhammad Kamran, Mona H. Soliman, Muhammad Rizwan, Haifa Abdulaziz S. Alhaithloulf, and Shafaqat Ali 28.1 Introduction  563 28.2 Sources of Nitric Oxide Biosynthesis in Plants  565 28.3 Effects of Nitric Oxide on Plants Under Abiotic Stresses  566 28.3.1 Heavy Metals  566 28.3.2 Drought  567 28.3.3 Temperature  567 28.3.4 Salinity  568 28.3.4.1 Nitric Oxide–Mediated Mechanism of Salt Tolerance in Plants  571 28.4 Role of Calcium Signaling During Abiotic Stresses  571 28.4.1 Heavy Metals  572 28.4.2 Drought Stress  573 28.4.3 Salinity  574 References  575

Contents

29 29.1 29.2 29.3 29.4 29.5 30 30.1 30.2 30.2.1 30.2.2 30.2.3 30.2.4 30.2.5 30.3 30.3.1 30.3.2 30.3.3 30.3.4 30.3.5 30.3.6 30.3.7 30.4 30.5 30.6 30.6.1 30.6.2 30.6.3 30.7 30.8 31

31.1 31.2 31.3 31.4

Iron, Zinc, and Copper Application in Overcoming Environmental Stress  582 Titash Dutta, Nageswara Rao Reddy Neelapu, and Challa Surekha Introduction  582 Iron  586 Zinc  587 Copper  588 Conclusion  590 References  590 Role of Selenium and Manganese in Mitigating Oxidative Damages  597 Saket Chandra and Aryadeep Roychoudhury Introduction  597 Factors Augmenting Oxidative Stress  599 Pollutants  600 Herbicides  600 Metals  600 Drought  601 Photosensitizing Toxins  601 Effects of Heavy Metals on Plants  601 Chromium (Cr)  602 Manganese (Mn)  602 Selenium (Se)  602 Aluminum (Al)  603 Nickel (Ni)  603 Copper (Cu)  604 Zinc (Zn)  604 Role of Manganese (Mn) in Controlling Oxidative Stress  604 Role of Selenium (Se) in Controlling Oxidative Stress  607 Role of Antioxidants in Counteracting ROS  608 Glutathione Peroxidase  608 SOD Enzyme  608 Additional Antioxidants  609 Role of Se in Re-establishing Cellular Structure and Function  609 Conclusion  610 Acknowledgment  611 References  611 Role of Silicon Transportation Through Aquaporin Genes for Abiotic Stress Tolerance in Plants  622 Ashwini Talakayala, Srinivas Ankanagari, and Mallikarjuna Garladinne Introduction  622 Aquaporins  623 Molecular Mechanism of Water and Si Transportation Through Aquaporins  624 AQP Gating Influx/Outflux  624

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31.5 31.6 31.7 31.8 31.9 31.10 31.11 31.12

Si-induced AQP Trafficking  627 Roles of Aquaporins in Plant–Water Relations Under Abiotic Stress  627 Role of Silicon in Abiotic Stress Tolerance  627 Si-mediated Drought Tolerance Through Aquaporins  627 Si-mediated Salinity Tolerance Through Aquaporins  628 Si-mediated Oxidative Tolerance Through Aquaporins  629 Si Mediated Signal Transduction Pathway Under Biotic Stress  630 Conclusion  630 References  630

32

Application of Nanoparticles in Overcoming Different Environmental Stresses  635 Deepesh Bhatt, Megha D. Bhatt, Manoj Nath, Rachana Dudhat, Mayank Sharma, and Deepak Singh Bisht 32.1 Introduction  635 32.2 Physicochemical Properties of Nanoparticles  637 32.2.1 Physical Properties  637 32.2.2 Optical Properties  637 32.2.3 Chemical Properties  637 32.2.4 Electrical Properties  637 32.3 Mode of Synthesis of Nanoparticles  638 32.3.1 Physical Approach  638 32.3.2 Chemical Approach  638 32.3.3 Biological Approach (Green Synthesis)  639 32.3.3.1 Nanoparticle Synthesis Using Bacteria  639 32.3.3.2 Nanoparticle Synthesis Using Fungi  639 32.3.3.3 Nanoparticle Synthesis Using Plants  639 32.4 Types of Nanoparticles and Their Role in Stress Acclimation  639 32.4.1 Silver Nanoparticles (AgNP)  639 32.4.2 Gold Nanoparticles (AuNP)  641 32.4.3 Silica Nanoparticles  642 32.4.4 Silicon Nanoparticles (SiNP)  642 32.4.5 Aluminum Nanoparticles (AlNP)  643 32.4.6 Titanium Dioxide Nanoparticles (TiO2)  644 32.4.7 Zinc Nanoparticles (ZiNP)  644 32.4.8 Iron Nanoparticles (FeNP)  645 32.4.9 Selenium Nanoparticles (SeNP)  646 32.5 Types of Environmental Stresses  646 32.6 Possible Protective Mechanism of Nanoparticles  649 32.7 Conclusion and Future Perspectives  650 References  650

Index  655

xix

List of Contributors Ghulam Hassan Abbasi Department of Soil Science University College of Agriculture and Environmental Sciences The Islamia University of Bahawalpur Bahawalpur Pakistan Krishnendu Acharya Molecular and Applied Mycology and Plant Pathology Laboratory Department of Botany University of Calcutta Kolkata India Sobia Afzal Department of Soil Science University College of Agriculture and Environmental Sciences The Islamia University of Bahawalpur Bahawalpur Pakistan Farhan Ahmad Department of Bioengineering Integral University Lucknow India

Haseen Ahmed Laboratory of Photobiology and Molecular Microbiology Centre of Advanced Study in Botany Institute of Science Banaras Hindu University Varanasi India Haifa Abdulaziz S. Alhaithloulf Biology Department College of Science, Jouf University Sakaka Kingdom of Saudi Arabia Shafaqat Ali Department of Environmental Sciences and Engineering Government College University Faisalabad Allama Iqbal Road, Faisalabad Pakistan and Department of Biological Sciences and Technology China Medical University Taichung Taiwan Rohaya Ali Department of Biochemistry University of Kashmir Srinagar India

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List of Contributors

Nimisha Amist Plant Physiology Laboratory Department of Botany University of Allahabad Allahabad India Srinivas Ankanagari Department of Genetics Osmania University Hyderabad India Mohammad Israil Ansari Department of Botany University of Lucknow Lucknow India Mohd. Asgher Plant Physiology and Biochemsitry Lab Department of Botany Baba Ghulam Shah Badshah University, Rajouri, Jammu and Kashmir India Aditya Banerjee Post Graduate Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata West Bengal, India Saswati Bhattacharya Department of Botany Dr. A. P. J. Abdul Kalam Govt. College New Town, Rajarhat, West Bengal India

Pooja Bhatnagar-Mathur International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru, Hyderabad, Telangana Indıa Eijaz Ahmed Bhat Life Science Institute Zhejiang University Hangzhou, Zhejiang PR China Deepesh Bhatt Department of Biotechnology Shree Ramkrishna Institute of Computer Education and Applied Sciences Veer Narmad South Gujarat University Surat, Gujarat India Megha D. Bhatt GSFC AgroTech Ltd., Gujarat State Fertilizers & Chemicals Ltd. Vadodara India M.H.M. Borhannuddin Bhuyan Laboratory of Plant Stress Response Department of Applied Biological Sciences Faculty of Agriculture, Kagawa University Takamatsu, Kagawa Japan and Citrus Research Station Bangladesh Agricultural Research Institute Jaintapur, Sylhet Bangladesh Deepak Singh Bisht ICAR—National Research Centre on Plant biotechnology IARI Pusa, New Delhi India

List of Contributors

Syed Asad Hussain Bukhari Department of Agronomy Bahauddin Zakariya University Multan Pakistan Naresh V. Butani Shree Ramkrishna Institute of Computer Education and Applied Sciences Veer Narmad South Gujarat University Surat, Gujarat India Debasis Chakrabarty Council of Scientific and Industrial Research—National Botanical Research Institute (CSIR—NBRI) Rana Pratap Marg Lucknow India Nilanjan Chakraborty Department of Botany Scottish Church College Kolkata India Saket Chandra Department of Bio-Engineering Birla Institute of Technology Mesra, Ranchi, Jharkhand India and Gulf Coast Research & Education Center IFAS University of Florida Wimauma, Florida USA

Muhammad Danish Department of Soil Science University College of Agriculture and Environmental Sciences The Islamia University of Bahawalpur Bahawalpur Pakistan Kingsuk Das Department of Botany Serampore College Serampore Hooghly, West Bengal India Muhammad Dawood Department of Environmental Sciences Bahauddin Zakariya University Multan Pakistan Murat Dikilitas Department of Plant Protection Faculty of Agriculture Harran University S. Urfa Turkey Rachana Dudhat Shree Ramkrishna Institute of Computer Education and Applied Sciences Veer Narmad South Gujarat University Surat, Gujarat India Titash Dutta Department of Biochemistry and Bioinformatics Institute of Science GITAM (Deemed to be University) Visakhapatnam Andhra Pradesh India

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Masayuki Fujita Laboratory of Plant Stress Response Department of Applied Biological Sciences Faculty of Agriculture Kagawa University Takamatsu, Kagawa Japan Mallikarjuna Garladinne Plant Molecular Biology laboratory Agri Biotech Foundation Hyderabad India Satabdi Ghosh Department of Botany Scottish Church College Kolkata India Mirza Hasanuzzaman Department of Agronomy Faculty of Agriculture Sher-e-Bangla Agricultural University Dhaka, Bangladesh Sumaya Hassan Department of Biochemistry University of Kashmir Srinagar India Syed Uzma Jalil Amity Institute of Biotechnology Amity University Uttar Pradesh, Lucknow Campus, Lucknow India Lakshmi Chandra Lekha Jalli International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru, Hyderabad, Telangana Indıa

Aisha Kamal Department of Bioengineering Integral University Lucknow India Muhammad Kamran Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River) Ministry of Agriculture, Huazhong Agriculture University Wuhan, Hubei P. R. China Meenu Kapoor University School of Biotechnology Guru Gobind Singh Indraprastha University Dwarka, New Delhi India Muhammad Imran Khan Department of Soil and Environmental Sciences University of Agriculture Faisalabad Faisalabad Pakistan Maria Kidwai Council of Scientific and Industrial Research—National Botanical Research Institute (CSIR—NBRI) Rana Pratap Marg, Lucknow India Neha Kumari Laboratory of Photobiology and Molecular Microbiology Centre of Advanced Study in Botany Institute of Science Banaras Hindu University Varanasi India

List of Contributors

Anirban Kundu P.G. Department of Botany Ramakrishna Mission Vivekananda Centenary College Rahara, West Bengal India Jubayer Al Mahmud Department of Agroforestry and Environmental Science Faculty of Agriculture Sher-e-Bangla Agricultural University Dhaka, Bangladesh Zaffar Malik Department of Soil Science University College of Agriculture and Environmental Sciences The Islamia University of Bahawalpur Bahawalpur Pakistan Sayed Mohammad Mohsin Laboratory of Plant Stress Response Department of Applied Biological Sciences Faculty of Agriculture Kagawa University Takamatsu, Kagawa Japan and Department of Plant Pathology Faculty of Agriculture Sher-e-Bangla Agricultural University Dhaka, Bangladesh Kamrun Nahar Department of Agricultural Botany Faculty of Agriculture Sher-e-Bangla Agricultural University Dhaka, Bangladesh

Manoj Nath ICAR—Directorate of Mushroom Research Chambaghat, Solan, Himachal Pradesh India Nazish Nazir Centre of Research for Development University of Kashmir Srinagar, Jammu & Kashmir India Nageswara Rao Reddy Neelapu Department of Biochemistry and Bioinformatics Institute of Science GITAM (Deemed to be University) Visakhapatnam, Andhra Pradesh India Ganesh C. Nikalje Department of Botany R. K. Talreja College of Arts, Science and Commerce Ulhasnagar, Thane, Maharashtra India Abha Pandey Laboratory of Photobiology and Molecular Microbiology Centre of Advanced Study in Botany Institute of Science Banaras Hindu University Varanasi India Khursheda Parvin Laboratory of Plant Stress Response Department of Applied Biological Sciences Faculty of Agriculture Kagawa University Takamatsu, Kagawa Japan

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and Department of Horticulture Faculty of Agriculture Sher-e-Bangla Agricultural University Dhaka, Bangladesh Vikas Yadav Patade Defence Institute of Bio-Energy Research Haldwani, Nainital, Uttarakhand India Jainendra Pathak Laboratory of Photobiology and Molecular Microbiology Centre of Advanced Study in Botany Institute of Science Banaras Hindu University Varanasi India and Department of Botany Pt. Jawaharlal Nehru College Banda India Rajneesh Laboratory of Photobiology and Molecular Microbiology Centre of Advanced Study in Botany Institute of Science Banaras Hindu University Varanasi India Muhammad Rizwan Department of Environmental Sciences and Engineering Government College University Faisalabad Allama Iqbal Road, Faisalabad Pakistan

Arpita Rout Plant Molecular Biology and OMICS Laboratory Regional Plant Resource Centre Bhubaneswar, Odisha India Aryadeep Roychoudhury Post Graduate Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata, West Bengal India Nasreena Sajjad Department of Biochemistry University of Kashmir Srinagar India Santanu Samanta Post Graduate Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata, West Bengal India Parankusam Santisree International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru, Hyderabad, Telangana India Anik Sarkar Molecular and Applied Mycology and Plant Pathology Laboratory Department of Botany University of Calcutta Kolkata India

List of Contributors

Durdana Shah Centre of Research for Development University of Kashmir Srinagar, Jammu & Kashmir India

Pradyumna Kumar Singh Council of Scientific and Industrial Research—National Botanical Research Institute (CSIR—NBRI) Rana Pratap Marg, Lucknow India

Kiran K. Sharma International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru, Hyderabad, Telangana India

and

Mayank Sharma Martin Luther University of HalleWittenberg Halle Germany Eray Simsek Department of Plant Protection Faculty of Agriculture Harran University S. Urfa Turkey Ananya Singh Department of Biosciences Integral University Lucknow India Ankur Singh Post Graduate Department of Biotechnology St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani Kolkata, West Bengal India

Academy of Scientific and Innovative Research (AcSIR) Ghaziabad India N. B. Singh Plant Physiology Laboratory Department of Botany University of Allahabad Allahabad India Deepak Singh Bisht ICAR—National Research Centre on Plant Biotechnology Pusa Campus New Delhi India Rajeshwar P. Sinha Laboratory of Photobiology and Molecular Microbiology Centre of Advanced Study in Botany Institute of Science Banaras Hindu University Varanasi India Gunjan Sirohi University School of Biotechnology Guru Gobind Singh Indraprastha University Dwarka, New Delhi India

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Mona H. Soliman Biology Department Faculty of Science Taibah University Al-Sharm, Yanbu El-Bahr Saudi Arabia and Department of Botany and Microbiology Faculty of Science Cairo University Giza Egypt Dipali Srivastava Council of Scientific and Industrial Research—National Botanical Research Institute (CSIR—NBRI) Rana Pratap Marg Lucknow India Sudhakar Srivastava Institute of Environment and Sustainable Development Banaras Hindu University Varanasi India Giridara-Kumar Surabhi Plant Molecular Biology and OMICS Laboratory Regional Plant Resource Centre Bhubaneswar, Odisha India Challa Surekha Department of Biochemistry and Bioinformatics Institute of Science GITAM (Deemed to be University) Visakhapatnam, Andhra Pradesh India

Ashwini Talakayala Plant Molecular Biology Laboratory Agri Biotech Foundation Hyderabad India and Department of Genetics Osmania University Hyderabad India Madhu Tiwari Council of Scientific and Industrial Research—National Botanical Research Institute (CSIR—NBRI) Rana Pratap Marg Lucknow India Rudra Deo Tripathi Council of Scientific and Industrial Research—National Botanical Research Institute (CSIR—NBRI) Rana Pratap Marg, Lucknow India B. Vidya Vardhini Faculty of Science and Computer Science Telangana University Dichpally Nizamabad, Andhra Pradesh India Shabir H. Wani Mountain Research Centre for Field Crops Sher-e-Kashmir, University of Agricultural Sciences and Technology of Kashmir Khudwani, Anantnag Jammu & Kashmir India

List of Contributors

Ishfaq Ahmad Wani Plant Physiology and Biochemsitry Lab Department of Botany Baba Ghulam Shah Badshah University Rajouri, Jammu and Kashmir India Abubakar Wani Indian Institute of Integrative Medicine Jammu India

Abbu Zaid Plant Physiology and Biochemistry Section Department of Botany Aligarh Muslim University Aligarh India

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1

1 Role of Proline and Glycine Betaine in Overcoming Abiotic Stresses Murat Dikilitas1, Eray Simsek 1, and Aryadeep Roychoudhury 2 1 2

Department of Plant Protection, Faculty of Agriculture, Harran University, S. Urfa, Turkey Department of Biotechnology, St. Xavier’s College (Autonomous), Kolkata, West Bengal, India

1.1 ­Introduction Plants are continuously under exposures of many kinds of abiotic and biotic stresses starting from vegetative to reproductive stages (Roychoudhury et al., 2011; Hajihosseinlo et al., 2015; Aksakal et al., 2017; Aamer et al., 2018; Cheng et al., 2018; Dawood and El‐Awadi 2018; Duhan et al., 2018; Choudhary et al., 2019; Ghaffari et al., 2019; Sahitya et al., 2018; Paul and Roychoudhury 2019). Salinity, waterlogging, chilling or cold stress, drought, heat, light, heavy metal stress, pesticide wastes, nutrient deficiency, UV‐B damages, and pathogen stress and their combinations might lead to more devastating consequences on crop plants (Roychoudhury et al. 2011). Even if crop plants may exhibit tolerance to stress arising from either biotic or abiotic stress agents or to both, they may not be productive as desired in terms of crop production and quality. Although plants are able to accumulate osmolytes to defend themselves against stress, the synthesis of osmolytes could be reduced under severe stress conditions. In general, increases of osmolytes have been regarded as the reflection of stress tolerance. There are quite a few osmolytes commonly measured in cells such as proline, polyamines, glycine betaine (GB), sugar and sugar products, glycerol, sorbitol, mannitol, etc., that have significant roles in protecting cells from cell‐damaging stress factors (Chen and Jiang 2010; Rabbani and Choi 2018). Therefore, increasing amino acid or osmolyte contents under stress conditions would be a proper approach to tackle at least one of the stress factors. However, if one of the stress agents is biotic, the mechanisms for tolerance would be more complex due to the adaptation of attacking pathogens to these stressful conditions. It is possible that the pathogens may use the compounds having low molecular weights such as sugars, polyamines, or low‐molecular‐weight amino acids as substrates. These “ready to take‐in substrates” could even increase the virulence of the pathogens and result in more devastating effects on crop production. Therefore, modulation of abiotic stress tolerance under the combined stress conditions may not be satisfactory for crop plants. Applications of proline, sugar, and amino acid might increase the Protective Chemical Agents in the Amelioration of Plant Abiotic Stress: Biochemical and Molecular Perspectives, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

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1  Role of Proline and Glycine Betaine in Overcoming Abiotic Stresses

pathogen dissemination via secretion of secondary metabolites under abiotic or combined stress occurrences. For example, Dunn et  al. (1998) reported that NaCl stress led to increased production of arginine levels and decreased phenylalanine ammonia lyase (PAL) activity in citrus plants, thus causing increased susceptibility to nematode attack caused by Tylenchulus semipenetrans. The authors also stated that the reduction in PAL activity due to an increase in arginine levels increased higher infection rates. It was concluded that the increased level of arginine following salinity stress led to reduction in host defense responses against the attacking mites. In another study, Mathur et al. (2013) stated that the elevated CO2 (550 ppm) led to reduction in disease severity of Alternaria blight and downy mildew caused by Alternaria brassicae and Hyaloperonospora brassicae, respectively, in Brassica juncea cv. Pusa Tarak (mustard plants). They concluded that elevated CO2 resulted in the accumulation of higher amounts of epicuticular wax with the increase of total phenolics and PAL activity levels, which might have enabled the mustard plants to resist the infection caused by those pathogens. A reduction in pore size and stomatal density with reduced stomatal conductance might have played significant roles in decreasing the disease index of downy mildew caused by H. brassicae, which is a stomata‐invading pathogen. However, the same authors also stated that the pathogenicity of Albugo candida, a causal agent for white rust infection, increased in the same plant. The authors suggested that three times higher sugar concentration levels than plants grown in ambient CO2 might have played significant roles and led to higher incidence and severity of the white rust disease. Therefore, we have to consider the biotic stress cases while we aim to develop and improve the level of tolerance or resistance of crop plants under stress. In this chapter, we mainly focus on the modulation of abiotic stress issues through proline and GB application, but we would discuss on the biotic stress involvement as well.

1.2 ­Responses of Crop Plants Under Abiotic Stresses Abiotic stress factors significantly cause reductions in crop production and deteriorate the crop quality, which eventually results in the depletion of food source. In recent years, climate changes in terms of increase in air and surface temperatures along with the enhanced accumulation of CO2 and the environmental pollution make the situation worse. The stress agents whether abiotic or biotic would disrupt the biochemical and molecular pathways of crop plants and physiologically deteriorate the functions of crop plants (Ramegowda and Senthil‐Kumar 2015; Dikilitas et al. 2018). Abiotic stress combinations or abiotic and biotic stress combinations cannot be easily solved because of their different characteristics. Although the generation of crop plants for multiple stress factors is an urgent need, the addition of different stressors in the present scenario as well as the evolution of pathogenic and insect races would make the development of resistant crop plants difficult. Many approaches have been adopted to eradicate stress in crop plants; however, none of them has achieved complete success in agricultural systems. Plant breeding, genetic modifications, or application of signaling molecules as well as polyamines have all been attempted to remediate the stressful environments for the crop plants. Until we find the best strategy to improve the crop plants against all types of stresses, we have to improve the crop plants via cost‐effective methods. Plant growth regulators (PGRs) (Khan et  al. 2017), plant growth

1.3  ­Mechanisms of Osmoprotectant Functions in Overcoming Stres

promoting rhizobacteria (PGPR) (Kumar et  al. 2018), polyamines (Alcazar and Tiburcio 2018), proline (Singh et al. 2018), GB (Rasheed et al. 2018), or sugars (Martinez‐Noel and Tognetti 2018) could be applied to plants exogenously to counteract the negative effects of one of the stress factors. Such applications may modulate the defense responses through the lignification, synthesis of phytoalexins, hormonal homeostasis, and other stress‐related defense metabolites. However, while we plan to apply those chemicals to crop plants under abiotic stresses, we have to consider the issues regarding other stress factors including abiotic and biotic ones that might interfere with the applied chemicals. Abiotic stress factors, in general, cause limitation in the crop productivity and quality as well as disrupt defense mechanisms of crop plants depending on their severity, duration, and nature. One of the earliest responses in terms of biochemical and physiological parameters is the production of reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide (O2−˙), hydroxyl (OH−) ions, and nonradical hydrogen peroxide (H2O2) (Camejo et al. 2016; Waszczak et al. 2018). Due to incomplete reduction of O2 during plant metabolic processes, ROS are continuously generated during cell metabolism (Rejeb et al. 2014). ROS, in general, damage all sorts of organic molecules including proteins, lipids, and carbohydrates and disrupt the functions of cell membrane via modifying enzyme system and eventually cause death via damaging or methylating DNA (Demidchik 2015). The ROS are scavenged via various enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR), and low‐molecular‐weight nonenzymatic antioxidant molecules, such as glutathione (GSH), ascorbic acid (vitamin C), carotenoids, tocopherol, proline, polyamines, etc. (Gill and Tuteja 2010; Roychoudhury and Basu 2012). Although accumulation of proline has been considered as a marker of stress response, it may also contribute to the formation of ROS in mitochondria, which trigger hypersensitive response (HR) in plants (Rejeb et al. 2014). Many plant researchers have stated that proline accumulation is considered to be beneficial. The high accumulation of osmoprotectants including proline, polyamines, sugars, and GB under stress is generally correlated with their increased biosynthesis and is regarded to be associated with stress tolerance (Kaur et al. 2017; Ashraf et al. 2018). Proline has a dual role in plants, as its low or moderate doses have remediation effects with signaling roles, while its higher doses are toxic to living cells.

1.3 ­Mechanisms of Osmoprotectant Functions in Overcoming Stress Compatible solutes such as proline, GB, or polyamines as well as sugars are considered to be osmoprotectants that are produced in plants during stress. These are neutrally charged, low‐molecular‐weight compounds, playing important roles for the stabilization and protection of cell membranes and proteins during stress (Khan et al. 2015; Hasanuzzaman et al. 2019). The compatible solutes can be classified as amino acids (proline, arginine, phenylalanine, melanin, glycine, etc.), sugars (sucrose, glucose, trehalose), and polyamines. These osmoprotectants are also considered to be involved in signaling mechanisms and ­eventually

3

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1  Role of Proline and Glycine Betaine in Overcoming Abiotic Stresses

they are associated with tolerance or resistance mechanisms involved in production of lignin, suberin, phytoalexins, and other structural defense‐related compounds. Osmolytes, in general, are considered soluble compounds within a cell that play significant roles in maintaining fluid homeostasis (Khan et al. 2017). One of the most studied osmoprotectants in crop plants is proline, which is involved in the regulation of ion homeostasis and redox balance (Kaur and Asthir 2015; Roychoudhury et  al. 2015; Zandalinas et  al. 2018). Proline and reducing or nonreducing sugars are abundant in cells in contrast to GB in stress conditions (Ben Ahmed et  al. 2010). For example, sugars are accumulated during cold stress and frequently observed in leaves of many plants including Arabidopsis, tomato, pepper, petunia, etc. (Nagele et  al. 2011; Bauerfeind et al. 2015; Gharsallah et al. 2016). Sugars such as glucose or fructose could be transported from the apoplast into the cytosol to regulate the osmotic balance in plant cells under stress at the expense of energy (Khan et al. 2017). In general, sensitive genotypes of crop plants accumulate fewer osmoprotectants with low concentrations than tolerant genotypes under stress. From this point of view, it could be possible to apply osmoprotectants to nonaccumultaing or low‐accumulating crop plants to prevent or reduce the adverse effects of stress‐related responses (Yang and Lu 2005). Apart from the osmotic regulation, osmoprotectants have other roles, such as scavenging free radicals (Szabados and Savoure 2010; Puthur 2016), inducing osmotic stress‐related genes, and stabilization of proteins and cell membranes (Theocharis et al. 2012; Roychoudhury and Nayek 2014). They also sustain leaf turgidity and impede chlorophyll biodegradation (Pottosin et al. 2014).

1.3.1  Proline Biosynthesis and Mechanism of Action in Plants Proline is a five‐carbon amino acid involved in osmotic balance during stress and is also essential in scavenging of free radicals, stabilization of macromolecules, and signaling mechanism pathways (Verslues and Sharma 2010; Hayat et al. 2012). Other functions of proline include promoting embryo/seed evolvement, extending stem length, as well as moving plants from vegetative growth to reproductive stage (Emamverdian et al. 2015). As stated, proline is the main amino acid that increases osmotic pressure and regulates water potential under various abiotic stress conditions (Zandalinas et al. 2017; Junior et al. 2018; Singh et al. 2018). Various organisms such as protozoa, eubacteria, fungi, invertebrates, and many plant species under stress conditions have been known to accumulate proline (Liang et  al. 2013; Hasanuzzaman et  al. 2018). Its break down following stress may create an opportunity to sustain sufficient reducing agents and energy for the synthesis of adenosine triphosphate (ATP) for plants to recover from stress and stress‐induced damages (Sasaki‐ Sekimoto et al. 2005). Proline accumulation under stress shows variations, depending on the species of plants, and can occupy major parts of the cellular amino acid pool (Roychoudhury et al. 2015). It, therefore, serves as a major source of carbon and nitrogen. Biosynthesis of proline occurs in a reductive pathway, and requires NADPH for the reduction of glutamate as the starting point during osmotic stress to Δ1‐pyrroline‐5‐carboxylate (P5C) and P5C to proline. Two enzymes are involved in the synthesis; P5C is first catalyzed by the enzymes Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS) and Δ1‐pyrroline‐5‐carboxylate reductase (P5CR) to produce proline (Khan et al. 2015). An alternative

1.3  ­Mechanisms of Osmoprotectant Functions in Overcoming Stres

pathway for the synthesis of proline occurs via ornithine (Orn), which can be transaminated to P5C by ornithine‐δ‐aminotransferase (OAT) enzyme present in mitochondria. This pathway mainly occurs under nitrogen limitation (Delauney and Verma 1993). However, in a recent study, De la Torre‐Gonzalez et  al. (2018) reported that proline accumulation, via ornithine pathway, is associated with salt tolerance in tomato (Solanum lycopersicum L.); however, synthesis of proline increased the production of H2O2 and did not act as a compatible solute in improving salt tolerance in tomato plants. During stress occurrence, low NADPH:NADP+ ratio should be maintained with an increased rate of proline synthesis in chloroplasts to stabilize redox balance and to reduce the damage on photosynthetic parts (Hare and Cress 1997). The accumulation of proline under stress is associated with tolerance of stress, and its increased concentrations have generally been stated in tolerant species as compared to sensitive ones. Iqbal (2018) stated that the activities of P5CR and OAT enzymes increased in wheat lines along with the decrease of proline oxidase enzymes under stress conditions (Iqbal 2018). Proline takes great part in ROS scavenging via either protecting the glutathione–ascorbate cycle or enhancing the activities of enzymes involved during osmotic stress (Szekely et al. 2008). Although proline metabolism has been widely studied in response to abiotic stresses, in recent years, studies related to proline accumulation and mechanisms have also been characterized by pathogen stress. The mechanism or the accumulation of proline is not straightforward as in the abiotic stress cases. For example, Haudecoeur et al. (2009) reported that the accumulation of proline in tobacco (Nicotiana tabacum) was found to be related to susceptibility to Agrobacterium tumefaciens. The authors stated that proline antagonized the plant defense responses via interfering with the γ‐aminobutyrate‐mediated degradation of bacterial quorum‐sensing signals. Under combined stress conditions, sucrose and low‐molecular‐weight proteins are mainly accumulated to relieve the negative effects of stresses to protect mitochondria and DNA from increased concentrations of toxic pyrroline‐5‐carboxylate (Rizhsky et al. 2004; Mittler et al. 2006; Kumar et al. 2013; Nanda and Agrawal 2016; Siddiqui et al. 2018). For example, Nanda and Agrawal (2016) stated that malondialdehyde (MDA) level increased in seedlings of Cassia angustifolia plants in response to zinc or copper stress along with the increase of proline and low‐molecular‐weight proteins to ease the severity of the metallic stress. The authors suggested that increased proline accumulation in Zn‐treated and Cu‐treated seedlings might be due to the increased activity of enzymes responsible for proline synthesis rather than protein degradation. They stated that the accumulation of proline had a protective role in maintaining DNA integrity. Similarly, Kumar et al. (2013) showed that Talinum triangulare (Jacq.) exposed to various concentrations of Pb(NO3)2 (lead) exhibited increased amounts of MDA, ROS, protein oxidation, and DNA damages, along with decrease in protein content in a dose‐dependent manner. Proline accumulation in response to lead toxicity was correlated with the DNA integrity. However, decrease in proline content was positively correlated with the increase of DNA damages in Talinum plants. Similarly, Siddiqui et al. (2018) stated that magnesium application improved plant tolerance (Vicia faba) to heat stress by suppressing cellular damage induced by ROS through the enhancement of the accumulation of proline and GB and the antioxidant enzymes. The application of magnesium along with the increase of proline and GB significantly decreased DNA damage. Similar findings were made by Siddiqui et al. (2017) using

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indole acetic acid (IAA) and sodium nitroprusside (SNP) on tomato seedlings under heat stress. The authors stated that the coapplication of SNP and IAA improved the conditions of plants via promoting CAT, peroxidase (POX), and SOD activities along with the increased accumulation of photosynthetic pigments such as chlorophyll a and b and proline with a concomitant decrease in H2O2 and other ROS elements. Increases in proline contents had a preventive role for remediation of DNA damages. Similar observations were also made by Zhang et al. (2017) who reported that genomic template stability (GTS) of Lemna minor L. plants exposed to mercury toxicity was severely affected. They reported that the accumulation of heat shock protein such as HSP70 along with the activation of defense mechanisms including nonenzymatic and enzymatic defense responses and upregulation of proline improved and protected DNA and the cell from further stress. Zhang and Becker (2015) reported that the ability of proline to delay leaf and petal senescence was associated with oxidative stress. Therefore, they stated that signaling mechanisms should be evaluated for a better understanding of proline metabolism in senescing leaves to delay the stress‐induced senescence. Proline accumulation was also reported under waterlogging conditions. Singh et  al. (2017) stated that waterlogging tolerance in pigeon pea was found associated with proline accumulation. Increases in some metabolites may be beneficial to remediate the severity of stress levels; on the other hand, they might lead to susceptibility to pathogenic attacks. For example, Dikilitas (2003) stated that NaCl‐tolerant Medicago sativa callus (cv. Kabul) responded differently under low (50 mmol l−1) and very high (200 mmol l−1) NaCl stress in terms of proline and PAL activity, when inoculated with the carbohydrate fraction of Verticillium albo‐atrum fungus, a causal agent for wilting. NaCl‐tolerant callus responded to the low concentration of NaCl via accumulating high proline and synthesizing high PAL activity under the pathogen stress. Further increase was noticed when the level of NaCl concentration increased. However, under high salinity and pathogen stress, a drastic reduction in both proline and PAL was evidently exhibited so that even a NaCl‐tolerant lucerne variety might show susceptibility when attacked by the wilt fungus. When high NaCl stress was combined with the fungus, the NaCl‐tolerant lucerne variety was not able to produce proline and PAL, although fungal sporulation was positive in in vitro test at high NaCl conditions. Similar findings have also been reported by Akhtar et al. (2016) who showed that germination and seedling growth of mash bean (Vigna mungo L.) were significantly decreased when treated with either Macrophomina phaseolina or various levels of copper (50–100 ppm). Combination of copper and pathogen stresses significantly reduced total chlorophyll, sugar, and carotenoid content, while proline and total phenolic contents were increased. However, at 100 ppm of copper toxicity and pathogen stress, both proline and total phenolic contents decreased significantly. Huang et al. (2017) concluded that nitrogen fertilization increased the susceptibility of rice plants to fungal attacks caused by Magnaporthe oryzae, a causal agent for rice blasts, although increased expression of several defense‐related genes were evident. They hypothesized that an increase in nutrient availability for plants also sustained the growth of the fungus. Although glutamine accumulation enhanced the defense response, it is still to be evaluated which metabolite is responsible for fungal pathogenicity. These findings, in general, showed that metabolites such as amino acids and sugars played significant roles under

1.4  ­Application of Osmoprotectants in Stress Condition

combined stress conditions; however, high accumulation of these metabolites may also increase the pathogen virulence and dissemination. For example, Quaglia et  al. (2016) found a significant negative relation between phenolic contents of pomegranate (Punica granatum L.) and mycelial growth of Penicillium adametzioides. They stated that the purified phenolic extract with its low sugar contents had more preventive effects on mycelial growth than those of the extract with high sugar contents. Similarly, Liebe and Varrelmann (2016) stated that the intensity in tissue necrosis caused by microbial agents was significantly correlated with invert sugar accumulation in stored sugar beet after storage rots caused by Botrytis, Fusarium, and Penicillium.

1.3.2  Glycine Betaine (GB) Biosynthesis and Mechanism of Action in Plants GB accumulation mostly occurs as a response to water stress or drought in which the accumulation is mainly within chloroplast to protect thylakoid membrane via adjusting osmotic regulation and maintaining photosynthetic efficiency. GB is widely accumulated along with the other osmoprotectants in plants under stress conditions. As a low‐molecular‐ weight organic metabolite, it can have a crucial role against abiotic stressors (Rasool et al. 2013; Roychoudhury and Banerjee 2016). Among the many quaternary ammonium compounds, GB is generally characterized under salinity and water stress conditions. GB is synthesized in chloroplast for the protection of thylakoid membranes; therefore, it helps to maintain photosynthetic efficiency (Gupta and Thind 2018). It is synthesized via two different pathways using two substrates, such as choline and glycine (Rasool et al. 2013; Gupta and Thind 2018). As a compatible solute, it is easily soluble in water and it does not exert any toxicities at high concentrations (Giri 2011). Its main role in plants is the protection of plant cells through stabilization of proteins, osmotic adjustment, and detoxification of ROS (Roychoudhury and Banerjee 2016). Many researchers suggested that GB at low concentrations was able to protect macromolecules including nucleic acids, proteins, and lipids, which are rich in nitrogen and carbon to be utilized as energy sources (Umezawa et al. 2006). Increases in GB could also be associated with stress tolerance via increased CAT and SOD activities along with the reduction of cell membrane damage after regulation of lipid peroxidation and ion homeostasis pathway (Alasvandyari et al. 2017).

1.4 ­Application of Osmoprotectants in Stress Conditions Applications of osmoprotectants have increased in recent years for crop improvement. The role of proline and GB in this connection is summarized in Table 1.1.

1.4.1  Application of Proline de Freitas et  al. (2018) observed that exogenous proline (30 mmol l−1) counteracted the effects of salt (NaCl, 80 mmol l−1) in 10‐day‐old maize (Zea mays) seedlings. The most significant outcomes of exogenous proline application were associated with reducing Na+ and

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Table 1.1  Exogenous applications of osmoprotectants in crop plants exposed to various stress factors. Plant species

Stress type

Application of proline/GB

References

Solanum lycopersicum L.

Salinity stress

Foliar spray of low doses of proline increased the tolerance of tomato in field conditions.

Kahlaoui et al. (2018)

Glycine max L.

Salinity stress

GB increased the tolerance of soybean via increasing antioxidant capacity and scavenging ROS.

Malekzadeh (2015)

Carthamus tinctorius L.

Salinity stress

GB treatment increased salinity tolerance in safflower and reduced MDA and improved homeostasis.

Alasvandyari et al. (2017)

Saccharum officinarum L.

Salinity stress

Proline supplementation improved growth rate significantly.

Patade et al. (2014)

Lens culinaris

Drought stress

Proline provided a protective role by reducing H2O2 levels and increasing the antioxidant defense systems.

Molla et al. (2014)

Zea mays L.

Drought stress

GB accumulation in transgenic maize Quan et al. (2004) provided greater protection by preserving cell membrane integrity.

Nigella sativa L. Water deficit stress

Proline preserved the water balance through osmotic adjustment.

Rezaei‐Chiyaneh et al. (2018)

Crataegus monogyna

Chilling injury

GB treatment enhanced the antioxidant enzyme activity and reduced ROS accumulation.

Razavi et al. (2018)

Camellia sinensis L.

Cold stress

Kumar and Yadav Proline provided protection by reducing MDA content and activating (2009) antioxidant enzymes.

Solanum lycopersicum L.

Waterlogging stress

GB improved the antioxidant mechanism.

Vigna angularis Waterlogging stress

Rasheed et al. (2018)

Waterlogging significantly stimulated Ullah et al. (2017) proline accumulation.

Gossypium hirsutum L.

Heavy metal GB improved photosynthesis and stress (cadmium) reduced oxidative damages.

Farooq et al. (2016)

Olea europaea L.

Heavy metal stress (lead)

Proline supply increased the enzymatic and nonenzymatic antioxidant parameters.

Zouari et al. (2018)

Glycine max L.

Arsenic‐induced oxidative stress

Proline directly scavenged ˙OH radicals and lowered arsenic accumulation.

Chandrakar et al. (2018)

Vigna aconitifolia

Heat stress

Proline increased the activities of CAT, POX, and SOD enzyme levels.

Harsh et al. (2016)

Tagetes erecta

Heat stress

GB lowered the oxidative stress mechanism.

Sorwong and Sakhonwasee (2015)

Nicotiana tabacum L.

Drought

Transgenic tobacco plants (P5CR) showed increased tolerance via accumulating more proteins.

Pospisilova et al. (2011)

1.4  ­Application of Osmoprotectants in Stress Condition

Cl− ion accumulation along with the increase of K+ ion content. Exogenous application of proline in stressed plants was associated with reduced P5CS activity and increased proline dehydrogenase (PDH) activity, along with significant decreases in H2O2 and MDA contents. Kahlaoui et al. (2018) stated that low doses of exogenous foliar application of proline in S. lycopersicum cultivars grown under saline conditions during the flowering stage in field conditions increased proline and total soluble protein contents as well as decreased proline oxidase activities. Osmoprotectant activities induced resistance or tolerance in plants exposed to abiotic or biotic stress factors. Hasanuzzaman et al. (2014) showed that exogenous proline and GB were found to be very effective to improve short‐term salt tolerance both in salt‐sensitive Bangladesh Rice Research Institute (BRRI) dhan49 and salt‐tolerant BRRI dhan54 rice seedlings. Proline or GB application alleviated the oxidative damages via increasing the antioxidant levels and glyoxalase systems. Application of osmoprotectants showed better performance in salt‐tolerant rice seedlings, and proline was found to be the better protectant than GB. A similar observation was also made during postharvest chilling stress. Yao et al. (2018) stated that GB application (10 mmol l−1) led to significant reduction in postharvest chilling injury of zucchini fruit during a 15‐day period of storage at 1°C followed by additional 3 days at 20°C. The increased antioxidant enzyme activities such as CAT, SOD, and APX were evident along with the increase of regulatory gene transcription. It was suggested that both antioxidant enzyme activities and gene expression levels were remarkably higher than those of control fruit. GB‐applied zucchini fruits accumulated more proline, associated with P5CS and OAT enzymes and less MDA contents, and exhibited lower levels of palmitic acid and lipoxygenase (LOX) activity. Preharvest application of osmoprotectants also participates in alleviating the conditions of postharvest fruits. For example, Rodriguez‐Zapata et al. (2015) stated that the preharvest foliar application of GB (100 mmol l−1) on banana plants reduced the chilling injury via regulating biochemical and physiological mechanisms in postharvest banana fruits. Similar findings were also obtained with preharvest osmoprotectant applications. For example, Wang et al. (2015) concluded that the application of GB to button mushrooms preserved weight loss and reduced the respiration rate and browning. Treated button mushrooms maintained a high content of polyphenol and ascorbic acid and exhibited better integrity in cell membranes. Demiralay et al. (2017) stated that exogenously applied proline was more effective when given through the roots as compared to those of foliar spray or seed‐soaking applications on maize seedlings under short‐term drought stress. Exogenous proline application (1 mmol l−1) effectively increased electron transport, photosynthetic efficiency, chlorophyll content, and water potential, and lowered the MDA content. The combined application of osmoprotectants was also found to be quite effective in protecting plants from various environmental stresses. For example, Zhang et al. (2013) reported that GB or humic acid (HA) treatments decreased the stomatal conductance and MDA content in Malus robusta (an apple rootstock) seedlings exposed to drought stress. The combination of GB and HA had greater beneficial effects, which offered an efficient, economical, and cost‐effective way to tolerate the apple rootstock. Zhang et al. (2016) stated that the combination of hot water dipping (HW, 45°C for 10 minutes) treatment with GB (10 mmol l−1) on loquat fruit stored at 1°C significantly alleviated chilling

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injury as compared to control. The combination of HW and GB treatments was found to be more effective in alleviating chilling stress and preserving quality parameters than either treatment alone. Significant reductions in ion leakage and MDA contents and increased antioxidant enzyme activities such as SOD, APX, and CAT were higher in the combined treatment as compared to those of nontreated fruits. The accumulation of γ‐ aminobutyric acid (GABA) and proline contents was evident in loquat fruits following the combination of HW and GB treatments, which was associated with increased P5CS and OAT activities. Koc (2017) reported that foliar proline application helped the pepper plants inoculated with Phytophthora capsici, which is a highly destructive pathogen of pepper. Proline had quite a significant potential to directly scavenge free radicals and promoted enzyme activity in pepper seedlings under the pathogen stress. Liu et al. (2011) observed that exogenous GB treatment alleviated the viability of Cystofilobasidium infirmominiatum, an antagonistic yeast cell, under oxidative stress. GB‐treated yeast cells accumulated less ROS and exhibited lower protein oxidation as compared to those of untreated control cells. GB treatment also led to greater biocontrol activity against Penicillium expansum and faster growth rate of the biocontrol agent was evident in wounds of apple fruits stored at 25°C. The antioxidant enzyme activities such as CAT, SOD, and GPX of C. infirmominiatum were enhanced following GB treatment.

1.4.2  Application of GB Exogenous application of GB has also been commonly applied to crop plants under various stresses. For example, Park et al. (2006) stated that plants took up GB easily when applied foliarly. Uptake of GB from roots is also reported (Park et al. 2003). Although GB is not directly associated with scavenging of ROS, it contributes to the increase of antioxidant enzymes and reduces ROS production possibly acting as a reducing agent. Lavanya and Amruthesh (2017) reported that GB treatment activated the host defense responses of pearl millet (Pennisetum glaucum L.) during downy mildew pathogen infection caused by Sclerospora graminicola. GB treatment at 30 mg ml−1 concentration for 6 hours significantly increased the seed germination and seedling vigor as well as inhibited sporangial sporulation, zoospore release, and motility. The authors stated that seed treatment with GB was more effective than foliar application to reduce the disease incidence. Similar findings were also made by Bakhoum and Sadak (2016) who reported that sunflower (Helianthus annuus L.) seed treatments with GB enhanced salt tolerance via improving photosynthetic pigments, growth, and developmental stages. They also stated that exogenously applied GB improved the oil quality of sunflower by protecting cellular structures during fatty oil biosynthesis and storage. However, other authors showed a negative correlation between GB concentration and salt tolerance, as this was the case during proline application in tomato seedlings (Heuer 2003). GB also enhanced fruit quality via improvement of protein biosynthesis and increased accumulation of calcium and reduction in sodium contents during waterlogging conditions (Rasheed et al. 2018). Wang et al. (2016) also stated that GB alleviated chilling tolerance of sweet pepper via increasing antioxidant gene expression and enzyme activity.

1.4  ­Application of Osmoprotectants in Stress Condition

1.4.3  Transgenic Approaches Due to the increase of world population and a decrease in arable lands, global agriculture is under pressure to supply adequate food and food products for the next generations. Environmental pollution and increases in surface temperature are the major challenges so that the conventional plant‐breeding methods and inducing resistance or tolerance via chemical application may not be adequate to cope with the stresses. Transgenic approach has developed for the production of improved crop varieties against biotic or abiotic stresses (Khan et al. 2015). Some stressors such as biotic ones have been controlled by single genes, and some by many genes; however, most of the abiotic stresses have been controlled by multiple genes. One of the most popular approaches in the production of transgenic lines is the upregulation of genes associated with the synthesis of osmoprotectants (Paul and Roychoudhury 2018). Various plant species have been engineered via employing several genes to increase the tolerance of stress in crop plants. Genes involved in proline synthesis have been mostly evaluated in Arabidopsis thaliana plants. Genetic engineering of crop plants upregulating the proline‐related genes has given us an opportunity to reduce the osmotic stress caused by abiotic or biotic stress factors. Kishor et al. (1995) reported that biomass of root under water stress was enhanced in transgenic tobacco plants expressing high proline synthesis. Similarly, Hmida‐Sayari et al. (2005) stated that transgenic potato expressing P5CS gene from Arabidopsis spp. accumulated remarkably high proline as compared to those of control plants grown under high salt stress (100 mmol l−1 NaCl). Similar studies were also made with transgenic petunia (Petunia hybrida cv. Mitchell) plants containing P5CS genes (AtP5CS from A. thaliana L. or OsP5CS from Oryza sativa L.) that conferred drought tolerance (Yamada et al. 2005). Transgenic plants accumulated 1.5–2.6 times more proline contents leading to drought tolerance for a period of 14 days as compared to those of wild‐type plants grown under normal conditions. Similar cases were also reported by Zhang et al. (2014) who showed that tobacco plants expressing OsP5CS1 and OsP5CS2 genes accumulated higher proline thereby suffering less oxidative damages under abiotic stress conditions as compared to those of control plants. Similarly, Surekha et al. (2014) transformed pigeon pea with the mutagenized version (P5CSF129A) of wild P5CS gene from Vigna aconitifolia leading to higher proline contents than those of nontransgenic counterparts. Trehalose, a nonreducing disaccharide of glucose, has also been suggested to enhance stress tolerance in many kinds of cells, such as bacteria, fungi, and plants. Due to its role in protecting membranes and proteins, a transgenic approach was also made on trehalose biosynthetic pathway. Various genes involved in trehalose biosynthesis were isolated from prokaryotes and from different crop plants (Zentella et al. 1999; Lunn et al. 2014). Yeo et al. (2000) reported that transgenic plants exhibited significantly enhanced drought tolerance. Transgenic plants including Arabidopsis, rice, eucalyptus, wheat, tobacco, tomato, and potato overexpressing GB biosynthetic genes were associated with enhanced GB accumulation and stress tolerance (Huang et al. 2000; Kurepin et al. 2017; Tian et al. 2017; Zhang et al. 2019). Transgenic crop plants with more GB accumulation not only exhibited stress tolerance but also improved reproductive components including flowers and fruits (Sulpice et al. 2003; Park et al. 2004). Li et al. (2019), stating that codA‐transgenic lines of tomato (S. lycopersicum cv. Moneymaker) plants exhibited more tolerance to low phosphate stress.

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The authors reported that the wild‐type plants showed severe symptoms such as stunting due to low Pi levels than transgenic lines under low phosphate conditions. This was attributed to the increased Pi uptake ability in the transgenic lines in which the translocation of Pi into the plant cell increased with the enhanced plasma membrane H+‐ATPase activity. With this approach, GB accumulation in transgenic lines altered the uptake of Pi, modulated carbohydrate signaling, as well as expressions of low phosphate‐response genes involved in Pi signaling. Increased mobilization and translocation of Pi maintained Pi homeostasis and enabled a higher photosynthetic rate with the increased production and translocation of sucrose for better adaptation to low phosphate stress. Fan et  al. (2012) reported that the gene encoding betaine aldehyde dehydrogenase (BADH) enzyme (involved in the biosynthesis of GB) improved abiotic stress tolerance in sweet potato (Ipomoea batatas). The increased gene expression and GB accumulation as a result of an increase in BADH activity in the transgenic sweet potato lines resulted in increased protection via increased cell membrane integrity and photosynthetic activity along with the reduction in ROS accumulation. Genes involved with the enhancement of proline and GB accumulation led to the upregulation of ROS‐scavenging genes stimulating the ROS‐scavenging system. The authors showed that increased GB biosynthesis in sweet potato led to the improvement in tolerance against multiple abiotic stresses without causing phenotypic defects. Similar applications were also made with polyamines. For example, Patel et al. (2019) reported that the postharvest shelf‐life of green bell pepper (Capsicum annuum L.) was extended with the exogenous application of spermidine and putrescine. They observed that the titratable acidity, protein content, POX and CAT activities, chlorophyll and capsaicin contents gradually decreased in all untreated and treated fruits throughout the storage period of 40 days; however, proline content and antioxidant 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) radical‐scavenging activity increased continuously during storage.

1.4.4  Negative Effects of Proline Application Although the beneficial effects of osmoprotectants have been documented in many studies, we should consider the negative aspects of application as well. The concentrations used for application must be well optimized and considered with other biochemical and stress parameters. Previously, Heuer (2003) stated that exogenous application of proline was characterized with a decreased accumulation of Na+ and Cl− ions in tomato seedlings. However, they reported that growth of the seedlings was significantly inhibited following addition of GB and proline to the media. They suggested that toxic effect could play significant roles in reducing the growth of seedlings. Similar issues were also made by Roy et al. (1993) who showed that the concentrations over optimum dose proved to be toxic in rice seedlings. High proline contents in cells were also associated with destabilization of DNA. Rajendrakumar et al. (1997) stated that proline destabilized the double helix of DNA (Calf thymus) and lowered the melting point (Tm) of DNA in a dose‐dependent manner. Susceptibility of DNA to nucleases increased at high proline concentrations. Recently, Pazuki et al. (2018) stated that application of proline over 0.3 mmol l−1 doses on sugar beet explant had a deleterious effect, while at 0.3 mmol l−1 concentration, proline induced more

1.4  ­Application of Osmoprotectants in Stress Condition

proliferation and propagation. Similarly, Borgo et al. (2015) reported that high proline accumulation in leaves of transgenic tobacco plants induced by water stress did not cause any morphological damages in the chloroplasts and mitochondria ultrastructure. However, they reported that high proline production in transgenic tobacco plants did not contribute to the osmotic adjustment in plants under water stress conditions. Hayat et al. (2012) stated that increased endogenous overproduction of proline imparted stress tolerance via preserving cell turgor and osmotic balance as well as reducing electrolyte leakage and lowering ROS and preventing oxidative burst in plants. However, quite a few reports have mentioned that exogenously applied high concentrations of proline were toxic, while low concentrations were beneficial and increased stress tolerance in plants. For example, Kiyosue et al. (1996) stated that PDH genes were upregulated with the application of exogenous proline. On the other hand, Hare et al. (2001) reported that exogenously applied proline at high concentrations inhibited the growth of Arabidopsis explants, while low concentrations enhanced the in vitro shoot organogenesis. At high levels of exogenous proline, P5CS inhibited organogenesis (Zhang et al. 1995). The toxic effect of exogenously applied proline was attributed to activation of a cycle of cytosolic proline synthesis from glutamate and mitochondrial proline degradation (Hare 1998). The toxic effects of high concentrations of exogenous proline application were also previously reported by Rodriguez and Heyser (1988), who showed that the growth in suspension culture of saltgrass (Distichlis spicata) was seriously inhibited. This treatment also caused reduction in proline biosynthesis. Shahbaz et al. (2013) stated that exogenous proline applications as a foliar spray at different concentrations (0, 10, 20 mmol l−1) could overcome the adverse effects of salt stress on shoot fresh weight of two eggplant cultivars, viz., L‐888 and Round grown under saline regimes (150 mmol l−1) with significant assimilation rate/water use efficiency in the cv. Round. Overall, proline was reported to be ineffective in alleviating the damages caused by salt stress. Similar reports were also made by Deivanai et al. (2011), who stated that a high level of proline was not found effective in overcoming growth retardation of rice under salinity stress. Although many works and findings are available regarding proline and GB, more research is needed to further clarify the role of these osmoprotectants in plant defense system. It is probable that the role of osmoprotectants depends on the species and concentrations studied. So far, very few works have been conducted on the role of proline in terms of the pathogen and abiotic stress combinations. It is possible that plant pathogens, in general, are more tolerant to environmental stresses than crop plants, and they might metabolize the exogenously applied proline or GB for their growth and sporulation. High proline or GB accumulation in response to environmental stress might encourage the growth and development of attacking pathogens. Therefore, studies to clarify the role of pathogens in in vitro conditions need to be performed in detail if virulence or sporulation is to be associated with the concentrations of proline or GB. Detailed works should be needed dealing with in situ localization of toxic metabolites and gene expression studies regarding proline accumulation under abiotic stress conditions. Detailed analysis of cross talks between different osmoprotectants and other signaling molecules also needs to be elucidated in order to develop tolerant crop varieties (Roychoudhury et al. 2015).

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Although remarkable performances have been made with transgenic approach studies, the success of genes associated with osmoprotectant accumulation has been limited due to constraining effects such as climate change, environmental pollution, and global increase in world temperature. Under such conditions, transgenic plants have been examined at the early growth stages with short‐term stress exposure. The addition of biotic stress factors would create more stressful environments for the crop plants because most of the plant pathogens have more resistance or tolerance to abiotic stress factors than those of crop plants. These handicaps may negatively affect the success of transferred genes (Khan et al. 2015).

1.5 ­Conclusion and Future Perspectives One of the major challenges for agriculture in future is to generate strongly resistant or tolerant crop plants for biotic or abiotic stress factors or both. Simultaneous or sequential stress combinations would make the case more difficult. Recent advancements in molecular biology and biochemistry have enabled us to examine the complex signaling network occurring in plant cells, which could be improved under stress conditions via the use of molecular or biochemical tools. Use of phytohormones, PGRs, or chemicals eliciting defense reactions as well as using signaling molecules help restoring the defense responses and aim to increase the crop production under such conditions; however, negative issues such as increasing of virulence and propagation of pathogens or insects should not be ignored but taken into consideration. With the changing environmental conditions and global temperature, the agricultural production system should be improved to feed the ever‐increasing world population. Cost‐effective approaches, use of marginal lands, phytoremediation processes, and good agricultural practices should be carried out; however, we should consider the plant– microbe interactions under abiotic stress conditions, while we focus on the abiotic stress issues and explore the interactions from every angle. Mimicking the field conditions in laboratory or greenhouse conditions while we generate the tolerance mechanism would be necessary to evaluate the complex case, if we plan to apply the osmoprotectants. Many approaches including biochemical and transgenic have been put forward to develop stress tolerance or resistance in plants, although most of them have been found partially successful. Therefore, future experimental plans regarding biochemical and transgenic approaches should be carefully designed taking into consideration other signaling pathways and mechanisms.

­Acknowledgment Financial assistance from Science and Engineering Research Board, Government of India, through the grant [EMR/2016/004799], and Department of Higher Education, Science and Technology and Biotechnology, Government of West Bengal, through the grant [264(Sanc.)/ ST/P/S&T/1G‐80/2017], to Dr. Aryadeep Roychoudhury is gratefully acknowledged.

 ­Reference

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2 Glycine Betaine and Crop Abiotic Stress Tolerance: An Update Giridara-Kumar Surabhi and Arpita Rout Plant Molecular Biology and OMICS Laboratory, Regional Plant Resource Centre, Bhubaneswar, Odisha, India

2.1 ­Introduction Environmental stress factors such as drought, high salinity, extreme temperatures, heavy metals, ultraviolet-B radiation, waterlogging, mineral deficiency, and toxicity severely limit plant growth and crop production worldwide (Kumar 2013; Cabello et al. 2014; Surabhi 2018). Abiotic stresses are projected to reduce crop yields to less than half of that possible under ideal growing conditions, across a range of cropping systems around the globe (Surabhi 2018). Plants have evolved diverse morphological structures, physiological functions, molecular changes, and ecological interactions to adapt to their environment (Surabhi 2018; Zhang et al. 2019). When the crop plants get exposed to abiotic stress factors, they get stimulated to accumulate certain organic compounds of low molecular mass in the cytoplasm. These organic compounds are collectively known as “compatible solutes” (Surabhi et al. 2000; Roychoudhury et al. 2015). Since plants are immobile, their metabolism is the only weapon to survive under such stress conditions. Glycine betaine (GB), proline, trehalose, and other compatible solutes adjust the osmotic homeostasis of crops against different abiotic stresses (McCue and Hanson 1990; Kishitani et al. 1994; Surabhi et al. 2000, 2003; Surabhi 2018; Xu et al. 2018). Although compatible solutes fall in different biochemical groups, similar roles have been assigned to them in plant protection against stresses (Giri 2011). Among different compatible solutes, one of the most extensively studied osmoprotectants is GB. Many taxonomically distant plant species normally contain low levels of GB (these plants are known as natural accumulators of GB), but can also accumulate larger amounts of GB when subjected to abiotic stress. In many other species, GB has not been detected under normal or stressful conditions (natural nonaccumulators) (Chen and Murata 2011). Levels of GB accumulation vary considerably among plant species and organs (Chen and Murata 2011). GB is a low-molecular-mass-compatible solute, which is a soluble quaternary ammonium compound without cellular toxicity (McDonnell and Wyn

Protective Chemical Agents in the Amelioration of Plant Abiotic Stress: Biochemical and Molecular Perspectives, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

2.2  ­Biosynthesis of G

Jones 1988; Cleland et al. 2004). It is dipolar but electrically neutral at physiological pH (Rhodes and Hanson 1993). GB effectively stabilizes the quaternary structures of enzymes and complex proteins, protects various components of the photosynthetic machinery (Shahbaz et al. 2011), such as ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) and the oxygen-evolving photosystem II (PSII) (Murata et al. 1992; Rhodes and Hanson 1993; Lee et al. 1997; McNeil et al. 1999; Nayyar et al. 2005a,b), maintains highly ordered state of membranes at nonphysiological temperatures (Zhao et al. 1992; Rajashekar et al. 1999; Sakamoto and Murata 2002; Nayyar et al. 2005a,b; Fariduddin et al. 2013), and mitigates oxidative damage (Chen and Murata 2011) and high salt concentrations (Papageogiou and Murata 1995). Several studies have interpreted the relationship between GB and tolerance to abiotic stress in different crop plants either through exogenous application of GB, natural accumulation, or through transgenic expression of GB pathway genes that have appeared recently, such as under salt stress in tomato (Umar et al. 2018), soybean (Malekzadeh 2015), wheat (Salama et  al. 2015), and sunflower (Khan et  al. 2016); under drought stress in cotton (Gossypium hirsutum L.) (Ahmad et al. 2014), corn (Zea mays L.) (Miri and Armin 2013), and wheat (Raza et  al. 2014); under heavy metal stress in cotton (Farid et  al. 2013; Bharawana et al. 2014); and under low temperatures in Arabidopsis (Hayashi et al. 1997; Alia et al. 1998). However, a precise role of compatible solutes, including GB, in abiotic stress tolerance is largely unknown (Giri 2011). With the increasing efforts of whole genome sequencing for several crop plants coupled with recent advances in high throughput, omics approaches and genome editing can shed light on understanding GB biosynthetic pathway more thoroughly toward abiotic stress tolerance. In this review, we briefly update different aspects of GB, such as natural accumulation, exogenous application, and genetic engineering of crop plants for GB biosynthesis, with particular emphasis on abiotic stress tolerance. Further, we also discussed GB effects on reproductive and yield parameters of different crop species under varied stress conditions.

2.2 ­Biosynthesis of GB Using choline and glycine as respective substrates, GB is synthesized via two pathways. In plants, phosphoethanolamine N-methyltransferase (PEAMT) is the key enzyme for synthesis of choline. All three of the methylation steps that are required to convert phosphoethanolamine to phosphocholine are catalyzed by a cytosolic enzyme PEAMT, the precursor to choline biosynthesis (McNeil et al. 2001). The choline is then transported into the chloroplast in plants where it undergoes a two-step oxidation reaction. First, choline is oxidized to betaine aldehyde, a toxic intermediate, which then is oxidized to GB. The first oxidation is catalyzed by choline monooxygenase (CMO), an unusual ferredoxin-dependent soluble protein with a motif characteristic of Rieske-type iron–sulfur proteins (Hibino et al. 2002). This oxidation reaction in animals and bacteria is catalyzed by choline dehydrogenase (CDH), but some bacteria may also use choline oxidase (COD) for the first step of GB synthesis. The nicotinamide adenine dinucleotide (NAD)+dependent betaine aldehyde ­dehydrogenase (BADH) catalyzes the second oxidation step,

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in most organisms (Rathinasabapathi et al. 1997), although in some bacteria CDH and COD can also catalyze the second step. The GB synthesis can also occur in chloroplast of higher plants, from serine via ethanolamine and betaine aldehyde (Rhodes and Hanson 1993). Both CMO and BADH are encoded by nuclear genes and contain transit sequences targeting them to chloroplasts, though they are localized in the stroma of chloroplasts. An alternate biosynthetic pathway of betaine from glycine was reported in cyanobacterium and Arabidopsis, and two N-methyltransferase enzymes catalyze the reaction. It was found that the coexpression of N-methyltransferase genes caused accumulation of betaine that confers stress tolerance. The second pathway has only been found in the extreme halophytic phototrophic bacteria Actinopolyspora halophila and Ectothiorhodospira halochloris, in which the bacteria use glycine instead of choline for GB synthesis (Nyyssola et al. 2000; Ahmed et al. 2013). These bacteria accumulate GB up to 33% of total cell dry weight (Nyyssola et al. 2000). Thus, for improving stress tolerance in crops, it becomes interesting to compare the efficiency of the methyltransferase pathway with the choline oxidation pathway. The cell extracts of both E. halochloris and A. halophila have methyltransferase activity on glycine, and AdoMet (S-Adenosyl methionine) acts as the methyl group donor. Further, analysis of the reaction products confirms that the organisms synthesize betaine from glycine in a three-step methylation reaction. Glycine is first methylated to sarcosine and then further to dimethylglycine and betaine.

2.3 ­Accumulation of GB Under Abiotic Stress in Crop Plants Among the compatible solutes, GB is particularly an effective protectant under abiotic stress. Many crops are known to accumulate GB at low levels, but can also accumulate larger amount of GB when subjected to abiotic stress environments. These types of crops are called natural accumulators of GB (Hayashi et al. 1997; Baloda et al. 2017). GB accumulation is usually associated with the range of increased tolerance in different crops under abiotic stress conditions, such as in spinach (Spinacia oleracea) and sugar beet (Beta vulgaris) from chenopodiaceae family (Rhodes and Hanson 1993; Chen and Murata 2008; Khan et al. 2009; Fariduddin et al. 2013). It was noticed that GB accumulated at a high concentration in maize (Z. mays) and barley (Hordeum vulgare) from poaceae family (Khan et al. 2009) [4–40 μmol g−1 fresh weight (FW)] similar to naturally accumulating plants such as spinach and sugar beet, and acted as an osmoprotectant under abiotic stress conditions (Giri 2011). Interestingly, in some naturally GB-accumulating plants, including mangrove (Hibino et al. 2001) and barley (Fujiwara et al. 2008), no CMO activity was detected in chloroplasts (Koyro et al. 2012). It was observed that naturally synthesized/accumulated GB is not enough to augment osmotic stress caused by salt stress for many plant species (Sakamoto and Murata 2002; Subbarao et  al. 2001; Kuttana et  al. 2018). The Mediterranean halophytes Plantago crassifolia and Inula crithmoides also accumulate GB under salt stress environments. However, it is found that I. crithmoides is more salt-tolerant than P. crassifolia, in agreement with the distribution of the two species in nature (Pardo-Domenech et al. 2015). Under high temperature stress, crops were seen to accumulate GB to stimulate synthesis of the D1 protein, which supports the repair of photodamaged PSII (Chen and Muruta

2.4  ­Exogenous Application of GB in Crop Plants Under Abiotic Stres

2011; Allakhverdiev et  al. 2007). Further, accumulation of GB prevented the seizure of Rubisco activase to the thylakoid membrane, thereby maintaining the activity of Rubisco at a high temperature (Yang et al. 2005). Moreover, it has been found that the accumulation of GB improves the inhibition of net photosynthetic rate under heat stress (Wang et  al. 2010). During dehydration (drought) stress, GB localized in chloroplasts shows increase in concentration and plays an important role in chloroplast adjustment and protects thylakoid membranes, which leads to maintenance of photosynthetic efficiency and membrane integrity (Yokoi et al. 2002; Ahmed et al. 2013).

2.4 ­Exogenous Application of GB in Crop Plants Under Abiotic Stress Exogenous application of GB technique has been developed to make crops resistant against abiotic stress and to increase the crop production. The limited accumulation of GB naturally and in transgenic plants enables exogenous applications, which enhance internal GB levels in numerous species of low or nonaccumulating crops (Chen and Murata 2008; Fariduddin et al. 2013). This indeed results in reducing critical effects due to abiotic stresses and in turn enhances the yield and growth of plants (Reddy et al. 2014). Exogenously, GB has been applied to foliar tissues to make the crop plants stress tolerant and leaf tissues readily uptake the GB when applied on it (Park et al. 2006; Chen and Murata 2008). GB was applied foliarly in different crop plants under abiotic stress conditions, such as in alfalfa (Medicago sativa) (Fariduddin et al. 2013), sunflower (Helianthus) (Iqbal et al. 2005), soybean (Glycine max), (Agboma et al. 1997; Rezaei et al. 2012), and wheat (Triticum aestivum) (Gupta and Thind 2017). Park et al. (2006) have applied GB exogenously on leaves of tomato (Lycopersicon esculentum), and major quantity of the GB is localized in the cytosol and only a small fraction of the cytosolic GB is translocated to chloroplasts when leaves encounter GB, while large amount of foliar-applied GB gets translocated to meristem-containing tissues, including the flower buds and shoot apices (Park et al. 2006). In another study, Mäkelä et al. (1996) have demonstrated with the aid of phloem that the GB gets actively translocated to growing and expanding plant parts (Mäkelä et al. 1996). The exogenous foliar application of GB on soybean plants (low accumulator of GB with average accumulation of around 5 μmol g−1 dry weight) showed enhanced accumulation of GB by 12-fold (60 μmol g−1 dry weight). This led to an overall increase in leaf area expansion, photosynthetic yield, nitrogen fixation, and seed production (Mäkelä et al. 1996; Roychoudhury and Banerjee 2016).

2.4.1  Drought The soybean plants subjected to drought stress have reduced reproductive yield, leaf area, and biomass accumulation; and these effects were coupled with a reduction in photosynthesis and N2 fixation. By contrast, foliar spray of GB on field-grown soybean plants showed increased reproductive yield and leaf area, both effects being accompanied by an increased rate of photosynthesis and enhanced N2 fixation. Exogenous pretreatment of droughtstressed pot-grown tobacco plants using a spray of GB enhanced their stress tolerance, and

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these effects were associated with greater stomatal conductance, an increased efficiency of PSII, higher carboxylation efficiency of CO2 assimilation, and better photosynthesis (Kurepin et al. 2015). Foliar application of GB was done at tillering and anthesis stages in wheat (T. aestivum) by taking 19 genotypes under drought stress. Among the 19 GB-treated wheat genotypes, maximum reduction was observed in PBW 343 (29.4%) and minimum in BW 9025 (12.2%). Foliar application of GB improved number of grains per spike and thousand grain weight (TGW) to a certain extent in most genotypes under stress, but BW 9183, BW 9097, and PBW 550 responded significantly to GB treatment by greater magnitude by percent increase of yield components (Gupta and Thind 2017). While in case of drought stress cotton (G. hirsutum L.) plants, exogenous application of GB to the foliar parts showed higher photosynthetic ability, transpiration rate, and improved intercellular CO2 concentration, better yield, and quality as compared to wildtype plants. The maximum chlorophyll content (51.01%) was recorded for the cotton plants those were treated GB exogenously, whereas minimum chlorophyll content (39.17%) was recorded for untreated cotton (Ahmad et al. 2014). Two tobacco (Nicotiana tabacum L.) cultivars, drought-tolerant DHJ5210 and drought-sensitive ZY100, were taken to study the effects of foliar-applied GB (80 mM) under well-watered and waterdeficit conditions. Cv. DHJ5210 absorbed more GB than Cv. ZY100 in the leaves under stress conditions. Therefore, foliar GB application at the rapid growth stage favors plants growth in drought-stressed plants, mainly by improving water status and increasing PSII activity (Ma et al. 2007). In another study, GB was applied foliarly in sunflower (Helianthus annuus L. Hysun-33) under drought stress to improve the yield of hybrid sunflower. Exogenous GB application improved the head diameter, number of achenes, 1000-achene weight, achene yield, and oil yield under water stress. However, exogenous GB application at the flowering stage was more effective than other treatments. Water stress in combination with foliar application of GB has shown greater impact in flowering stage than in the vegetative stage (Hussain et  al. 2008). GB application on pepper (Capsicum annuum) under water stress conditions increased shoot dry weight by 27%, leaf area by 42%, photosynthetic rate by 30%, and transpiration rate by 40% of pepper seedlings under water stress as compared with non-GB-treated plants (Korkmaz et al. 2014). The seeds and leaves of cumin (Cuminum cyminum cv. Mashhad) were treated with GB at flowering stage under drought stress and the yield was found to be higher in GB-treated cumin. Plant height at irrigated treatment was 9.34% higher than rain-fed planting. Application of GB with seed and leaf treatment augmented yield by 32% in rain-fed condition while this value was 24% more in irrigation condition (Armin and Miri 2014).

2.4.2  Salt Stress One of the world’s important cereal food crops, rice (Oryza sativa) is unable to synthesize GB naturally, as it lacks CMO, which is involved in the synthesis of betaine aldehyde, the immediate precursor of GB (Lutts 2000). Application of GB exogenously protected rice from salt stress by maintaining PSII integrity and relative water content (RWC) (Lutts 2000). The exogenous application of GB mitigated the detrimental effects of salt stress, through better activity of antioxidant enzymes involved in NaCl-induced oxidative stress

2.4  ­Exogenous Application of GB in Crop Plants Under Abiotic Stres

on tobacco (N. tabacum “Bight Yellow-2”) suspension-cultured cells (Hoque et al. 2007). Likewise, exogenous application of GB-ameliorated salt stress effects in GB nonaccumulating/low GB-accumulating crop plants, such as in wheat (T. aestivum) (Raza et al. 2007), maize (Z. mays) (Yang and Lu 2005), kidney beans (Phaseolus vulgaris) (Lopez et al. 2002), turnip rape (Brassica rapa) (Makela et al. 1999), and tomato (Solanum lycopersicum) (Park et al. 2006).

2.4.3  Temperature Stress The exogenous application of GB has been successfully used to improve heat stress tolerance in tomato (Makela et al. 1998; Li et al. 2011) and sugarcane (Rasheed et al. 2011). GB was applied foliarly to the seedlings of marigold (Tagetes erecta cv. “Narai Yellow,” “Bali Gold,” and “Columbus Orange”) and it effectively mitigated the effect of heat stress. In another study, cotton (G. hirsutum cv. Lumianyan19) plants were treated with GB to make it cold-tolerant. The GB content in leaves was increased by 25.6% in treated plants as compared to control plants. The final germination percentage was higher in seeds treated with 400 μg ml−1 GB (93.3%) as compared to the seeds treated with 200 μg ml−1 GB (92.7%). The chlorophyll content of GB-treated plants was higher than that of control plants, by 14.3% after 1 day, 33.3% after 2 days, and 13.7% after 3 days of GB treatment. This indicated a greater resistance to low-temperature damage in treated plants (Cheng et al. 2018). In cold-stressed tomato (L. esculentum Mill. cv. Moneymaker) plants, exogenous application of GB ameliorated the adverse effects and improved the growth. Further, foliar application of GB was readily taken up and translocated to various organs, with the highest levels of accumulation observed in meristematic tissues (shoot apices and flower buds). In leaves, the majority of endogenous GB was observed in the cytosol and only 0.6–22% of the total leaf GB was localized in chloroplasts. In both GB-treated and control plants, a steady increase in H2O2 levels was observed, till day 3. The H2O2 contents were 2.4-fold higher than their original values (day 0) in control plants, whereas up to twofold more H2O2 was accumulated in GB-treated plants (Park et  al. 2006) (Table  2.1), after 3 days of chilling stress. VollenWahid and Gunthardt-Goerg (2005) have pretreated the barley seeds with GB, and GB-treated seedlings showed greater shoot biomass and increased rate of net photosynthesis, when the seeds were germinated under heat stress conditions, compared to untreated barley seeds (VollenWahid and Gunthardt-Goerg 2005).

2.4.4  Heavy Metal Stress The exogenous application of GB suppressed cadmium (Cd)-induced reactive oxygen species (ROS) production by increasing the activities of ASC–GSH (ascorbate–glutathione) cycle enzymes and significantly restored the membrane integrity under Cd stress in tobacco bright yellow-2 cells (Islam et al. 2009). Cotton (G. hirsutum genotype MNH 886) plants were subjected to Cd stress (1 and 5 μM Cd concentrations) to notice its effects. When compared to control, plant height was reduced by 28.19% at 1 μM Cd and 61.17% with 5 μM Cd concentrations. When plants were treated with GB at 5 μM concentration, there was increase in the dry weight of leaves by 30%, stem by 21.21%, and roots by 28.31%. There was increase in Chla by 33.31%, total chlorophylls by 31.56%, and carotenoids content by 45.41%

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2.5  ­Transgenic Approach to Enhance GB Accumulation in Crop Plants Under Abiotic Stres

as compared to 5 μM Cd stress alone (Farooq et al. 2016). Similarly, in Cu-stressed mustard plants, exogenous GB application counteracted the oxidative stress. Cotton plants were treated exogenously with GB under lead (Pb) stress (Bharawana et al. 2014) and control plants exhibited stunting of growth in the absence of GB as compared to those treated with GB. Similarly, Pb-treated plants at both Pb levels (50 and 100 μM) were smaller than those of the plants grown with GB under Pb stress. The positive effect of GB was greater for the plants that were treated with Pb as compared to control plants. The exogenous application of GB enhanced the overall performance of different crop plants under varied stress conditions. However, it is required to establish correlation between exogenous application of GB and plant reproductive stage performance and yield-related aspects under different stress conditions.

2.5 ­Transgenic Approach to Enhance GB Accumulation in Crop Plants Under Abiotic Stress Several GB biosynthetic pathway genes were used to develop transgenic plants, which accumulated GB and showed enhanced tolerance to abiotic stresses. Certain major cereals such as wheat, barley, and maize do not accumulate significant amount of GB naturally. This could be due to the production of truncated transcripts in these crop plants for GB-synthesizing enzyme (BADH) (Niu et al. 2007). Among these, rice is the only cereal that does not accumulate GB naturally (Shirasawa et al. 2006). These necessitate development of transgenics and enhance crop tolerance to different abiotic stress. Among such transgenic plants, codA (cox gene for choline oxidase from Arthrobacter globiformis, the enzyme that converts choline into GB) transgenic plants have been investigated most thoroughly in terms of the morphological, physiological, and reproductive aspects, such as production of fruits and seeds. The cox gene for choline oxidase from Arthrobacter pascens was used to generate transgenic lines that synthesize GB, and fused to a chloroplast-targeting sequence that is expressed under the control of either an abscisic acid (ABA)-inducible promoter or the promoter of a constitutively expressed gene for ubiquitin (UBI) (Su et al. 2006; Chen and Muruta 2008). The GB accumulation was targeted in chloroplast, where its increased accumulation warranted plant tolerance to different abiotic stresses (Khan et al. 2015). GB has three main pathways in different organisms (Sakamoto and Murata 2000). Therefore, different methods could be used to introduce a synthetic system into non-GB-accumulating plants to improve their abiotic stress tolerance (Cheng et al. 2013). Numerous crops species have been engineered, using these different genes to enhance their abiotic stress tolerance (Khan et al. 2015). One such method is by inducing codA gene from A. globiformis, a soil bacterium, to the crops that do not accumulate GB naturally such as mung bean (Vigna radiata L.Wilczek) (Baloda et  al. 2017), potato (Solanum tuberosum L.  cv. Superior) (Cheng et  al. 2013), tomato (S. lycopersicum cv. “Moneymaker”) (Wei et  al. 2017), poplar (Populus alba × Populus glandulosa) (Ke et al. 2016), and alfalfa (M. sativa L. cv. Xinjiang Daye) (Li et al. 2014) under various abiotic stress conditions. In general, compared to other osmoprotectant genes, the GB biosynthetic genes in transgenic plants proved very effective in enhancing abiotic stress tolerance. The accumulated

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GB content and the resultant stress tolerance are believed to be influenced by three factors in transgenic plants: choline (precursor for GB) availability (Hunag et  al. 2000), type of transgene of the GB biosynthetic pathway (Hibino et al. 2002; Khan et al. 2009), and the type of promoter (constitutive and stress inducible) (Su et al. 2006).

2.5.1  Drought Transgenic approaches have been adopted to produce crops to sustain during water-stressed conditions. It involves manipulations in the qualitative as well as quantitative traits through transfer of desired genes (Ashraf 2010). Higher plants have been successfully engineered to develop drought-tolerant crops (Zhang et  al. 2008), such as transgenic rice, potato and alfalfa (Perl et al. 1993; McKersie et al. 1996; Wang et al. 2005), tobacco (Gubis et al. 2007), and soybean (DeRonde et al. 2004). Cox gene from another soil bacterium A. pascens was induced to Arabidopsis thaliana (ecotype RLD), tobacco (N. tabacum cv. Xanthi), and mustard (Brassica napus cv. Westar) under drought conditions. Under abiotic stress, transgenic lines (8–18.6 μmol g−1 dry weight [DW]) showed tenfold higher betaine accumulation than in wild-type (WT) plants (approximately 1 μmol g−1 DW) in all three species. The shoot weight was increased in transgenic tobacco, as compared to parental line. COX+ tobacco showed improved tolerance during water-stressed conditions as related with transgenic Arabidopsis lines. Further, photosynthetic rate was better in B. Napus transgenics in comparison with Arabidopsis transgenic (Hunag et al. 2000).

2.5.2  Salt Stress Through the introduction and overexpression of a COD gene, salt stress tolerance was achieved successfully in Brassica juncea and Japanese persimmon (Diospyros kaki) (Gao et al. 2000; Prasad et al. 2000). Transgenic tobacco plants overexpressing OsCMO gene from Japonica rice (O. sativa cv. Nipponbare) result in increased GB content and elevated tolerance to salt (Luo et al. 2012). In another study, BADH gene was transformed into alfalfa (M. sativa L. cv. Sanditi) through Agrobacterium-mediated transformation method to make the plant salt tolerant. Compared to the wild-type control, peroxidase (POD) activity was increased by 149% in transgenic line B106, and over 20% in the other transgenic lines, except for line B227. The superoxide dismutase (SOD) activity in the transgenic lines was increased by 50.55 ± 25.4% on average with a range of 0.7–92.4% over the wild-type control. The alfalfa transgenic plants grew vigorous under salt stress condition, whereas the wildtype plants were retarded and did not survive after cradle. The study revealed that the SOD and POD activities in the transgenic plants were higher than the wild-type plants. Thus, salt tolerance of the transgenic plants was further evidenced through the improvement of antioxidative activity (Liu et al. 2011). CMO from spinach (S. oleracea) was transformed to rice (O. sativa var japonica cv. “Sasanishiki”) by Agrobacterium-mediated transformation and tested under salt stress. There was a tenfold increase in levels of GB in transgenic rice plants that expressed CMO under stress as compared to the wild-type plants (Shirasawa et al. 2006). In another study BADH gene from mountain spinach (Atriplex hortensis L.) was induced to wheat (T. aesti-

2.6  ­Effect of GB on Reproductive Stage in Different Crop

vum L.) under salt stress conditions. After 4 days of stress, chlorophyll and carotenoid contents of WT declined significantly, than in transgenic lines (Tian et al. 2017). BADH gene from spinach (S. oleracea L.) was induced to tobacco (N. tabacum L.) to make it saline-tolerant and chloroplast accumulated 61–85% of total leaf GB under stresses. For the first time, Yang et  al. (2008) have demonstrated that the accumulation of GB in transgenic tobacco plants also protected Rubisco, FBPase (fructose-1,6-bisphosphatase), FBP aldolase (fructose-1,6-bisphosphate aldolase), and PRKase (phosphoribulokinase) against salt stress (Yang et al. 2008). BADH gene from spinach (S. oleracea; SoBADH) was introduced into sweet potato (Ipomoea batatas cv. Sushu-2) to make it tolerant to various abiotic stress conditions. Transgenic lines (2.56 mmol g−1 FW) showed fivefold higher GB content as compared with wild-types plants (0.63 mmol g−1 FW) (Fan et al. 2012).

2.5.3  Temperature Stress Transgenic tomato (Park et al. 2007; Chen and Muruta 2008) and rice (Sakamoto and Alia 1998; Chen and Murata 2008) plants have been developed by inducing codA gene in the chloroplast to accumulate GB under cold stress. Transgenic tomato plants developed expressing codA gene, and wild-type tomato plants that were supplemented with GB exogenously showed a greater heat stress tolerance than wild-type plants that did not receive exogenous application GB (Li et al. 2011). Similarly, overexpression of codA gene in A. thaliana plants (and thus accumulating GB) exhibited an increased tolerance to a short-term heat stress (Hayashi et al. 1998; Kurepin et al. 2015). The tobacco plants expressing BADH transgene from spinach were investigated by Yang et  al. (2005). The transgenic tobacco plants accumulated more GB than WT plants, and coincidentally they exhibited increased heat stress tolerance, both events being associated with increased CO2 assimilation, higher Rubisco activity, and a more stable PSII (Yang et al. 2005). Li et al. (2013) took BADH gene from spinach (S. oleracea L.) and introduced it into tomato (L. esculentum cv. “Moneymaker”) via Agrobacterium-mediated transformation. Their results confirmed that GB accumulation in vivo increased the tolerance of PSII to heat stress (Table 2.2).

2.6 ­Effect of GB on Reproductive Stage in Different Crops Crop plants are very subtle to abiotic stresses at reproductive stage compared to other stages of plant development, such as vegetative stage. However, such studies are rather scanty. Studies by Agboma et al. (1997) have revealed that GB could compensate grain yield for a reduction in the amount of water needed for irrigation in maize (Z. mays L.) and sorghum (Sorghum bicolor L.).

2.6.1  Role of GB on Flower Initiation Drought stress during flowering can interrupt floret initiation, translocation of assimilates to the grain that lowers grain filling, and size (Akram et al. 2013; Ahadiyat et al. 2014). Previous studies revealed that in codA-transgenic Arabidopsis plants different organs

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2.7  ­Pyramiding GB Synthesizing Genes for Enhancing Abiotic Stress Tolerance in Plant

a­ ccumulated different levels of GB (with COD targeted to chloroplasts) when codA gene was constitutively driven by a CaMV 35S promoter (Sulpice et al. 2003; Chen and Murata 2011). In fully matured plants, levels of GB in flowers, siliques, and inflorescence apices were about threefold to fivefold higher than those in leaves in transgenic COD-rice chloroplasts (Sakamoto and Murata 2002). Application of GB under cold stress in bud stage of chickpea (Cicer arietinum L.) resulted in improvement of flower functioning in terms of pollen viability, pollen germination (in vitro and in vivo), pollen tube growth, stigma receptivity, and ovule viability. As a result, increases in floral retention by 47%, pod set by 38%, and pod retention by 23% were observed, as compared to wild-type plants (Nayyar et al. 2005a,b). To date, very few studies have focused on GB initiation in flower in different plants and more focus is required in this aspect.

2.6.2  GB on Seed Set and Yield Stability Reduction in yield by as much as 40% was observed for maize and 21% for wheat under water-stressed conditions (Daryanto et al. 2016). In case of cowpea, an important food legume worldwide, yield reduction was between 34% and 68% depending on the onset of developmental stage of the grain under drought stress (Farooq et al. 2017). Foliar application of GB in wheat under drought stress improved the grains/spike in the GB-applied plants (Gupta and Thind 2017). Rice crop demonstrated 22.6% yield reduction under drought stress (Carrijo et  al. 2017). During reproduction, the yield of transgenic tomato (L. esculentum Mill. cv. Moneymaker) plants was 10–30% higher than those of wild-type following chilling stress (Park et al. 2004). While in case of cotton (G. hirsutum L.) under drought stress, exogenous application of GB to the foliar parts showed higher photosynthetic ability, transpiration rate, improved intercellular CO2 concentration, better yield, and quality as compared to wild-type plants. The maximum chlorophyll content (51.01%) was recorded for the cotton plants that were treated with exogenous GB, whereas minimum chlorophyll content (39.17%) was recorded for untreated cotton (Ahmad et al. 2014). In another study, GB was applied foliarly in sunflower (H. annuus L. Hysun-33) under drought stress to improve the yield of hybrid sunflower. Exogenous GB application improved the head diameter, number of achenes, 1000-achene weight, achene yield, and oil yield under water stress. However, exogenous GB application at the flowering stage was more effective than other treatments. Water stress in combination with foliar application of GB has shown greater impact on flowering stage than in the vegetative stage (Hussain et al. 2008).

2.7 ­Pyramiding GB Synthesizing Genes for Enhancing Abiotic Stress Tolerance in Plants Abiotic stress tolerance is a multigenic trait; therefore, it requires a coordinated function of multiple genes to impart a viable tolerance against single stress or combination of stresses. The method that aims at assembling multiple desirable genes from multiple parents (cross or same crop plants) into a single genotype (single crop plant) is known as gene pyramiding. The end product of a gene-pyramiding program is a genotype with all of the target genes (Malav and Indu 2016). There have been few reports on transformation of multi-

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genes into an individual higher plant and evaluation of their physiological performance under abiotic stress. Recently, researchers have tried to discover synergistic effects of gene pyramiding utilizing GB-synthesizing genes and other stress-protecting genes (Zhou et al. 2008; Ahmed et al. 2013; Niu et al. 2014; Song et al. 2018). The betA + AtNHX1 transgene pyramiding tobacco was developed by Duan et al. (2009) using sexual crossing, and the salt tolerance was evaluated at the cellular and plant levels. Their studies revealed that the overexpression of betA (encoding choline dehydrogenase from Escherichia coli) or AtNHX1 (a vacuolar Na+/H+ antiport from A. thaliana) genes showed salt tolerance in tobacco transgenic plants. Further, transgene pyramiding lines exhibited higher percentage of seed germination, better seedling growth, and higher fresh weight than lines that had betA or AtNHX1 alone. Wei et al. (2011) have developed maize plants through cross pollination that were transgenic for both betA (encoding choline dehydrogenase from E. coli) and TsVP (encoding V-H+-PPase from Thellungiella halophila). Their study demonstrated that the pyramided transgenic plants had higher GB contents and H+-PPase activity compared with the parental lines, which had either betA or TsVP, and exhibited higher RWC, greater solute accumulation, and lower cell damage under drought stress treatment. Further, the pyramided plants grew more vigorously with less growth reduction, shorter anthesis-silking interval, and higher yields than their parental lines and the wild-type plants (Wei et al. 2011). The transgenic cotton coexpressing ApGSMT2g and ApDMT2g was developed by Agrobacterium-mediated transformation (Song et  al. 2018). Coexpression of ApGSMT2g and ApDMT2g in cotton resulted in higher GB amounts, compared to WT plants. Further, higher RWC, lower osmotic potential, more K+, and less Na+ under salt stress, which contributes to maintaining intracellular osmoregulation and K+/Na+ homeostasis, and thus confers higher salt tolerance and more vigorous growth. Coexpressing ApGSMT2g and ApDMT2g in cotton also reduces membrane damage and increases SOD activity compared to WT under salt stress. Further, their study confirmed that transgenic cotton with ApGSMT2g and ApDMT2g exhibited higher salt tolerance and more seed cotton yield in saline fields compared to wild-type plants. In another study, tobacco (N. tabacum L. cv. Wisconsin 38) was transformed by taking two methyltransferase genes named ApGSMT2 (Aphanothece halophytica glycine sarcosine methyltransferase) and ApDMT2 (A. halophytica dimethylglycine methyltransferase) to develop drought-resistant tobacco plants. After imposing drought stress for 5 days, the maximum photochemical efficiency of PSII in the GSD (transgenic tobacco coexpressing ApGSMT2 and ApDMT2) plants only decreased about 23%, whereas the maximum photochemical efficiency of PSII in the BL (betA transgenic tobacco) decreased about 47% and in wild-type plants by 80%. Thus, the GSD plants had enhanced stress tolerance and better photosynthetic performance under drought stress, than the other plants tested. The leaf RWC decreased from about 87–55% in the wild-type plants. It decreased to 60% in the BL plants and about 65% in the GSD plants. On the seventh day of drought stress treatment, leaf RWC in the GSD plants was significantly higher than in the wild-type and BL plants (He et al. 2011a). Niu et al. (2014) have utilized Agrobacterium-mediated gene transformation to produce transgenic rice (O. sativa L., cv. Nipponbare) plants containing ApGSMT and ApDMT genes, which were isolated from A. halophytica. The coexpression of both ApGSMT and

­Acknowledgmen

ApDMT transgenes resulted in a significant increase of GB biosynthesis and enhanced tolerance to salt and cold stresses in the transgenic rice plants. Zhou et al. (2008) have cointroduced two genes (BADH and SeNHX1) that underlie different mechanisms of salt tolerance into tobacco (N. tabacum cv. Wisconsin 38) plants. A binary plasmid (Agrobacterium tumefaciens (LBA4404) carrying a binary vector pBinBS) was constructed containing a vacuolar Na+/H+ antiporter gene, SeNHX1, which was isolated from halophyte Salicornia europaea and a betaine synthesis gene BADH from salt-tolerant plant A. hortensis. Transgenic lines S1 and S15 transformed with SeNHX1, lines of B8 and B23 transformed with BADH, and lines of BS5 and BS15 cotransformed with BADH and SeNHX1. The two transgenes functioned additively in the transgenics. The transgenic plants exhibited enhanced accumulation of betaine and Na+ under salt stress due to additive expression of BADH and SeNHX1 genes. However, when these plants were exposed to 200 mM NaCl, the plants overexpressing BADH displayed a sixfold to 30-fold increase in betaine contents with line B8 being the highest (7.2 μmol g−1). For instance, exposure to NaCl led to increase in Na+ contents in wild-type leaves by 3.5-fold, whereas the Na+ contents in leaves of the dual-gene-transformed lines of BS5 and BS15 increased by 5.7-fold and 6.7-fold when treated by the 200 mM concentration of NaCl, respectively (Zhou et  al. 2008). However, till date a very few studies are focused on applying gene pyramiding of GB biosynthesis genes and validating those for abiotic stress tolerance in crop plants.

2.8 ­Conclusion and Future Prospective Exogenous application of GB on different crop plants has shown enhanced stress tolerance and increased yield. A variety of genes have been employed to generate transgenic plants that accumulate GB and are tolerant to abiotic stresses. Among the properties of these transgenic plants, physiological and morphological aspects of stress tolerance have been investigated most extensively in codA transgenic plants. Further, extensive studies are required to increase stress tolerance in different crop species to enhance abiotic stress tolerance, coupled with better crop yield and productivity. The application of newer technologies such as synthetic biology, gene pyramiding, and genome editing tools are helpful in engineering GB pathway further and to enhance abiotic stress tolerance in crop plants. In addition, high throughput global “omics” approaches have a promising role in exploring gene function in connection with GB biosynthetic pathway and crop abiotic stress responses, in a more holistic way. The ultimate practical goal of all these studies is to enhance abiotic stress tolerance of crop plants that can grow better under adverse environmental stress conditions worldwide.

­Acknowledgment Work in the laboratory of Giridara-Kumar Surabhi was supported by the Forest and Environment Department, Government of Odisha, India, and is gratefully acknowledged. The authors apologize for being unable to cite all relevant papers.

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Ma, X.L., Wang, Y.J., Xie, S.L. et al. (2007). Glycinebetaine application ameliorates negative effects of drought stress in tobacco. Russian Journal of Plant Physiology 54: 472–479. Mäkelä, P., Peltonen-Sainio, P., Jokinen, K. et al. (1996). Uptake and translocation of foliarapplied glycinebetaine in crop plants. Plant Science 121: 221–230. Makela, P., Jokinen, K., Kontturi, M. et al. (1998). Foliar application of glycinebetaine-a novel product from sugar beet- as an approach increase tomato yield. Industrial Crops and Products 7: 139–148. Makela, P., Kontturi, M., Pehu, E., and Somersalo, S. (1999). Photosynthetic response of drought- and salt-stressed tomato and turnip rape plants to foliar-applied glycinebetaine. Physiologia Plantarum 105: 45–50. Malav, A.K. and Indu, C.K.S. (2016). Gene pyramiding: an overview. International Journal of Current Research in Biosciences and Plant Biology 3: 22–28. Malekzadeh, P. (2015). Influence of exogenous application of glycinebetaine on antioxidative system and growth of salt-stressed soybean seedlings (Glycine max L.). Physiology and Molecular Biology of Plants 21: 225–232. McCue, K.F. and Hanson, A.D. (1990). Drought and salt tolerance; towards understanding and application. Trends in Biotechnology 8: 358–362. McDonnell, E. and Wyn Jones, R.G. (1988). Glycinebetaine biosynthesis and accumulation in unstressed and salt stressed wheat. Journal of Experimental Botany 39: 421–430. McKersie, B.D., Bowley, S.R., Harjanto, E., and Leprince, O. (1996). Water deficit tolerance and field performance of transgenic alfalfa overexpressing superoxide dismutase. Plant Physiology 111: 1177–1181. McNeil, S.D., Nuccio, M.L., and Hanson, A.D. (1999). Betaines and related osmo-protectants targets for metabolic engineering of stress resistance. Plant Physiology 120: 945–949. McNeil, S.D., Nuccio, M.L., Ziemak, M.J., and Hanson, A.D. (2001). Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N-methyltransferase. Proceedings of the National Academy of Sciences of the United States of America 98: 10001–10005. Miri, H.R. and Armin, M. (2013). The interaction effect of drought and exogenous application of glycine betaine on corn (Zea mays L.). European Journal of Experimental Biology 3: 197–206. Murata, N., Mohanty, P.S., Hayashi, H., and Papageorgiou, G.C. (1992). Glycine betaine stabilizes the association of extrinsic proteins with the photosynthetic oxygen-evolving complex. FEBS Letters 296: 187–189. Nayyar, H., Bains, T., and Kumar, S. (2005a). Low temperature induced floral abortion in chickpea: relationship with abscisic acid and cryoprotectants in reproductive organs. Environmental and Experimental Botany 53: 39–47. Nayyar, H., Chander, K., Kumar, S., and Bhains, T. (2005b). Glycinebetaine mitigates cold stress damage in chickpea. Agronomy for Sustainable Development 25: 381–388. Niu, X., Zheng, W., Lu, B.R. et al. (2007). An unusual post-transcriptional processing in two betaine aldehyde dehydrogenase (BADH) loci of cereal crops directed by short-direct repeats in response to stress conditions. Plant Physiology 143: 1929–1942. Niu, X., Xiong, F., Liu, J. et al. (2014). Co-expression of ApGSMT and ApDMT promotes biosynthesis of glycine betaine in rice (Oryza sativa L.) and enhances salt and cold tolerance. Environmental and Experimental Botany 104: 16–25.

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Rezaei, M.A., Kaviani, B., and Masouleh, A.K. (2012). The effect of exogenous glycine betaine on yield of soybean [Glycine max (L.) Merr.] in two contrasting cultivars Pershing and DPX under soil salinity stress. Plant Omics Journal 5: 87–93. Rhodes, D. and Hanson, A.D. (1993). Quaternary ammonium and tertiary sulphonium compounds in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 44: 357–384. Roychoudhury, A. and Banerjee, A. (2016). Endogenous glycine betaine accumulation mediates abiotic stress tolerance in plants. Tropical Plant Research 3: 105–111. Roychoudhury, A., Banerjee, A., and Lahiri, V. (2015). Metabolic and molecular-genetic regulation of proline signaling and its cross-talk with major effectors mediates abiotic stress tolerance in plants. Turkish Journal of Botany 39: 887–910. Sakamoto, A. and Alia, M.N. (1998). Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold. Plant Molecular Biology 38: 1011–1019. Sakamoto, A. and Murata, N. (2000). Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance. Journal of Experimental Botany 51: 81–88. Sakamoto, A. and Murata, N. (2002). The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant, Cell & Environment 25: 163–172. Salama, K.H.A., Mansour, M.M.F., and Al-Malawi, H.A. (2015). Glycinebetaine priming improves salt tolerance of wheat. Biologia 70: 1334–1339. Shahbaz, M., Masood, Y., Perveen, S., and Ashraf, M. (2011). Is foliar-applied glycinebetaine effective in mitigating the adverse effects of drought stress on wheat (Triticum aestivum L.). Journal of Applied Botany and Food Quality 84: 192–199. Shams, M., Yildirim, E., Ekinci, M. et al. (2016). Exogenously applied glycine betaine regulates some chemical characteristics and antioxidative defence systems in lettuce under salt stress. Horticulture, Environment and Biotechnology 57: 225–231. Shirasawa, K., Takabe, T., and Kishitani, S. (2006). Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation of their tolerance to abiotic stress. Annals of Botany 98: 565–571. Song, J., Zhang, R., Yue, D. et al. (2018). Co-expression of ApGSMT2g and ApDMT2g in cotton enhances salt tolerance and increases seed cotton yield in saline fields. Plant Science 274: 369–382. Sorwong, A. and Sakhonwasee, S. (2015). Foliar application of glycine betaine mitigates the effect of heat stress in three marigold (Tagetes erecta) cultivars. The Japanese Society for Horticultural Science 38: 1–11. Su, J., Hirji, R., Zhang, L. et al. (2006). Evaluation of the stress-inducible production of choline oxidase in transgenic rice as a strategy for producing the stress-protectant glycine betaine. Journal of Experimental Botany 57: 1129–1135. Subbarao, G.V., Wheeler, R.M., Levine, L.H., and Stutte, G.W. (2001). Glycine betaine accumulation, ionic and water relations of red-beet at contrasting levels of sodium supply. Journal of Plant Physiology 158: 767–776. Sulpice, R., Tsukaya, H., Nonaka, H. et al. (2003). Enhanced formation of flowers in saltstressed Arabidopsis after genetic engineering of the synthesis of glycinebetaine. The Plant Journal 36: 165–176.

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Surabhi, G.K. (2018). Update in root proteomics with special reference to abiotic stresses: achievements and challenges. Journal of Proteins and Proteomics 9 (1): 31–55. Surabhi, G.K., Madhusudhan, K.V., Sreenivasulu, N., and Sudhakar, C. (2000). Stress responses in two genotypes of mulberry (Morus alba L.) under NaCl salinity. Indian Journal of Experimental Biology 38: 192–195. Surabhi, G.K., Reddy, A.M., and Sudhakar, C. (2003). NaCl effects on proline metabolism in two high yielding genotypes of mulberry (Morus alba L.) with contrasting salt tolerance. Plant Science 165: 1245–1251. Tian, F., Wang, W., Liang, C. et al. (2017). Overaccumulation of glycine betaine makes the function of the thylakoid membrane better in wheat under salt stress. The Crop Journal 5: 73–82. Umar, J., Aliyu, A., Shehu, K., and Abubakar, L. (2018). Influence of salt stress on proline and glycinebetaine accumulation in tomato (Solanum lycopersicum. L). Journal of Horticulture and Plant Research 1: 19–25. Vollenweider, P. and Gunthardt-Goerg, M.S. (2005). Diagnosis of abiotic and biotic stress factors using the visible symptoms in foliage. Environmental Pollution 137: 455–465. Wang, Y.J., Hao, Y.J., Zhang, Z.G. et al. (2005). Isolation of trehalose-6- phosphate phosphatase gene from tobacco and its functional analysis in yeast cells. Journal of Plant Physiology 162: 215–223. Wang, Q.B., Xu, W., Xue, Q.Z., and Su, W. (2010). Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tolerance to extreme temperature and high salinity. Journal of Zhejiang University Science 11: 851–861. Wei, A.Y., He, C.M., Li, B. et al. (2011). The pyramid of transgenes TsVP and bet a effectively enhances the drought tolerance of maize plants. Plant Biotechnology Journal 9: 216–229. Wei, D., Zhang, W., Wang, C. et al. (2017). Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants. Plant Science 257: 74–83. Xu, Z., Sun, M., Jiang, X. et al. (2018). Glycinebetaine biosynthesis in response to osmotic stress depends on jasmonate signaling in watermelon suspension cells. Frontiers in Plant Science 6: 1–14. Yang, X. and Lu, C. (2005). Photosynthesis is improved by exogenous glycinebetaine in salt-stressed maize plants. Physiologia Plantarum 124: 343–352. Yang, X., Liang, Z., and Lu, C. (2005). Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants. Plant Physiology 138: 2299–2309. Yang, X., Liang, Z., Wen, X., and Lu, C. (2008). Genetic engineering of the biosynthesis of glycinebetaine leads to increased tolerance of photosynthesis to salt stress in transgenic tobacco plants. Plant Molecular Biology 66: 73–86. Yokoi, S., Quintero, F.J., Cubero, B. et al. (2002). Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporters in the salt stress response. The Plant Journal 30: 529–539. Zhang, G.H., Su, Q., An, L.J., and Wu, S. (2008). Characterization and expression of a vacuolar Na+/H+ antiporter gene from the monocot halophyte Aeluropus littoralis. Plant Physiology and Biochemistry 46: 117–126.

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Zhang, H., Dong, H., Li, W. et al. (2009). Increased glycine betaine synthesis and salinity tolerance in AhCMO transgenic cotton lines. Molecular Breeding 23: 289–298. Zhang, T., Liang, J., Wang, M. et al. (2019). Genetic engineering of the biosynthesis of glycinebetaine enhances the fruit development and size of tomato. Plant Science 280: 355–366. Zhao, Y., Aspinall, D., and Paleg, L.G. (1992). Protection of membrane integrity in Medicago sativa L. by glycinebetaine against the effect of freezing. Journal of Plant Physiology 140: 541–543. Zhou, S., Chen, X., Zhang, X., and Li, Y. (2008). Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene BADH and a vacuolar Na+/H+ antiporter gene SeNHX1. Biotechnology Letters 30: 369–376.

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3 Osmoprotective Role of Sugar in Mitigating Abiotic Stress in Plants Farhan Ahmad 1, Ananya Singh 2, and Aisha Kamal 1 1 2

Department of Bioengineering, Integral University, Lucknow, India Department of Biosciences, Integral University, Lucknow, India

3.1 ­Introduction The expanding human population, industrialization and urbanization, destruction of agricultural land with harmful chemicals present in irrigation water, and climatic ups and downs have been considered trending challenges to plants (Gangola and Ramadoss 2018). Extreme ecological conditions such as drought (Basu et al. 2016), salinity (Roychoudhury et  al. 2008; Ismail and Horie 2017; Ahmad et  al. 2018a), extreme temperatures (Hasanuzzaman et al. 2013; Pereira 2016), nutrient unavailability (Khan et al. 2015), metal toxicity (Roychoudhury et al. 2012; Emamverdian et al. 2015), ultraviolet‐B radiation (UV‐ BR) (Banerjee and Roychoudhury 2016), flooding, atmospheric pollutants, and pesticide usage (Fatma et al. 2018) altogether constituting abiotic stress further worsen the problem. Such stresses together affect photosynthesis, water and osmotic balances, nutrient adequacy, plasma membrane instability, and oxidative stress, leading to oxidation of biomolecules and finally cell demise (Sofo et al. 2015; Ahmad et al. 2017). Plants deal with abiotic stress by well‐coordinated network sensing, a critical phase where plants experience a disturbance in ionic/osmotic balances through various mechanisms (Duque et  al. 2013; Roychoudhury and Chakraborty 2013). The second phase is signaling cascade where plants detect the presence of signaling molecules, such as reactive oxygen species (ROS) and Ca2+, and adjustment of osmoprotectants (proline, glycine betaine, soluble sugars, amines, etc.) that activate defense systems to attain normal physiological and metabolic process. To counter the influence of abiotic stress, plants have developed a well‐coordinated network that regulates physio‐biochemical response, modulates antioxidant systems, and alters the resistance gene(s). In addition to this, the interaction of growth regulators and their cross talk to improve tolerance has been extensively studied in the context of abiotic disturbance (Khatoon et  al. 2017; Yadu et  al. 2017; Ahmad et  al. 2018b). Recently, sugars and their derivatives have turned up as potential components for enhancing tolerance to biotic stress (Sami et al. 2016). Thus, it is not astounding that plants have advanced mechanisms that empower them to face adverse atmospheres with the accumulation of specific osmolytes Protective Chemical Agents in the Amelioration of Plant Abiotic Stress: Biochemical and Molecular Perspectives, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

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and sugars. However, this cannot be simply stated that all sugar molecules in plants have a significant role in signaling molecules under stress condition. At least, glucose, sucrose, fructose, and trehalose‐6‐P directly or indirectly attain the role of strength molecules (Li  and Sheen 2016; Ceusters et  al. 2017). This chapter emphasizes on the multidimensional role of different sugars in several physiological processes and explores sugar‐­ regulated sensing, response, and mechanisms linked to improving tolerance on exposure to abiotic stress.

3.2 ­Involvement of Sugar in Plant Developmental Process Being autotrophic, plants utilize light energy, CO2, and water through photosynthesis to produce sugar. Sugars are active biochemical molecules providing adequate nourishment to plants and govern photosynthesis, respiration, seed germination, flowering, and senescence. Besides this, glucose and sucrose are prime sources of energy reservoir for various metabolic pathways, biosynthesis, and production of important metabolites and have strong control of source–sink regulation. Soluble sugars shed an essential part in sustaining the general organization and development of plants under normal condition (Tardieu 2013) that regulates in well‐organized manner due to the target specificity (Lemoine et al. 2013). The active involvement of sugars in numerous roles in growth metabolism of plants under normal and stress conditions has been well known and termed as a next‐generation metabolite that regulates important growth parameters. Mason et  al. (2014) found that sugar signals stimulate lateral bud outgrowth after apex decapitation. Under high concentration of sugars, adventitious root formation significantly increases in Arabidopsis, while glucose enhances cell division and initiates progression of embryogenesis and sucrose initiates cell growth and storage in beans cotyledons (Gibson 2004). Both glucose and sucrose are directly or indirectly involved in the regulation and enhancement of reserved carbohydrates in developing embryos (Yaseen et al. 2013). Contrary to this, a highly active role of glucose was found as compared to sucrose in cell division of the nondifferentiated cell (Eveland and Jackson 2011). Another molecule, trehalose, a nonreducing disaccharide, is also actively involved in the formation and functioning of the organs (inflorescence, leaf, and tuber) and seed development (Paul and Dijck 2011). Exogenous application of trehalose enhances the activity of AGPase or ATPase by increasing the manifestation of ADP‐ glucose pyrophosphorylase, a critical regulator in the biosynthesis of starch (Wingler 2002). Universally, carbohydrates are derived from photosynthesis that either directly or indirectly governs growth and development. A low sugar concentration enhances photosynthesis and transportation of reserved carbohydrates, though significant enhancement of sugar level induces cell enlargement and simultaneously increases storage of other sugars (Smeekens and Rook 1997; Koch 2004). In source leaves of Phaseolus vulgaris, carbohydrate accumulation significantly decreases the photosynthesis by inhibiting the activity of Rubisco enzymes (Araya et al. 2006). The higher content of sugar‐derived reactive carbonyls (RCs), including methylglyoxal (MG) (produced in chloroplasts by Calvin cycle as a by‐product), enhances higher concentration of CO2 by accelerating the metabolic turnover of Calvin cycle (Takagi et al. 2014). Thus, accumulation of soluble sugars in source

3.3  ­Multidimensional Role of Sugar Under Optimal and Stressed Condition

tissues down‐regulates the photosynthesis maintaining homeostasis (Banerjee and Roychoudhury 2018). The juvenile‐to‐adult transition is controlled by mature transcripts (miR156) that in turn are down‐regulated by the accumulation of sugars. For example, sucrose accumulation in young primordia from mature leaves inhibits the transcriptional process of MIR156A, and MIR156C that results in decreasing of miR156 levels, thus promoting the expression of adult traits (Proveniers 2013). Research work carried out at Max Planck Institutes in Potsdam and Tubingen by Vanessa Wahl and co‐workers unconcealed that in shoot meristems, trehalose‐6‐phosphate (T6P) acts as an area signal that associates with sugar accessibility to biological process (Wahl et al. 2013). In Arabidopsis, early flowering was induced by enhanced activity of T6P (Gibson 2004). But, sucrose that shows dual role in floral transition, either inhibitory or stimulatory effect, depends upon the time and concentration of sugars (Ohto et  al. 2001). In many angiospermic families, such as Gramineae, Compositae, Liliaceae, Amaryllidaceae, fructans and their derivatives act as reserved carbohydrates and initiate flowering (Rolland et  al. 2006; da Silva et  al. 2013; Martínez‐Vilalta et al. 2016). Soluble sugars also mediate normal senescence process (Sheen et al. 1999; Doorn 2008). In a microarray analysis with the Arabidopsis Affymetrix, ATH1 array revealed that high sugar/low nitrogen combination and excluding under nourishment along with dark condition trigger changes in gene expression that are the distinct features of developmental leaf senescence (Wingler et al. 2009). It has been seen that in the course of senescence, higher volume of sugar aggregation is found in tobacco and Arabidopsis leaves (Wingler et  al. 2012). In addition to this, higher CO2 concentration initiates the senescence process by  reducing nitrogen availability, and Rubisco activity results in sugar accumulation (Rodziewicz et al. 2014). Another factor, apoplastic sucrose/hexose ratio is also responsible for delaying leaf senescence in tobacco and tomato plants by altering cell wall invertase activity (Lara et al. 2004; Gregersen et al. 2013). In contradiction with above result, accumulation of T6P resulted in delayed senescence, as indicated by maintaining leaf structure and color, declined expression of senescence‐associated genes, and lack of anthocyanin accumulation (Wingler et al. 2012). Thus, all developmental transition is well‐coordinated with sugar signaling pathways including germination, dormancy, apical meristem growth, floral induction, and senescence process.

3.3 ­Multidimensional Role of Sugar Under Optimal and Stressed Conditions 3.3.1  Sugar as Sensing and Signaling Molecules Sugars are also recognized as chemically signaling molecules for a specific receptor and as sensing molecules that can sense specific stimulus for normal functioning of the plant under stress and normal conditions (Figure 3.1). The protective role of sugar involves acting upon membrane and or macromolecules provoking signaling cascade system that regulates gene expression in response to both internal/external stimuli under stress conditions, such as drought, salinity, and oxidative stress (Martínez and Tognetti 2018).

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3  Osmoprotective Role of Sugar in Mitigating Abiotic Stress in Plants Invertase

Sucrose

Glucose

Fructose

Signals Sensor

Sensor

Sensor carrier

carrier

Sucrose

Glucose

Cell wall

carrier

Fructose

as er t Inv e

Hexokinase-1

Energy source Enzymatic and Metabolic activity

Gene expression

Plant growth and Development

MAPK kinase

Abiotic stress

NADPH oxidase

SnRK1 Kinase

G protein

Cytosol

Stress tolerance

Figure 3.1  Schematic diagram of sugar signals, receptors, and signal transmitting system operative in a typical plant cell.

Hexose sensing: Hexokinase (HXK), a multigene family with a different locus in a plant, comprises sugar phosphorylation, but HXK‐independent pathways can directly use sugar as a signal molecule. There are many different classes of HXK depending upon their location in the cell, but the most active and highly regulated HXK is localized in the nuclear region that generates glucose signal stimuli upon high/low accumulation of sugar and directs the photosynthetic machinery accordingly under stress condition. However, specific intracellular sugar sensing and the underlying mechanism by hexokinase sensing still require extensive study (Keunen et al. 2013). Other specific sugar signaling pathways regulated through fructose with recognized sensors are fructose 1,6‐bisphosphatase and fructokinase (FRK), but signaling pathway is independent of its enzymatic activity (Ruan 2014; Stein and Granot 2018). The components of HXK and FRK sensors hold isozymes together with enzymatic activity complicating their association among sugar signaling stability (Tiessen and Padilla‐Chacón 2013).

3.3.2  Sucrose and Trehalose Sensing The most abundant transportable disaccharide is sucrose, which also act as signaling molecule and regulate starch synthesis through the sucrose‐specific pathway. Sucrose also activates transcriptional factor MYB75 and facilitates anthocyanin and fructan biosynthesis through steadying of DELLA proteins (Li et al. 2016). It was also predicted that sucrose might be involved in plant responses to elevated CO2 levels as a signal which is transported

3.3  ­Multidimensional Role of Sugar Under Optimal and Stressed Condition

from CO2‐fed source leaves to the S‐adenosyl methionine (SAM) where stomata enlargement is repressed was entirely attuned with sucrose (Coupé et  al. 2006). Furthermore, down‐regulation of the gene associated with the photosynthetic process is linked with the accumulation of sugar under chilling stress (Rolland et  al. 2002). Another disaccharide molecule trehalose has also been found in negative feedback regulation of sucrose and exhibited the same response of insulin and glucagon in animals (Yadav et al. 2014; John et al. 2017). SnRK1 (protein kinase sucrose nonfermenting related kinase‐1) catalytic activity is involved in T6P signaling as well as growth and metabolism of the plant in response to starvation (O’Hara et al. 2013).

3.3.3  Sugar Alcohol (Polyol) Sensing Generally, sugar alcohols are distributed among acyclic mannitol and cyclic pinitol, commonly termed as polyols. The increased polyol content has a dual mode of action by assisting osmotic regulation and maintaining redox hemostasis (Slama et al. 2015). Mannitol is widely distributed sugar alcohol derived from a modification of mannose and its synthesis depends upon the synthetic pathway of sucrose (Rumpho et al. 1983), or with raffinose family of oligosaccharides (RFO) synthesis (Haritatos et  al. 1996). Sugar repression of mannitol utilization enhances the accumulation of mannitol storage as reserved carbohydrate. Under salinity stress, mannitol dehydrogenase (MTD, a catabolic enzyme) activity decreases due to stress and sugar repression (Williamsons et  al. 2002). Apart from mannitol, the second most abundant polyol sugar in angiospermic plant species is cyclitol myo‐inositol (Bachmann et al. 1994), which is synthesized from glucose‐6‐ phosphate (G‐6‐P) (Loewus and Loewus 1983). This myo‐inositol plays a critical role in cell wall development, membrane integrity, seed germination, hormonal action, and stress tolerance additionally (Loewus and Loewus 1983). Besides this, myo‐inositol also works as a carrier molecule for galactose that is changed to sucrose or raffinose or derivatives of other sugar of higher range (Nadwodnik and Lohaus 2008).

3.3.4  Sugar and Redox Homeostasis The equilibrium between ROS formation and their decontamination by antioxidant defense coordination system has been considered critical redox hemostasis under optimal plant metabolism (Kwak et al. 2006). But, abiotic stresses cause disturbances in the production and scavenging of ROS and in the long run lead to oxidative stress (Khan et al. 2015). The antioxidant system in plants includes a nonenzymatic system and enzymatic model comprises of mainly catalase, superoxide dismutase, and peroxidases (Ahmad et  al. 2018b). Nowadays, plant researches consider sugar molecules as a new tool to protect the plants from the adverse conditions due to possession of enormous potential in the antioxidant property. Monosaccharide sugars are more susceptible and easily affected by hydroxyl radicals than disaccharides, hence their role in ROS scavenging is somewhat unusual (Morelli et al. 2003; Das and Roychoudhury 2014). However, monosaccharides are assured to influence the antioxidative potential of a plant cell in a backhanded methodology by the formation of bioactive polymers or as optional mediators to facilitate the communication or action of different antioxidants (Gangola and Ramadoss 2018). An in vitro study shows that

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substantial free‐radical quenching effect is also exhibited by disaccharide sugars (Wehmeier and Mooradian 1994; Morelli et al. 2003). Sucrose seems in accordance with a dimension that is moderate in the antioxidative limit; however, because of its little magnitude in addition to the simplicity of transport, it may assume a progressive role in ROS regulator. Peshev and Van den Ende (2013) used fructan as plant insulin that has the ability to detoxify ROS more efficiently than disaccharides. The primary role of plant insulin is to scavenge hydroxyl radical and inhibit membrane lipid peroxidation by tonoplast stabilization (Van den Ende and Valluru 2009). Subsequently, fructans radically change into their radicals to the amount that can also be used with the aid of fanatical vacuolar antioxidants (Bolouri‐ Moghaddam et al. 2010; Peshev and Van den Ende 2013). RFOs act as hydroxyl radical scavengers in Arabidopsis by oxidized free radical by interacting with ascorbic acid, flavonoids, and nonenzymatic antioxidants (Nishizawa et  al. 2008; Bolouri‐Moghaddam et al. 2010). Sucrosyl oligosaccharides including RFOs and fructans synergistically mediate ROS signaling pathways by their metabolic enzymes (Bolouri‐Moghaddam et  al. 2010). Furthermore, extracellular invertase is additionally up‐controlled to supply sugars to sink organs amid abiotic stress conditions (Roitsch et al. 2003). It has been also found that in alfalfa roots oligogalacturonides stabilize the antioxidant organization and improve metabolic process under stress condition (Ramírez‐Camejo et al. 2012), feasibly crossing point sugar conductance including plant tolerance toward oxidative stress.

3.3.5  Sugars as Osmoprotectants Excessive dehydration of plant cell due to extreme drought and salinity prompts disturbed hydrophilic communication, the disintegration of biomolecules structure, and organelles structure and membranes (Anjum et  al. 2014; Singh et  al. 2015). To protect plants and enhance tolerance by minimizing dehydration throughout abiotic stress, production and accumulations of diversified osmolytes provide strength to organization structure, promote metabolic pathway, and maintain turgor pressure (Roychoudhury et al. 2009). In this context, both monosaccharides, disaccharides, and other sugars proved their potential in the osmoprotective role (Slama et al. 2015). The underlying mechanism to sugars during dehydration is the replacement of water molecule with hydroxyl groups of the sugars to withstand the hydrophilic interactions at the cellular level as stable natural macromolecules and membrane assembly (Koster 1991; Pukacka et al. 2009). The dehydration tolerance initiated by sugars through synthesis of sheath‐like structure or viscous solution on cell is termed as vitrification. These adaptations ensure permanency by preventing tools mandatory for diffusion, protect cellular breakdown, and make strong hydrogen attachment within the cell (Tahir et al. 2011; Angelovici et al. 2010). The most capable osmoprotective trehalose sugars are generally regarded concentration independent in metabolic activity and efficient in a very lower amount (Nahar et al. 2016), and also be substituted by sucrose and their derivatives in plants. The combined action of RFO and late embryogenesis abundant (LEA) proteins inhibits Maillard’s reaction, decreasing biosynthesis of monosaccharides (Martinez Villaluenga et  al. 2008; Kannan 2014). Also, under salinity, exogenous application of glucose removes some extent of oxidative strain by regulating stomatal movement and transpirational process and sustaining

3.3  ­Multidimensional Role of Sugar Under Optimal and Stressed Condition

photosynthetic machinery in wheat plants (Hu et al. 2012). Increased level of proline and soluble sugar content and their prominent role in tolerance under various stresses have already been widely discussed, for example, salt stress in Pisum sativum (Ahmad et  al. 2018b); drought stress in Nicotiana tabacum (Borgo et  al. 2015); and metal toxicity in Brassica juncea upon Cadmium stress (Ahmad et al. 2015).

3.3.6  Sugars and Abiotic Stress Tolerance in Plants Sugars are essential components of abiotic stress tolerance in plants along with a multifaceted role in plant growth and development, viz. germination, photosynthesis, and flowering senescence. The accumulation of sugars in plants has been widely reported as a response to abiotic stresses, listed in Table 3.1, in several crops. The mechanism and mode of plant also depend upon the type of stress and its intensity. The role of sugars and their involvement in abiotic stress also vary according to plant’s growing condition as well as plant organization. In this section, role of sugar in mitigating or enhancing tolerance under major abiotic stress and involved mechanism or pathway will be briefly discussed. 3.3.6.1  Salinity Stress

Salt stress has excessive amounts of Na+ and Cl− ions in soil that show deadly effects on plants and cause metabolic alterations, such as damage of chloroplast content and photosynthetic activity, decreased photosynthetic and increased photorespiration rate, disturbed nitrogen assimilation, overproduction of reactive oxygen production, and disturbed osmatic equilibrium (Parida and Das 2005; Ismail and Horie 2017; Paul and Roychoudhury 2019). Sugars are not only responsible for energy production and solutes for osmotic regulation but also controlling expressions of numerous genes as governing messengers (Fu et al. 2010). The study conducted by Peng et al. (2016) demonstrated the increase in soil salinity accumulation of soluble sugar and decrease in starch contents, while the ratio of sucrose synthase and sucrose phosphate synthase increased in a main‐stem leaf of cotton. An enhanced biosynthesis of mannose‐containing polysaccharides on exposure to salinity was reported at the time of seed germination and seedling development (He et al. 2017). It was additionally discovered that starches including raffinose family oligosaccharides and sugar alcohols are available at abnormal states under optimal as well as distressing conditions, and go about as antioxidant agents to shield plant cells from oxidative harm and keep up redox homeostasis (Nishizawa‐Yokoi et  al. 2008). Trehalose aggregation also protects plants against a few physical and synthetic/chemical exposures and salinity stress too (Gupta and Kaur 2005). 3.3.6.2  Drought Stress

Drought is also one of the most critical factors that inhibit photosynthesis significantly (Ashraf and Harris 2013). Glucose accumulation induces stomatal closure, reduces the rate of photosynthesis, maintains the leaf water content and osmotic adjustment, prevents the oxidation of cell membranes, and enhances plant’s adaptability under drought stress (Liu et al. 2004; Xu et al. 2007; Arabzadeh 2012; Osakabe et al. 2013). Previous studies show significant accumulation of raffinose, stachyose, and verbascose sugars at the time of seed

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Table 3.1  Sugars and their associated genes to stress tolerance in various crops under stress. Sugars

Fructans

Transgene

Stress

Species

References

Sucrose:sucrose 1‐fructosyltransferase

Freezing

Nicotiana tabacum

Li et al. (2007)

Sucrose:sucrose 1‐fructosyltransferase and sucrose:fructan 6‐fructosyltransferase

Chilling

Oryza sativa

Kawakami et al. (2008)

SacB

Drought

Nicotiana tabacum

Pilon‐Smits et al. (1999)

Beta vulgaris

Pilon‐Smits et al. (1999)

Nicotiana tabacum

Parvanova et al. (2004)

Trehalose‐6‐phosphate Drought/salinity synthase

Lycopersicon esculentum

Cortina and Culiáñez‐Maciá (2005)

Trehalose‐6‐phosphate Drought/salinity/ temperature synthase and phosphatase

Arabidopsis thaliana

Miranda et al. (2007)

Trehalose phosphorylase

Drought

Nicotiana tabacum

Han et al. (2005)

Levansucrase

Trehalose

Freezing

otsA, otsB

Salinity

Oryza sativa

Garg et al. (2002)

Galactinol

Galactinol synthase

Drought/salinity/ oxidative

Arabidopsis thaliana

Nishizawa et al. (2008)

Raffinose

α‐Galactosidase

Freezing

Petunia spp.

Pennycooke et al. (2003)

UDP‐glucose 4‐ epimerase

Drought, freezing

Arabidopsis thaliana

Liu et al. (2007)

Mannitol‐1‐phosphate dehydrogenase

Oxidative stress

Nicotiana tabacum

Shen et al. (1997)

Drought/salinity

Oryza sativa

Pujni et al. (2007)

Salinity

Pinus taeda

Tang et al. (2005)

Arabidopsis thaliana

Zhifang and Loescher (2003)

Mannitol

D‐ononitol

IMT1 (myo‐inositol O‐ methyltransferase) of common ice plant

Drought/salinity

Nicotiana tabacum

Sheveleva et al. (1997)

Sorbitol

Glucitol‐6‐phosphate dehydrogenase

Salinity

P. taeda

Tang et al. (2005)

Diospyros kaki

Deguchi et al. (2004)

Aldehyde reductase

MsALR

Chemical

Nicotiana tabacum

Oberschall et al. (2000)

Invertase

Apoplastic invertase

Salinity

Fukushima et al. (2001)

3.3  ­Multidimensional Role of Sugar Under Optimal and Stressed Condition

desiccation (Peterbauer and Richter 1998; Tuberosa 2012; Kaushal and Wani 2016), and they also accumulate markedly in leaves when the plant is exposed to chilling, temperature, or saline stress (Janska et al. 2009). In addition to this, cyclic carbohydrates (cyclitols) and a linear polyhydric alcohol content have been increased, and thus inhibit photoinhibition and metabolic detoxification under stress condition (Keller and Ludlow 1993; Wanek and Richter 1997; Pharr et al. 1995). During anthesis, sugar alcohol, mannitol, soluble protein, and other osmolytes significantly increase under drought stress in drought‐sensitive variety of wheat, thus improving growth performance (Saeidi et al. 2017). A double adjustment methodology in potato under long‐term drought stress environments improves membrane stability combined with an increased osmolality due to an increased accumulation of starch (Rudack et al. 2017). Above‐discussed substances are mostly recommended to perform as well‐suited solutes or osmoprotectants to tolerate osmotic tuning of plant cells open to water scarcity. 3.3.6.3  Heat/Cold Stress

Temperature is one of the primary environmental factors that limit plant distribution and crop productivity. Cold stress causes significant disruption of membranes, ROS accumulation, protein denaturation, etc. (Yuanyuan et al. 2009; Mahajan and Tuteja 2005). Moreover, it was accounted for that high intracellular dimensions of galactinol and raffinose in Arabidopsis plants overexpressing the heat shock transcription factor or galactinol synthase, which expanded resistance to methyl viologen treatment and saltiness or chilling, and also galactinol and raffinose likewise, are found to viably shield salicylate from damage caused by hydroxyl radicals in vitro. In higher plants, various assortments of solvent sugars, for example, glucose, sucrose, fructose, raffinose, and stachyose, are adequately known to give freezing tolerance (Yuanyuan et al. 2009). Fructans have high water dissolvability that gives protection from crystallization under chilling (Livingston et al. 2009). Furthermore, fructans likewise balance out layers and by implication are related with an osmotic change under states of freezing and lack of hydration (Krasensky and Jonak 2012). In rice, the improved content of trehalose is seen under chilling pressure (Garg et al. 2002). High‐temperature stress likewise alters the plant metabolic activity and higher accumulation of sugar, yet its role in regard to osmotic change is, in any case, still under discussion. 3.3.6.4  Mineral Nutrient Deficiency

The primary roles of sugar are providing energy, regulating metabolic actions, and acting as molecular signals regulating different genes. Furthermore, nutrient deficiency is not an abiotic stress, but an example of a stress where its excess and deficiency affect overall metabolism of the plant. For instance, nitrogen deficiency disables plant development without affecting photosynthesis, and may occur at a moderately high rate. In previous studies, Sugar accumulation was additionally detailed for grain leaves (Wang and Tillberg 1996), barley seedlings (Comadira et al. 2015), and radish hypocotyls (Su et al. 2016) under N insufficiency. Additionally, lacking of phosphorus, potassium, and magnesium was found to result in sucrose gathering in leaves, in parallel with a decline of this sugar in phloem, which is demonstrative of development hindrance (Marschner et al. 1996).

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3.3.7  Limitations and Future Prospects Sugars play an important role in the life of a plant as the main source of energy and food reserve materials, and also as transitional metabolites of numerous biochemical procedures. In current ecological condition, sugars are treated as a potent mitigating tool to overcome abiotic stress by detoxifying toxic compound, maintaining osmotic adjustment and membrane stabilization. A brief role of sugar in the main metabolic processes such as germination, photosynthesis, flowering, and senescence under normal and stress conditions has been discussed. The detailed studies on the involvement of sugar as osmoprotectant, as signaling molecules, as a detoxifying agent, and main signaling pathway have also been explored. Yet, the molecular and physiological investigation is looked for to comprehend the process of sugar signaling in facilitating the developments preliminary from embryogenesis and ending up ultimately to senescence. The significant role of sugar‐associated gene related to physiological procedures and up‐control or down direction of stress quality additionally requires an effective and scientific approach. The development of abiotic stress‐tolerant species by advance plant breeding techniques by targeting sugar molecules should also be taken into active consideration. Recent advances in molecular biology, especially next‐generation sequences, have alleviated the problem of identifying crucial compounds or genes participating in abiotic stress tolerance, yet there are very few examples of developing a consistent or stable crop variety against ­abiotic stresses.

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Roychoudhury, A., Basu, S., and Sengupta, D.N. (2009). Effects of exogenous abscisic acid on some physiological responses in a popular aromatic indica rice compared with those from two traditional non‐aromatic indica rice cultivars. Acta Physiologiae Plantarum 31: 915–926. Roychoudhury, A., Pradhan, S., Chaudhuri, B., and Das, K. (2012). Phytoremediation of toxic metals and the involvement of Brassica species. In: Phytotechnologies: Remediation of Environmental Contaminants (eds. N.A. Anjum, M.E. Pereira, I. Ahmad, et al.), 219–251. Boca Raton: CRC press/Taylor and Francis Group. Ruan, Y.L. (2014). Sucrose metabolism: gateway to diverse carbon use and sugar signaling. Annual Review of Plant Biology 65: 33–67. Rudack, K., Seddig, S., Sprenger, H. et al. (2017). Drought stress‐induced changes in starch yield and physiological traits in potato. Journal of Agronomy and Crop Science 203 (6): 494–505. Rumpho, M.E., Edwards, G.E., and Loescher, W.H. (1983). A pathway for photosynthetic carbon flow to mannitol in celery leaves. Activity and localization of key enzymes (Apiumgraveolens). Plant Physiology 73: 869–873. Saeidi, M., Moradi, F., and Abdoli, M. (2017). Impact of drought stress on yield, photosynthesis rate, and sugar alcohols contents in wheat after anthesis in semiarid region of Iran. Arid Land Research and Management 31 (2): 204–218. Sami, F., Yusuf, M., Faizan, M. et al. (2016). Role of sugars under abiotic stress. Plant Physiology and Biochemistry 109: 54–61. Sheen, J., Zhou, L., and Jang, J.C. (1999). Sugars as signaling molecules. Current Opinion in Plant Biology 2 (5): 410–418. Shen, B.O., Jensen, R.G., and Bohnert, H.J. (1997). Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts. Plant Physiology 113: 1177–1183. Sheveleva, E., Chmara, W., Bohnert, H.J., and Jensen, R.G. (1997). Increased salt and drought tolerance by D‐ononitol production in transgenic Nicotiana tabacum L. Plant Physiology 115: 1211–1219. da Silva, F.G., Cangussu, L.M.B., de Paula, S.L.A. et al. (2013). Seasonal changes in fructan accumulation in the underground organs of Gomphrena marginata Seub (Amaranthaceae) under rockfield conditions. Theoretical and Experimental Plant Physiology 25: 46–55. Singh, M., Kumar, J., Singh, S. et al. (2015). Roles of osmoprotectants in improving salinity and drought tolerance in plants: a review. Reviews in Environmental Science and Biotechnology 14: 407–426. Slama, I., Abdelly, C., Bouchereau, A. et al. (2015). Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress. Annals of Botany 115 (3): 433–447. Smeekens, S. and Rook, F. (1997). Sugar sensing and sugar‐mediated signal transduction in plants. Plant Physiology 115 (1): 7. Sofo, A., Scopa, A., Nuzzaci, M., and Vitti, A. (2015). Ascorbate peroxidase and catalase activities and their genetic regulation in plants subjected to drought and salinity stresses. International Journal of Molecular Sciences 16 (6): 13561–13578. Stein, O. and Granot, D. (2018). Plant fructokinases: evolutionary, developmental, and metabolic aspects in sink tissues. Frontiers in Plant Science 9: 339.

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Su, N., Wu, Q., and Cui, J. (2016). Increased sucrose in the hypocotyls of radish sprouts contributes to nitrogen deficiency‐induced anthocyanin accumulation. Frontiers in Plant Science 7. Tahir, M., Vandenberg, A., and Chibbar, R.N. (2011). Influence of environment on seed soluble carbohydrates in selected lentil cultivars. Journal of Food Composition and Analysis 24 (4–5): 596–602. Takagi, D., Inoue, H., Odawara, M. et al. (2014). The Calvin cycle inevitably produces sugar‐ derived reactive carbonyl methylglyoxal during photosynthesis: a potential cause of plant diabetes. Plant and Cell Physiology 55 (2): 333–340. Tang, W., Peng, X., and Newton, R.J. (2005). Enhanced tolerance to salt stress in transgenic loblolly pine simultaneously expressing two genes encoding mannitol‐1‐phosphate dehydrogenase and glucitol‐6‐phosphate dehydrogenase. Plant Physiology and Biochemistry 43: 139–146. Tardieu, F. (2013). Plant response to environmental conditions: assessing potential production, water demand, and negative effects of water deficit. Frontiers in Physiology 4: 17. Tiessen, A. and Padilla‐Chacon, D. (2013). Subcellular compartmentation of sugar signaling: links among carbon cellular status, route of sucrolysis, sink‐source allocation, and metabolic partitioning. Frontiers in Plant Science 3: 306. Tuberosa, R. (2012). Phenotyping for drought tolerance of crops in the genomics era. Frontiers in Physiology 3: 347. Van den Ende, W. and Valluru, R. (2009). Sucrose, sucrosyl oligosaccharides, and oxidative stress: scavenging and salvaging? Journal of Experimental Botany 60: 9–18. van Doorn, W.G. (2008). Is the onset of senescence in leaf cells of intact plants due to low or high sugar levels? Journal of Experimental Botany 59 (8): 1963–1972. Wahl, V., Ponnu, J., Schlereth, A. et al. (2013). Regulation of flowering by trehalose‐6‐ phosphate signaling in Arabidopsis thaliana. Science 339 (6120): 704–707. Wanek, W. and Richter, A. (1997). Biosynthesis and accumulation of d‐ononitol in Vigna umbellata in response to drought stress. Physiologia Plantarum 101: 416–424. Wang, C. and Tillberg, J.E. (1996). Effects of nitrogen deficiency on accumulation of fructan and fructan metabolizing enzyme activities in sink and source leaves of barley (Hordeum vulgare). Physiologia Plantarum 97: 339–345. Wehmeier, K.R. and Mooradian, A.D. (1994). Autooxidative and antioxidative potential of simple carbohydrates. Free Radical Biology and Medicine 17: 83–86. Williamson, J.D., Jennings, D.B., Guo, W.W. et al. (2002). Sugar alcohols, salt stress, and fungal resistance: polyols—multifunctional plant protection? Journal of the American Society for Horticultural Science 127 (4): 467–473. Wingler, A. (2002). The function of trehalose biosynthesis in plants. Phytochemistry 60 (5): 437–440. Wingler, A., Masclaux‐Daubresse, C., and Fischer, A.M. (2009). Sugars, senescence, and ageing in plants and heterotrophic organisms. Journal of Experimental Botany 60 (4): 1063–1066. Wingler, A., Delatte, T.L., O’Hara, L.E. et al. (2012). Trehalose 6‐phosphate is required for the onset of leaf senescence associated with high carbon availability. Plant Physiology 158 (3): 1241–1251. Xu, S.M., Liu, L.X., Woo, K.C., and Wang, D.L. (2007). Changes in photosynthesis, xanthophyll cycle and sugar accumulation in two North Australia tropical species differing in leaf angles. Photosynthetica 45: 348–354.

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Yadav, U.P., Ivakov, A., Feil, R. et al. (2014). The sucrose‐ Tre6P nexus: specificity and mechanisms of sucrose signalling by trehalose‐6‐phosphate. Journal of Experimental Botany 65: 1051–1068. Yadu, S., Dewangan, T.L., Chandrakar, V., and Keshavkant, S. (2017). Imperative roles of salicylic acid and nitric oxide in improving salinity tolerance in Pisum sativum L. Physiology and Molecular Biology of Plants 23 (1): 43–58. Yaseen, M., Ahmad, T., Sablok, G. et al. (2013). Role of carbon sources for in vitro plant growth and development. Molecular Biology Reports 40 (4): 2837–2849. Yuanyuan, M., Yali, Z., Jiang, L., and Hongbo, S. (2009). Roles of plant soluble sugars and their responses to plant cold stress. African Journal of Biotechnology 8: 2004–2010. Zhifang, G. and Loescher, W.H. (2003). Expression of a celery mannose 6‐phosphate reductase in Arabidopsis thaliana enhances salt tolerance and induces biosynthesis of both mannitol and a glucosyl‐mannitol dimer. Plant, Cell and Environment 26: 275–283.

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4 Sugars and Sugar Polyols in Overcoming Environmental Stresses Saswati Bhattacharya 1 and Anirban Kundu 2 1 2

Department of Botany, Dr. A.P.J. Abdul Kalam Government College, New Town, Rajarhat, West Bengal, India P.G. Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, West Bengal, India

4.1 ­Introduction Plants require an array of abiotic factors such as sunlight, temperature, moisture, water, mineral salts, nutrients, as well as O2 and CO2 in order to attain optimal growth. Each of these abiotic factors has a precise effect on plant growth and development, which depends on its magnitude and concentration. Any deviation in the intensity of these optimal abiotic factors in their chemical or physical environment may lead to a condition known as abiotic stress (Bray et al. 2000; Paul and Roychoudhury 2019). Under natural environments, plants are very often exposed to these abiotic stresses that adversely affect plant growth, development, and reproduction (Rosa et  al. 2009; Roychoudhury et  al. 2009). Drought, salinity, heat, and freezing temperature are important examples of such environmental adversities that restrict the plants from displaying their full genetic potential and account for extensive crop loss (Cramer et al. 2011; Keunen et al. 2013; Roychoudhury et al. 2008). In plants, abiotic stress involves three primary phases comprising stress sensing, signaling, and exhaustion. Early phases of stress involve sensing or perception of the adverse abiotic stimuli through various sensors and induce a signaling cascade that relays the stress signal to the interior of the cell (Roychoudhury and Banerjee 2017). This eventually leads to an alteration in gene expression, thereby changing the host physiology and metabolism referred to as exhaustion (Rosa et al. 2009; Duque et al. 2013). Additionally, a fourth phase known as regeneration may come into existence that involves partial or total normalization of the physiological aspects of the plants upon removal of the stress factors. Continuous agitations of these stress factors may result in reduced photosynthesis, impaired water transport, osmotic imbalance, disturbed ion homeostasis, membrane instability, and oxidative stress through excessive generation of reactive oxygen species (ROS) that collectively hinder growth and development of plants (Rosa et al. 2009; Van den Ende and El‐Esawe 2014; Roychoudhury et al. 2016). To counter the impact of these hostile environmental conditions, plants have evolved complex molecular and physiological stratagems that aid in their survival under these Protective Chemical Agents in the Amelioration of Plant Abiotic Stress: Biochemical and Molecular Perspectives, First Edition. Edited by Aryadeep Roychoudhury and Durgesh Kumar Tripathi. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

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stressful conditions. These mechanisms become operative with the onset of stress situations and their tolerance through biochemical and physiological adaptations that improve plant performance under stressed conditions. In the recent past, understanding of the host biochemical responses to abiotic stimuli has improved and sugars and sugar alcohols have emerged out as key molecular players pertaining to abiotic stress tolerance (Gupta and Kaur 2005; Sami et al. 2016). Plants being autotrophs perform both as producer and consumer of carbohydrate molecules. They generate an array of sugars through photosynthesis that serve as building blocks, supply energy, and provide biomass to support plant life. Apart from their well‐ established role in various physiological processes, they serve as important regulators of abiotic stress responses in the cell. Accumulation of soluble sugar molecules such as sucrose, trehalose, raffinose, and fructans and sugar polyols such as mannitol, sorbitol, and inositols plays a critical role to confer tolerance against the various environmental stressors (Sami et al. 2016). Production of these compatible sugars and sugar polyols stabilizes cellular proteins and membranes, maintains cell turgor by either acting as an osmolyte or osmoprotectant, participates in stress signaling pathways, and scavenges excess ROS to maintain redox homeostasis (Krasensky and Jonak 2012; Roychoudhury et  al. 2015). Therefore, the present chapter summarizes a comprehensive overview of the types and metabolism of each of these sugars and sugar polyols acting as abiotic stress busters and the role they play in abiotic stress responses. Additionally, an update on the genetic engineering of sugar metabolic pathways to enhance tolerance to salt, drought, heat, and cold stress in different crop species has also been included.

4.2 ­Types of Sugars and Sugar Alcohols Abiotic stresses affect the cellular carbohydrate metabolism that results in an alteration in the levels of various sugars and their alcohol derivatives (polyols). Plants respond to these conditions by accumulation of sugars (trehalose, sucrose, raffinose family oligosaccharides [RFOs], and fructans) and some polyols (mannitol, sorbitol, and inositols) performing a myriad of functions. A schematic representation of the biosynthesis of these compatible solutes has been depicted in Figure 4.1. This section reviews the various types of compatible sugars and sugar alcohols and their role in conferring tolerance to stresses.

4.2.1  Trehalose The nonreducing disaccharide trehalose (d‐glucopyranosyl, D‐glucopyranoside), also known as tremalose or mycose, comprises of two glucose residues bonded together by α,α‐1,1′‐o‐glycosidic linkage. It is one of the most stable disaccharides occurring in nature for its low‐energy glycosidic bond (less than −4.2 kJ mol−1). Since its discovery in ergot of rye, it has been found to be ubiquitously distributed in biological systems including fungi, bacteria, nematodes, crustaceans, insects, as well as in higher plants. Trehalose performs a myriad of functions in these organisms, which makes it difficult to focus on a particular one that often varies from one species to the other. In bacteria, trehalose serves as the source of energy during developmental processes (Elbein et al. 2003) and accumulates

4.2  ­Types of Sugars and Sugar Alcohol Pinitol Raffinose

Stachyose

Verbascose

OEP

Mannitol

Sucrose

Ononitol Galactinol GolS

UDP-Galactose

IMT

M1PP

Myo-inositol

Mannitol-1-P

IMP

Fructose-6-P

Myo-inositol-1-P MIPS

UDP-Glucose

Glucose-6-P S6PDH HXK

Glucose TPS

Sorbitol-6-P

S6PP

Sorbitol SDH

Trehalose-6-P TPP

Trehalose

Sucrose

Fructose

1-SST 1-FFT

Fructans

Figure 4.1  Schematic representation of the biosynthesis of various types of sugars and sugar alcohols implicated in abiotic stress tolerance. The sugars are highlighted in light grey, while the sugar alcohols are highlighted in dark grey.

during osmotic stress responses, while in mycobacteria, it acts as a structural component that remains impregnated in cell wall glycolipids (Elbein 1974). Trehalose protects yeast cells from high temperature and desiccation and may act as a free radical scavenger (Benaroudj et al. 2001). Insects utilize trehalose as a source of energy during their flight (Elbein 1974). Until recently, its presence has been confirmed in angiosperms predominantly in some dehydration‐tolerant plants (Bianchi et al. 1993; Albini et al. 1994). Five major pathways of trehalose synthesis have been reported in living organisms that utilize various substrates. However, in plants, only a single pathway is operative commonly known as the TPS–TPP pathway. Here, the enzyme trehalose‐6‐phosphate synthase (TPS) combines ­glucose‐6‐phosphate and uridine diphosphate (UDP)‐glucose to generate trehalose‐6‐phosphate (T6P), which is further dephosphorylated to trehalose by the action of the enzyme trehalose‐6‐phosphate phosphatase (TPP).

4.2.2  Sucrose The disaccharide sucrose is one of the principal transport and storage carbohydrates synthesized primarily from photosynthetically fixed triose phosphates. Chemically, it is a nonreducing sugar where glucose and fructose units are combined together by an α(1,2) glycosidic bond. After its synthesis in the photosynthetic source tissues, it is transported to

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the non‐photosynthetic sink tissues and metabolized to yield simpler sugars in order to procure energy and sustain metabolic reactions. Due to its high solubility even at low temperature, it is more appropriate as a translocated sugar in the phloem sap and its concentration may reach up to 1 M (Koch 2004). The glycosidic bond of sucrose releases a high free energy on hydrolysis (−7 kcal mol−1), which is considered very high in case of carbohydrates. In plants, the principal pathway for sucrose biosynthesis involves UDP‐glucose and fructose‐6‐phosphate acted upon by the enzymes sucrose phosphate synthase and sucrose phosphate phosphatase. Alternative pathways in sucrose biosynthesis include degradation of starch or through interaction with nicotinamide adenine dinucleotide phosphate (NADP)‐glucose. Following synthesis, sucrose is loaded into the phloem apoplastically or symplastically and translocated and stored in the vacuoles of the sink organs. Sucrose mobilization in the sink tissues provides substrate and energy required for growth and development and involves two major pathways. The most common reaction involves the activity of the enzyme invertase that hydrolytically cleaves sucrose into its monomers, glucose and fructose (Roitsch et al. 2003). Sucrose synthase, a soluble or membrane‐associated enzyme complex, plays a vital role in sucrose catabolism and reversibly produces NDP‐­ glucose and fructose consuming a molecule of nucleoside diphosphate. In higher plants under stress condition, sucrose acts as an osmoregulatory molecule that prevents dehydration and maintains turgor pressure (Keunen et al. 2013).

4.2.3 Fructans Fructans are water‐soluble polymers of fructose and are one of the principal reserve carbohydrates in temperate grasses (French and Waterhouse 1993). Besides bacteria and fungi, about 15% of the higher plants have been reported to accumulate fructans (Hendry 1993). They have also been reported from both monocots and dicots, while their distribution varies in different tissues of the plants. Fructan accumulation takes place either in vacuolar or in prevacuolar vesicles where it is synthesized primarily from sucrose (Frehner et  al. 1984). Based on the glycosidic linkage between fructose units and the position of glucose, five major structural types can be recognized in plants (Lewis 1993): (i) Inulins: unbranched molecules having 2,1 linkages between their fructofuranosyl units and a terminal glucose; (ii) Levan (or phlein): unbranched molecules of 2,6‐linked fructosyl units having terminal glucose residue; (iii) Graminan: branched‐type fructans containing both 2,1 and 2,6 linkages with terminal glucose unit; (iv) Neo‐inulin: unbranched linear polymers showing 2,1 linkages having an internal glucose residue; (v)  Neo‐levan: fructans comprising 2,6 linkages having an internal glucose molecule. Fructans are synthesized by the cooperation of specific groups of enzymes known as fructosyltransferases that transfer fructose units from sucrose molecules catalyzing the formation of fructans with varied chain lengths. Two enzymes are operative during the synthesis, the first one, sucrose: sucrose 1‐fructosyltransferase (1‐SST) transfers the fructose moiety of one sucrose to another sucrose molecule, thereby resulting in the production of a trisaccharide, 1‐kestose (G1‐2F1‐2F). The fructan 1‐fructosyltransferase (1‐FFT) catalyzes another important step in the biosynthetic pathway, which promotes chain elongation by adding fructose units from one chain to another fructan or to sucrose molecule (Konstantinova et al. 2002).

4.2  ­Types of Sugars and Sugar Alcohol

4.2.4  Raffinose Family Oligosaccharides (RFOs) RFOs comprise a class of soluble carbohydrates that are derived from sucrose through transfer of galactosyl moieties from galactinol. These nonreducing sugars include raffinose (trisaccharide; Galα1, 6Glcα1, 2βFru), stachyose (tetrasaccharide; Galα1, 6Galα1, 6Glcα1, 2βFru), verbascose (pentasaccharide), ajugose (hexasaccharide), etc. Although ubiquitously distributed in the plant kingdom, RFOs with a greater degree of polymerization (DP) have also been reported from Ajuga reptans, a member of Lamiaceae under cold stress (Bachmann et al. 1994). Among all, raffinose and stachyose have been found to be present in all plant organs. However, distribution of verbascose and ajugose remains restricted to the storage tissues of certain plants only (Kandler and Hopf 1984; Janeček et  al. 2011). Some cucurbits, legumes, mint, grapes, and some cereals accumulate RFOs during tolerance to abiotic stresses (Sheveleva et al. 1997). RFOs also serve as photosynthate transporters and take part in various physiological processes such as cellular signaling, membrane transport, and transport of mRNA. They are further involved as storage molecules and osmolytes during seed desiccation. In plants, UDP‐glucose acts as the precursor molecule for the biosynthesis of RFOs catalyzed by various enzymes, such as galactinol synthase, raffinose synthase, stachyose synthase, etc. Galactinol synthase induces the producing of galactinol, the key regulatory molecule in RFO‐biosynthesis involving myo‐inositol and UDP‐galactose. Subsequently, raffinose synthase transfers galactose moiety of galactinol to sucrose catalyzing raffinose synthesis. Similarly, galactinol interacts with raffinose in presence of stachyose synthase to produce stachyose, and interaction of galactinol with stachyose results in verbascose catalyzed by verbascose synthase (Keller and Pharr 1996).

4.2.5  Sugar Alcohols Chemically, sugar alcohols are aldose or ketose sugars that are reduced to their respective hydroxyl (─OH) residues. They are commonly known as polyols, poly‐alcohols, or polyhydric alcohols, since sugar molecules are themselves polyhydroxy compounds and their respective alcohol derivatives have one extra ─OH group. Polyols are water soluble, broad class of compounds and can be further classified into cyclic (myo‐inositol, pinitol, ononitol, etc.) or acyclic (mannitol, sorbitol, and inositols) structures. These molecules have been detected in diverse groups of organisms when subjected to salinity, dehydration, and osmotic stress. They are principally involved in osmotic adjustments as compatible solutes, in mitigating oxidative damage or as molecular chaperones (Bohnert et al. 1995; Sengupta et  al. 2008). A concise list of the various osmolytes along with their functions has been tabulated in Table 4.1. 4.2.5.1  Mannitol

Mannitol is a widely distributed, water‐soluble hexitol structurally similar to the aldohexose sugar mannose (Stoop et al. 1996). Presence of mannitol is widespread and has been reported from more than 100 plants belonging to 70 angiosperm families (Lewis and Smith 1967; Ruijter et al. 2003), as well as observed in fungi, bacteria, lichens, algae, and apicomplexa. In some members of Apiaceae, Combretaceae, Oleaceae, and Rubiaceae, mannitol is synthesized as a phloem‐translocated photosynthate. Mannitol also serves

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Table 4.1  Role of different sugars and sugar polyols under various abiotic stress tolerances. Compatible solutes

Role in stress tolerance

References

Trehalose

Stabilizes cell membranes, protects cell from oxidative damage

Romero et al. (1997)

Sucrose

Osmolyte, maintains turgor pressure

Keunen et al. (2013)

Fructans

Stabilizes cell membranes, protects cell from oxidative damage

Li et al. (2007)

Raffinose family oligosaccharides (RFO)

Osmotic tolerance, alleviation of oxidative stress

Nishizawa et al. (2008)

Mannitol

Reduces ROS species, stabilizes biological macromolecules

Tarczynski et al. (1992)

Sorbitol

Osmotic adjustment, maintains redox homeostasis

Sheveleva et al. (1998)

Myo‐inositol

Osmotic adjustment, scavenges ROS

Smart and Flores (1997)

Ononitol

Osmotic adjustment, maintains redox homeostasis

Sheveleva et al. (1997)

Pinitol

Osmotic adjustment, maintains redox homeostasis

Sheveleva et al. (1997)

Sugars

Sugar alcohols

multiple functions including osmoregulation, scavenging of ROS during stress periods, storage of reducing power (Stoop and Pharr 1992), as a carbon stockpiling compound (Lewis 1984), as a compatible osmolyte in photochemical impulses, and functions as a major translocated carbohydrate when sucrose is exhausted (Davis and Loescher 1990). Higher plants synthesize mannitol from mannose‐6‐phosphate, acted upon by nicotinamide adenine dinucleotide phosphate (NADPH)‐mannose‐6‐phosphate reductase, resulting in the formation of mannitol‐1‐phosphate. Subsequently, mannitol‐1‐phosphate phosphatase enzyme catalyzes dephosphorylation of mannitol‐1‐phosphate to yield mannitol (Rumpho et al. 1983; Loescher et al. 1992). The catabolic enzyme, mannitol dehydrogenase (MTD), is involved in the conversion of mannitol to mannose that serves as a key determinant of mannitol pool within the cell and activates plant defense. 4.2.5.2  Sorbitol

Sorbitol is a widely distributed six‐carbon sugar alcohol that has been reported from bacteria, yeast, fungi, animals, and plants (Touster and Shaw 1962; Bielski 1982). It is synthesized as a photosynthetic assimilate in mature leaves along with sucrose translocated through phloem. Sorbitol represents the main form of photosynthetically fixed carbon, translocated through phloem in some members of Rosaceae and Plantaginaceae. In apple, it is synthesized as the major photosynthate. Sorbitol‐6‐phosphate dehydrogenase (S6PDH) initiates sorbitol biosynthesis in the source tissues, which converts glucose‐6‐phosphate to sorbitol‐6‐phosphate. Consequently, sorbitol‐6‐phosphate phosphatase (S6PP) converts

4.3  ­Mechanism of Action of Sugars and Polyol

sorbitol‐6‐phosphate to sorbitol. In the sink tissues, sorbitol dehydrogenase converts sorbitol to fructose. 4.2.5.3  Inositols

Inositols represent a family of essential cyclohexitols containing nine members, of which myo‐inositol is the most common form. They are widely distributed throughout the plant kingdom and are functionally required for normal growth and development, membrane biosynthesis, and in signal transduction. Myo‐inositol biosynthesis is highly conserved and synthesized from an offshoot of the glycolytic pathway from glucose‐6‐phosphate. At first, the rate‐limiting enzyme myo‐inositol‐1‐phosphate synthase (MIPS) produces myo‐­ inositol‐1‐phosphate from glucose‐6‐phosphate. Subsequently, myo‐inositol is produced by dephosphorylation of myo‐inositol‐1‐phosphate by myo‐inositol mono phosphatase (IMP). Apart from acting as an osmolyte, it may function as a signaling intermediate or regulate metabolic responses under stress. Further derivatization of myo‐inositol leads to the formation of inositol derivatives such as D‐ononitol and D‐pinitol that also function as osmoprotectants. While myo‐inositol methyltransferase catalyzes the methylation of myo‐inositol resulting in the formation of O‐methyl inositol (D‐ononitol), D‐pinitol is derived from myo‐inositol by the action of the two enzymes inositol‐o‐methyltransferase (IMT1) and ononitol epimerase (OEP1).

4.3 ­Mechanism of Action of Sugars and Polyols 4.3.1  As Osmolytes Osmotic regulation is one of the most preferred tolerance mechanisms that a plant adopts in response to drought and salinity mainly through maintaining cell turgor and fortification of cellular components. Such osmotic adjustments are achieved by accumulation of osmotically active substances called osmolytes and osmoprotectants that guard the cell machinery against denaturing environmental conditions (Yancey 2005; Liang et al. 2013). Osmoprotectants are electrically uncharged, highly soluble, low‐molecular‐weight molecules that rarely interfere with cellular metabolism, showing nontoxicity to the cellular components. They aid in survival under extreme osmotic conditions either by maintaining osmotic balance by increasing their concentration to avert dehydration or by maintaining membrane fluidity and stabilizing cellular proteins by keeping them hydrated (Wani et al. 2013; Roychoudhury et al. 2012). Implication of sugars and sugar alcohols as osmoprotectants has been widely recognized in tolerance to stresses that include disaccharides (trehalose, sucrose), RFOs, and fructans, while glycerol, sorbitol, mannitol, ononitol, etc., are noteworthy among the sugar alcohols (Keunen et al. 2013). Experimental evidences demonstrate greater accumulation of sugars under environmental perturbations, such as salinity, drought, low temperature, and flooding (Murakeozy et al. 2003), while their accumulation was reduced under high light intensity, nutrient deficiency, and heavy metals (Gill et al. 2001). Response to these conditions appears to be accompanied by an upsurge in the levels of glucose, sucrose, and fructose, while fructan gets accumulated as a delayed response (Kerepesi and Galiba 2000).

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In some plants, accumulation of fructan is observed under severe drought and cold stresses, since it is resistant to crystallization at low temperatures (Krasensky and Jonak 2012). Additionally, it acts as a source of reserve carbohydrate that gets utilized during the recovery phase after withdrawal of the stress (Konstantinova et al. 2002). Fructans maintain membrane integrity under abiotic stress by incorporating themselves within the membrane lipids (Keunen et al. 2013). It is associated with profuse growth and branching of roots, which is considered as an adaptive mechanism under water scarcity. One of the central players in abiotic stress tolerance is trehalose, which protects the plants against the harmful effects of a variety of abiotic stressors. One of the key features is their accumulation in anhydrobiotic organisms and resurrection plants under extended periods of drought. This protective disaccharide can prevent destabilization of biological membranes that takes place due to transition of lipid phase and fusion of vesicles under desiccation. Trehalose accumulation reduces phase transition temperature, thereby maintaining lipids in a crystalline liquid phase, even under water deficiency. In addition, it also serves as a protein integrity protectant (Crowe et  al. 1992). It stabilizes biological membrane by forming hydrogen bonds with phosphate groups of membrane lipids owing to its high hydration potential (Kawai et al. 1992). Additionally, trehalose may be vitrified, by which it forms a highly stable, hygroscopic, glass‐like structure that is exceptionally stable under complete dryness and high temperature and may help biomolecules to return to their native state during recovery (Crowe and Crowe 2000). RFOs are galactosyl derivatives of sucrose and rank second only after sucrose as soluble sugars. Their role as an osmolyte in alleviating the ill effects of low and high temperatures or desiccation has now been widely accepted (ElSayed et al. 2014). Under conditions of freezing and desiccation, RFOs have been directly implicated in providing tolerance by combining sugars and biomolecules through hydrogen bonding that results in stabilization of membranes and enzymes/proteins (Carpenter and Crowe 1989). Besides functioning as  an antistress agent, they have also been reported to inhibit crystallization of sucrose (Caffrey et  al. 1988). Moreover, a combination of sucrose and raffinose has been found more effective during stress‐induced vitrification of cytosol (Koster and Bryant 2006). Although widely distributed in plants, it has been reported that sucrose is a more frequently accumulated osmolyte in monocots than in dicots upon condition of salinity and dehydration injury (Keunen et  al. 2013). Besides its popular role as an osmolyte, this disaccharide also takes part in stabilizing cellular proteins during abiotic stress. Sucrose is preferentially excluded from the protein surface upon solvent exposure by virtue of its hydrophobic nature, thereby favoring its native state conformation. Besides the prominent role of sugars as osmoprotectants, sugar polyols such as mannitol, sorbitol (acyclic) or myo‐inositol, ononitol, and pinitol (cyclic polyols) serve parallel role in higher plants (Bohnert and Jensen 1996). These compounds perform two distinct tasks in water‐stressed tissues, viz., osmotic adjustment and osmoprotection. RFOs not only promote osmotic adjustment facilitating water retention in stressed tissues, but also enable sequestration of Na2+ ions to the apoplast (cell wall) or vacuoles. Functioning as osmoprotectants, they maintain integrity of cellular components mainly by their interaction with cell membranes, protein, and enzyme complexes (Parida and Das 2005). For example, in olive, accumulation of mannitol was noted as a mechanism of tolerance under conditions of salinity and water deficit. Under conditions of salinity, a higher ratio of sorbitol to

4.3  ­Mechanism of Action of Sugars and Polyol

sucrose was observed in the phloem sap in Plantago with concentrations as high as 10 mg g−1 fresh weight (FW) (Pommerrenig et  al. 2007). Additionally, accumulation of sorbitol augments salt and desiccation tolerance in tomato, peach, and persimmon (Escobar‐Gutiérrez et al. 1998; Gao et al. 2001). Accumulation of the cyclic sugar alcohol myo‐inositol and its methylated products pinitol and ononitol has been reported in Mesembryanthemum crystallinum and other halotolerant species when subjected to salinity or desiccation (Vernon and Bohnert 1992; Sengupta et al. 2008).

4.3.2  As Antioxidants Living cells are constantly confronted to the toxicity of notorious reactive forms of molecular oxygen, in particular, free radicals such as superoxide anion (O2˙−) and hydroxyl radical (HO˙) and the nonradicals such as singlet oxygen (1O2) and hydrogen peroxide (H2O2). Referred to as ROS, these molecules are generated either as a by‐product of aerobic metabolism or due to adverse conditions (Keunen et al. 2013). Plants generate ROS through leakage of electrons from the metabolic activities occurring within mitochondria, chloroplast, and peroxisomes, or in other cellular compartments such as endoplasmic reticulum, plasma membrane, or apoplastic regions of the cell wall (Mittler et al. 2004). Generation of ROS is unavoidable and they are constantly being scavenged by the action of antioxidant machinery of the cell that leads to redox homeostasis (Das and Roychoudhury 2014). However, exposure to various biotic and abiotic stressors disturbs this equilibrium and results in greater generation of ROS and leads to a situation known as “oxidative stress.” Increased accumulation of ROS disrupts redox homeostasis resulting in peroxidation of lipids, oxidation of nucleic acids, and inactivation of proteins/enzymes, triggering programed cell death (PCD) (Torres et al. 2006; Paul and Roychoudhury 2016). Survival in this stressful situation demands a stable redox state through attainment of necessary equilibrium between ROS generation and detoxification by the action of the antioxidant machinery. Within the cell, damaging effects of excess ROS are minimized by the scavenging action of various enzymatic and nonenzymatic antioxidants. Until recently, soluble sugars and sugar alcohols have received special attention as contributors of the nonenzymatic antioxidant machinery (Couée et al. 2006; Bolouri‐Moghaddam et al. 2010). Accumulation of compatible sugars is associated with various forms of biotic and abiotic stresses and is assumed to be a type of protective response (Roitsch 1999). However, sugars are also implicated in generation of ROS. Photosynthesis, an important source of ROS, is also associated with accumulation of soluble sugars. Additionally, excess accumulation of glucose may elevate cytosolic H2O2 concentrations via activation of the membrane‐bound NADPH oxidase. Moreover, sugar‐induced interruption of the respiratory metabolism may evoke production of ROS in the mitochondrial electron transport chain (ETC) (Xiang et al. 2011). It has been experimentally demonstrated that sugars and sugar polyols counter oxidative stress as ROS scavengers both in in vivo (Nishizawa et al. 2008) and in vitro (Stoyanova et  al. 2011) conditions. These compatible solutes minimize hydroxyl radical toxicity through Fenton reactions involving hydrogen peroxide and Fe2+, resulting in formation of less toxic oxidized sugar radicals. Soluble sugars can nourish the oxidative pentose phosphate pathway that may stimulate ROS detoxification (Debnam et al. 2004). The key

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player contributing to this pathway is glucose‐6‐phosphate dehydrogenase that governs redox homeostasis in the plastids. Sugars are also implicated in recycling of NADPH that serves as a cofactor for oxidoreductase enzymes, such as glutathione reductase (GR) and monodehydroascorbate reductase (MDAR). Availability of sugars such as sucrose can also elicit synthesis of ascorbate, a molecule needed for ROS detoxification as a result of enhanced respiration (Millar et al. 2003; Nishikawa et al. 2004). In addition, compatible sugars also modulate stress‐associated gene expression through sugar‐specific signaling pathways. Variations in sugar levels can alter expression of genes implicated in maintenance of redox balance, for instance, glutathione‐S‐transferases and superoxide dismutase (Koch 1996; Price et al. 2004). ROS detoxification properties of sucrose have been experimentally proven, which largely advocates the existence of a parallel in planta reaction under stressful situation (Uemura and Steponkus 2003). Free radicals such as HO˙ are highly reactive that preferentially target the HO─C─H linkages of sugars and produce water after combining with H˙ (Morelli et al. 2003). Accordingly, the free radical‐scavenging activity of sugars can be correlated to the number of ─OH groups. This clarifies how effective is the disaccharide sucrose (having eight ─OH groups) over its component monosaccharides, glucose and fructose (with five ─OH groups each). In line, polyhydroxy sugars such as fructans having higher degree of polymerization display a better free radical‐scavenging activity. Compounds such as galactinol, RFOs, and sugar polyols have also been proven as scavengers of free radicals (Nishizawa et al. 2008; Peshev et al. 2013) that serve as better foragers of hydroxyl radicals than superoxide anions (Stoyanova et al. 2011). This is of particular interest as plants lack enzymatic ˙OH‐scavenging property and rely on the nonenzymatic antioxidants to nullify the effect of these toxic ROS species. Under in vitro conditions, the ˙OH‐scavenging capacity of galactinol and raffinose is comparable to the well‐known antioxidant systems such as glutathione and ascorbic acid (Nishizawa et al. 2008). Role of sugar alcohols in quenching oxidative stress has also been studied (Smirnoff and Cumbes 1989; Shen et al. 1997). At low concentrations, mannitol and sorbitol inhibit salicylate hydroxylation by the action of ˙OH radicals, thereby protecting the proteins/enzymes from inactivation.

4.3.3  As Signaling Molecule Sugars are the primary products of photosynthesis that participate in plant growth and development either as source of energy or by synthesizing storage/structural components. However, their role in abiotic stress tolerance is largely ignored particularly in the context of stress signaling. Extensive studies, especially during the past decade, have elaborated the mechanism of sugar sensing, depicting their contribution in signal transduction and regulating gene expression. In plants, sugars may serve the role of primary messengers and can modulate expression of genes. Unfavorable environmental conditions alter the source–sink metabolism and result in differential expression of genes involved in photosynthesis, respiration, starch and sucrose biosynthesis, immune mechanisms, and cell cycle regulation that ensues optimal utilization of carbon and energy. In these cases, adaptive responses are initiated only after perception of stress‐induced changes in the ratio of hexose to sucrose concentration by distinct sensors and relay this information into a signal transduction cascade. Hexokinase 1 (HXK1) is the principal glucose sensor that responds to glucose

4.3  ­Mechanism of Action of Sugars and Polyol

concentrations under stress conditions and modulates expression of genes accordingly (Van den Ende and El‐Esawe 2014). Hexokinase represents an evolutionarily conserved gene family with six members in Arabidopsis, two in potato, four in tomato, nine in tobacco, five in grapevine, and ten in rice (Aguilera‐Alvarado and Sánchez‐Nieto 2017). On the basis of its subcellular localization, HXK can be categorized into four groups of which the type‐B HXK having a nuclear localization signals is worth mentioning in this background as it can sense and transduce sugar signal directly to the nucleus in stressful situations. It has been demonstrated that HXK1 in association with VHA‐B1 and RPT5B may function in a glucose‐independent manner and modulate expression of photosynthetic genes, which advocates the involvement of metabolic enzymes in signal transduction (Chen 2007). Although not well characterized, HXK‐independent glucose sensing has also been observed in plants (Ramon et al. 2008). Moreover, several plants harbor fructokinases that may also participate in stress‐induced sugar sensing. In barley, it has been shown that α‐amylase expression is influenced by the fructose component of nonmetabolizable sugars, such as palatinose, turanose, and fluorosucrose (Loreti et al. 2000). Although no sucrose‐sensing mechanism has so far been discovered in plants, it is assumed that sucrose signal may modulate anthocyanin biosynthesis by the activation of MYB75 via a trehalose‐6‐­phosphate signal (Van den Ende and El‐Esawe 2014). Besides intracellular sensors, existence of extracellular sugar sensors has also been accepted. Application of the disaccharide palatinose, which is not absorbed by cells, enhances the activity of invertase that favored synthesis of starch in potato (Fernie and Willmitzer 2001). Since invertases are intricately associated with abiotic stress tolerance, glucose obtained from invertase activity maintains the HXK activity, thereby balancing the ROS homeostasis in mitochondria (Valluru and Van den Ende 2011). Sucrose nonfermenting 1‐related protein kinase 1 (SnRK1) is another important mediator of stress signaling that results in accumulation of protective metabolites and defense compounds in abiotic stress responses (Lin et al. 2014; Emanuelle et al. 2016). In response to stress‐associated energy deficit, SnRK1 initiates widespread transcriptional reprograming that restores cellular homeostasis, supporting plant survival and ensuring long‐ standing responses to stress adaptation. Two such members KIN10 and KIN11 regulate gene expressions of transcription factors, calcium modulators, protein phosphatases, and kinases in response to stress signals. In Arabidopsis apart from SnRK1, ten members of SnRK2s and 29 members of SnRK3s have been identified. Expressional inductions of nine and five members of SnRK2 family have been reported during water deficit and abscisic acid (ABA) treatments, respectively (Nakashima et al. 2009). Additionally, salt overly sensitive 2 (SOS2), a member of the SnRK3 family, is induced upon salinity stress that regulates ion homeostasis in plants. Furthermore, members of HXK and SnRK1 may link sugar signaling with phytohormones, thereby aiding in plant protection from environmental perturbations (Ljung et al. 2015). Stress‐induced sugar imbalance can elicit ABA accumulation and activate a unique sugar‐signaling machinery. ABI4 is one important downstream effector of ABA‐sugar signaling that governs regulation of sugar‐ responsive gene expression. ABI4 also induces the expression of ANAC060 (Arabidopsis n‐acetyl‐l‐cysteine [NAC]‐domain transcription factor 060), which suppresses the sugar‐ ABA signaling pathway (Ljung et  al. 2015). Furthermore, sugars such as glucose and sucrose are also involved in auxin signaling and biosynthesis. Disaccharide sucrose is

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associated with gibberellin (GA)‐signaling pathway by stabilizing the DELLA proteins, thereby acting as a negative regulator of gibberellin signaling and explaining the antagonism of sucrose‐dependent anthocyanin production induced by gibberellin under stress (Ljung et al. 2015). Sucrose‐GA signaling also influences jasmonate pathway via activation of bHLH and MYB transcription factors, a requisite for anthocyanin biosynthesis. Besides, the DELLA repressor interacts directly with Brassinazole resistant 1 (BZR1) transcription factor as a downstream regulator of the GA and brassinosteroid signaling. In these circumstances, sucrose stabilizes the DELLA repressors resulting in its accumulation, thereby chelating BRZ1 and hence antagonizing growth and development (Ljung et al. 2015).

4.4 ­Involvement of Sugars and Polyols in Abiotic Stress Tolerance 4.4.1  Cold Acclimation Low temperature is one of the major constrains that limit agricultural productivity of crop plants (Chinnusamy et al. 2007). Two different aspects of cold stress, viz. chilling (15–0°C) and freezing (