Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses (Plant in Challenging Environments, 1) 3030736776, 9783030736774

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Plant in Challenging Environments  1

M. Nasir Khan Manzer H. Siddiqui Saud Alamri Francisco J. Corpas  Editors

Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses

Plant in Challenging Environments Volume 1

Series Editors Dharmendra K. Gupta, Ministry of Environment, Forests and Cli,  New Delhi, India José Manuel Palma, Estación Experimental del Zaidín, Granada, Granada, Spain Francisco J. Corpas, Estación Experimental del Zaidín, Granada, Spain

The proposed series of books provides recent advancements in wide areas related to higher plants and how they adapt/evolve under environmental changes in a scenario of climate change. Thus, the series investigates plants under the complementary point of views including agronomy aspects (vegetables and fruits), nutrition and health (food security), “omics,” epigenetics, contamination by heavy metals, environmental stresses (salinity, drought, high and low temperatures), interaction with beneficial or pathogenic microorganisms, and application of exogenous molecules (nitric oxide, melatonin, chitosan, silicon, etc.) to palliate negative effects and also includes changes due to climatic condition (high/low rainfall) taking into account that the climate change is often the reason why plants evolve in a challenging environment. Thus, the impulse of this series of books will cover molecular-/cellular-level responses of plants under different climatic reasons. Families of molecules derived from hydrogen peroxide (H2O2), nitric oxide (NO) and hydrogen sulfide (H2S) designated as reactive oxygen, nitrogen and sulfur species (ROS, RNS and RSS, respectively) are included since, depending on the production level, they function both as signal molecules and as a mechanism of response against adverse/changing environmental conditions that can produce multiple cellular damages, alter the redox state or even trigger cell death. During these ensued metabolic processes, some antioxidative/oxidative enzymes are also disturbed or triggered abruptly, but there are adequate mechanisms of regulation/homeostasis in the different subcellular compartments to keep these enzymes under control. In the last decades, the progression in this field has been enormous, but still there is so much in this field to understand the plethora of phenomena behind. The series serves as an important reference material for students at different levels (undergraduate, master and PhD), teachers, developers, policymakers and implementers who are dealing with similar topics. More information about this series at http://www.springer.com/series/16619

M. Nasir Khan  •  Manzer H. Siddiqui Saud Alamri  •  Francisco J. Corpas Editors

Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses

Editors M. Nasir Khan Department of Biology, College of Haql University of Tabuk Tabuk, Saudi Arabia Saud Alamri Department of Botany and Microbiology, College of Science King Saud University Riyadh, Saudi Arabia

Manzer H. Siddiqui Department of Botany and Microbiology, College of Science King Saud University Riyadh, Saudi Arabia Francisco J. Corpas Department of Biochemistry, Cell and Molecular Biology of Plants, Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture Estación Experimental del Zaidín (Spanish National Research Council, CSIC) Granada, Spain

ISSN 2730-6194     ISSN 2730-6208 (electronic) Plant in Challenging Environments ISBN 978-3-030-73677-4    ISBN 978-3-030-73678-1 (eBook) https://doi.org/10.1007/978-3-030-73678-1 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Early studies on hydrogen sulfide (H2S) exhibit its injurious effects on plants. However, over the last several years, H2S has emerged as a potent regulator of plant growth and development. A vast number of physiological and biochemical processes are regulated by H2S influencing plants at every stage of their life cycle. In plants, desulfuration of cysteine (Cys), by Cys desulfhydrases (CDes), synthesizes H2S in various cell compartments. In addition, several other enzymes have been reported to synthesize H2S in plants; however, L-Cys desulfhydrase 1 (DES1) has been recognized as the key enzyme involved in the generation of H2S from Cys in the cytosol. The existence of a dedicated system of H2S synthesis, diffusion, sensing, and scavenging acknowledges the H2S as a signaling molecule in plants. The signaling function of H2S encompasses through its crosstalk with other signaling molecules like nitric oxide, carbon monoxide, abscisic acid, reactive oxygen and reactive nitrogen species, and calcium. Role of H2S has been well investigated in mediating plant adaptive responses to a plethora of abiotic stresses such as drought, heat, cold, salinity, heavy metals, dark, and post-harvest stress. Also, the onset of these stresses induces endogenous synthesis of H2S, which generates a downstream signaling cascade and mediates plant responses to the stressful conditions. The initial signaling mechanism of H2S is operated through post-translational modification of target proteins, a process known as persulfidation. In this process, reactive Cys residues on target proteins are modified via conversion of the thiol group into a persulfide group. Persulfidation is believed to play crucial role in the protective mechanisms against stress-induced impairments. However, the underlying molecular mechanism through which H2S employs its action is not completely comprehended. Also, it has yet to be recognized the pathway(s) in which H2S might be involved, and how and in what sequence H2S functions in association with other signaling molecules. Therefore, to expand our understanding of H2S in plant biology under adverse conditions, the present book titled Hydrogen Sulfide and Plant Acclimation to

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Abiotic Stresses was compiled. In this book, information on the biosynthesis of H2S and its role in mediating plant responses to abiotic stress, mechanism of action of H2S, its interaction with other signaling molecules, and their sequence of action in plants under various abiotic stresses is covered in the 12 chapters in an orderly manner. The Chap. 1 deals with the various role of H2S in plant physiological processes and its crosstalk with other molecules. Chapter 2 of this book presents a generalized view of H2S functions in plants under various abiotic stresses. In Chap. 3, functional crosslinks of H2S with other mediators in the formation of concrete adaptive reactions of plants, in particular, in the activation of antioxidative system, is discussed. Chapter 4 deals with the metabolism of H2S and its protective role during metal toxicity in plants. Chapter 5 covers the role of H2S in osmotic adjustment of plants under different abiotic stresses. Effect of H2S on the accumulation of a range of plant osmolytes in response to the prevailing abiotic stresses was elaborated. Chapter 6 updates the readers with current knowledge on the role of H2S in the signaling network that commands stomatal movement in response to external and endogenous stimuli. Chapter 7 presents an updated comprehensive overview of the H2S metabolism and its implication in the ripening of climacteric and non-­ climacteric fruits. Additionally, the beneficial effects exerted by the exogenous application of H2S during the ripening period and postharvest storage are also overviewed. Chapter 8 sheds light on the possible functions of H2S during seed germination, with an emphasis on the mechanisms through which H2S mitigates abiotic stress effects and maintains high germination efficiency under penalizing conditions. Chapter 9 provides the key roles of H2S in plants under abiotic stresses such as metals, high salinity, drought, and extreme temperatures. The crosstalk among H2S, phytohormones, second messengers, and metabolites is also addressed. In Chap. 10, the authors present a proteomic and transcriptomic overview of the mechanisms of H2S signaling network involved in plant adaptive responses to abiotic stresses. Chapter 11 is focused on the significance of Cys in H2S-mediated protective mechanisms and their interactive role in alleviating abiotic stress-induced impairments. Besides, the role of Cys and its allied molecules and products in the mechanisms responsible for plant acclimation to environmental stresses is also discussed. Chapter 12 sheds light on the role of H2S in posttranslational modification of proteins and the importance of persulfidation in H2S-meidated plant adaptive responses to abiotic stresses. Also, the methods for the detection of protein persulfidation are discussed. The diversity of chapters presented in this book certainly expands the understanding of the readers. We hope the book will play a pivotal role in closing the gap of information and in opening new avenues in the field of H2S research in plant biology.

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We express our heartfelt gratitude to all the authors and reviewers of this book. We acknowledge the assistance from Dr. D. K. Gupta, Prof. J. M. Palma, and Prof. Francisco J. Corpas, editors of the book series Plant in Challenging Environments. Moreover, we are highly thankful to Springer Nature, Switzerland, and Ms. Zuzana Bernhart, Executive Editor Plant Sciences – Books, for the professional support and cooperation during the preparation of this volume. Tabuk, Saudi Arabia

M. Nasir Khan

Riyadh, Saudi Arabia

Manzer H. Siddiqui

Riyadh, Saudi Arabia

Saud Alamri

Granada, Spain

Francisco J. Corpas

Contents

1 Hydrogen Sulfide on the Crossroad of Regulation, Protection, Interaction and Signaling in Plant Systems Under Different Environmental Conditions������������������������������������������    1 Zahid H. Siddiqui, Zahid K. Abbas, M. Wahid Ansari, and M. Nasir Khan 2 Hydrogen Sulfide: A Road Ahead for Abiotic Stress Tolerance in Plants����������������������������������������������������������������������������������   13 Mehmet Tufan Oz and Fusun Eyidogan 3 Functional Interaction of Hydrogen Sulfide with Nitric Oxide, Calcium, and Reactive Oxygen Species Under Abiotic Stress in Plants����������������������������������������������������������������   31 Yu V. Karpets, Yu E. Kolupaev, and M. A. Shkliarevskyi 4 Hydrogen Sulfide and Redox Homeostasis for Alleviation of Heavy Metal Stress����������������������������������������������������   59 Ankur Singh and Aryadeep Roychoudhury 5 Effect of Hydrogen Sulfide on Osmotic Adjustment of Plants Under Different Abiotic Stresses��������������������������������������������   73 Aryadeep Roychoudhury and Swarnavo Chakraborty 6 Hydrogen Sulfide and Stomatal Movement������������������������������������������   87 Denise Scuffi and Carlos García-Mata 7 Hydrogen Sulfide and Fruit Ripening����������������������������������������������������  109 Francisco J. Corpas, Salvador González-Gordo, and José M. Palma 8 Hydrogen Sulfide Impact on Seed Biology Under Abiotic Stress��������������������������������������������������������������������������������  123 Emmanuel Baudouin

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9 Hydrogen Sulfide Signaling in the Defense Response of Plants to Abiotic Stresses��������������������������������������������������������������������  139 Cristiane J. Da-Silva, Ana Cláudia Rodrigues, and Luzia V. Modolo 10 A Transcriptomic and Proteomic View of Hydrogen Sulfide Signaling in Plant Abiotic Stress ������������������������  161 Susana González-Morales, Raúl Carlos López-Sánchez, Antonio Juárez-­Maldonado, Armando Robledo-Olivo, and Adalberto Benavides-Mendoza 11 Cysteine and Hydrogen Sulfide: A Complementary Association for Plant Acclimation to Abiotic Stress������������������������������  187 M. Nasir Khan, Manzer H. Siddiqui, Mazen A. AlSolami, Riyadh A. Basahi, Zahid H. Siddiqui, and Saud Alamri 12 Hydrogen Sulfide and Posttranslational Modification of Proteins: A Defense Strategy Against Abiotic Stress������������������������  215 Dengjing Huang, Changxia Li, Chunlei Wang, and Weibiao Liao Index������������������������������������������������������������������������������������������������������������������  235

Chapter 1

Hydrogen Sulfide on the Crossroad of Regulation, Protection, Interaction and Signaling in Plant Systems Under Different Environmental Conditions Zahid H. Siddiqui, Zahid K. Abbas, M. Wahid Ansari, and M. Nasir Khan

Abstract  Due to climate change, the severity of the damage caused by the biotic and abiotic stress is unprecedented. In order to overcome the loss of productivity plant scientists are trying to elucidate the mechanisms of organism’s response to environmental changes worldwide. Their findings recorded that the organisms make physiological adjustments and genetic changes to adapt in a new environment. These adjustments and changes require the participation of an array of signaling molecules like reactive oxygen species (ROS), reactive nitrogen species (RNS), carbon monoxide (CO), nitric oxide (NO), hydrogen sulfide (H2S), calcium (Ca), salicylic acid, phospholipids etc. The understanding of how these molecules cross talk within the organism will elucidate the mechanisms through which they adapt to their external environment. Among these molecules, H2S is on the crossroad of regulation of the processes at key cellular, sub-cellular, and molecular level in plants and known to play a vital role in plant growth and development. The signaling molecule H2S, protects plants from various types of biotic and abiotic stresses. Moreover, H2S also interacts with different phytohormones and other signaling molecules like Ca and NO in different metabolic processes in plants under different environmental conditions. Keywords  Abiotic stress · Autophagy · Climate change · Hydrogen sulfide · Photosynthesis · Signaling Z. H. Siddiqui (*) · Z. K. Abbas Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Kingdom of Saudi Arabia e-mail: [email protected] M. W. Ansari Department of Botany, Zakir Husain Delhi College, University of Delhi, New Delhi, India M. N. Khan Department of Biology, College of Haql, University of Tabuk, Tabuk, Saudi Arabia e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_1

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1.1  Introduction In this era of climate change, every living organism on the planet Earth is experiencing stress in its life cycle. The stress falls into two categories, biotic and abiotic of both natural and anthropogenic origin, affecting the physiological metabolism, growth cycle, and epigenetic modifications of the organisms. Perhaps, the plants and other organisms have natural mechanism to overcome these stress factors but due to climate change, the severity of the damage caused by the stress is unprecedented. In order to get higher productivity from plants, researchers are trying to elucidate the mechanisms of organism’s response to environmental changes worldwide. So far, it is well known that organisms adjust their physiology and genetic status to adapt in a changing environment (Kroll et al. 2014). Perhaps, these physiological and genetic changes require the participation of an array of signaling molecules like reactive oxygen species (ROS) (Ha et al. 2018), reactive nitrogen species (RNS) (Hancock et al. 2011; Zhang et al. 2018), carbon monoxide (CO) (Wareham et al. 2018), nitric oxide (NO) (Khan et al. 2017, 2020), calcium (Ca) (Dodd et al. 2010; Wang et al. 2016), salicylic acid (Guo et al. 2017), and phospholipids (Liu et al. 2017). Similar to NO and CO, hydrogen sulfide (H2S) is the third gasotransmitter, which is present in bacteria, plants, invertebrates, and vertebrates, including mammals, and control their physiological and biochemical activities (Hancock et  al. 2011; Mancardi et al. 2009; Wang 2012). The understanding of these molecules and their cross talk within the system elucidate the mechanisms through which organisms adapt to their external environment. However, the H2S is an enigmatic molecule but at the same time, it is one of the important compounds from Stanley Miller’s spark discharge experiments for origin of life on Earth (Bada 2013), and has surplus functions in all forms of life. A few decades ago, H2S was a gaseous pollutant, produced primarily by burning of fossil fuels. However, over the years several reports recognized its endogenous production as a signaling molecule. Being a foul, colorless gas its study was exceedingly difficult, however, the emission of H2S in plants was discovered from squash, cucumber, cantaloupe, pumpkin, cotton, soybean, and corn using sulfur-­specific flame photometric detector (Wilson et al. 1978). Due to small size and neutral nature of H2S, it can easily move between the cells and does not require any transporters, through the hydrophobic cellular membranes (Mathai et al. 2009). The H2S regulates numerous physiological processes in plants and known to play an important role in plant growth and development as well as in the protection of plants against various environmental stresses (Jin and Pei 2015; Jin et al. 2017; Khan et al. 2018). In animals, the role of H2S as a signaling molecule is in details with its own limitations and open-end questions. The scenario in case of plant is quite different because of the late recognition of signaling role of H2S in plants (Zhang et al. 2008a). Primarily, H2S was known for its phytotoxic effects and its function in plant sulfur metabolism. Not long ago, new functions of this gas has been reported like as an alternative of sulfur source in plant nutrition, an instrument to manage excess sulfur in the system and most importantly its role in regulation

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Fig. 1.1  Role of hydrogen sulfide (H2S) in various processes of plant biology. ABA absciscic acid, GA gibberellic acid, H2O2 hydrogen peroxide, NO nitric oxide

and signaling (Lisjak et  al. 2013; García-Mata and Lamattina 2013; Calderwood and Kopriva 2014). It acts as an important regulator of secondary messengers in different stress responses, plant developmental processes and activation of signal transduction cascades similar to NO (Zhang et al. 2008b; Shi et al. 2012; GarcíaMata and Lamattina 2013). Besides that, H2S is a member of a crosstalk network which is shared with Ca, NO and abscisic acid (ABA) (Fotopoulos et  al. 2015, Zhang et al. 2015a, b, Aroca et al. 2018). In this chapter, various roles of H2S in plant systems and its crosstalk with other molecules will be explored (Fig. 1.1).

1.2  Biosynthesis and Role of H2S in Plant System Initially, H2S was considered as a pollutant and a lethal gas for living organisms. In the plant system, biosynthesis of H2S is predominantly occurring in chloroplast and to a lower extend in mitochondria and cytosol. Unlike animal system, in plants this process is controlled by multiple enzymatic systems, different from the sulfate assimilation pathway (Aroca et al. 2018). Mainly, following enzymes are responsible for the biosynthesis of H2S in plants: L-cysteine desulfhydrase (LCD), D-cysteine desulfhydrase (DCD), β-cyanoalanine synthase (β-CAS), cysteine synthase (CS), sulfite reductase (SiR) and carbonic anhydrase (Rausch and Wachter 2005; Yamasaki and Cohen 2016). However, non-enzymatic pathway also contributes to the partial synthesis of H2S as compared to the enzymatic pathway. In short, the process is overly complex and for further updated understanding the following reviews can be consulted (Calderwood and Kopriva 2014; Shivaraj et al. 2020). There are numerous reports that describe the phytotoxic effects of H2S on plants (Rodriguez-Kabana et al. 1965; Lamers et al. 2013). In the report by Thompson and Kats (1978) high concentration of H2S triggered injuries in the leaves, defoliation and reduced growth

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of the plants, however its low concentration was reported to be beneficial for the growth of Medicago, lettuce and sugar beet. Over the years, H2S and plant -based studies have undergone in various directions and opened several avenues in plant biology (Fig. 1.1).

1.3  H2S and Regulation of Physiological Processes in Plants A vast array of literature is now available to confirm the role of H2S in the regulation of various physiological/biochemical processes namely photosynthesis (Wang 2012; Chen et al. 2013), opening and closing of stomata (Papanatsiou et al. 2015; Jin et al. 2017; Chen et al. 2020; Shen et al. 2020), seed germination (Zhang et al. 2008a), root morphogenesis (Zhang et al. 2009; Jia et al. 2015; Mei et al. 2019), fruit ripening (Ge et al. 2017; Muñoz-Vargas et al. 2018), and senescence in leaves, flowers, and fruits (Zhang et al. 2011; Zheng et al. 2016). The role of H2S during photosynthesis in Spinacia oleracea seedlings was studied in detail by Chen et al. (2011). Their results suggested that photosynthetic parameters such as maximum net photosynthetic rate (Pmax), maximal photochemical efficiency of photosystem II [F(v)/F(m)], carboxylation efficiency (CE), and the light saturation point (Lsp) reached to peak values under sodium hydrosulfide (NaHS) treatment. Besides that, the number of grana lamellae and activity of RuBisCO was significantly increased by NaHS treatment. Recently, Liu et al. (2019) reported that H2S regulates photosynthesis of tall fescue under low-light stress. In their results the photosynthetic parameters, which are supposed to be reduced during low light conditions like net photosynthetic rate, chlorophyll content, photochemical efficiency of PSII, intercellular CO2 concentration, enzymatic activity of RuBisCO etc., were significantly increased under NaHS treatment. Moreover, the antioxidant activities were enhanced by the NaHS treatment. Plant system ensures its bulk and selective recycling, protection from pathogen and senescence by autophagy (Liu and Bassham 2012). Autophagy is a catabolic process in which consumption of the body’s own tissues occurs in starvation or in diseases. In animals, the role of lysosomes and thier hydrolyzing enzymes are key factors in the process of autophagy. In plant system, the regulation of autophagy is associated with H2S (Álvarez et al. 2010; Laureano-Marin et al. 2016). In starving Arabidopsis, H2S prevents the ATG8 (autophagy-related ubiquitin-like protein) accumulation, thereby inhibits autophagy (Álvarez et al. 2012). This regulation was achieved by persulfidation of the enzymes involved in the autophagosome formation (Gotor et  al. 2013). In Arabidopsis leaves, a high throughput proteomic approach reported persulfidation of some autophagy (ATG)-related proteins, ATG18a, ATG3, ATG5, and ATG7 (Aroca et al. 2017). Besides that, in yeast and algae, thioredoxin regulates ATG4 (Pérez-Pérez et al. 2014), in mammals ATG4b and ATG1 are controlled by phosphorylation and S-nitrosylation (Li et  al. 2017; Pengo et al. 2017; Sanchez-Wandelmer et al. 2017), and Caspase-3, which is indispensable for autophagic activity, is persulfidated at Cys163 associating this

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amendment to the cytoprotective outcome of sulfide (Marutani et al. 2015). These reports suggest that persulfidation is the molecular mechanism through which sulfide regulates autophagy in Arabidopsis. Nevertheless, the recognition of other targets associated with autophagy needs additional detailed studies, and the constructive part of persulfidation in autophagy requires further investigation (Aroca et al. 2018).

1.4  H2S and Protection of Plants Under Stress As we discussed earlier, the adverse environmental conditions are responsible for negative growth, development, and loss of crop productivity. However, plants develop several mechanisms to overcome these adverse conditions. These mechanisms are regulated by generation of various types of ROS/RNS (Mantri et al. 2012). Further, new researches are suggesting the role of H2S in response to adverse biotic and abiotic environmental conditions (Shi et al. 2015; Banerjee et al. 2018) along with a significant correlation between the functions of H2S and NO (Corpas et al. 2019). The systematic understanding of the defensive mechanism of H2S is relatively complex and challenging. The role of H2S in plant protection from chilling stress (Fu et al. 2013), drought stress (Jin et al. 2017), osmotic stress (Khan et al. 2017), salt stress (Lai et al. 2014), and heavy metal stress (Guo et al. 2017; Khan et al. 2020) has been reported. The metal toxicity of copper, aluminum, chromium, cadmium, lead, arsenic, and zinc are effectively eased by H2S (Zhang et al. 2008a; Chen et al. 2013; Mostofa et al. 2015; Liu et al. 2016; He et al. 2018, Khan et al. 2020). The role of exogenous H2S in alleviating metal toxicity in plants is related to the concentration of H2S supply (He et al. 2018). H2S relieved the harmful effect of elements on plants by accumulating cell wall-related pectin methylesterase (PME), expansions, citrate, or plasma membrane (PM) H+-ATPase (Wang et al. 2010; Chen et al. 2013). Recently, Corpas and Palma (2020) compiled the beneficial effects of the exogenous application of H2S on a wide range of agricultural crops (rice, soybean, barley, wheat, maize, pea, cucumber etc.) affected by different kinds of abiotic stresses including heavy metals, salinity, drought as well as heat and cold stresses. Generally, the exogenous application of H2S tends to increase the various components of antioxidant systems like catalase, ascorbate peroxidase, and superoxide dismutase which modulate the level of hydrogen peroxide (H2O2) and decrease lipid peroxidation. In Brassica napus the exogenous application of H2S improved plant growth, root morphology, photosynthetic rate, and chlorophyll content. It also brings positive ultra-structural changes in the chloroplast (well-developed thylakoid membrane), mature Golgi bodies, mitochondria, and large endoplasmic reticulum in the root tip cells of Brassica napus (Ali et al. 2014). In alfalfa seedlings, H2S enhanced Cd tolerance by regulating reduced (homo)-glutathione and ROS homeostasis (Cui et al. 2014). The protective role of H2S against Cd in rice was managed by reducing oxidative stress and maintaining mineral homeostasis (Mostofa et al. 2015). He et al. (2018) proposed an action mechanism of H2S-induced metal tolerance in plants. Perhaps in the initial step, H2S regulates the metal transport proteins

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and thereby reduces the metal accumulation in the plants. It is followed by the improvement in the antioxidant capacity resulting in the scavenging of metal-­ induced ROS in plants by H2S (Khan et al. 2020). In the final step, H2S interacts with other signaling pathways, microRNAs (miRNAs) regulation and protein persulfidation under toxic metal stress. In banana fruit under low temperature, H2S treatment can ease chilling injury by augmenting antioxidant system and the Δ1-­ pyrroline-­5-carboxylate synthetase activity which mainly attributed to an elevation in proline content (Luo et al. 2015).

1.5  H2S Signaling and Interaction in Plants The signaling molecule H2S is involved in plant cross-adaptation, a known wild phenomenon of nature. There are evidences that suggest the signaling role of H2S to activate downstream signal transductions and to reduce the heavy metals stress (Shi et al. 2014; Singh et al. 2020). If a plant is exposed to a mild stress, it can elicit the resistance to other stresses (Hossain et al. 2016). In case of winter rye, cold pretreatment can improve the heat tolerance, and UV-B can improve the heat tolerance in cucumber and the cold tolerance in Rhododendron. Similarly, mechanical stimulus can increase the heat tolerance and the chilling tolerance in tobacco cells (Knight 2000; Li and Gong 2013; Li et  al. 2016). Furthermore, cross-adaptation can be induced between abiotic and biotic stresses (Foyer et  al. 2016). In tomato, sunflower, pea, and rice infection by mycorrhizal fungi can increase the resistance to different abiotic stresses (Grover et al. 2011) and drought stress decreases the aphid fertility in Arabidopsis (Pineda et al. 2016). The intricacies of cross-adaptation are related to a complex of signal network that includes different secondary messengers like H2O2, NO, ABA, and Ca as well as their cross talk. The mild environmental stress or external use of signal molecules or their donors activate the cross-­adaptation signaling, which in turn stimulates cross-adaptation by accumulating osmolytes, augmenting antioxidant system activity, producing heat shock proteins, as well as ameliorating ion and nutrient balance (Li et al. 2016). There are reports that suggest that both H2S and NO are involved in multiple pathways (Corpas et al. 2019b) and known to perform posttranslational modifications such as persulfidation and S-nitrosation, respectively (Aroca et al. 2018). The outcome of S-nitrosation regulates the role of different types of proteins and suggest the direct action of NO in various signaling processes. Similarly, the H2S-assisted protein persulfidation provides protection against over-oxidation (Filipovic 2015; Aroca et al. 2018). The role of H2S signaling is very promising in the regulation of stomatal movement. The stomata are the windows for gas exchange between plant and the atmosphere and H2S is a gaseous molecule. Besides that, the NO signals stomatal closure with the help of ABA molecule is already reported (García-Mata and Lamattina 2001; Neill et  al. 2002), but in another report García-Mata and Lamattina (2010) suggest the role of H2S as a novel gasotransmitter involved in guard cell signaling. The H2S induces stomatal closure and that inhibition of H2S

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production partially blocks the ABA effect on stomata. However, in another report, H2S induces stomatal opening and reduces NO accumulation (Lisjak et al. 2010). In drought tolerance experiments, H2S treatment induces closure of stomata (Jin et al. 2011; Shen et al. 2013). Jin et al. (2013) confirmed that by analyzing an lcd mutant with reduced H2S accumulation; these mutants were more prone to drought stress and displayed increased stomatal opening in untreated mature leaves. The LCD gene encodes a PLP-binding protein with possible L-cysteine desulfhydrase activity, but in this line the T-DNA is inserted downstream the gene and disturbs the expression of another gene with unknown function. Therefore, it is not clear if LCD protein is another H2S producing enzyme (Jin et  al. 2013). Recently, Chen et  al. (2020) reported that the enzyme SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6), which undergoes persulfidation, promotes ABA signaling and thereafter ABA-induced stomatal closure. Persulfidation-based modification of cysteine desulfhydrase and NADPH oxidase [Respiratory burst oxidase homolog protein D (RBOHD)] controls guard cell abscisic acid signaling (Shen et al. 2020). In light of these reports, the complete mechanism of action on stomatal aperture is still a long case, H2S acts via regulation of ABC channels (García-Mata and Lamattina 2010), but the similarity in action of NO and H2S cannot be ignored (García-Mata and Lamattina 2001). Further, it becomes more complex as H2S interacts with ethylene to induce stomatal closure in Arabidopsis thaliana (Hou et  al. 2013). In pepper plants, it is suggested that both NO and H2S check catalase activity and supplement or antagonize each other in controlling H2O2 content by modulating the antioxidant enzymes (Kaya et al. 2020). As compared to the association between H2S and NO, the relationship between H2S and H2O2 in plants is less understood. Recently, Liu et al. (2020) tried to reveal the mechanism underlying the interaction between H2S and H2O2 in cucumber in response to photosynthesis. Their results suggest that H2S and H2O2 increased the photosynthetic carbon assimilation, carbon metabolism, and photoprotection for both PSII and PSI in cucumber seedlings under chilling stress. They further suggest that H2O2 may act as a downstream signal in H2S-induced protection as 1.0 mM NaHS considerably improved the relative gene expression of RBOH, which in turn contributes to raise the endogenous H2O2 accumulation in cucumber seedlings. However, H2O2 had little effect on gene expression of LCD/DCD and endogenous H2S level (Liu et al. 2020). Further, in tomato seedlings H2O2 is involved in H2S induced lateral root formation (Mei et al. 2017). In bermudagrass, exogenous application of H2S improved Cd tolerance by modulating ROS and osmolytes together with NO (Shi et al. 2014) whereas, in case of arsenic, H2S ease its toxic effects by up-regulation of ascorbate-glutathione cycle in pea seedlings with the involvement of NO that led to the reduced accumulation of arsenic (Singh et al. 2015). Also, in cucumber seedlings, H2S relieve chilling damage by interacting with NO signaling (Wu et al. 2016).

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1.6  Conclusion Presence of H2S in the cellular system initiates a signaling mechanism and induces different constituents of the antioxidant system at both the gene and protein level and protects the plants from abiotic stress, oxidative stress, and postharvest senescence. Further, H2S regulates several physiological processes such as photosynthesis modulation, stomatal movement, seed germination, root organogenesis, etc. Besides, H2S plays an active role in the transduction pathways of several other cellular signaling molecules including ABA, GA, ethylene, H2O2 and NO. Nowadays, H2S is in focus to illuminate the crossroad of regulation, protection, interaction and signaling in various plant systems under different environmental conditions. However, the exact biochemical and molecular mechanisms to elucidate the role of H2S in these processes still need more investigation. Therefore, the use of H2S alone or combined with other molecules, such as NO, Ca, chitosan, thiourea, melatonin, and silicon, which seems to constructively improve crop productivity, should be explored in light of climate change.

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Chapter 2

Hydrogen Sulfide: A Road Ahead for Abiotic Stress Tolerance in Plants Mehmet Tufan Oz and Fusun Eyidogan

Abstract  Hydrogen sulfide (H2S), a phytotoxic gas, is considered a signaling molecule at low concentrations with multiple physiological functions in plants during growth, development, germination, and response mechanisms to abiotic stress. Several reports have indicated that H2S is released in plant cells as a crucial signal for the survival under different abiotic stress conditions. H2S provides systemic resistance to different abiotic stress conditions mainly by reestablishing redox homeostasis, enhancing osmolyte accumulation, maintaining ion balance, and regulating gene expression. It also improves the plant tolerance to abiotic stress with its capacity to react with thiol groups. Like other gaseous signal molecules, H2S is integrated in complex signaling networks with various second messengers such as calcium, hydrogen peroxide (H2O2), nitric oxide (NO), and abscisic acid (ABA). The objective of this review is to summarize the potential physiological functions of H2S under various abiotic stresses. Keywords  Abiotic stress · Antioxidant system · Hydrogen sulfide · Redox homeostasis · Signalling molecules

2.1  Introduction Changes in environmental conditions together with the climate change affect all ecosystems in the world. As a result of these changes, plants are faced with different stress factors which limit plant growth and productivity. Salinity, heavy metals (HMs), heat, drought, and other mechanical stresses are the most common M. T. Oz Earlham Institute, Norwich Research Park, Norwich, United Kingdom F. Eyidogan (*) Institute of Food, Agriculture and Livestock Development, Baskent University, Ankara, Turkey e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_2

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environmental stress factors (Pandey et  al. 2016; Zhang and Sonnewald 2017). When plants are exposed to abiotic stress factors, vital metabolic reactions are affected. These altered processes include osmotic and ionic imbalance and excessive accumulation of major reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radicals (•OH) and superoxide ions (O2⦁―) which induce irreversible cellular damage during these stress conditions. High concentrations of ROS in cells inhibit photosynthesis through stomatal closure and slow down key biochemical processes. Plants develop different mechanisms to protect themselves from these unwanted stressful conditions. The protection mechanism starts with the perception of stress signal by receptors and that information is carried out by signaling pathways to generate a response in the cell. The main responses under stress conditions are the accumulation of organic solutes, both enzymatic and non-enzymatic antioxidants, and transcription factors (Pandey and Gautam 2020). However, timely and accurate perception of the stress signal and defense response to that signal, before the onset of stress-induced damage, is crucial for the endurance of plant under stressful conditions. Priming of the plants with various chemical compounds has been found a promising way to enhance the stress tolerance mechanism in various plant species. After priming with a specific chemical or biological agent, plant defense system is activated under stress conditions. Among the priming agents, H2O2, polyamines, and nitric oxide (NO) occupy a prominent position that enhance stress tolerance in plants. Similarly, hydrogen sulfide (H2S), another endogenous gaseous transmitter, is formed under various stresses and is found to be one of the effective priming agents. In addition, H2S is a lipophilic molecule and is accepted as the third gas transmitter after NO and carbon monoxide (CO) (Wang 2002). In the late 1990s researchers showed that H2S might have important functions in humans (Kimura 2015). In mammalian cells, H2S was indicated as a possible signaling component in cells (Abe and Kimura 1996). Later, it was shown that H2S is also produced by bacteria and plants where it functions as a signaling molecule (Da-Silva and Modolo 2018). Plants synthesize H2S endogenously both in normal and stress conditions. The crosstalk between signaling pathways and H2S indicates the role of H2S in tolerance to different abiotic stresses (Niu and Liao 2016). Other studies showed that H2S may have roles in antioxidant systems (Yu et al. 2013; Da-Silva et al. 2017), maintenance of K+/Na+ ratio (Lai et al. 2014; Deng et al. 2016), and osmolyte accumulation (Shi et al. 2013) under stress conditions. Analysis of the effect of H2S on different physiological processes in cells led to the use of potential donors of H2S such as (p-methoxyphenyl)morpholino-­ phosphinodithioic acid (GYY4137) and sodium hydrosulfide (NaHS). Exogenous application of a H2S donor can improve tolerance to various abiotic stress in plants (Corpas and Palma 2020). Since plant genotype, concentration and duration of the treatment, and application methods affect plant behavior to H2S, it is important to investigate endogenous dynamics of H2S and potential effects of exogenous H2S donors. The key role of H2S in regulation of plant responses to unwanted environmental conditions in plants is given in Fig. 2.1.

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Fig. 2.1  Generalized illustration of key role of hydrogen sulfide in the regulation of response to environmental stress in plants. ABA abscisic acid, ABC ATP-bindind cassette, APX ascorbate peroxidase, AsA ascorbate, CAT catalase, CS cysteine synthase, DCD D-cysteine desulfhydrase, GPX glutathione peroxidase, GR glutathione reductase, GSH glutathione, H2S hydrogen sulfide, LCD L-cysteine desulfhydrase, NO nitric oxide, POD peroxidase, Pro proline, ROS reactive oxygen species, SiR sulfite reductase, SOD superoxide dismutase, Tre trehalose

2.2  Biosynthesis of H2S in Plants The synthesis and emission of H2S have been known long ago in corn, cucumber and soybean leaves (Wilson et al. 1978), and the enzymes involved in the biosynthesis of H2S have also been identified (Riemenschneider et al. 2005). It was shown that compared to young plants, leaves from old plants contain higher H2S levels (Rennenberg and Filner 1983). In plants, H2S is mainly synthesized from L/D-­cysteine or sulfide with enzymes including D-cysteine desulfhydrase (DCD),

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L-cysteine desulfhydrase (LCD), cysteine synthase (CS), sulfite reductase (SiR) and 𝛽-cyanoalanine synthase (CAS) (Li 2015). It was indicated that LCD activity was altered under stress conditions in Arabidopsis thaliana (Lai et  al. 2014; Da-Silva et  al. 2017). Similarly, DCD activity was regulated by different stress conditions in different crops (Cui et  al. 2014; Hu et  al. 2015; Liu et  al. 2016). L-cysteine converted into pyruvate by LCD releases ammonia, pyruvate, and H2S (Romero et al. 2013). In plants, H2S biosynthesis from D-cysteine is catalyzed by DCD (Huo et al. 2018). DCD activity is also regulated by abiotic stress conditions in different plants (Cui et al. 2014; Hu et al. 2015; Liu et al. 2016). The mitocondrial enzyme CAS, catalyses the reaction of L-cysteine with cyanide (CN−) releasing H2S (García et al. 2010; Yi et al. 2012). During ethylene production, CN− level is regulated by CAS enzyme in plants (O’Leary et  al. 2014, Li 2015). Cysteine synthetase (CS) found in mitochondria, cytosol and chloroplasts, is responsible for the reversible reaction between L-cysteine and acetate to synthesize O-acetyl-Lserine and release of H2S (Li 2015). It has been well observed that all the enzymes involved in H2S biosynthesis play decisive roles under different stress conditions (Singh et al. 2020).

2.3  Physiological Functions of H2S in Plants It is well established that H2S regulates a plethora of physiological processes in plants (Corpas and Palma 2020). Studies showed that H2S has a direct or indirect role in various physiological processes like root organogenesis (Zhang et al. 2009a; Mei et al. 2019), seed germination (Zhang et al. 2008), stomatal movement (García-­ Mata and Lamattina 2001; Zhang et al. 2020a), photosynthesis (Chen et al. 2011), senescence (Zhang et al. 2011), and fruit ripening (Ge et al. 2017; Muñoz-Vargas et  al. 2018). H2S has a role  not only in physiological processes but also in the response to the abiotic and biotic stress conditions (Shi et al. 2015; Banerjee et al. 2018). Being nucleophilic in nature, H2S can react with O2⦁―, H2O2 and/or peroxynitrite playing significant role in reducing cellular oxidative stress (Paul and Roychoudhury 2020). It has been observed that enzymatic and non-enzymatic antioxidant defense systems are activated by both endogenous increase in H2S levels and exogenously applied H2S that assist the plant in improving tolerance to abiotic stress conditions (Shi et al. 2015). Interaction of H2S has also been observed with thiol (-SH) group of cysteine residues of proteins via post-translational modification, termed as persulfidation (Corpas and Palma 2020). Enzymes such as ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO), LCD, ascorbate peroxidase (APX), glyceraldehyde 3-­phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), actin and catalase (CAT) are some of the plant protein targets which H2S affect their function through persulfidation (Corpas and Palma 2020). Under in vitro conditions, the behavior of a protein depends on the application method of the H2S donor to the plant. Functional interaction/competition of H2S with other molecules like

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melatonin, NO, and phytohormones determines its action in the cellular system (Corpas and Palma 2020). In the forthcoming pages the enhancement of plant tolerance through H2S under drought, salinity, heavy metal accumulation and temperature stress is discussed.

2.4  Effect of H2S on Plants Under Salt Stress Plant growth, development and yield are adversely affected by salt and non-ionic osmotic stresses in semi-arid and arid regions of the world. Salt stress causes oxidative stress which leads to DNA damage, protein inactivation, and lipid peroxidation. It has been shown that H2S allows plants to alleviate the detrimental effects of high salinity through decreasing the impact of oxidative stress leading to reduced lipid peroxidation, electrolyte leakage, and protein oxidation under salt stress conditions in plant cells. Decreased oxidative stress and lipid peroxidation by H2S has been found to be associated with enhanced activities of antioxidant enzymes (Yu et al. 2013; Khan et al. 2017, 2018). After pretreatment of wheat seeds with different concentrations of H2S donor NaHS, the injuries resulted from salinity were ameliorated (Bao et al. 2011). When the endogenous H2S was inhibited by scavengers, the activities of CAT, superoxide dismutase (SOD) and APX were decreased under salt stress (Da-Silva et al. 2017). Regarding the sequence of action, H2S has been observed to perform downstream of NO against oxidative stress, and this effect was shown to alleviate salt stress in tomato plants (Da-Silva and Modolo 2018) and metal stress in mung bean (Khan et al. 2020). In maize leaves, an increase in the ratio of reduced and oxidised form of ascorbate (ascorbate/dehydroascorbate, respectively) and glutathione (reduced glutathione/glutathione disulfide) was induced by NaHS pretreatment under salt application (Shan et  al. 2014). When H2S was supplied exogenously, oxidative stress effect declined in bermudagrass, alfalfa, cucumber, and strawberry (Wang et al. 2012; Christou et al. 2013; Shi et al. 2013; Lai et al. 2014; Sun and Luo 2014). It was also shown that, in rice, H2S donor NaHS increased glyoxalase I and II activities which affected methylglyoxylate levels (Mostofa et al. 2015a). Lipooxygenase activity was also decreased with NaHS application. In mangrove plants, NaHS treatment promoted the enhancement of the membrane lipid stability and quantum efficiency of photosystem II under salinity (Liu et al. 2019b). Exogenous H2S application helped to keep high K+/Na+ ratio under salt stress. After pre-application of 50μM NaHS, it was shown that K+/Na+ ratio was increased in NaCl treated wheat plants (Deng et al. 2016). The NaHS application enhanced the expression of plasma membrane Na+/H+ antiporter genes in strawberry plants under high salinity (Christou et al. 2013). In alfalfa, under salinity NaHS treatment maintained K+/Na+ homeostasis (Lai et al. 2014) and improved seed germination (Wang et al. 2012). The root growth inhibition under salinity was also eliminated by NaHS in Arabidopsis (Li et  al. 2014). An increase was observed in stomatal conductance, relative water

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content (RWC) and photosynthetic rate in the leaves of strawberry with pre-­ application of NaHS before exposure to salt (Christou et al. 2013). NaHS also raised total protein, carotenoid, and chlorophyll contents under salt conditions in rice (Mostofa et al. 2015a), and also, sucrose, proline, and other soluble carbohydrates were accumulated with H2S application under salt stress in bermudagrass (Shi et al. 2013). The Salt Overly Sensitive (SOS) pathway is important for the regulation of cytosolic ion levels (Abid et al. 2017). Transcription levels of genes encoding plasma membrane H+-ATPase, vacuolar H+-ATPase subunit β and vacuolar Na+/H+ antiporter were increased with H2S, and low Na+ levels were maintained in the cytosol of barley root cells (Chen et al. 2015). Plasma membrane Na+/H+ antiporter (SOS1) expression maintained the Na+ and K+ homeostasis together with a decline in lipid peroxidation and ROS generation. SOS3 gene encodes Ca2+ binding protein which activates SOS3-SOS2 protein kinase. This enzyme phosphorylates SOS1 (Na+/H+ antiporter) protein (Prajapati and Vadassery 2016). When H2S was applied, osmotic stress and salt tolerance were promoted via the SOS pathway (Chen et al. 2015). Under salt stress, H2S treatment upregulates SOS2, SOS3, and SOS4 gene expressions that assist in the influx and efflux of ions (Yang and Guo 2018).

2.5  Response of Plants to H2S Under Drought Stress Prolonged shortage of water or decrease in soil moisture content leads to drought that negatively affects growth and yield of plants. Drought stress causes a decrease in leaf area, photosynthesis, stomatal movement, and stem elongation. It was indicated that H2S regulates drought resistance in plants. Role of H2S in alleviating drought stress has also been observed in plants at various stages of their life cycle (Khan et al. 2018; Zhang et al. 2010c). It was shown that in germinating wheat seedlings APX and CAT activities were increased while H2O2 and malondialdehyde (MDA, oxidative stress marker) levels were decreased (Zhang et al. 2010c). In another study, dehydroascorbate reductase (DHAR), glutathione reductase (GR), APX, and γ-glutamylcysteine synthetase (γ-ECS) activities were enhanced and H2O2 and MDA levels were decreased under drought with NaHS priming in wheat (Shan et al. 2011). Exogenously applied H2S reduced the MDA and H2O2 levels, and antioxidant enzyme activities were regulated by lowering the ROS accumulation in wheat (Ma et al. 2016). Related with the participation of H2S in ABA biosynthesis, it was indicated that ABA biosynthetic gene (TaZEP, TaNCED, TaAAO, and TaSDR) expression levels were up-regulated in roots and leaves of wheat under drought (Ma et al. 2016). In Arabidopsis, H2S fumigation and NaHS enhanced expression of drought-­ associated genes that contributed to drought resistance, and a reduction in stomatal aperture was observed (Jin et  al. 2011). When treated with 80μM H2S, higher expression of drought-responsive genes, C-repeat binding factor 4 (CBF4), dehydration-­responsive element-binding 2A (DREB2A), and dehydration-responsive

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element-­binding 2B (DREB2B) was determined in Arabidopsis (Jin et  al. 2011). Stomatal closure is one of the key responses of plants to drought. In Vicia faba, stomatal closure was induced by H2S donors, NaHS or GYY4137 in guard cells (GarcíaMata and Lamattina, 2001). It was shown that H2S participates in ABA-dependent signaling pathways under drought and enhances RWC in V. faba plants (García-Mata and Lamattina 2001). In Spinacia oleracea application of NaHS counteract the drought-induced changes and restored RWC, photosynthesis and efficiency of PSII. It was suggested that H2S-induced drought tolerance was implemented through improved osmoprotectant levels and stimulation of polyamine biosynthesis (Chen et al. 2016a). Various sulfur-containing compounds (NaHS, Na2SO4, Na2SO3, Na2S) were used to understand the role of NaHS under stress conditions in sweet potato plants. It was proved that only NaHS mitigates the stress compared to other sulfur-containing chemicals. When sweet potato seedlings that were grown under PEG (15% w/v) were sprayed with 0.05  mM NaHS, leaves showed increased chlorophyll content and improved SOD, CAT and APX, with low MDA and H2O2 levels with respect to control (Zhang et al. 2009b). Pretreatment with NOSH (donates NO and H2S to plants) resulted in acclimation to drought stress in Medicago sativa plants (Antoniou et  al. 2020). Under drought stress, H2S induced the KEGG pathways  of ‘protein processing in endoplasmic reticulum’, ‘ribosome biogenesis in eukaryotes’, and ‘cyanoamino acid metabolism’. Higher expression levels of functional genes involved in signal transduction, drought-induced transcription factors and protein kinases were detected in wheat plants when pretreated with NaHS (Li et al. 2017). In Arabidopsis, it was proposed that LCD, NFS1, NFS2, DCD1 and DES1 genes were upregulated after drought exposure in order to generate H2S which then regulates the ABA-dependent signaling pathway, modulates a set of drought responsive genes and drought associated miRNAs, and improves tolerance to drought (Shen et al. 2013). H2S regulates the ATP-binding cassette (ABC) transporters in A. thaliana, Impatiens walleriana, and V. faba. It improves drought tolerance through regulation of the ABA signaling pathway (García-Mata and Lamattina 2001; Aimar et al. 2011). It was shown that Arabidopsis plants exhibited H2S regulated expression of miR393 and its target genes including transport inhibitor response 1 (TIR1) and auxin signaling F-box protein (AFB1, AFB2 and AFB3) under drought (Shen et al. 2013; Shi et al. 2015).

2.6  Effect of H2S Under Heavy Metal Stress Elements like chromium (Cr), cadmium (Cd), zinc (Zn), and copper (Cu) are HMs with a density greater than 6 g·cm−3 (Li et al. 2016). The main sources for the heavy metal accumulation in the environment are dust from smelters, fertilizers, industrial wastes, and sewage sludge which affect the cultivation area and therefore growth and productivity of crop plants. Heavy metal contamination of soil also poses negative effects on the food chain because of the biological accumulation (Khan et al. 2008). Accumulation of HMs causes protein oxidation, enzyme inactivation and lipid peroxidation in plants (Jaishankar et al. 2014; Shahid et al. 2014). Under Cd

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stress, plants synthesize H2S as a common response (Tiwari and Lata 2018). The enhanced level of endogenous H2S decreases oxidative stress and therefore improves growth of rice plants under Cd stress. In H2S-treated Cd-stressed rice plants, RWC was enhanced with stomatal conductance and transpiration resulting in improvement of photosynthesis (Mostofa et al. 2015b). In foxtail millet seedlings, the H2S biosynthesis occurred after exposure to Cr6+ ion, and expression levels of DCD and LCD were increased during the first 12 h of Cr6+ exposure while expression levels of genes involved in calcium (Ca2+) transport and signaling were enhanced in the same stress period. It was indicated that H2S dependent pathway interacts with Ca2+ signaling in order to activate the antioxidant system and improve Cr tolerance (Fang et al. 2014). In another study, activity of antioxidant enzymes, amylase, and esterase, and germination rates were improved with NaHS application under Cr toxicity (Zhang et al. 2010a). NaHS treatment resulted in a decrease in Cr content, and a decline in H2O2 and MDA levels together with a rise in antioxidant enzyme activities in cauliflower (Ahmad et al. 2020). In tomato, Cr toxicity caused an increase in ROS, and altered cysteine and H2S metabolisms (Alamri et al. 2020a). Additionally, it was demonstrated that endogenous H2S triggered by NO conferred tolerance of tomato seedlings to Cr stress and improved photosynthesis and plant growth (Alamri et al. 2020a). The inhibition of Al, Cu and Zn stress by NaHS has been indicated in various plants (Zhang et al. 2008; Chen et al. 2013; Liu et al. 2016). In Chinese cabbage roots, endogenous H2S levels were increased with Cd stress. Additionally, after NaHS pretreatment, excessive ROS were scavenged by upregulation of antioxidant system, and cell death induced by Cd stress was inhibited (Zhang et  al. 2015). Although the effect of H2S in the regulation of heavy metal transport is not clearly determined, a decrease in Cd accumulation in the cytoplasm and vacuolar Cd influx was observed in Populus euphratica. Pretreatment of P. euphratica cells with NaHS resulted in up-regulated activities of antioxidant enzymes, reduced Cd entry to the cytoplasm, and increased Cd sequestration into the vacuole. Also, this pretreatment brought about a decrease in Cd influx by H2O2-activated plasma membrane Ca2+ channels in Populus plants (Sun et al. 2013). An increase in endogenous H2S with different concentrations of Cd was observed in bermudagrass (Shi et  al. 2014). Pretreatment with NaHS and sodium nitroprusside (SNP, a NO donor) in alfalfa roots caused a decrease in Cd toxicity. Both applications in the medium ameliorated oxidative damage through changes in the SOD (Cu/Zn), POD and APX enzymes. It was also shown that the NO scavenger cPTIO (2–4-carboxyphenyl-4,4,5,5-­ tetramethylimidazoline-­ 1-oxyl-3-oxide) reversed the effect of NaHS (Li et  al. 2012a). These results suggested the crosstalk between H2S and NO. In another study endogenous NO and H2S levels were increased with Cd stress. Exogenous treatments of NaHS and SNP enhanced the antioxidant enzyme activities (SOD, CAT, and POD) that maintained the membrane integrity, and therefore provided plant acclimation to oxidative stress in wheat plants (Kaya et al. 2020). In tomato critical role of endogenous H2S during Cd stress was demonstrated by differential biochemical responses to Cd and higher LCD activity after low dose of Cd (Alamri et al. 2020b). The overexpression of the DCD gene in Arabidopsis, caused endogeneous H2S accumulation and decreased Cd and ROS concentrations (Zhang

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et al. 2020b). NaHS treatment promoted a reduction in electrolyte leakage, decrease in MDA and H2O2 content and induction of antioxidant enzyme activities together with the improvement of iron uptake in strawberry (Kaya and Ashraf 2019). In pepper plants, NaHS treatment increased the growth, yield, proline content and water status and enhanced the activity of antioxidant enzymes under Zn toxicity (Kaya et al. 2018). In Solanum nigrum seedlings the growth inhibition caused by Zn stress was reversed by H2S. Under Zn toxicity, H2S regulates the expression of metallothioneins which chelate excess Zn (Liu et al. 2016). While expression of Zn-regulated transporter (ZRT), iron-regulated transporter gene (IRT)-like protein (ZIP) and natural resistance associated macrophage protein (NRAMP) were declined, antioxidant enzymes expression were increased with H2S (Liu et al. 2016). It was shown that the accumulation of mercury (Hg) was inhibited by H2S in the roots of rice plants. H2S mitigated Hg toxicity in leaves of rice and strengthened non-protein thiol and metallothionein synthesis to fix Hg in roots, and also reduced Hg transport to shoots (Chen et  al. 2017). Cucumber seedlings exhibited higher pectin methylesterase (PME) activity under boron (B) stress (Wang et al. 2010). The regulatory role of H2S under B toxicity was reported but that effect was only in the first 4 h of B toxicity (Wang et al. 2010). When seedlings of wheat were primed by NaHS under Cu stress, a decrease in MDA levels and electrolyte leakage was observed. The expression levels and activities of DHAR, APX, monodehydroscorbate reductase (MDHAR), GR, L-galactono-1,4-lactone dehydrogenase, and γ-ECS were inclined. Therefore, it was suggested that H2S regulates ascorbate-glutathione (AsA-GSH) metabolism under Cu stress (Shan et al. 2012). When wheat plants were pretreated with NaHS under aluminum (Al) stress, antioxidant capabilities were increased (Zhang et al. 2010b). In barley plants, NaHS ameliorated Al toxicity through suppression of Al uptake (Dawood et al. 2012). H2S also improved the antioxidative system of barley plants through the expression of Al-activated citrate transporter (HvAACT1) and protein expression level of H+-ATPase (Chen et al. 2013). The antioxidant capacity was enhanced and therefore oilseed rape seedling growth was promoted by H2S under Al stress (Qiao et  al. 2015). When NaHS was exogenously applied with arsenate, the arsenate induced toxic effect on growth and photosynthesis was ameliorated in pea seedlings, and components of AsA–GSH cycle were also upregulated by NaHS treatment (Singh et al. 2015). The enzyme activities of AsA-­ GSH cycle were also increased after NaHS application (Singh et al. 2015). These results demonstrate important role of H2S in alleviating metal-induced toxicity.

2.7  Effect of H2S Under Temperature Stress Temperature stress is divided into three categories: high, chilling and freezing temperature. Temperature stress causes retardation in growth, germination rate, and photosynthesis that can result in the death of the plant. The underlying mechanisms related with the plant adaptive responses to temperature stress is imperative to

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understand. There are various studies in literature, related to the role of H2S in the induction of tolerance of plants to different temperature stresses.

2.7.1  Low Temperature Stress Low temperature stress (freezing: 0 °C) significantly inhibit plant growth and development (Tang et  al. 2020). Plants exhibit differential responses to low temperatures. In agricultural areas, unexpected and unseasonal cold or frost can damage or kill plants. Extreme temperature conditions induce over-­ production of ROS. However, in bermuda grass, H2S production was also observed in addition to ROS at 4  °C (Shi et  al. 2013). LCD overexpressing transgenic Arabidopsis plants produced higher H2S and had higher overall abiotic stress tolerance (Shi et al. 2015). When Lamiophlomis rotata plants were grown at different altitudes, expression levels of DCD, LCD, CAS, and O-acetylserine (thiol) lyase (OAS-TL) genes were increased. It was suggested that H2S production elevates with the rise in atmospheric pressure (Ma et  al. 2015). When cucumber leaves were exposed to 4 °C for 12 h, the H2S content was increased via increased expression of H2S generating enzymes. The expression of genes encoding cucurbitacin C, a triterpenoid secondary metabolite, were up regulated by exogenous H2S at 4 °C. It was also shown that H2S modified proteins through persulfidation and therefore acted as a positive regulator of cucurbitacin C synthesis (Liu et al. 2019a). The chilling tolerance of Vitis vinifera L., was promoted by exogenous H2S. This effect was related to the increase in the SOD activity and mRNA abundance of ICE1 and CBF3 and decrease in MDA and O2⦁― radicals (Fu et al. 2013). During chilling storage, treatment with NaHS resulted in an increase in antioxidant capacity and proline content of banana fruit (Luo et al. 2015). It was shown that H2S was included in the cold stress-dependent upregulation of mitogen-activated protein kinase (MAPK) gene in Arabidopsis. MAPK4 expression level was highly affected by H2S. The expression levels of the cold responseive genes CBF3, ICE1, COR15A, and COR15B were regulated by both by MPK4 and H2S. It was also observed that MPK4 was involved in H2S-related inhibition of stomatal opening in response to cold stress (Du et al. 2017). The degradation of carotenoids and chlorophyll was alleviated and the photoinhibition of PSII and PSI were prevented in Lowbush blueberry with the treatment of NaHS at low temperatures (Tang et al. 2020). Transgenic Arabidopsis plants, overexpressing LCD, produced higher H2S and had higher stress tolerance. But it was also shown that LCD knockdown mutants were sensitive to the stress (Shi et al. 2015).

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2.7.2  High Temperature Stress Together with global warming, heat stress is started to be more damaging in plantable areas. Heat stress induces cellular damage through protein degradation, changes in gene expressions and enzyme inactivation (Yang et al. 2016). Heat shock proteins (HSPs) are produced as a general response under high temperature stress (Singh et  al. 2020). At high temperatures, H2S biosynthesis was also triggered in many plants (Fu et al. 2013). Expression of LCD which lead to biosynthesis of H2S was elevated when tobacco seedlings were exposed to 35 °C. The consequent level of H2S continued after 3 days (Chen et al. 2016b). Exposure of strawberry plants to 42 °C after pretreatment with NaHS resulted in a rise in the expression levels of CAT, APX, Mn-SOD and GR enzymes, heat shock proteins and aquaporins (Christou et al. 2014). Heat tolerance was improved by NaHS pretreatment in tobacco suspension cultured cells, through the entry of extracellular Ca2+ into the cells across the plasma membrane and activity of intracellular calmodulin (CaM) (Li et  al. 2012b). Endogenous H2S generation was regulated by Ca2+ and CaM by activating LCD activity in tobacco cells. Furthermore, it was indicated that thermotolerance can be improved by H2S donor NaHS, Ca2+ and CaM treatment (Li et al. 2015). Irrigation of maize seedlings with H2S showed higher temperature stress -tolerance with the accumulation of proline. Upon H2S treatment, proline 5 carboxylase synthease activity was increased, whereas proline dehydrogenase activity was decreased (Li et al. 2013). NaHS treatment improved endogenous H2S levels in roots and coleoptiles under normal conditions. NaHS pretreatment also increased the seed germination ratio at 38 °C in maize (Li et al. 2013). Under high temperature, in germinating maize seedlings, NaHS application brought about not only activation of antioxidant enzymes but also induction of non-­ enzymatic antioxidants (AsA and GSH) levels. The levels of osmolytes were also improved in NaHS pretreated seedlings (Zhou et al. 2018). Pretreatment of wheat seedlings with NaHS, resulted in an increase in expression levels and activities of antioxidant enzymes and soluble sugars with an antiparellel reduction in H2O2 and MDA levels under heat (38 °C) stress (Yang et al. 2016). Exogenous application of NaHS or GYY4137 to poplar plants increased the S-nitrosoglutathione reductase activity and diminished reactive oxygen/nitrogenspecies-induced damage at high temperature (Cheng et al. 2018). NaHS or GYY4137 application in Arabidopsis, enhanced the seed germination rate and increased the ABI5 gene expression at high temperatures (Chen et al. 2019).

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2.8  Conclusion As an important gaseous molecule, H2S is effective at low concentrations in signaling cascades in plants. H2S provides protection against various abiotic stresses by induction of the antioxidant system and signaling pathways via the process of persulfidation. Exogenously applied H2S has a positive effect on agronomically important plants under stress conditions. However, it is necessary to further investigate the effect of H2S-induced tolerance mechanisms and H2S signaling during plant adaptive responses using transcriptome, proteome and metabolome approaches. The usage of these comprehensive approaches will be informative to understand the effect of H2S on plant tolerance to abiotic stresses. Interaction of H2S with other signaling molecules at biochemical and molecular levels under various environmental conditions need to be elucidated.

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Liu Z, Li Y, Cao C et  al (2019a) The role of H2S in low temperature-induced cucurbitacin C increases in cucumber. Plant Mol Biol 99(6):535–544 Liu YL, Shen ZJ, Simon M et al (2019b) Comparative proteomic analysis reveals the regulatory effects of H2S on salt tolerance of mangrove plant Kandelia obovata. Int J Mol Sci 21(1):118 Luo Z, Li D, Du R et  al (2015) Hydrogen sulfide alleviates chilling injury of banana fruit by enhanced antioxidant system and proline content. Sci Hortic 183:144–151 Ma L, Yang L, Zhao J et al (2015) Comparative proteomic analysis reveals the role of hydrogen sulfide in the adaptation of the alpine plant Lamiophlomis rotata to altitude gradient in the Northern Tibetan Plateau. Planta 241(4):887–906. https://doi.org/10.2307/43564214 Ma D, Ding H, Wang C et al (2016) Alleviation of drought stress by hydrogen sulfide is partially related to the abscisic acid signaling pathway in wheat. PLoS One 11:e0163082 Mei Y, Zhao Y, Jin X et al (2019) L-Cysteine desulfhydrase-dependent hydrogen sulfide is required for methane-induced lateral root formation. Plant Mol Biol 99(3):283–298 Mostofa MG, Saegusa D, Fujita M et al (2015a) Hydrogen sulfide regulates salt tolerance in rice by maintaining Na+/K+ balance, mineral homeostasis and oxidative metabolism under excessive salt stress. Front Plant Sci 6:1055 Mostofa MG, Rahman A, Ansary MMU et  al (2015b) Hydrogen sulfide modulates cadmium-­ induced physiological and biochemical responses to alleviate cadmium toxicity in rice. Sci Rep 5:14078 Muñoz-Vargas MA, González-Gordo S, Cañas A et al (2018) Endogenous hydrogen sulfide (H2S) is up-regulated during sweet pepper (Capsicum annuum L.) fruit ripening. In vitro analysis shows that NADP-dependent isocitrate dehydrogenase (ICDH) activity is inhibited by H2S and NO. Nitric Oxide 81:36–45 Niu L, Liao W (2016) Hydrogen peroxide signaling in plant development and abiotic responses: crosstalk with nitric oxide and calcium. Front Plant Sci 7:230 O’Leary B, Preston GM, Sweetlove LJ (2014) Increased β-cyanoalanine nitrilase activity improves cyanide tolerance and assimilation in Arabidopsis. Mol Plant 7:231–243 Pandey AK, Gautam A (2020) Stress responsive gene regulation in relation to hydrogen sulfide in plants under abiotic stress. Physiol Plant 168:511–525 Pandey G, Sharma N, Sahu PP et al (2016) Chromatin-based epigenetic regulation of plant abiotic stress response. Curr Genomics 17:490–498 Paul S, Roychoudhury A (2020) Regulation of physiological aspects in plants by hydrogen sulfide and nitric oxide under challenging environment. Physiol Plant 168:374–393 Prajapati R, Vadassery J (2016) Role of calcium in perception of salt stress. Endocytobiosis Cell Res 27:21–24 Qiao ZJ, Jing T, Liu ZQ et al (2015) H2S acting as a downstream signaling molecule of SA regulates Cd tolerance in Arabidopsis. Plant Soil 393:137–146 Rennenberg H, Filner P (1983) Developmental changes in the potential for H2S emission in cucurbit plants. Plant Physiol 71:269–275 Riemenschneider A, Wegele R, Schmidt A et  al (2005) Isolation and characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana. FEBS J 272:1291–1304 Romero LC, García I, Gotor C (2013) L-cysteine desulfhydrase 1 modulates the generation of the signaling molecule sulfide in plant cytosol. Plant Signal Behav 8:e24007 Shahid M, Pourrut B, Dumat C et al (2014) Heavy-metal-induced reactive oxygen species: phytotoxicity and physicochemical changes in plants. Rev Environ Contam Toxicol 232:1–44 Shan CJ, Zhang SL, Li DF et al (2011) Effects of exogenous hydrogen sulfide on the ascorbate and glutathione metabolism in wheat seedlings leaves under water stress. Acta Physiol Plant 33:2533–2540 Shan C, Dai H, Sun Y (2012) Hydrogen sulfide protects wheat seedlings against copper stress by regulating the ascorbate and glutathione metabolism in leaves. Aust J Crop Sci 6:248–254 Shan C, Liu H, Zhao L et al (2014) Effects of exogenous hydrogen sulfide on the redox states of ascorbate and glutathione in maize leaves under salt stress. Biol Plant 58:169–173

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Shen J, Xing T, Yuan H et al (2013) Hydrogen sulfide improves drought tolerance in Arabidopsis thaliana by microRNA expressions. PLoS One 8:e77047 Shi H, Ye T, Chan Z (2013) Exogenous application of hydrogen sulfide donor sodium hydrosulfide enhanced multiple abiotic stress tolerance in bermudagrass (Cynodon dactylon [L]. Pers.). Plant Physiol Biochem 71:226–234 Shi H, Ye T, Chan Z (2014) Nitric oxide-activated hydrogen sulfide is essential for cadmium stress response in bermudagrass (Cynodon dactylon (L). Pers.). Plant Physiol Biochem 74:99–107 Shi H, Ye T, Han N et al (2015) Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis. J Integr Plant Biol 57(7):628–640 Singh VP, Singh S, Kumar J et al (2015) Hydrogen sulfide alleviates toxic effects of arsenate in pea seedlings through up-regulation of the ascorbate–glutathione cycle: possible involvement of nitric oxide. J Plant Physiol 181:20–29 Singh S, Kumar V, Kapoor D et al (2020) Revealing on hydrogen sulfide and nitric oxide signals co-ordination for plant growth under stress conditions. Physiol Plant 168(2):301–317 Sun YD, Luo WR (2014) Effects of exogenous hydrogen sulphide on seed germination and seedling growth of cucumber (Cucumis sativus) under sodium bicarbonate stress. Seed Sci Technol 42:126–131 Sun J, Wang R, Zhang X et al (2013) Hydrogen sulfide alleviates cadmium toxicity through regulations of cadmium transport across the plasma and vacuolar membranes in Populus euphratica cells. Plant Physiol Biochem 65:67–74 Tang X, An B, Cao D et al (2020) Improving photosynthetic capacity, alleviating photosynthetic inhibition and oxidative stress under low temperature stress with exogenous hydrogen sulfide in blueberry seedlings. Front Plant Sci 11:108 Tiwari S, Lata C (2018) Heavy metal stress, signaling, and tolerance due to plant-associated microbes: An overview. Front Plant Sci 9:452 Wang R (2002) Two’s company, three’s a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J 16(13):1792–1798 Wang BL, Shi L, Li YX et al (2010) Boron toxicity is alleviated by hydrogen sulfide in cucumber (Cucumis sativus L.) seedlings. Planta 231(6):1301–1309 Wang Y, Li L, Cui W et al (2012) Hydrogen sulfide enhances alfalfa (Medicago sativa) tolerance against salinity during seed germination by nitric oxide pathway. Plant Soil 351:107–119 Wilson LG, Bressan RA, Filner P (1978) Light-dependent emission of hydrogen sulfide from plants. Plant Physiol 61:184–189 Yang Y, Guo Y (2018) Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytol 217:523–539 Yang M, Qin B, Ma X et al (2016) Foliar application of sodium hydrosulfide (NaHS), a hydrogen sulfide (H2S) donor, can protect seedlings against heat stress in wheat (Triticum aestivum L.). J Integr Agric 15:2745–2758 Yi H, Juergens M, Jez JM (2012) Structure of soybean 𝛽-cyanoalanine synthase and the molecular basis for cyanide detoxification in plants. Plant Cell 24:2696–2706 Yu LX, Zhang CJ, Shang HQ et  al (2013) Exogenous hydrogen sulfide enhanced antioxidant capacity, amylase activities and salt tolerance of cucumber hypocotyls and radicles. J Integr Agric 12:445–456 Zhang H, Sonnewald U (2017) Differences and commonalities of plant responses to single and combined stresses. Plant J 90:839–855 Zhang H, Hu LY, Hu KD et  al (2008) Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. J Integr Plant Biol 50:1518–1529 Zhang H, Tang J, Liu XP et al (2009a) Hydrogen sulfide promotes root organogenesis in Ipomoea batatas, Salix matsudana and Glycine max. J Integr Plant Biol 51:1084–1092 Zhang H, Ye YK, Wang SH et al (2009b) Hydrogen sulphide counteracts chlorophyll loss in sweetpotato seedling leaves and alleviates oxidative damage against osmotic stress. Plant Growth Regul 58:243–250

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Zhang H, Hu LY, Li P et al (2010a) Hydrogen sulfide alleviated chromium toxicity in wheat. Biol Plant 54:743–747 Zhang H, Tan ZQ, Hu LY et al (2010b) Hydrogen sulfide alleviates aluminum toxicity in germinating wheat seedlings. J Integr Plant Biol 52:556–567 Zhang H, Wang MJ, Hu LY et al (2010c) Hydrogen sulfide promotes wheat seed germination under osmotic stress. Russ J Plant Physiol 57:532–539 Zhang H, Hu SL, Zhang ZJ et al (2011) Hydrogen sulfide acts as a regulator of flower senescence in plants. Postharvest Biol Technol 60:251–257 Zhang L, Pei Y, Wang H et  al (2015) Hydrogen sulfide alleviates cadmium-induced cell death through restraining ROS accumulation in roots of Brassica rapa L ssp. Pekinensis. Oxid Med Cell Longev 2015:804603 Zhang J, Zhou M, Ge Z et al (2020a) Abscisic acid-triggered guard cell L-cysteine desulfhydrase function and in situ hydrogen sulfide production contributes to heme oxygenase-modulated stomatal closure. Plant Cell Environ 43:624–636 Zhang Q, Cai W, Ji TT et  al (2020b) WRKY13 enhances cadmium tolerance by promoting D-CYSTEINE DESULFHYDRASE and hydrogen sulfide production. Plant Physiol 183:345–357 Zhou ZH, Wang Y, Ye XY et al (2018) Signaling molecule hydrogen sulfide improves seed germination and seedling growth of maize (Zea mays L.) under high temperature by inducing antioxidant system and osmolyte biosynthesis. Front Plant Sci 9:1288

Chapter 3

Functional Interaction of Hydrogen Sulfide with Nitric Oxide, Calcium, and Reactive Oxygen Species Under Abiotic Stress in Plants Yu V. Karpets, Yu E. Kolupaev, and M. A. Shkliarevskyi

Abstract  In response to the action of various abiotic stresses, increase in endogenous content of hydrogen sulfide (H2S) has been reported in the plants, that is associated with the upregulation of gene expression and increase in the activity of L-cysteine desulfhydrase and other enzymes of H2S synthesis. The knowledge about the role of H2S in the functioning of signaling network of plant cells are required for understanding of fundamental mechanisms of plant adaptation and creation of new technologies of induction of plant resistance to stress factors. In plants, H2S is in complex functional interaction with other mediators of signaling network. Its synthesis is induced with the participation of calcium (Ca2+) and calmodulin. At the same time, Ca2+ ions are involved in H2S signal transduction to the genetic apparatus. Reactive oxygen species (ROS) can also induce H2S synthesis and take part in signal transduction during plant adaptive responses. Intensification of ROS generation by plant cells under the influence of H2S is substantially caused by increase in the activity of NADPH oxidase. H2S and nitric oxide as signal mediators are in remarkably close functional interaction. They can react chemically with each other, compete during the interaction with thiol groups of proteins by participation in reactions of persulfidation and S-nitrosation, and influence the synthesis of each other. In this review the role of functional crosslinks of H2S with other mediators in the formation of concrete adaptive reactions of plants, in particular, in the activation of antioxidative system, is discussed. Keywords  Hydrogen sulfide · Calcium · Reactive oxygen species · Nitric oxide · Persulfidation

Y. V. Karpets (*) · Y. E. Kolupaev · M. A. Shkliarevskyi Dokuchaev Kharkiv National Agrarian University, Kharkiv, Ukraine e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_3

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3.1  Introduction Hydrogen sulfide (H2S), along with nitric oxide (NO) and carbon monoxide (CO), belongs to key gasotransmitters in cells of plants and animals (He and He 2014; Yamasaki and Yamasaki and Cohen 2016; Singh et al. 2020). Activation of adaptive reactions of plants is one of the brightest physiological effects of H2S (Singh et al. 2020; Li et  al. 2016b; Shi et  al. 2015; Khan and Alzuaibr 2019). However, the mechanisms of induction of stress-protective systems of plants under the influence of H2S, direct targets of its influence, signaling, and hormonal mediators providing physiological effects, remain poorly studied. Studying the mechanisms of action of H2S, as well as other gasotransmitters, is complicated due to absence of concrete receptors and multilevel functional interactions with other signaling molecules, in particular with NO and reactive oxygen species (ROS) (Hancock 2019), and also calcium ions (Ca2+) (Li 2019). Possibly, in many cases ROS act as the mediators in realization of signaling processes, in which H2S is involved. So, the adaptive reactions of wheat plants to dehydration stress can be formed with the participation of consecutive increase in the content of H2S and ROS in cells (Shan et al. 2018). On the other hand, ROS, in particular hydrogen peroxide (H2O2), can be the mediators in the realization of signaling effects of exogenous H2S (Kolupaev et al. 2017a). The crosstalk between H2S and NO can be also caused by the presence of general binding sites with protein targets, namely, thiol groups (-SH). Nitric oxide is capable to change the state of these groups by S-nitrosation, and H2S by persulfidation (Hancock 2019). One more mechanism of interaction between H2S and NO is their influence on the synthesis of each other. In a number of studies, it is reported that physiological effects of H2S can be mediated by NO and vice versa (Wang et al. 2012a, b; Singh et al. 2015). The bivalent Ca2+ is known as the universal second cellular messenger (Kaur and Gupta 2005; Kolupaev et  al. 2015). The data are available on the participation of Ca2+ in H2S formation, and in the transduction of H2S signals into genetic apparatus (Li et al. 2015a, b). Interplay of H2S with the complex network of hormonal signaling, including key stress phytohormones has been well studied (Li et  al. 2015d; Chen et  al. 2016b; Shan et  al. 2017; Ziogas et  al. 2018). The experimental data indicate the role of abscisic acid (ABA) in the biosynthesis of H2S (Shi et al. 2015). On the other hand, the role of H2S, as mediator in the realization of ABA effects is also shown (Shan et al. 2017). Also, H2S can participate in the implementation of protective action of salicylic acid on plants (Li 2015b; Li et al. 2015d). The awareness on the position of H2S in the signaling network of plant cells is required to understand the fundamental mechanisms of adaptation of plants and creation of new tools of induction of plant resistance to stress factors. The analysis of such data was also the main objective of the present review.

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3.2  Biosynthesis of H2S in Plants The transformation of L-cysteine (L-Cys) into pyruvate with the release of H2S and ammonium is considered as one of the main pathways of H2S synthesis in plants (Romero et al. 2013). This reaction is catalyzed by L-cysteine desulfhydrase (LCD; EC 4.4.1.1), which is localized in cytoplasm, plastids, and mitochondria (Li 2013; Riemenschneider et al. 2005). Formation of H2S from D-Cys under the influence of D-cysteine desulfhydrase (DCD; EC 4.4.1.15), localized in cytoplasm, is also possible (Riemenschneider et  al. 2005; Guo et  al. 2016). Besides, H2S can be synthesized by the reduction of sulfite with the participation of sulfite reductase (EC 1.8.7.1) (Li 2013), using ferredoxin as reductant of sulfur (Table 3.1). Biosynthesis of H2S in plants can also occur with the participation of β-cyanoalanine synthase (EC 4.1.1.9) (Li et al. 2016b). This mitochondrial enzyme catalyzes the condensation reaction of L-cysteine and cyanide (CN−) with the release of H2S (Li 2015b). It is considered that its main function is to control toxic CN− content in the cells. The enzyme Cys-synthase (EC 2.5.1.47), localized in the cytosol, mitochondria, and chloroplasts, usually synthesizes Cys from O-acetyl-L-­ serine (OAS) and sulfide (Lisjak et  al. 2013), but the enzyme can also make the contribution to H2S formation. It catalyzes reversible reaction between L-Cys and acetate with the formation of OAS and H2S (Wirtz Hell 2006; Alvarez et al. 2010; Li 2015b). At last, synthesis of H2S is also possible with the participation of carbonic anhydrase (EC 4.2.1.1) (Wang et  al. 2012b), which catalyzes decomposition of carbonyl sulfide to carbon dioxide and H2S. It is remarkable that transformation of tobacco plants, with the gene encoding O-acetylserine(thiol)lyase (OASTL, also known as cysteine synthase), caused the resistance to high concentration of

Table 3.1  Ways of synthesis of hydrogen sulfide in plants Enzymes L-cysteine desulfhydrase (EC 4.4.1.1) D-cysteine desulfhydrase (EC 4.4.1.15) β-Cyanoalanine synthase (EC 4.1.1.9) Sulfite reductase (EC 1.8.7.1) Cysteine synthase (EC 2.5.1.47)

Reactions L-cysteine + H2O→H2S + NH3 + pyruvate

Localization Plastids, mitochondria

References Romero et al. (2013), and Li (2015a, b)

D-cysteine + H2O→H2S + NH3 + pyruvate

Cytoplasm

Romero et al. (2013) and Li (2015a)

L-cysteine + CN−→H2S + β- cyanoalanine

Mitochondria

Li et al. (2016b)

SO32− + ferredoxin red. → H2S + ferredoxin ox. + H2O L-cysteine + acetate → H2S + O-acetylserine

Chloroplasts

Li (2013)

Cytoplasm, chloroplasts

Wirtz and Hell (2006) and Li (2015a) Rudenko et al. (2015), and Yamasaki and Cohen (2016)

Carbonic anhydrase Carbonyl sulfide + (EC 4.2.1.1) H2O→H2S + CO2

Chloroplasts, cytoplasm

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exogenous H2S. In this connection it is supposed that this enzyme participates in the regulation of endogenous content of H2S and in the detoxification of exogenous H2S (Tai and Cook 2000).

3.3  C  hanges in Endogenous Level of H2S in Plants in Response to Stresses To date, an increase in the synthesis of H2S in plants under the influence of various stresses has been shown (Singh et al. 2020), and also possibility of the induction of resistance by exogenous H2S. In recent years, the point of view, according to which the universality of main cellular protective systems is the basis of plants resistance to stresses of various nature, finds more and more experimental confirmations (Corpas and Palma 2020). In particular, the antioxidative system, which includes the wide spectrum of compounds both the proteinaceous and low-molecular nature, belongs to such general protective systems, formed in the course of long evolution (Blokhina et al. 2003; Shao et al. 2008; Kolupaev et al. 2019d). Also the accumulation of multifunctional low-molecular protectors, first of all, proline and some other amino acids (Szabados and Savoure 2010; Liang et  al. 2013), and also betaines (Sakamoto and Murata 2002; Fitzgerald et  al. 2009; Wu et  al. 2012) and sugars, including oligosaccharides (Asada 1999; Ramel et al. 2009), are considered as the important factors for the plants resistance to various stresses. These compounds perform not only osmoprotective, but also membrane-protective, antidenaturative and antioxidative functions. Along with the osmoprotective and antioxidative systems, the stress proteins (heat shock proteins – HSP), which primarily have a chaperone effect, are considered as the universal protective components of plant cells (Basha et al. 2004; Wang et al. 2004; Wahid et al. 2007; Su and Li 2008). The functioning of all these protective systems in the stress conditions of various natures is modified to some extent with the participation of H2S (Bhuyan et al. 2020). In the following pages an attempt is made to shed light on the role of H2S in plants in response to various abiotic stresses, and also effect of these stresses on endogenous functioning of H2S is explored.

3.3.1  Low Temperature Stress and H2S Low temperature stress significantly affects the metabolism of plants. In response to low temperatures, the upregulation of gene expression of LCD/DCD and increase in content of H2S occurred in leaves of Arabidopsis plants (Shi et al. 2015). The same effect was also found in cucumber leaves in response to the temperature of 4 °C (Liu et al. 2019). The increased synthesis of H2S in grapes has also been reported to play crucial role in the adaptation of plants to cold stress (Fu et al. 2013). The increase in

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plants resistance to low temperature stress by the treatment with H2S donors has also been reported (Table  3.2). So, under the influence of H2S donor, sodium hydrosulfide (NaHS), the frost resistance of Bermuda grass (Cynodon dactylon L.) increased (Shi et  al. 2014). At the same time, the rise of activity of antioxidant enzymes – catalase (CAT), guaiacol peroxidase (GPOX), and glutathione reductase (GR) was registered in leaves. The pretreatment with NaHS, under the usual conditions and under the chilling hardening, caused the increase in frost resistance of wheat and rye plants (Kolupaev et al. 2019b) with a concomitant increase in the activity of GPOX and CAT, and the content of sugars and proline (Table  3.2). It is also found that under the chilling hardening of wheat plants, H2S donor caused significant increase in the activity of phenylalanine ammonium lyase (PAL), an enzyme that converts L-phenylalanine into trans-cinnamic acid, which is the precursor of most secondary metabolites (Kolupaev et al. 2018). And also, under the influence of H2S donor, the total content of flavonoid compounds (Aghdam et  al. 2018), which have extremely high antioxidative activity, increased in the plants (Khlestkina 2013). The fumigation of banana fruits with H2S, released from NaHS at the low-­ temperature storage, raised their storing and reduced the accumulation of malondialdehyde (MDA), the product of lipid peroxidation (Luo et al. 2015). At the same time, the treatment with H2S caused increase in the activity of PAL and total content of phenolic compounds. Besides, the activity of superoxide dismutase (SOD), GPOX, CAT, ascorbate peroxidase (APX), and glutathione reductase (GR) increased in fruits (Luo et al. 2015). The authors also associate such an increase in the resistance of banana fruits to low-temperature storage with H2S-induced alterations in proline metabolism i.e. increase in the activity of Δ1-pyrroline-5-­carboxylate synthetase and decrease in the activity of proline dehydrogenase (Luo et al. 2015).

3.3.2  High Temperature Stress and H2S The rise in the content of H2S in response to high temperature has been reported in various plants (Chen et al. 2016b). But also, in a number of plants, the increase in heat resistance under the influence of exogenous H2S is shown (Table  3.2). The application of NaHS solution elevated the survival of corn plantlets after the action of potentially lethal high temperature (Li et al. 2014; Li and Zhu 2015; Zhou et al. 2018). The treatment of wheat plants with NaHS caused upregulation of gene expression of antioxidant enzymes – APX and CAT - followed by heat resistance (Yang et  al. 2016). The induction of heat resistance of wheat coleoptiles by H2S donor caused elevation in the activity of SOD, CAT, and GPOX (Kolupaev et al. 2017a, b). In the experiments with the plants of strawberry, NaHS treatment caused induction of gene expression of HSP90, HSP80, HSP70, and aquaporins in roots (Christou et al. 2014). Such effect was followed by the increase in heat resistance of

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Table 3.2  Induction of plant protective systems by hydrogen sulfide

Stress Low temperature

H2S donors (mM) Species Vitis vinifera NaHS L. (0.1)

Low temperature

Cynodon dactylon L.

NaHS (0.5)

Low temperature

Triticum aestivum L.

NaHS (0.1; 0.5)

Low temperature

Secale cereale L.

NaHS (0.1; 0.5)

High temperature High temperature

Nicotiana tabacum L. Triticum aestivum L.

NaHS (0.1) NaHS (0.1)

High temperature

Triticum aestivum L.

NaHS (0.15)

High temperature High temperature

Zea mays L.

GYY4137 (0.1) NaHS (0.7)

Zea mays L.

High temperature

Fragaria x ananassa

NaHS (0.1)

High temperature

Spinacia oleracea

NaHS (0.1)

Dehydration Triticum aestivum L.

NaHS (0.3)

Dehydration Triticum aestivum L.

NaHS (0.1; 0.5)

Physiological effects Reduction of cold-induced oxidative damages, increased expression of cold-sensitive genes VvICE1 and VvCBF3 Increased activity of antioxidative enzymes, increased plant survival after freezing Increased activity of antioxidative enzymes and phenylalanine ammonialyase, rise in content of proline and sugars, increased plant survival after freezing Increased activity of antioxidative enzymes, rise in content of proline and sugars, increased plant survival after freezing Reduction of heat-induced oxidative damages of cells Increased survival of coleoptile segments, increased activity of antioxidative enzymes Increased gene expression of cu/Zn-, , Mn-, and Fe-superoxide dismutases, and ascorbate peroxidase Reduction of heat-induced oxidative damages Increased activity of catalase, guaiacol peroxidase, superoxide dismutase, and ascorbate peroxidase, rise in content of glutathione (GSH) and ascorbic acid Induction of gene expression of heat shock proteins (HSP70, HSP80, HSP90) and aquaporins Increased level of osmoprotectants and upregulation of several genes related to soluble sugar biosynthesis. Intensifying of gene expression and increase in activity of ascorbate peroxidase, glutathione reductase and monodehydroascorbate reductase at the osmotic stress Enhanced plant growth during soil drought, increased superoxide dismutase activity, content of proline and anthocyanins

References Fu et al. (2013)

Shi et al. (2013, 2014) Kolupaev et al. (2018, 2019b)

Kolupaev et al. (2019b) Li et al. (2015b) Kolupaev et al. (2017b) Yang et al. (2016) Li et al. (2013) Li et al. (2014)

Christou et al. (2014) Chen et al. (2016a, b) Shan et al. (2018)

Kolupaev et al. (2019a) (continued)

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Table 3.2 (continued)

Stress Species Dehydration Triticum aestivum L.

H2S donors (mM) NaHS (0.1)

Dehydration Cynodon dactylon L.

NaHS (0.5)

Dehydration Fragaria × ananassa

NaHS (0.1)

Salt stress

Cd2+

Triticum aestivum L.

NaHS (0.1) NaHS (0.1) NaHS (0.1) NaHS (0.005– 0.02) NaHS (0.9)

Cd2+

Hordium vulgare L.

NaHS (0.2)

Ni2+

Cucurbita pepo L. Zea mays L.

Triticum aestivum L. Arabidopsis thaliana L. Medicago sativa L. Cucumis sativus L.

Cr6+

Brassica oleracea L.

NaHS (0.1) NaHS (0.5) NaHS (0.2)

Pb2+

Brassica oleracea L. Glycine max L.

NaHS (0.2) NaHS (0.025)

Cr6+

Al3+

Physiological effects Increase in relative water content, reduction of oxidative damages, rise in content of glycine-betaine, sugars and polyamines Reduction of oxidative stress and cell damages, via modulating of antioxidative enzymes and accumulation of osmolytes (proline, sucrose and soluble total sugars) Mitigation of PEG-induced oxidative damages, increase in gene expression of ascorbate peroxidase, catalase and Mn-SOD, rise in content of ascorbate and GSH, as well as increase in ratio of GSH/GSSG Enhanced seed germination in NaCl presence Increased activity of antioxidative enzymes Maintaining ion homeostasis Maintaining ion

Intensifying seeds germination, mitigation of oxidative stress, increase in activity of antioxidative enzymes Intensifying growth, increase in activity of superoxide dismutase and peroxidase in roots homeostasis Increased activity of antioxidative enzymes, enhanced plant growth Increased activity of antioxidative enzymes, reduced oxidative damage Mitigation of oxidative damages, increase in chlorophyll content and activity of antioxidative enzymes Intensifying seeds germination, mitigation of oxidative damages Reducing accumulation of aluminum ions in tissues, mitigation of growth-­ inhibiting effect

References Chen et al. (2016a)

Shi et al. (2013)

Christou et al. (2013)

Ye et al. (2015) Shi et al. (2015) Wang et al. (2012a, b) Jiang et al. (2019) Huang et al. (2016) Fu et al. (2019) Valivand et al. (2019) Kharbech et al. (2017) Ahmad et al. (2020) Chen et al. (2018) Wang et al. (2019)

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the plants. It is remarkable that the synthesis of H2S, induced by the osmotic stress, increased under the influence of exogenous ABA (Shan et al. 2017).

3.3.3  Dehydration Stress and H2S The upregulation of gene expression of LCD and DCD with a concomitant increase in H2S production in Arabidopsis plants under drought conditions has been established (Jin et  al. 2011). In wheat plants in response to the treatment of polyethylene glycol (PEG), the rise of H2S level was also registered (Shan et  al. 2018). It is remarkable that synthesis of H2S, induced by the osmotic stress, was further increased by the exogenous application of ABA (Shan et al. 2017). In the etiolated wheat plant, treated with NaHS, the upregulation of gene expression and increase in the activity of APX, GR, and monodehydroascorbate reductase (MDHAR) was observed under osmotic stress (Shan et al. 2018). At the same time, treatment with the H2S biosynthesis inhibitor, aminooxyacetic acid (AOAA), reduced the increase in activity of APX, GR, dehydroascorbate reductase, and MDHAR under osmotic stress (Shan et al. 2018). The treatment of wheat plants with NaHS solution before the soil drought promoted the increase in SOD activity and prevented the decrease in the activity of CAT and GPOX in the leaves (Kolupaev et al. 2019a). Also, under drought stress, the H2S donor increased the content of low-molecular weight protectors – proline and anthocyanins in the leaves of wheat plants. Pre-treatment with NaHS also causes increase in peroxidase (POX), CAT, and GR activity, and also elevation in reduced glutathione (GSH) pool in Bermuda grass under PEG 6000-induced osmotic stress (Shi et  al. 2013). Application of the same H2S donor provoked a significant increase in the content of ascorbate (AsA) and GSH, and also a rise of the ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) under the conditions of osmotic and salt stresses in strawberry plants (Christou et al. 2013). Glycinebetaine and trehalose, having osmoprotective and antioxidative properties, exhibited elevation in their levels in drought stressed plants of Spinacia oleracea L. when treated with H2S donor (Chen et al. 2016a, b).

3.3.4  Salt Stress and H2S Salt stress is considered as one of the menaces that limit crop production worldwide. Role of H2S in plants under the regime of salinity has also been studied and an increase in the number of transcripts of LCD and the endogenous H2S content in alfalfa under the influence of stress concentrations of sodium chloride was observed (Lai et al. 2014). At the same time, the activity of DCD in these conditions practically did not change. In bean plants, the activation of LCD and DCD and the increase in H2S content were necessary for salt-induced stomatal closure (Ma et al. 2019).

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The positive influence of exogenous H2S under salt stress is revealed in the experiments with plants of different taxonomic groups (Table 3.2). The activation of SOD, CAT, APX, and GPOX by the donor of H2S was found in wheat plants under salt stress (Shan et  al. 2018; da-Silva and Modolo 2018; Ding et  al. 2019). The treatment of Arabidopsis plants with NaHS increased their survival rate under the long-time exposure to salt stress and the rise in the activity of SOD, GR, and nonspecific POX (Shi et al. 2015). Maintenance of higher K+/Na+ ratio is one of the vital strategies that plants adopt to counter salinity. The application of H2S to salt-­ stressed plants of lucerne and cucumber positively influenced the ionic homeostasis and increased the K+/Na+ ratio (Wang et al. 2012b; Jiang et al. 2019).

3.3.5  Heavy Metals (HMs) and H2S Increased industrial expansion and use of various metallic items in human life led to the discharge of their waste products to arable lands. These waste products contain various toxic chemical compounds including heavy metals (HMs). Excessive accumulation of HMs in agricultural soil led to their entry in the plants. Presence of HMs in the plant system not only affect growth and development of the crops but they also affect human health through entering the food chain. Role of H2S in the protection of plants from these toxic HMs has been studied by a number of researchers. The rise in H2S content in plant tissues, exposed to toxic HMs, is shown. The upregulation of gene expression encoding both LCD and DCD and increase in the content of H2S was observed in Italian millet (Setaria italica L.) under the influence of toxic concentration of chromium (Cr6+) ions (Fang et al. 2014). The increase in H2S content in response to cadmium (Cd2+) in Bermudas grass (Cynodon dactylon L.) and cucumber was also revealed (Shi et al. 2014; Kabala et al. 2019). In soy plants, increase in the activity of LCD/DCD and β-cyanoalanine synthase under the influence of aluminium ions (Al3+) was shown (Wang et  al. 2019). Apparently, due to activation of these enzymes there was a rise in the endogenous content of H2S. In roots and leaves of zucchini (Cucurbita pepo L.) the increase in the content of H2S under the nickel (Ni2+) toxicity was reported (Valivand et al. 2019). With the use of plants of different species, the increase in resistance to Cd2+, Ni2+, 6+ Cr , lead (Pb), and other toxic metals under the influence of exogenous H2S is shown (Table 3.2). The increased germination of wheat seeds, caused by H2S donor under the toxic influence of Cd2+, was followed by increase in the activity of GPOX, APX, and CAT (Huang et al. 2016). The protective effect of NaHS against Cd2+ in barley plants was also followed by the activation of the components of antioxidative system – superoxide dismutase and peroxidase (Fu et al. 2019). Stress-protective influence of NaHS on the corn plants, exposed to Cr6+, was manifested in the rise of the activity of SOD and POX, and also in the stabilization of CAT activity (Kharbech et al. 2017). In various concentrations NaHS exerted the positive impact on the plants of Brassica oleracea L., exposed to Pb toxicity at the early stages of development.

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Protective effects of H2S were expressed in intensifying seed germination in stress conditions, activation of plant growth and reduction of their oxidative damages (Chen et al. 2018). The treatment of plants of the same species with NaHS exerted positive impact on the toxic action of Cr6+ too (Table 3.2). At the same time, the decrease of Cr6+ content and also the reduction in the amount of H2O2 and MDA in the plant organs was registered (Ahmad et al. 2020).

3.4  Functional Interactions of H2S with Ca2+ Ions Calcium (Ca2+) is recognized as the universal mediator in cellular responses of plants and animals (Kaur and Gupta 2005; Kim et  al. 2009). Precisely, cytosolic Ca2+ serves as the link for many signaling pathways, providing the formation of signaling network of plant cell (Kaur and Gupta 2005; Johnson et al. 2014). Influx of Ca2+ ions into the cytosol takes place through various types of Ca2+ channels, located in the plasmalemma, tonoplast, membranes of endoplasmic reticulum, chloroplasts, and nuclear membrane (Demidchik et al. 2002; Demidchik 2012; Kim et al. 2009). Transduction of stress signals with the participation of Ca2+ occurs due to its ability to interact differentially with cellular proteins. It has been observed that about 700 various proteins can be directly and indirectly involved in the Ca2+ signaling (Hepler 2005). As it is known, CaM is extremely important highly conserved receptor of Ca2+ (Chin and Means 2000) and plants have a specific set of CaM isoforms and CaM-like proteins (Ranty et al. 2006). Thus, apparently, the different molecular forms of CaM have unequal affinity to proteins, which they modify, primarily, to protein kinases. CaM-regulated proteins also include some transcription factors, proteins of ion channels, enzymes of common metabolic cycles, proteins of cytoskeleton, chaperones and proteins taking part in the transduction of hormonal signals (Ranty et al. 2006; Kim et al. 2009). Calcium is involved in both, the regulation of H2S synthesis, and the transduction of its signals (Fig. 3.1). It is established that exogenous Ca2+, which increases the heat resistance of cells of suspension culture of tobacco, caused the rise in H2S concentration that was associated with the elevation in the activity of LCD (Li et al. 2015b). The activation of LCD enzyme in the cells of suspension culture of tobacco in the conditions of heat stress was suppressed by the pretreatment with the Ca2+chelator EGTA [ethylene glycol-bis(b-aminoethylether)-N,N,N′,N′-tetraacetic acid] that testifies the role of Ca2+ in inducing the synthesis of H2S under heat stress. And also, high-temperature induction of H2S formation was suppressed by the application of the CaM antagonists chlorpromazine (CPZ) and trifluorpromazine (TFP) that indicates the importance of CaM in H2S synthesis (Li et al. 2015b). In Arabidopsis, Cr6+-induced synthesis of H2S was dependent on Ca2+ and CaM (Fang et al. 2017). Treatment of plants with EGTA alleviated the expression level of LCD encoding gene and increased in H2S content (Fang et  al. 2017). Similarly, it is shown that Ca2+, in association with CaM, interplays with the transcriptional factor of TGA3

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H2S

Catalase

L/D-cysteinedesulfhydrase

Ca2+-channels

H2O2

NADPHoxidase

Ca 2+

Nitrate reductase

NO

Changes in redox homeostasis

Induction of antioxidative and other protective systems Fig. 3.1  Functional interaction of hydrogen sulfide (H2S) with the other signal mediators. The activity and/or gene expression of key enzymes of H2S synthesis L- and D-cysteine desulfhydrases can be activated with the participation of calcium (Ca2+), hydrogen peroxide (H2O2), and also nitric oxide (NO). At the same time, H2S can activate NADPH oxidase by the influence on the Ca2+ homeostasis, and directly inhibit the catalase, causing thereby the rise in the content of H2O2 and other ROS. In turn, ROS can induce NO formation by activating nitrate reductase enzyme. The changes in the redox homeostasis, caused by the rise in H2O2 and NO content, can induce the antioxidant and other protective systems of plants

that is required for its binding with the promoter of gene of LCD and upregulates its expression. Similar mechanisms were also reported to operate during the elevation in H2S concentration in response to Ni2+ in zucchini roots and leaf cells . The increase in the content of H2S, induced by Ni2+, was abolished by EGTA, verapamil (a Ca2+-channel blocker), and a CaM-antagonist TFP (Valivand et  al. 2019). Protective effect of NaHS on the Italian millet plants, exposed to Cr6+ toxicity, was increased after their treatment with Ca2+ (Fang et  al. 2014). On the contrary, the application of EGTA manifested in the reduction of physiological influence of H2S (Valivand et al. 2019). The LCD mutant of Arabidopsis plants differed by the weak influx of Ca2+ into the cytosol in response to drought stress (Jin et  al. 2013), suggesting that H2S exerts its impact through affecting Ca2+ channels.

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The increase in heat resistance of suspension culture of tobacco (Nicotiana tabacum) cells under the influence of H2S donor NaHS was abolished by the Ca2+chelator EGTA, the Ca2+-channel blocker lanthanum chloride (LaCl3, La3+), and also the CaM antagonists – CPZ and TFP (Li et al. 2012b). On the other hand, the stress-­ protective action of H2S donor was increased by the simultaneous application of the Ca2+ ionophore A23187 or exogenous Ca2+. These results are in agreement with the data on the suppression of H2S influence on the heat resistance of wheat coleoptile cells, ROS generation, and activity of antioxidative enzymes by the treatment with EGTA or inhibitor of phospholipase C, which participates in the regulation of Ca2+ homeostasis (Kolupaev et al. 2017a). It is remarkable that there is information on the increase in the activity of a number of antioxidant enzymes viz. SOD, CAT, APX, and GR under the influence of CaM (Li 2019). A significant increase in gene expression and activity of SOD, GPOX, and GR by the application of H2S donor and exogenous Ca2+ was also observed in Setaria italica (Fang et  al. 2014). Moreover, the treatment of strawberry plants with NaHS in the conditions of iron deficiency led to the increase in Ca2+ content in their leaves (Kaya and Ashraf 2019). The influence of H2S on the stomatal movement is also mediated by Ca2+. It is shown that treatment of Arabidopsis leaves with the Ca2+ chelator, permeable for cells, eliminated the stomatal closing caused by the organic donor of H2S CYY4137 [morpholin-4-ium-4-methoxyphenyl-(morpholino)-phosphinodithioate] (Honda et al. 2015). Wang et al. (2016), reported that Ca2+ influx into the cytosol during the activation of anion channels of S-type in guard cells of Arabidopsis was influenced by exogenous H2S. Besides, it was established that the protein kinase OST1 mediates the H2S-induced activation of these channels and stomatal closing (Wang et al. 2016). It was shown that the treatment of Arabidopsis leaves with the extracellular Ca2+chelator, EGTA or with the Ca2+-channel blocker, La3+, completely eliminated the influence of H2S donor on the state of stomata (Yastreb et al. 2019). And also, the influence of H2S donor on stomata of Arabidopsis leaves was suppressed by the treatment with neomycin  – an inhibitor of phosphatidylinositol-dependent phospholipase C, which is involved in the formation of 1,4,5-inositol-3-phosphate, which is capable to influence the state of intracellular Ca2+ channels (Yastreb et al. 2019). The antagonist of CaM, CPZ caused a little reduction on the effect of H2S on stomata. Thus, various antagonists of Ca2+ differentially influence the impact of H2S on the size of stomatal aperture. Their presence not only effected the effect of NaHS on aperture width, but also partial opening of stomata was registered (Yastreb et al. 2019).

3.5  Crosstalk of H2S with ROS The term ROS is referred to the plurality of crossly turning chemically active species of oxygen, the majority of which have a short life span. Among these are free-­ radical species – superoxide anion-radical (О2•–), hydroxyl (•ОН) and hydroperoxyl

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(HO2▪), etc., and also neutral molecules, such as Н2О2, singlet oxygen (1О2) etc. (Gill and Tuteja 2010). Generation of ROS in plants takes place during the functioning of electron-­ transport chains of chloroplasts and mitochondria (Kolupaev and Karpets 2014). But the major level of Н2О2 is synthesized in peroxisomes (Foyer and Noctor 2003). The phosphoglycolate, which enters the peroxisomes from chloroplasts, is converted into the glycolate with the participation of phosphoglycolate phosphatase, and then under the influence of glycolate oxidase – into glyoxylate, and at the same time the Н2О2 is formed. Hydrogen peroxide can be also formed during β-oxidation of fatty acids in peroxisomes (Gerhardt 1983; Kreslavski et al. 2012). In the generation of ROS, the NADPH oxidase (EC 1.6.3.1), localized in the plasma membrane, has special value. This enzymatic complex reduces the molecular oxygen with the formation of О2•– using as electron donor NADPH (Glyan’ko and Ischenko 2010; Gautam et al. 2017). The genome of Arabidopsis contains 10 representatives gene families of membrane-bound subunit of RBOH (Respiratory Burst Oxidase Homologs), designated as AtRboh (A, B, C, D, E, F, G, H, J, L) (Torres et al. 2002; Kaye et  al. 2011). In the genome of rice, nine genes coding catalytic subunit of NADPH oxidase are revealed. Genes of RBOH are also identified in the genomes from some other plants (Demidchik 2012). The plant membrane-bound subunit contains N-terminal link, which binds Ca2+ ions. The NADPH oxidase activity is regulated by the direct and indirect participation of Ca2+ ions and phosphatidic acid, and by phosphorylation of catalytic subunit (Kolupaev et al. 2015). Along with the NADPH oxidase, the extracellular peroxidases of class III can make contribution to the formation of ROS in the apoplast. These enzymes can show the oxidase activity bound to transfer electrons from reductants [for example, NAD(P)H] to the oxygen, with the formation of О2•– and H2O2 (Minibayeva et al. 2009). Besides, ROS are formed in the reactions catalyzed by polyamine oxidases and other flavin oxidases. It is well known that all the ROS in higher concentration cause oxidative stress in the plant cells that trigger damage to biomacromolecules and cellular structures (Gill and Tuteja 2010; Kolupaev et al. 2019d). At the same time, ROS in physiological concentration are obligatory participants of the cellular signaling. Recently, the obtained data suggest that activity of almost all classes of known effector proteins of signaling systems are influenced by the increasing concentration of ROS (Pradedova et  al. 2017). Functions of these proteins are regulated by these ROS either directly or indirectly by the redox regulation of their phosphorylation/ dephosphorylation, and also due to the change of content of other signal mediators (Ca2+, NO, H2S) (Kolupaev et  al. 2015; Li et  al. 2015c2016a, b; Pradedova et al. 2017). The main signaling functions of ROS are associated with the role of H2O2, which is recognized as a secondary messenger (Tkachuk et al. 2012). Molecules of Н2О2 have considerable life span and have ability to move in cells on significant distances. It possesses rather low reaction ability and high penetration through membranes due to lack of the charge (Tkachuk et al. 2012). Besides, the evidence of the possibility

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of facilitated diffusion of Н2О2 with the help of proteins aquaporins are appearing (Bienert et al. 2007; Miller et al. 2010). So far, a few (especially for plant objects) information has also been obtained on the participation of other ROS in signaling (Kreslavski et al. 2012; Trchounian et al. 2016; Waszczak et al. 2018; Hasanuzzaman et al. 2020). Possibly, ROS in many cases act as the mediators in the regulation of signaling processes involving H2S. ROS, in particular Н2О2, can be mediators in the generation of protective effects of exogenous H2S (Fig. 3.1). It has been reported that increase of heat resistance of cells of wheat coleoptiles, caused by H2S and to which the transitional increase of ROS generation preceded, was eliminated under the influence of antioxidants butylhydroxytoluene and dimethyl thiourea (DMTU) (Kolupaev et  al. 2017b). By inhibitory methods it is shown that exogenous H2S causes increase in the generation of О2•– which is dependent on the NADPH oxidase, but not on extracellular peroxidase, and its subsequent transformation into the Н2О2 by SOD (Kolupaev et al. 2017a). Probably, the induction of NADPH oxidase is not the only mechanism that intensifies the accumulation of Н2О2 under the influence of H2S. The increase in activity of glucose-6-phosphate dehydrogenase can also be the reason for increased accumulation of ROS in cells under the influence of H2S (Hancock 2019). It has also been shown the H2S, present in plant peroxisomes, inhibits CAT activity that leads to the rise in cellular concentration of H2O2 (Corpas et al. 2019). Authors also tested that H2S reduced the CAT activity, exerted from various objects in both plants (Aroca et al. 2015) and animals (Yang et al. 2013). Such effect can be associated with the persulfidation of enzymes molecules (Corpas et al. 2019), a process in which reactive Cys residues on target proteins are modified via conversion of -SH to a persulfide group (-SSH). The role of Н2О2 as a signaling molecule, which may be downstream to H2S in the signaling cascade, was shown under UV-B radiation in barley plants (Li et al. 2016a). In response to UV-B irradiation, plants accumulate increased level of diamine putrescine that causes the rise in synthesis of H2S and Н2О2. It is remarkable that the rise in content of H2S was not eliminated with DMTU, the scavenger of Н2О2. At the same time, the effect of stabilization of activity of antioxidant enzymes (SOD, CAT, APX, and GPOX), caused by putrescine, was eliminated by both DMTU, and the scavenger of H2S hypotaurine. In this regard, authors consider that the signal couple «H2S–H2O2» is necessary for regulation of the action of UV-B and exogenous putrescine, thus the H2O2 functions downstream to H2S in the signaling chain (Li et al. 2016a). Apparently, H2S is also the component of signaling chain, which is induced by the influence of another polyamine – spermidine (Li et al. 2019). It is remarkable that this polyamine is the link, which is required for the manifestations of signaling effects of H2O2 and H2S on plants under the influence of drought. The intensifying synthesis of H2S in clover plants, caused by dehydration, was reduced by the inhibitor of spermidine synthesis. However, the increase in the concentration of H2O2, affected by spermidine, was not removed by hypotaurine, the scavenger of H2S. At the same time, scavenger of H2O2, DMTU eliminated the increase in the

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content of H2S, caused by spermidine (Li et al. 2019). Probably, the H2O2 can induce the synthesis of H2S in plant cells. The treatment of seeds of Jatropha curcas with H2O2 activated the germination and also caused the rise in LCD activity and content of H2S (Li et al. 2012a). Arabidopsis plants also exhibited enhanced expression of genes related with LCD/DCD in response to H2O2 treatment (Shi et al. 2015). In the processes of signaling involved in stomatal closure under the influence of ABA or jasmonic acid, H2S functions downstream to H2O2 (Jin, Pei 2015). Apparently, the signal of H2S is secondary in relation to H2O2 during stomatal closure under darkness. Thus, the effect of stomatal closure was induced both by darkness, and by exogenous H2O2, and H2S. Antioxidants eliminated the influence of darkness on the stomatal aperture, however in the presence of H2S donor such effect was not shown (Ma et al. 2019). Also, antioxidants eliminated the increase in activity of LCD/DCD and rise of contents of H2S in the cells, affected by darkness. In mutants atrbohD, atrbohF, atrbohD/F the enhanced synthesis of H2S in the drought conditions was not shown (Wang et al. 2012a, b). The treatment of wheat plants with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) decreased the content of H2S, caused by the action of osmotic stress (10% of PEG 6000) (Shan et al. 2018). Under the influence of DPI, as well as under the treatment with the H2S biosynthesis inhibitor AOAA, the increase in the activity of antioxidant enzymes, caused by dehydration, was leveled. Authors make the conclusion about the reciprocal influence of H2S and H2O2 as the signaling mediators in the processes of plants adaptation to the osmotic stress (Shan et al. 2018). In general, there is reason to believe that forward and reverse crosslinks exist between H2S and H2O2 as signaling molecules, since the inhibitor of H2S synthesis, AOAA leveled the formation of H2O2, induced by drought in wheat plants (Shan et al. 2018). Under the action of salt stress on bean plants, the content of both H2S and H2O2 increased. Thus antagonists of H2S did not influence the effect of increase in the amount of H2O2, while exogenous antioxidant AsA and CAT, and also inhibitors of NADPH oxidase and POX (DPI and salicylhydroxamic acid, respectively), leveled the rise in the content of H2S, caused by the salt stress in guard cells of stomata (Ma et al. 2019). The authors suggested that H2S is located downstream to H2O2 in the signaling chain, induced by the salt stress. In general, there are grounds to suggest the complex interaction between H2O2 and H2S and they work synergistically and can increase the synthesis of each other. On the other hand, antagonistic relations between H2S and ROS can also be noticed where we can find the induction of antioxidant system by exogenous H2S (Shan et al. 2011; Hancock and Whiteman 2014). In this regard, it is supposed that the rise in the content of GSH, ascorbic acid and activity of a number of antioxidant enzymes, affected by H2S, should lead to the decrease of ROS content and to the modification of ROS signals (Hancock and Whiteman 2014). There are also data on direct reaction of H2S with ROS, including О2•–, •ОН, and H2O2 (Li and Lancaster 2013). However, the importance of direct contribution of H2S in the regulation of ROS remains doubtful, since its concentration in cells is much lower, than other antioxidants (Hancock and Whiteman 2014). In addition, it is known that the speed of reaction of H2S with oxidizers is exceptionally low.

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3.6  H2S and NO as Interdependent Signal Mediators Currently, NO is considered as one of the most important components of signaling network in plant and animal cells (Mur et al. 2013), which, as well as H2S, belong to gasotransmitters category (Kolupaev et al. 2019c). NO in plants can be formed by reductive or oxidative pathways (Corpas and Barroso 2017). The reductive pathway operates through the use of nitrate or nitrite as substrates in the reactions catalyzed by nitrate reductase, nitrite reductase, bound to the plasma membrane, and xanthine oxidoreductase, localized in peroxisomes (Gupta and Kaiser 2010; Farnese et  al. 2016). It is supposed that NO synthesis, dependent on nitrate reductase, plays a role in the adaptation of plants to various stresses, in particular, low temperatures, dehydration and hypoxia (Jeandroz et al. 2016; Khan et al. 2017, 2020a). In plants, the mechanism of NO formation through oxidative pathway from L-arginine still remains the subject of discussion, since the homologues of NO synthase of animals are found only in green algae, but not in the higher plants (Li and Lancaster 2013). Currently, it is considered that terrestrial plants do not have the NO synthase, typical for animals. There is an opinion, that this gene was lost during the course of evolution (Jeandroz et  al. 2016). In this regard the question of the mechanisms of L-arginine-dependent NO synthesis in higher plants remains open (Kolbert et al. 2019). It is possible that the proteins, those are different from NO synthase, but capable to generate NO using L-arginine as a substrate, are present in the peroxisomes of higher plants. This reaction, as well as the catalyzed by the NO synthase in animals, can occur in the presence of NADPH, FMN, FAD, CaM, and Ca2+ ions (Corpas and Barroso 2017; Gupta et  al. 2020). Recently, not only L-arginine, but also polyamines and hydroxylamine are considered as the main substrates for the formation of NO through oxidative pathway (Hancock and Whiteman 2014; Liu et al. 2019). It is supposed that these reactions may be catalyzed by polyamine oxidase (Flores et al. 2008). The signaling molecules H2S and NO are in exceptionally close functional interaction (Khan et al. 2018, 2020b). One of such mechanisms is direct reaction of these molecules where H2S can react with NO and peroxynitrite (ONOO−) (Lisjak et al. 2013; Carballal et al. 2011). The result of this interaction will be their mutual neutralization and weakening of signals, and also appearance of a new compound  – nitrosothiol, which may have signaling properties itself (Whiteman et al. 2006). The interaction between H2S and NO can also be caused by the presence of common binding sites with protein targets that includes thiol groups. NO is capable to change the state of these groups by S-nitrosation, and H2S – by the persulfidation (Hancock 2019) (Fig.  3.2). The trans-persulfidation or trans-nitrosation of thiol groups is also possible (Corpas 2019). One example of proteins whose state is regulated by this way is one of the key antioxidant enzymes, APX. It may be exposed to both S-nitrosation, and persulfidation (Corpas 2019). At the same time, apparently, both modifications lead to the increase in the resistance of enzyme to oxidative damages (Paul and Roychoudhury 2020). There is evidence indicating the possibility of regulating the activity of other enzymes by persulfidation too (Mukherjee 2019).

3  Functional Interaction of Hydrogen Sulfide with Nitric Oxide, Calcium, and…

tP-SSH

H2 S

NO

Persulfidation

S-nitrosation

tP-SH

Trans-persulfidation

rP-SSH

47

tP-SNO

Trans-S-nitrosation

rP-SH

rP-SNO

Fig. 3.2  Persulfidation and S-nitrosation of thiol groups. H2S can cause the persulfidation of SH-group of proteins. The same groups can be the targets for S-nitrosation under the influence of NO. The reactions of trans-S-nitrosation and trans-persulfidation of thiol groups of other proteins are possible. tP target (primary) proteins for persulfidation or S-nitrosation, rP restorative (secondary) proteins of target (primary) proteins

Another example of protein that can be modified by both S-nitrosation and persulfidation is glyceraldehyde-3-phosphate dehydrogenase, which plays a key role in glycolysis (Hancock et al. 2005; Aroca et al. 2017). The interaction of this protein with H2S leads to the appearance of its ability to be transported from cytoplasm into the nucleus and to act in other role, influencing the state of transcription factors and gene expression (Hancock 2019). Cross-influence of H2S and NO on the synthesis of each other is one better studied mechanism of their interaction (Fig. 3.1). In a number of studies, it is reported that physiological effects of H2S can be mediated by NO and vice versa. The positive influence of NaHS on the salt resistance of lucerne plants and expression of genes of antioxidant enzymes was eliminated with the NO scavenger cPTIO [2-(4-carboxyphenyl)-4,4,5,5-35 tetramethylimidazoline-1-oxyl-3-oxide] (Wang et al. 2012a, b). The increase in the resistance of pea plants to the toxic effect of arsenic under the treatment with NaHS was also mediated by the rise of NO content (Singh et al. 2015). Exogenous application of S-nitrosoglutathione, an NO donor, to osmotic-stressed wheat seedlings, significantly enhanced endogenous level of Cys and H2S together with increase in the activity of antioxidant enzymes (Khan et al. 2017). However, NO scavenger, cPTIO invalidated the effect of NO on endogenous H2S levels and Cys content which resulted in weak protection against osmotic stress (Khan et al. 2017). The induction of heat resistance of wheat plants by NaHS was followed by the transitional rise of H2O2 content and NO.  The H2O2 scavenger DMTU and the inhibitor of the NADPH oxidase imidazole completely eliminated the influence of

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H2S on the NO content, whereas the antagonists of NO poorly influenced H2O2 content (Karpets et al. 2020a). The increase in NO content, caused by H2S donor was followed by the rise in the activity of nitrate reductase, however this increase in enzyme activity was almost completely eliminated with its inhibitor sodium tungstate. In addition, the inhibitor of NO synthase, L-NAME (NG-nitro-L-arginine methyl ester), practically did not influence this effect (Karpets et al. 2020a). At the same time, H2S-induced increase in the activity of nitrate reductase was eliminated by DMTU and imidazole. Apparently, NO functions downstream to the H2O2 in the signaling pathway of H2S during the induction of heat resistance of wheat plants (Karpets et al. 2020a). Tomato plants, treated with high concentration of nitrate (100 mM), exhibited a decrease in oxidative stress by the influence NaHS (Liang et al. 2018). Thus, the H2S donor caused increase in the endogenous NO content in roots, which was associated with the rise of nitrate reductase activity, but not the enzyme, similar to the NO synthase of animals (Liang et al. 2018). NaHS affected the rise of endogenous content of NO up to the concentration that was sufficient to increase the resistance of Bermudas grass to toxic action of Cd2+ (Shi et al. 2014). At the same time, cPTIO partially leveled the positive influence of H2S on the activity of antioxidant enzymes and resistance of plants (Shi et al. 2014). On the other hand, the degree of resistance of Bermudas grass to Cd2+ was also improved by NO donor sodium nitroprusside (SNP). Thus, the increase in endogenous H2S content was registered, and the inhibitors of its synthesis completely eliminated the positive influence of NO donor on the plants resistant to Cd2+ toxicity. Simultaneously, involvement of NO and H2S in the adaptation of plants to HMs was probably through their ability to maintain redox homeostasis and to bind toxic ions (Singh et al. 2020). The increase in heat resistance of corn plants by NO donor SNP was associated with the rise in endogenous level of H2S (Li et  al. 2013). This H2S-mediated role of NO was confirmed by the use of inhibitors of H2S-synthesizing enzymes that abolished the positive effect of NO. At the same time, the H2S donor GYY4137 again induced the positive effect of NO on the heat resistance capacity of the plants. In general, H2S and NO are located in the signalling chains in different sequences but they can affect the synthesis and action of each other. The ambiguous notion of the relationship between two key gasotransmitters – H2S and NO – is verified by the witty name of a recent review: «Nitric oxide and hydrogen sulfide in plants: Which comes first?» (Corpas et al. 2019).

3.7  F  unctional Interaction of H2S with Other Signal Mediators During Adaptive Reactions in Plants Stress protective effects of H2S are shown mainly by its functional interaction with the key signal mediators, primarily, Ca2+, ROS and NO. As it was already noted, the activation of H2S synthesis associated with the upregulation of gene expression

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encoding LCD can be induced with the help of Ca2+ and CaM (Fang et al. 2017). On the other hand, Ca2+ acts as mediator in the transduction of H2S signals (Valivand et al. 2019) (Fig. 3.1). Many physiological effects of H2S are mediated by intensifying ROS generation in plant cells, but also H2S can act as the mediator in the realization of the effects of H2O2 (Li et al. 2015b). At last, the intensification of ROS generation, caused by H2S, apparently, in many cases depends on Ca2+. For example, the H2S-­ induced heat resistance of plant cells is preceded, apparently, with the intensification of Ca2+influx into the cytosol from both extracellular space, and from intracellular compartments, with the related increase in activity of NADPH oxidase and generation of О2•– (Kolupaev et al. 2017b). The О2•– is converted to H2O2 under the influence of SOD, which is activated by the action of H2S (Kolupaev et al. 2017a). It is possible that the rise in the content of H2O2 is promoted by direct inhibitory effect of H2S on CAT and other heme-containing antioxidant enzymes (Corpas et al. 2019). Such influence can act as a signal, resulting in the redox-dependent activation of expression of different genes of antioxidative defense system (Fig. 3.1). Quite naturally, this is not the only possible mechanism of activation of components of antioxidant system and other plant defense responses under the influence of H2S. It is possible that the activation of antioxidant enzymes or preservation of their activity under stress conditions can be promoted by the direct interaction of their thiol groups with H2S (persulfidation) (Fig. 3.1). So, it is shown that persulfidation increases the activity of APX. This enzyme can be inactivated by oxidation of Cys 32 (Aroca et al. 2018). Persulfidation not only increases its activity, but also, as it was already noted, protects from oxidative damages. On the other hand, since not only H2S interacts with thiol groups, but also NO and ROS, the state of the protein will depend on its local concentrations (Hancock, 2019). Unfortunately, the methodological possibilities for determining these indicators remain still limited. It is remarkable that the resistance to high temperature stress and other stresses (see above) by H2S depended on NO formation. At the same time, the increase in the NO content, caused by exogenous H2S, was eliminated with antioxidants and inhibitors of NADPH oxidase, while the elevation in H2O2 content was not affected by NO scavenger PTIO and inhibitors of enzymes of NO synthesis (Karpets et al. 2020a). Thus, there are the grounds to believe that, at least, some stress protective effects of H2S are mediated by ROS-dependent increase in the synthesis of NO (Fig. 3.1). According to the transcriptomic data, H2S as the signal mediator can be involved in the transduction of signals of practically all classes of phytohormones: auxin, ABA, cytokinins, gibberellins, ethylene, salicylic and jasmonic acids (Li et  al. 2017). Besides, the signal of H2S can activate the synthesis of phytohormones, for example jasmonic acid (Chen et  al. 2016b). Li et  al. (2015d) reported that the induction of heat resistance by exogenous salicylic acid was associated with the increase in H2S content. Also, the synergistic effect of salicylic acid and H2S caused the activation of antioxidant system in corn plants (Li 2015b). In our experiments, it is shown that induced activity of antioxidant enzymes in roots of salicylic acid-­ treated wheat plants was associated with the endogenous content of H2S (Karpets et  al. 2020b). Similarly, the treatment with the inhibitors of LCD prevented the

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activation of antioxidative system and the development of heat resistance in plants treated with salicylic acid. This indicates the role of H2S as a mediator in the realization of its stress-protective effects. Due to the functional interaction of H2S with other mediators, the combined use of H2S donors with Ca2+, NO donors, and also phytohormones (for example, salicylic acid), is of specific practical interest for improving protective effects of H2S against various stresses. It is shown that synergistic effect of H2S and NO protects soy and corn plants from Al3+ and Cr6+ toxicity (Kharbech et al. 2017; Wang et al. 2019). The combined action of H2S and Ca2+ significantly improved the resistance of zucchini plants to Ni2+ stress (Valivand et al. 2019). Also, a significantly positive effect of Ca2+ and H2S on heat resistance of corn plants was registered (Li et  al. 2015a; Banerjee et  al. 2018). The protective effect of combined application of H2S and salicylic acid was also shown on the plants of corn and wheat under heat stress (Li 2015b; Karpets et al. 2020b).

3.8  Conclusions In the stress signaling network, H2S occupies a central position. In response to various stresses, an upregulation of gene expression and increase in the activity of key enzyme involved in the biosynthesis of H2S is registered along with the increase in H2S content. In general, H2S is functionally closely related to ROS, NO, Ca2+, phytohormones, and several metabolites. The effects of crosstalk of these signaling molecules include chemical interaction, competition for the common targets and influence the synthesis of each other. The study of mechanisms of such crosstalk is still complicated due to methodological difficulties in the determination of endogenous H2S content in real time in the individual cellular compartments of plants. In this regard, the donors of H2S can be used as the inducers of plants resistance against various stresses. Also, H2S is involved in the realization of physiological effects of many compounds, which are used as priming agents for increasing tolerance of plants to the stresses. However, the development of such methods requires more clear view on the mechanisms of crosstalk of H2S with other components of signaling network in plant cells. Moreover, the role of pathways of H2S synthesis and the associated enzymes in the signaling processes and in plant adaptive responses are poorly investigated. Also, unravelling the role of enzymes and physiologically active compounds, involved in the degradation of H2S under abiotic stresses, will open new avenues for H2S research in plant biology.

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Chapter 4

Hydrogen Sulfide and Redox Homeostasis for Alleviation of Heavy Metal Stress Ankur Singh and Aryadeep Roychoudhury

Abstract  The industrial development has led to the release of heavy metals (HMs) in the environment beyond the safe limit. The effluents of industries carrying HMs like lead, mercury, cadmium, aluminium, zinc, copper, arsenic, etc. are admixed with the surrounding water bodies. Agricultural fields when irrigated with water from these polluted sources also get contaminated which ultimately has led to the toxicity in plants due to accumulation of metals in their tissues above the threshold limit. Metal toxicity reduces plant growth and affects crop yield. In addition, metal toxicity also induces the formation of reactive oxygen species (ROS) which disturb the redox homeostasis of the cells. ROS, when present above a threshold level in cells, damage the lipid membranes and other macromolecules. However, plants have some protective machinery such as osmolytes, and enzymatic and non-­ enzymatic antioxidants which protect them from metal stress and helps in the detoxification of ROS. As a potent endogenous gasotransmitter, hydrogen sulfide (H2S) can enhance the function of these protective machineries of plants when exogenously applied. H2S also protects proteins which are sensitive to damage by ROS through persulfidation of cysteine residues present in the protein. During metal stress, H2S can mediate the signalling pathways of calcium and nitric oxide (NO). This chapter mainly deals with the toxic effect of different HMs in plants, metabolism of H2S and its protective role during metal toxicity in plants. Keyword  Hydrogen sulphide · Metal stress · Redox homeostasis · Reactive oxygen species · Osmolytes · Antioxidants

A. Singh · A. Roychoudhury (*) Department of Biotechnology, St. Xavier’s College (Autonomous), Kolkata, West Bengal, India e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_4

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4.1  Introduction Metals having atomic weight above 20 and which are malleable, ductile with high conductivity, ligand specificity, high density and cation stability are considered as heavy metals (HMs) (Raskin et al. 1994), whereas elements having poor thermal and electrical conductivity and high electron affinity, ionization energy and electronegativity are considered as non-metals. Metals may exist as in mixed form with other soil components or as separate form. Geological and anthropogenic activities have led to the increased concentration of HMs which are naturally present in the soil. Several human activities such as burning of fossil fuels, smelting and mining of metals, production of HMs and batteries, application of fertilizers and pesticides, sewage sludge and waste disposal contribute to higher release of cobalt (Co), copper (Cu), iron (Fe), zinc (Zn), manganese (Mn), and nickel (Ni) in the soil which ultimately cause toxic effects on both plants and animals (Alloway 1990; Raskin et al. 1994; Shen et al. 2002). Metals present in combination with silicon do not cause pollution or contamination of soil, whereas metals in association with other compounds or as separate entities when present in high concentration cause environmental pollution (Ramos et  al. 1994). Several other factors like pH of the soil, presence of organic matter, content of moisture and water holding capacity of the soil govern the availability of the metal in the soil (Sharma and Raju 2013). The amount of one heavy metal in the soil may also affect the availability of other metals, but antagonistic or synergistic behaviour can be also noted with regard to metal availability in the soil. Salgare and Acharekar (1992) stated that mineralization of carbon was inhibited by the presence of Mn whose action was again interrupted in the presence of cadmium (Cd). In plants, Cu, Ni, Cd and Zn are taken by the same membrane carriers and thus they compete with each other for their uptake; it was also reported that the toxicity of Zn in barley was enhanced in presence of Cu (Luo and Rimmer 1995). Plants uptake metals from the soils which are present as soluble components. Plants need specific amount of HMs from the soil for their proper growth, but excessive concentration may cause damage to the plants directly or indirectly as they cannot be metabolized by them. Some of the direct toxicity caused by the HM stress in plants is inhibition of the enzyme activity and membrane damage (Jadia and Fulekar 2009; Assche and Clijsters 1990). The high concentration of HMs in the surrounding environment reduces the number of beneficial soil microorganisms which helps to decompose the organic waste matter that ultimately increases the nutrient content of the soil. Nutrient deficiency in soil adversely affects the growth of the plants and hampers their productivity. This direct and indirect toxicity of HMs results in a decline in plant growth and yield and may also result in plant death. Metals like Cd, mercury (Hg), arsenic (As) and lead (Pb) cause adverse effect to the plants even at low level. Growth of rice plants was significantly arrested in soil which was contaminated with 1  mg  kg−1 of Hg and concentration higher than 1  mg  kg−1 reduced the panicle formation and tiller of the plants (Kibra 2008). Ahmad (2012) reported that the presence of Cd higher than 5 mg L−1 in soil reduces

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the root and shoot length of wheat plants. Higher contamination of HMs in the soil affects several important physiological parameters of the plants such as the uptake of mineral nutrition, photosynthetic activities, and the functions of key enzymes (Kabata-Pendias 2001). Other metals which are beneficial to the plants at lower concentration have reverse effect when their concentration increases above a certain level in the soil. Jayakumar et  al. (2013) reported that the presence of Co up to 50 mg kg−1 in soil enhances the nutrient content of tomato plants, whereas level of Co higher than 100 mg kg−1 in soil reduces the nutrition level as compared to that of control. A similar effect was noted in case of Zn, where it was reported that a concentration of 25 mg L−1 in soil solution helps to enhance the growth and physiology of cluster bean, whereas adverse effect was observed when the amount of Zn in the soil solution increased above 50 mg L−1 (Manivasagaperumal et al. 2011). In reallife situation, effluents from factories and waste disposal comprise more than one metal contamination and the presence of these metals may cause synergistic or antagonistic effect on plant growth and development. Presence of Pb and Cu in soil negatively affects the growth of the stem and leaves of Lythrum salicaria (Nicholls and Mal 2003). Ghani (2010) reported the effect of six HMs (Cd, Co, Cr, Hg, Mn and Pb) on the growth of maize plants. The study showed a severe decrease in the growth and protein content of the plants as compared to that of control condition. The combined effect of the metals was comparable with the toxic effect of any two metals taken together. The toxicity order showed by the plants was Cd > Co > Hg > Mn > Pb > Cr. Plants can tolerate the toxic effects of metals up to certain level due to the presence of their internal protective mechanism such as osmolytes and antioxidants which can be enzymatic or non-enzymatic. In addition to these mechanisms, researchers have demonstrated that exogenous application of certain chemicals like hydrogen sulfide (H2S) and nitric acid (NO) enhance the function of these protective machineries of the plants which ultimately lowers the toxicity of HMs. H2S is a flammable and colourless gas smelling like rotten eggs. In early days, H2S was considered to be one of the toxic gases due to its obnoxious smell, but breakthrough came after the demonstration of neuromodulator role of H2S in brain of mammals (Zhang and Bian 2014). H2S is regarded as the third gas molecule involved in signalling after NO and carbon monoxide (CO) (Wang 2012). H2S can easily move between cells (Mathai et al. 2009) and its level in cells is strictly maintained by the action of specific enzymes (Papenbrock et al. 2007). Being a signalling molecule, H2S plays an important role during the development and growth of the plants by regulating several key processes like seed germination, photosynthesis and movement of stomata (Li and Liu 2012; Chen et al. 2011; Garcia-Mata and Lamattina 2010). H2S also protects the plants from abiotic stress conditions like drought, salinity, osmotic stress, dehydration stress, heavy metal stress, and temperature (Deng et al. 2016; Khan et al. 2017, 2018, 2020; Du et al. 2019). Interaction of H2S with molecules like CO, NO, Ca2+, abscisic acid, auxin, hydrogen peroxide (H2O2), salicylic acid, ethylene, and gibberellin helps the plants to regulate abiotic stress tolerance and maintain growth and development under unfavourable condition (Peng et al. 2016; Jin et al. 2013; Lv et al. 2017; Jia et al. 2015; Xie et al. 2014). H2S

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maintains the closure of the stomata by regulating the expression of genes encoding the proteins of Ca2+ and K+ channels (Jin et al. 2013). During salt stress in alfalfa, H2S regulates the ion channels and inhibits K+ leakage from the cells providing tolerance to plants by maintaining high K+/Na+ ratio (Lai et al. 2014). During As stress in pea seedlings, exogenous application of H2S helps the plants to maintain high endogenous cysteine level which are involved in the formation of phytochelatins (PCs) and enhanced the activities of the enzymes involved in ascorbate-­ glutathione cycle. Toxicity of As in the cells was reduced due to the formation of PCs which ultimately scavenged As within the cells (Banerjee and Roychoudhury 2018). A similar effect of H2S was also observed when wheat plants were exposed to Cu (Zhang et al. 2008) and Cr (Zhang et al. 2010) stress due to enhanced activities of antioxidant enzymes such as catalase, superoxide dismutase, amylase, and esterase. Although the reducing capacity of H2S is lower than that of glutathione (GSH) and cysteine, it can still directly scavenge ROS such as superoxide, H2O2, peroxynitrite and hypochlorite (He et al. 2018). The role of H2S during the growth and development of the plants under metal stresses was earlier demonstrated. This chapter mainly deals with the protective role of H2S under various heavy metal stresses, occurring due to various anthropogenic activities of humans. In addition, focus on the metabolic pathway of H2S involved in the plants.

4.2  Metabolism of H2S in Plants Like other signalling molecules, such as CO, NO and H2O2, the metabolism of H2S is tightly regulated in the plants. In plant cells, various pathways are present which lead to the synthesis of H2S.  Major enzymes involved in H2S metabolism are D-cysteine desulfhydrase (DCD; EC 4.4.1.15), L-cysteine desulfhydrase (LCD; EC 4.4.1.1), cysteine synthase (CS; EC 4.2.99.8), sulfite reductase (SiR; EC 1.8.7.1), and cyanoalanine synthase (CAS; EC 4.4.1.9) (Li 2015) (Fig.  4.1). D-cysteine is broken down by DCD to produce H2S in the mitochondria of plant cells, whereas LCD decomposes the L-form of cysteine to produce H2S along with amine and pyruvate in nucleus, mitochondria, and cytoplasm of plant cells. SiR uses ferredoxin as electron donor in the chloroplast to produce H2S from sulfite. In presence of hydrogen cyanide, CAS utilises the L-cysteine to produce H2S along with cyanuric acid in the cytoplasm and mitochondria. On the other hand, CS incorporates H2S into O-acetyl-L-serine to form cysteine. The reaction catalysed by CS is reversible; thus, it can again produce H2S by using cysteine as a substrate. Since L-form of the amino acids are only natural form of amino acids found in plants, during environmental stress conditions such as HMs, drought, temperature and pathogen infection, the activity and gene expression of LCD is enhanced which is specific for L-cysteine, the major form of cysteine found in plants (Hancock and Whiteman 2014). Along with this, many H2S donors in plants like sodium hydrosulfide (NaHS), calcium sulfide, dialkyldithiophosphates, diallyl trisulfide, morpholin-4-ium

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Fig. 4.1  Metabolic pathway of H2S in plants (Li et al. 2016). The major enzymes involved are DCD (D-cysteine desulfhydrase) which utilizes D-cysteine as a substrate and yield H2S as the final product; LCD (L-cysteine desulfhydrase) which is specific for L-cysteine; another enzyme, CS (Cysteine synthase), acts upon H2S to produce L-cysteine which maintains the H2S concentration in cells. It is a reversible reaction and CS can also use L-cysteine to generate back H2S. SiR (sulfite reductase) is involved in the formation of H2S from sulphite which is absorbed by the plants from the surrounding environment. APS Adenosine 5′-phosphosulfate, Fd Ferredoxin, OAS O-acetylserine

4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137), NOSH-aspirin, and (10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl) triphenylphosphonium bromide (AP39) have been identified (Huang et al. 2020).

4.3  Role of H2S in Alleviating Heavy Metal Stress As a gaseous molecule, H2S is involved in the signal process of the plants. Abiotic stress or exogenous application of H2S elevates the endogenous H2S content that effectively abrogate the toxicity effects of metal stress. Along with this, the sulfhydration of the Cys residues by H2S protects the catalytic activity of various proteins (Paul and Snyder 2012) which can modulate the activities of proteins like tubulin, glyceraldehyde-3-phosphate dehydrogenase, and actin (Mustafa et  al. 2009). Several reports have shown that the exogenous application of H2S can alleviate the toxic effect of several HMs. H2S induces metal tolerance in plants by several mechanisms including reduction of the metal accumulation in plant cells by regulating the transporter proteins. In addition, H2S application may enhance the production of antioxidants in plants which scavenge the reactive oxygen species (ROS) produced

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in cells due to metal toxicity. H2S, being a signalling molecule, interacts with other signalling pathways, microRNA (miRNA) pathways and protein persulfidation to inhibit the toxic effect of HMs in plants (He et al. 2018).

4.3.1  Abrogation of Al Toxicity in Plants by H2S Application Al is a non-essential element for the plants and is considered to be one of the limiting factors for their growth and development in acidic soils, and it covers one third of the total cultivable land present on the earth (Kochian et al. 2004). Several reports explain the protective role of H2S under Al toxicity. Chen et al. (2013) reported that Al toxicity in barely reduced the length of the root and drastically affected the height of the plants. Exogenous application of NaHS in plants alleviated the Al toxicity in barley by enhancing the activity of antioxidant enzymes along with lowering the accumulation of Al ions. Similarly, the protective role of H2S during Al toxicity in barley was earlier reported by Dawood et al. (2012). In soybean, Al toxicity was reduced by pre-treating with H2S which reduced the accumulation of Al in the cells by enhancing the production of citrate and up regulating the expression of GmMATE13 (multidrug and toxic compound extrusion 13) and GmMATE47 genes (Wang et al. 2019). Treatment of rice seeds with H2S also enhanced the expression of OsSATR1 (sensitive to aluminium rhizotoxicity), OsSATR2, and OsFRDL4 (ferric reductase defective like) which decreased the binding of Al to the cell wall (Zhu et al. 2018). Al toxicity inhibited the growth of oilseed rape along with cell and tissues damage. Pre-treatment of seeds with H2S inhibited the formation of ROS, protected the cells and thus maintained well-developed thylakoid membranes and healthy mesophyll cells (Basharat et al. 2015). Under Al toxicity, exogenous application of H2S in rice inhibited the activity of pectin methylesterase in roots which ultimately reduced the pectin and hemicellulose content thus lowering the negative charges on the cell walls.

4.3.2  Abrogation of Cd Toxicity in Plants by H2S Application Another toxic element to both plants and animals is Cd. Exogenous application of NaHS in rice seedlings exposed to Cd toxicity, elevate the endogenous content of H2S which ultimately lowered the Cd-induced toxicity in seedlings by reducing the uptake of Cd ions from the surrounding and also by enhancing the formation of protective metabolites, whereas on treatment of seedlings with hypotaurine, a H2S scavenger, the effect was reversed partially (Mostofa et al. 2014). In Populus euphratica, Cd toxicity led to programmed cell death by inducing chromatin condensation and DNA damage (Zhang et al. 2015). Exogenous application of H2S decreased the level of cell death by accumulating Cd in the vacuoles of the cells and immobilizing it into the cell wall (Jian et al. 2013; Guan et al. 2018). Increased level of H2S in the

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roots of Brassica rapa upon exposure to Cd toxicity can be explained by the enhanced expression of genes responsible for H2S synthesis such as DES1 (OAS-TL related family) and DCD1 (cysteine desulfhydrase) (Zhang et al. 2015); however, Alvarez et al. (2010) reported that Arabidopsis DES1 mutant showed higher tolerance toward Cd toxicity. The possible reason was that in absence of DES1 activity, the level of cysteine in the cytoplasm enhanced, and this led to a higher accumulation of antioxidants ultimately lowering the level of ROS in cells formed due to Cd toxicity (Herouart et  al. 1993). Qiao et  al. (2016) reported that LCD activity in Arabidopsis could be enhanced by calcium dependent protein kinase (CDPKs) which leads to higher formation of H2S and decreased the level of oxidized glutathione. This can enhance the resistance capability by S-sulfhydration under Cd toxicity. Exogenous application of NaHS and proline reduced the electrolyte leakage and H2O2 and MDA formation in foxtail millet seedlings when exposed to Cd toxicity. In addition, the activity of proline dehydrogenase (PDH) and proline-5-carboxylate reductase (P5CR) was also enhanced along with their gene expression (Tian et al. 2016). Lower concentration of Cd in the surrounding medium induced the formation of H2S by LCD and DCD dependent pathways which lower the superoxide and H2O2 level (Lv et  al. 2017). In Arabidopsis, exogenous application of salicylic acid induced the activity of LCD which again enhanced the formation of H2S using L-cysteine as a substrate, ultimately enhancing the rate of photosynthesis and decreasing the damages caused due to oxidative stress (Qiao et al. 2015). Alamri et al. (2020) demonstrated that endogenous H2S plays a pivotal role in maintaining the activity of enzyme involved in ascorbate-glutathione cycle, glutathione biosynthesis, redox state of glutathione and ascorbate and the level of non-protein thiols and phytochelatins that maintained the tolerance capability of tomato seedlings when exposed to lower dose of Cd stress (5 μM). Cd stress lowered the cellulose and pectin content in the cell wall of Isatis indigotica which was rescued by the exogenous application of NaHS. Along with this, exogenous application of NaHS also inhibited the long range translocation of Cd from root to shoot that ultimately lowered the effect of Cd-induced toxicity in the seedlings (Jia et al. 2020).

4.3.3  Mitigation of As Toxicity in Plants by H2S Application The heavy metal As belongs to the metalloid family. It is highly toxic for both plants and animals. Singh et al. (2015) reported that when pea seedlings were exposed to As stress, the ROS level was induced in plants which led to protein, lipid and bio-­ membrane damages along with enhanced cysteine level in the cells. Exogenous application of H2S reduced the symptoms of As toxicity and also relieved the activities of enzymes involved in ascorbate-glutathione cycle in comparison to that in non-treated stressed seedlings. The results showed that exogenous application of H2S reduced the accumulation of As in the cells and also enhanced the level of endogenous H2S, along with decreasing the level of ROS and enhancing the activity of enzymes involved in the ascorbate-glutathione cycle.

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4.3.4  Mitigation of Cr Toxicity in Plants by H2S Application In the environment, Cr exists in hexavalent and tetravalent state and is considered to be the second most common element responsible for land and water pollution (Zhao et  al. 2016). It is highly mutagenic and carcinogenic to both plants and animals (Seth et al. 2012). It affects the growth and development of plants and hampers the uptake of nutrition from the soil (Ali et al. 2012). Exogenous application of H2S in wheat plants improved the rate of germination by enhancing the activity of esterase, antioxidative enzymes, and amylase, and reduced the content of H2O2 and MDA when exposed to 0.5, 1.0, 2.0, 4.0, and 6.0  mM Na2CrO4 for 48  h (Zhang et  al. 2010). Similar effects were also noted in case of barley seedlings where the exogenous application of H2S donor, NaHS reduced Cr accumulation in shoots, roots, and leaves which ultimately reduced the toxicity symptoms triggered under 100 μM Cr. In addition, H2S application also protected the nuclei of the cells in the roots and maintained the integrity of chloroplasts (Ali et  al. 2013). On foliar treatment of cauliflower with 200  μM NaHS, the amount of Cr deposited in seedlings was reduced that ultimately lowered the symptoms of oxidative damage like electrolyte leakage, H2O2 and MDA formation and also upregulated the activity of antioxidative machineries on exogenous application of 10, 100 and 200 μM K2Cr2O7 (Ahmad et al. 2019). Kharbech et al. (2017) reported that exogenous application of H2S in maize seedlings reduced Cr toxicity by inhibiting ROS accumulation, membrane peroxidation, recycling of NADPH metabolism and increasing the activity of antioxidant enzymes. In Setaria italica, endogenous level of H2S and Ca2+ signalling was increased when exposed to Cr6+ toxicity. Ca2+ signalling induced the expression of phytochelatin synthase (PCS) and metallothionein 3A (MT3A), enhancing the formation of PCs and metallothioneins in a H2S-dependent manner. H2S alleviated Cr toxicity by inducing the ascorbate-glutathione cycle along with higher activity of antioxidant enzymes (Fang et al. 2016). Application of H2S up regulated the expression of glutamylcysteine synthetase (GSH1) and glutathione reductase (GR), ultimately enhancing the endogenous content of glutathione in the cells (Fang et al. 2014).

4.3.5  Mitigation of Cu Toxicity in Plants by H2S Application Proper growth of the plants is regulated by optimum concentration of Cu, but excessive amount of Cu in the surrounding medium inhibits the normal growth and development of the plants and also induces ROS production and DNA damage (Yadav et al. 2017; Poonam et al. 2014). Application of H2S in wheat seedlings reduced the symptoms of Cu toxicity such as ROS accumulation which disrupts the photosynthetic system and membrane lipids (Dai et al. 2016). Similar effect of H2S on the germination of wheat seeds, when exposed to Cu toxicity in a dose-dependent manner was also reported by Zhang et al. (2008). H2S enhanced the activity of esterase

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and amylase which improved the germination of seeds when exposed to Cu stress. Shan et al. (2012) reported that exogenous treatment of wheat seedlings with NaHS induced the activity of enzymes such as ascorbate peroxidase, glutathione reductase, L-galactono-1,4-lactone dehydrogenase, dehydroascorbate reductase and monodehydroascorbate reductase which enhanced the tolerance level of plants against Cu toxicity. Glutathione plays an important role in the protection of plants against heavy metal stress (Foyer and Noctor 2005). Plants generally maintain a high reduced glutathione/oxidised glutathione and ascorbate/dehydroascorbate ratios to protect the cells from oxidative damage.

4.3.6  M  itigation of Other Heavy Metal Toxicity in Plants by H2S Application Effect of H2S application is less studied on the plants exposed to other HMs like zinc (Zn), nickel (Ni) and lead (Pb). Higher ROS accumulation and Zn absorption was noted in Solanum nigrum when exposed to higher concentration of Zn. However exogenous application of H2S reduced the symptoms of toxicity and enhanced the activity of metallothioneins (Liu et  al. 2015). Similar effect was also noted in Capsicum annuum, where exogenous application of H2S reduced the H2O2, MDA and Zn content and enhanced the activity of enzymatic antioxidants (Kaya et  al. 2018). The effect of Ni toxicity in Oryza sativa and Cucurbita pepo was also alleviated when they were pre-treated with H2S (Rizwan et al. 2019; Valivand et al. 2019). In Brassica napus, Pb toxicity was reduced when the plants were treated with H2S. H2S acted as a protective agent by decreasing the Pb absorption, ultrastructure damage, and enhancing the absorption of micro and macro nutrients, improving the efficiency of ascorbate-glutathione cycle, and photosynthetic systems (Ali et  al. 2014a, b). Pb toxicity in Zea mays was similarly alleviated by reducing Pb accumulation and enhancing the protein and glutathione content, and activity of nitrate reductase (Zanganeh et al. 2019). In addition to H2S metabolism, sulfur metabolism, metallothioneins and PCs also play a pivotal role to abrogate the toxic effects of HMs in plants.

4.4  Conclusion and Future Perspectives H2S plays substantial role in alleviating the toxicity of HMs in plants. H2S suppresses the absorption of HMs and enhances the absorption of micro and macro nutrients which stimulates plant growth and development. In addition, exogenous application of H2S also induces the activity of several enzymatic antioxidants, ascorbate-glutathione cycle, metallothioneins and PCs and their gene expression. Being a signalling molecule, H2S interacts with Ca2+, NO, CO and H2O2. Several

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studies have reported the interaction of H2S as a signalling molecule with other hormones in plants during metal toxicity. However, the interaction of H2S with ABA, brassinosteroids, cytokinins, and ethylene is not well explored, and further studies are required to give a wide knowledge of pathways involved in the interaction of H2S with these signalling molecules for abrogating the toxicity of HMs in plants. Another important study which needs to be done in future is to reveal the link between H2S and protein persulfidation under heavy metal stress, since protein persulfidation has been reported to play a major role in the alleviation of heavy metal stress in plants. Acknowledgements 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.

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Kaya C, Ashraf M, Akram NA (2018) Hydrogen sulfide regulates the levels of key metabolites and antioxidant defense system to counteract oxidative stress in pepper (Capsicum annuum L.) plants exposed to high zinc regime. Environ Sci Pollut Res Int 25:1–7 Khan MN, Mobin M, Abbas ZK et al (2017) Nitric oxide-induced synthesis of hydrogen sulfide alleviates osmotic stress in wheat seedlings through sustaining antioxidant enzymes, osmolyte accumulation and cysteine homeostasis. Nitric Oxide 68:91–102 Khan MN, Al-Zuaibr FM, Al-Huqail AA (2018) Hydrogen sulfide-mediated activation of O-acetylserine (thiol) lyase and L/D-cysteine desulfhydrase enhance dehydration tolerance in Eruca sativa Mill. Int J Mol Sci 19:3981 Khan MN, Siddiqui MH, Al-Solami MA et  al (2020) Crosstalk of hydrogen sulfide and nitric oxide requires calcium to mitigate impaired photosynthesis under cadmium stress by activating defense mechanisms in Vigna radiata. Plant Physiol Biochem 156:278–290 Kharbech O, Houmani H, Chaoui A et al (2017) Alleviation of Cr (VI)-induced oxidative stress in maize (Zea mays L.) seedlings by NO and H2S donors through differential organ dependent regulation of ROS and NADPH-recycling metabolisms. J Plant Physiol 219:71–80 Kibra MG (2008) Effects of mercury on some growth parameters of rice (Oryza sativa L.). Soil Environ 27:23–28 Kochian LV, Hoekenga OA, Pineros MA (2004) How do crop plants tolerate acid soils? Mechanisms of aluminium tolerance and phosphorous efficiency. Annu Rev Plant Biol 55:459–493 Lai D, Mao Y, Zhou H et al (2014) Endogenous hydrogen sulfide enhances salt tolerance by coupling the reestablishment of redox homeostasis and preventing salt-induced K+ loss in seedlings of Medicago sativa. Plant Sci 225:117–129 Li ZG (2015) Analysis of some enzymes activities of hydrogen sulfide metabolism in plants. Methods Enzymol 555:253–269 Li ZG, Liu P (2012) Hydrogen sulfide is a mediator in H2O2 induced seed germination in Jatropha curcas. Acta Physiol Plant 34:2207–2213 Li ZG, Min X, Zhou ZH (2016) Hydrogen sulfide: a signal molecule in plant cross adaptation. Front Plant Sci 7:1621 Liu X, Chen J, Wang GH et al (2015) Hydrogen sulfide alleviates zinc toxicity by reducing zinc uptake and regulating genes expression of antioxidative enzymes and metallothioneins in roots of the cadmium/zinc hyperaccumulator Solanum nigrum L. Plant Soil 400:177–192 Luo Y, Rimmer DL (1995) Zinc-copper interaction affecting plant growth on a metal-contaminated soil. Environ Pollut 88:79–83 Lv W, Yang L, Xu C et al (2017) Cadmium disrupts the balance between hydrogen peroxide and superoxide radical by regulating endogenous hydrogen sulfide in the root tip of Brassica rapa. Front Plant Sci 8:232 Manivasagaperumal R, Balamurugan S, Thiyagarajan G et al (2011) Effect of zinc on germination, seedling growth and biochemical content of cluster bean (Cyamopsis tetragonoloba (L.) Taub). Curr Bot 2:11–15 Mathai JC, Andreas M, Philipp K et al (2009) No facilitator required for membrane transport of hydrogen sulfide. Proc Natl Acad Sci U S A 106:16633–16638 Mostofa MG, Rahman A, Ansary MMU et  al (2014) Hydrogen sulfide modulates cadmium induced physiological and biochemical responses to alleviate cadmium toxicity in rice. Sci Rep 5:14078 Mustafa AK, Gadalla MM, Sen N et al (2009) H2S signals through protein S-sulfhydration. Sci Signal 2:ra72 Nicholls AM, Mal TK (2003) Effects of lead and copper exposure on growth of an invasive weed, Lythrum salicaria L. (Purple Loosestrife). Ohio J Sci 103:129–133 Papenbrock J, Riemenschneider A, Kamp A et al (2007) Characterization of cysteine-degrading and H2S-releasing enzymes of higher plants from the field to the test tube and back. Plant Biol 9:582–588 Paul BD, Snyder SH (2012) H2S signalling through protein sulfhydration and beyond. Nat Rev Mol Cell Biol 13:499–507

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Peng R, Bian Z, Zhou L et al (2016) Hydrogen sulfide enhances nitric oxide induced tolerance of hypoxia in maize (Zea mays L.). Plant Cell Rep 35:2325–2340 Poonam RK, Shagun B, Ravinder S et al (2014) Treatment of 24-EBL to Brassica juncea plants under Cu metal stress lowers oxidative burst by activity of antioxidative enzymes. J Stress Physiol Biochem 10:315–327 Qiao Z, Jing T, Liu Z et al (2015) H2S acting as a downstream signaling molecule of SA regulates Cd tolerance in Arabidopsis. Plant Soil 393:137–146 Qiao Z, Jing T, Jin Z et al (2016) CDPKs enhance Cd tolerance through intensifying H2S signal in Arabidopsis thaliana. Plant Soil 398:99–110 Ramos L, Hernandez LM, Gonzalez MJ (1994) Sequential fractionation of copper, lead, cadmium and zinc in soils from or near Doñana National Park. J Environ Qual 23:50–57 Raskin I, Kumar PBAN, Dushenkov S et al (1994) Bioconcentration of heavy metals by plants. Curr Opin Biotech 5:285–290 Rizwan M, Mostofa MG, Ahmad MZ et al (2019) Hydrogen sulfide enhances rice tolerance to nickel through the prevention of chloroplast damage and the improvement of nitrogen metabolism under excessive nickel. Plant Physiol Biochem 138:100–111 Salgare SA, Acharekar C (1992) Effect of industrial pollution on growth and content of certain weeds. J Nat Conserv 4:1–6 Seth CS, Remans T, Keunen E et al (2012) Phytoextraction of toxic metals: a central role for glutathione. Plant Cell Environ 35:334–346 Shan CJ, Dai HP, Sun YF (2012) Hydrogen sulfide protects wheat seedlings against copper stress by regulating the ascorbate and glutathione metabolism in leaves. Aust J Crop Sci 6:248–254 Sharma MSR, Raju NS (2013) Correlation of heavy metal contamination with soil properties of industrial areas of Mysore, Karnataka, India by cluster analysis. Int Res J Environ Sci 2:22–27 Shen Z, Li X, Wang C et al (2002) Lead phytoextraction from contaminated soil with high-biomass plant species. J Environ Qual 31:1893–1900 Singh VP, Singh S, Kumar J et al (2015) Hydrogen sulfide alleviates toxic effects of arsenate in pea seedlings through up-regulation of the ascorbate–glutathione cycle: possible involvement of nitric oxide. J Plant Physiol 181:20–29 Tian B, Qiao Z, Zhang L et al (2016) Hydrogen sulfide and proline cooperate to alleviate cadmium stress in foxtail millet seedlings. Plant Physiol Biochem 109:293–299 Valivand M, Amooaghaie R, Ahadi A (2019) Seed priming with H2S and Ca2+ trigger signal memory that induces cross adaptation against nickel stress in zucchini seedlings. Plant Physiol Biochem 143:286–298 Wang R (2012) Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol Rev 92:791–896 Wang H, Ji F, Zhang Y et al (2019) Interactions between hydrogen sulphide and nitric oxide regulate two soybean citrate transporters during the alleviation of aluminium toxicity. Plant Cell Environ 42:2340–2356 Xie Y, Zhang C, Lai D et al (2014) Hydrogen sulfide delays GA-triggered programmed cell death in wheat aleurone layers by the modulation of glutathione homeostasis and heme oxygenase-1 expression. J Plant Physiol 171:53–62 Yadav P, Kaur R, Kanwar MK et al (2017) Castasterone confers copper stress tolerance by regulating antioxidant enzyme responses, antioxidants, and amino acid balance in B. juncea seedlings. Ecotoxicol Environ Saf 147:725 Zanganeh R, Jamei R, Rahmani F (2019) Role of salicylic acid and hydrogen sulfide in promoting lead stress tolerance and regulating free amino acid composition in Zea mays L. Acta Physiol Plant 41:94–99 Zhang X, Bian J-S (2014) Hydrogen Sulfide: a neuromodulator and neuroprotectant in the central nervous system. ACS Chem Neurosci 5:876–883 Zhang H, Hu LY, Hu KD et  al (2008) Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. J Integr Plant Biol 50:1518–1529

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Chapter 5

Effect of Hydrogen Sulfide on Osmotic Adjustment of Plants Under Different Abiotic Stresses Aryadeep Roychoudhury and Swarnavo Chakraborty

Abstract  Hydrogen sulfide (H2S), a potent gaseous transmitter in plants, has myriads of positive roles, which involves a boost on plant growth and development and execution of plant stress tolerance mechanisms. Initially this molecule was rather considered as a toxin to the plant system, but presently it has been proved that H2S has immense role to play in abiotic stress responses in plants. Exposure of a plant to any form of abiotic stress leads to alteration of the cellular osmotic potential and activation of oxidative stress circuits. H2S signalling and metabolism can modulate the stress tolerance response and osmotic adjustment in plants to combat the adverse circumstances of different forms of abiotic stresses including drought, high and low temperature, salinity, water logging, hypoxic conditions, metal toxicity, etc. In order to bring about osmotic adjustment in plants, H2S can induce the accumulation of a range of plant osmolytes like proline, glycine betaine, polyamines, sugars, inorganic ions, etc. These osmolytes can in turn lead to stabilization of the differences in the osmotic potential between the cellular interior and the surroundings in response to the prevailing abiotic stress. Keywords  Hydrogen sulfide · Osmotic potential · Osmotic adjustment · Osmolytes · Proline · Glycine betaine · Polyamines

A. Roychoudhury (*) · S. Chakraborty Department of Biotechnology, St. Xavier’s College (Autonomous), Kolkata, West Bengal, India e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_5

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5.1  Introduction With the rise in anthropogenic activities, plants are subjected to a range of abiotic stresses like drought, high and low temperature, salinity, water logging, hypoxic conditions, metal toxicity, etc. The most common effect of any kind of abiotic stress is associated with water deficit within the plant system. In response to different stressors, the solute content per cell of the plant gets altered, due to accumulation of different osmolytes that causes decrease in osmotic potential of the cells leading to osmotic adjustment in the plants (Zhang et  al. 1999). Several gaseous signalling molecules like carbon monoxide (CO), nitric oxide (NO) and hydrogen sulfide (H2S) have been demonstrated to be actively involved in generating plant tolerance to abiotic stress. These gaseous molecules are highly effective at minimal cellular concentrations, suggesting their potent roles in stress tolerance signalling cascades (Yamasaki and Cohen 2016). Initially hydrogen sulphide was rendered to be a toxic by-product generated within the plant system. This gaseous molecule has been shown to promote growth and act as a key regulator of stress-induced signalling within plants (Li 2013a, b, 2015a, b, c, d, 2016a, b, 2018). H2S is associated with signalling circuits of several important plant hormones like abscisic acid (ABA), ethylene, gibberellin, auxin, salicylic acid, etc (Sharma et al. 2019). Moreover, it also plays a pivotal role in the crosstalk networks involving NO, hydrogen peroxide (H2O2), and calcium. The key stress phytohormone ABA is universally recognised in plants. H2S, via interaction with this key hormone, can regulate a number of metabolic pathways involving the synthesis of osmolytes in plants (Guo et al. 2016). It is commonly observed that the genes responsible for encoding most plant osmolytes are abscisic acid-inducible, thus H2S can modulate the levels of these osmolytes due to exposure to different stresses. The major functions of these osmolytes are the detoxification of reactive oxygen species (ROS), prevention of the disruption of membrane integrity, maintenance of the cellular osmotic potential and conferring stability to different cellular proteins like enzymes. The common osmolytes include: (i) Glycine betaine: The levels of glycine betaine is commonly elevated in most plants commonly in response to salt stress and drought stress and to maintain the balance that exists between the intracellular and extracellular ions. This balance allows the conservation of water inside the plant cells, thereby reducing the adverse effects of excess salts on intracellular proteins and enzymes. The site of synthesis of glycine betaine in plant cells are the chloroplasts (Wang et al. 2014). Both choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are key enzymes involved the biosynthesis of glycine betaine in plants in plants (Ashraf and Foolad 2007; Chen and Murata 2008; Lutts 2000; Mäkelä et al. 2000; Wani et al. 2013). (ii) Proline: An important amino acid associated with plant resistance against abiotic stresses like osmotic stress and salinity is proline. Accumulation of this amino acid in plants can bring about the increased stability of plant subcellular organelles and other components other components (Alia et al. 1993; Hayat et al. 2012; Hoque et al. 2008), which serves as an active strategy to cope with

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osmotic stress. Moreover, proline can also maintain the osmotic potential and thus, mediates the osmotic adjustment in plants in response to salt stress (Khedr et  al. 2003). Two key enzymes associated with proline biosynthesis include Δ1-pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR). (iii) Glycerol: In many plants, glycerol can serve as one of the essential osmolytes in salinity stress. Glycerol is synthesised from glucose under the action of the enzyme glycerol-3–phosphate dehydrogenase (G3PDH). Glycerol is highly soluble, chemically inert, and non- toxic unlike many other compatible solutes. Moreover, glycerol is an end product of metabolic pathways in plants and is independent of the presence of nitrogen. Thus, accumulation of glycerol in plants is less likely to offset other important metabolic networks. The level of energy expenditure in glycerol synthesis is quite less, making it a very feasible osmolyte in many plants in combating different abiotic stresses (Eastmond 2004). (iv) Sugars: A variety of sugars including sucrose, fructose, trehalose, and glucose acts as efficient osmolytes in the osmotic adjustment in plants in response to salt and osmotic stresses. The total soluble sugar content in plants under stress is much greater than the plants grown in non-stressed environment (Dhir et al. 2012). It is often found that these sugars serve as key mediators in generating tolerance in plants against a variety of abiotic stresses. (v) Inorganic ions: Several ions including sodium, potassium and calcium ions (Li et  al. 2012), act as trigger for the increased accumulation of osmolytes under conditions of drought and high salinity. These ions control the osmotic adjustment in plants via increased water absorption into cells of the stressed plants. Many plants like some species of cassava display a heightened potassium concentration when subjected to water scarcity conditions (ElSharkawy 2007).

5.2  Metabolism of H2S in Plants The main source of H2S in plants is the amino acid cysteine. Both the D and L forms of cysteine can generate this gaseous molecule in plants. Additionally, sulfite, sulfate and sulphur dioxide can lead to generation of H2S in plants. The H2S metabolism in plants is associated with the enzymes involved in the synthesis and breakdown of cysteine and sulfite in plants. These enzymes include O-acetyl-L-serine lyase (OAS-TL; EC 2.5.1.47), L-cysteine desulfhydrase (L-CD; EC 4.4.1.1), cyanoalanine synthase (CAS; EC 4.4.1.9), cysteine synthase (EC 2.5.1.47), sulfite reductase (SIR; EC 1.8.7.1) and 3-mercaptopyruvate sulfurtransferase (MS; EC 2.8.1.2). The first reaction in Fig. 5.1 is catalysed by the enzyme L-CD and generates pyruvate and ammonia as the by-products (Wang 2012). The expression of this enzyme is greatly increased in response to a wide variety of stresses in plants. The conversion of D-cysteine to hydrogen sulfide gas is mediated by D-cysteine desulfhydrase (D-CD; EC 4.4.1.15). However, this enzyme will not catalyse the breakdown of

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Fig. 5.1  Various sources of H2S in plants

L- cysteine. It has been reported that both the L-CD and D-CD enzymes require molecules of water for their functionality. The third pathway is dependent on the presence of a reduced ferredoxin moiety, and the reduction of sulfite into H2S by the enzyme SIR is coupled to the generation of oxidised form of ferredoxin in plants (Wang 2012). Sulfates, sulfites and sulphur di oxide serve as the source of sulfite ions for H2S synthesis in many plants.

5.3  Roles of H2S in Different Forms of Abiotic Stresses Upon the inception of a particular abiotic stress or even on the exogenous application of H2S donor like sodium hydrosulfide (NaHS), the anabolic genes involved in the biosynthesis of H2S in plants are triggered. As the endogenous levels of H2S rise, this upregulates a series of signalling cascades involved in the osmotic adjustment and the antioxidative reactions in plants (Khan et al. 2014). The central theme of the signalling event involves the generation of a series of osmolytes, which are the compatible osmoprotectants in the plant system (Fotopoulos et al. 2015). These osmolytes maintain the water potential of the stressed plant cells and thereby maintain the normal metabolism and serves as the most important strategy for the plant to combat severe osmotic stresses (Singh et al. 2015a, b). The osmolytes maintain homeostasis by nullifying the differences in osmotic potential between the cell’s interior and exterior by preventing the generation of ROS, thus generating protection of cellular proteins and the lipid membrane envelopes against ROS (Yancey 1994).

5.3.1  Drought Stress The pronounced reduction in the water potential of leaf cells is one of the most significant symptoms in plants exposed to drought stress. A severe compromise in the chlorophyll biosynthetic pathways has been reported in many plant species in

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response to water scarcity (Din et al. 2011; Mäkelä et al. 2000). Due to reduction in chlorophyll content, the plants suffer from decreased photosynthetic rates thus leading to plant death. Exogenous spraying of the H2S donor NaHS on plants growing under water scarcity conditions led to increased tolerance to drought and elevated rates of seed germination. An array of osmolytes were found to be expressed at high rates, including the amino acid proline, many sugars, glycine betaine and a set of inorganic ions, in response to drought in plants (Serraj and Sinclair 2002). The accumulation of these osmolytes inside the plant cells changes their osmotic potential making it more and more negative. This high negative value of osmotic potential of the cell interior, leads to endosmosis that increase the water uptake, thereby maintaining the turgidity of the cells. Proline serves as the major osmolyte in plants, mitigating drought stress. Many reports have been made regarding the upregulation of proline in pea (Alexieva et al. 2001) and Petunia cultivars (Yamada et al. 2005) under water scarcity conditions. Polyamines like spermidine and glycine betaine are also elevated in drought-stressed plants. Interestingly, citrulline, a non-protein amino acid, is found to counteract drought stress in many watermelon cultivars (Akashi et al. 2001). The susceptibility of plants to drought was reduced considerably on exogenous application of H2S donors. This was demonstrated by the reduction in lipoxygenase (LOX) and malondialdehyde (MDA) levels and ROS generation in treated plants (Zhang et al. 2011). The reduced LOX activity decreases the chances of membrane lipid peroxidation during drought stress. The lowering of ROS generation is by virtue of the increased levels of the antioxidant enzymes ascorbate peroxidase (APX) and catalase (CAT) in these plants as compared to the untreated plants (Zhang et al. 2011). Antioxidant osmolytes like ascorbate and glutathione associated with ascorbate–glutathione cycle, one of the most important antioxidant machineries in plants, was highly up regulated in NaHS-treated plants (Banerjee et al. 2018; Ahmad et al. 2010). The treated seedlings displayed higher levels of APX, dehydroascorbate reductase (DHAR) and glutathione reductase (GR), which are involved in the ascorbate–glutathione cycle. The reduction in stomatal aperture via interaction of ABA and H2S served as important strategy for osmotic adjustment in NaHS treated plants, unlike the untreated plants under water scarcity conditions (Jin et al. 2013). Due to these effects, the relative water content (RWC) of the cells increased, thereby increasing plant longevity under drought conditions. Thus, H2S was found to be efficient in maintaining the normal growth and metabolism of plants exposed to conditions of water shortage (García-Mata and Lamattina 2010; Jin et al. 2011; Kathuria et al. 2009; Quan et al. 2004).

5.3.2  Salt Stress Almost one third of the earth’s landmass is affected by salt stress. The major outcome of salt stress in plants is the disruption of the efficiency of photosynthesis due to adverse effects of high salt on the photosynthetic apparatus. This results in the

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decline of chlorophyll and several accessory photosynthetic pigments in the leaves, thereby reducing plant productivity and yield (Mbarki et al. 2018). Plants under salt stress demonstrates loss of integrity of membranes due to lipid peroxidation (Banerjee and Roychoudhury 2018), disruption of activities of proteins and imbalance in water and nutrients, mainly due to generation of huge amounts of ROS. The high level of ROS in salt-stressed plants indicates that toxic levels of salts act as a trigger for osmotic stress symptoms inside a plant (Zhu 2001; Christou et al. 2011, 2013; Hasanuzzaman et al. 2013; Sudhakar et al. 2001). Plants pre-treated with NaHS, demonstrated higher tolerance to salt stress (Bao et al. 2011; Tang et al. 2015; Wang et al. 2012; Sumithra et al. 2006). Osmolytes like proline, polyamines, glycine betaine, etc. responsible for conferring membrane stability are commonly found to be highly expressed in salt-stressed plants pre-treated with NaHS. Maintenance of membrane homeostasis can bypass the adverse effects of high salts, thus regulating the normal growth, metabolism and cellular homeostasis of the plants. The amino acid proline acts as a regulator of many salt stress responsive genes, thereby inducing salt tolerance in plants grown under high salt conditions. Medicago seedlings, when treated with NaHS displayed an up regulation of antioxidative cascades involving a range of enzymes like catalase (CAT), superoxide dismutase (SOD), APX and peroxidases, under conditions of salt stress (Li et al. 2014a, b). Treated plants displayed higher activities of membrane transporters like Na+/H+ antiporters and H+ATPase (Li et al. 2014a, b). This up regulation of membrane transporters was necessary for the maintenance of sodium and potassium ion homeostasis inside the cells of the stressed plants. Usually, these plants maintained a higher level of potassium ions and lower level of sodium ions, to adjust themselves osmotically with the high salt content of the surrounding soil environment.

5.3.3  Temperature Stress Plants are continuously subjected to increased temperature than the normal range by virtue of global warming and greenhouse effect. As observed in most other forms of stress, during heat stress, hydrogen sulphide also acts as a trigger for antioxidative circuits in plants. High temperature leads to a fall in chlorophyll biosynthesis, thus serving as an indicator of heat stress in the plastids (Li et  al. 2010; Reda and Mandoura 2011; Tewari and Tripathy 1998). Enzymes like 5-aminolevulinate dehydratase (ALAD) associated with chlorophyll synthesis in plants, is severely downregulated at high temperature. However, strawberry seedlings pre-treated with H2S donor at higher temperature of around 42 °C exhibited higher rate of chlorophyll synthesis as compared to untreated plants. The treated plants demonstrated heightened activities of antioxidative enzymes like CAT, SOD, GR APX and glutathione peroxidase (GPX) for the scavenging of ROS produced as a response of high or low temperature exposure (Shi et al. 2013; Ma et al. 2015). Heat stressed seedlings displayed a rise in chaperone activities associated with heat shock proteins like hsp70, hsp80 and hsp90 (Christou et al. 2014). These treated plants also develop several

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strategies to combat the harmful consequences of heat stress. One of those strategies includes the accumulation and increased synthesis of several plant osmolytes like the amino acid proline, glycine betaine and calcium ions. Up regulation of Δ1pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (PDH) enzymes, involved in proline metabolism, brings about higher proline accumulation in heat as well as cold stressed seedlings (Fu et al. 2013; Vaida et al. 2012). Thus, these NaHS treated plants exhibited higher viability and yield as compared to untreated ones under heat stress. Proline also helps in generation of stress tolerance in treated seedlings exposed to chilling temperature (Kishor et al. 2005). So, proline acts as a master regulator in both heat and cold stress in NaHS exposed plants. Similarly, glycine betaine up regulation in treated plants helps in the establishment of osmotic adjustment under conditions of extreme temperature (Kishor et al. 2005). The endogenous release of H2S in NaHS sprayed plants, caused a rise in endogenous calcium levels. Calcium ions are found to exhibit protective roles and play a functional role in the maintenance of the osmotic balance inside plant cells, when subjected to temperature extremes (Li et al. 2012; Tan et al. 2011). Under both heat and chilling stress, the plants tend to produce more quantity of phenolics, accumulate more soluble sugars and proline and lower MDA (malondialdehyde) amounts to combat osmotic stress and rejuvenate the antioxidative machinery as a form of response against variations in temperature (Luo et al. 2015). Therefore, H2S promotes adaptation of plants to extremes temperature.

5.3.4  Heavy Metal Stress The increase in the levels of contamination of the environment by heavy metals like Cu, Al, Cd, Zn, Pb, As, Fe, Mn, Ni and Cr has hampered the normal development and yield of the plants (Chen et al. 2013, 2017; Fahad et al. 2019; Malik 2004; Sharma and Chakraverty 2013; Nagajyoti et al. 2010). The primary symptoms of plants under heavy metal stress are the activation of oxidative stress networks, leading to endogenous rise in levels of ROS. Rise in ROS levels leads to peroxidation of membrane lipids and oxidation of DNA, RNA and proteins inside plant cells, thus disrupting the function of many essential plant enzymes and other molecules (Sandhi et al. 2017). NaHS-­mediated increase in durability of pea seedlings to arsenic stress was demonstrated by Singh et  al. (2015a, b). The sulphur-containing amino acid cysteine played an immense role in the establishment of stress tolerance, by acting as a building block for heavy metal scavengers like phytochelatins. The elevated levels of phytochelatins in the treated plants actively chelate arsenic, thereby reducing their toxic effects on the plant system (Sandhi et al. 2017). Salvinia plants were found to accumulate large concentrations of osmolytes when exposed to a variety of heavy metals in the growth media (Dhir et al. 2012). The NaHS pre-­ treated plants showed elevated levels of sugars like sucrose, sugar alcohols like mannitol and glycine betaine, as compared to the untreated ones when exposed to high concentrations of heavy metals (Banerjee et al. 2018). These osmolytes can

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efficiently detoxify ROS and mediate osmotic adjustment within the plant system. Similarly, like all other forms of stress response, the activities of a range of antioxidative enzymes are elevated during heavy metal stress as well (Bhatti et al. 2013). These enzymes include CAT, SOD, GR, APX and GPX, along with up regulation of different forms of amylases and esterases in pre-treated wheat seeds under chromium and copper stress conditions (Zhang et al. 2008, 2010a, b, c). Metallothioneins, just like phytochelatins can also scavenge the accumulated heavy metals within the plant system (Khan et al. 2014; Sidhu et al. 2017). The NaHS sprayed plants displayed an increase in the metallothionein synthesis to combat different forms of heavy metal stress, for instance stress due to high zinc concentration in soil (Liu et al. 2016). Treated plants under heavy metal stress also demonstrated lowering of the lipoxygenase activities and MDA concentrations, and these can serve as one of the strategies to sustain the physiology and metabolism of the affected plants. The damages imposed by ROS on the membrane lipids, cellular enzymes and other proteins are quite reduced in the plants already treated with H2S donor (Banerjee et al. 2018).

5.3.5  Other Forms of Stress Plant growth and development is regulated by a number of environmental factors. Availability of oxygen and the intensity of sunlight are some of the prime factors that can alter the metabolic circuits within a plant system. If the availability of oxygen to the plant tissues is disturbed, commonly called hypoxia, plant tissues suffer from oxidative stress (Van Dongen and Licausi 2014; Cheng et al. 2013). Pea plants, when sprayed exogenously with H2S gas, exhibited greater resistance to hypoxic damages induced due to flooding of roots, as compared to untreated ones. This indicates the potential role of this gaseous molecule in hypoxic stress tolerance in some selected species of plants. The mechanism behind the action of H2S against stress is not well known. Mostly, in the treated plants, the antioxidative enzyme activities are quite high to detoxify ROS and prevent disruption of membrane lipids and essential enzymes and proteins (Shi et al. 2015). On the other hand, exposure of many plant species to higher light intensity and to wavelengths in the ultraviolet range can hamper the optimal functioning of the photosynthetic apparatus, thus reducing photosynthetic yield (Kirchhoff 2014). Elevated levels of ROS is the most potent cause of damage to membranes of the thylakoid and stroma. The osmolyte glycine betaine was found to be accumulated in these stressed plants and aiding the recovery of the affected chlorophyll content and combat the adverse effects of ROS on the electron transport chain of photosynthesis (Wang et al. 2014). Yet, in some other macrophyte species, the role of H2S seems to be rather contradictory, where exogenous H2S spray led to lowering of antioxidative enzymes and aggravated consequences of hypoxia mediated oxidative stress (Parveen et al. 2017).

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5.4  Conclusion and Future Perspectives Apart from its few negative roles in the plant system, H2S can be considered as a potent gaseous signalling molecule in plants. H2S serves as a positive regulator and enhancer of a series of plant osmolytes, which are the key performers in mediating abiotic stress tolerance. This gaseous transmitter is associated with crosstalk among several phytohormones, the most important being ABA. Much work has been done in this area; yet more detailed analysis of the crosstalk can throw light on the intricate stress signalling circuits that are involved. Upon exogenous application of H2S donor to different plant systems, an array of genes gets triggered; the pathways that these gene products modulate constitute active areas of study necessary to be carried out for better understanding of the role of H2S in osmotic stress tolerance and water uptake in plants. Acknowledgements  Financial assistance from Council of Scientific and Industrial Research (CSIR), Government of India, through the research grant [38(1387)/14/EMR-II], 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.

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Chapter 6

Hydrogen Sulfide and Stomatal Movement Denise Scuffi and Carlos García-Mata

Abstract  Hydrogen sulfide (H2S) is a small gaseous molecule currently recognized as a ubiquitous signal molecule in both plants and animals. In plants it can be produced enzymatically in both cytosol and organelles such as mitochondria and chloroplasts, and its production increases in response to multiple stimuli, participating in different signaling networks through the interaction with numerous signal molecules. Guard cells are among the plant systems where H2S is most studied. These specialized cells are arranged in pairs to conform the stomatal pores, through which plants exchange gases (mainly CO2 for photosynthesis and water vapor for evapotranspiration) with the environment. In this chapter we will update the current knowledge on the role of H2S in the signaling network that commands stomatal movement in response to external and endogenous stimuli. Keywords  Hydrogen sulfide · Guard cells · Persulfidation · Stomatal movement · Abiotic stress

6.1  Introduction The combination of climate change and human population growth is putting pressure to modern science, in particular to plant scientists who face the challenge to secure food production in order to feed an increasing world community. Considering that both world temperature and desertified areas are slowly but continuously increasing, and that about 70% of the fresh water stock is used for agriculture, one of the big challenges for plant science is to increase crops water use efficiency

D. Scuffi · C. García-Mata (*) Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Consejo Nacional de Investigaciones Científicas y Técnicas (IIB-UNMdP-CONICET), Mar del Plata, Argentina e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_6

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(WUE), and to do so we have to widen the knowledge about how plants use the available water and how they withstand the lack of it. One of the keys to hack plants “water economy” is to understand the function and regulation of the valves that administrate the exchange of H2O with the atmosphere. These tiny, osmotic driven, valves called stomata perforate the epidermis of the aerial part of most land plants, and not only regulate H2O release, but they also modulate CO2 uptake for photosynthesis, and represent the first physical barrier for pathogen microorganisms. Stomata are formed by pairs of specialized cells, called Guard Cells (GCs). The variation of the stomatal pore width is generated by volume changes of the GCs that are commanded by the relocation of osmotically active solutes (mainly K+ and malate). Stomatal opening is promoted by the activation of H+-ATPases which extrudes H+ generating a membrane potential shift towards more negative voltages. This hyperpolarization of the membrane generates the entry of K+ through the inward rectifying K+ channels (K+in) and uptake of Cl− and malate, as counterions, generating an increase in the osmotic potential that commands the influx of water and the increase of the cell volume (Blatt 2000; Schroeder et al. 2001). On the contrary, stomatal closure is initiated by the inhibition of plasma membrane H+ATPases, inactivation of K+in and the promotion of a Ca2+-dependent membrane depolarization; the activation of outward rectifying K+ channels (K+out), upon depolarization, induces K+ extrusion accompanied by the Cl− and NO3− anion efflux through slow activated anion channels (SLAC1, and homologs), favoring water extrusion. This osmotic driven reduction of cell volume relaxes guard cell walls and produces the closure of stomatal pore (Blatt 2000; Schroeder et al. 2001) (Fig. 6.1). In order to maintain this trade-off between C uptake and water homeostasis, GCs has the ability to sense external (biotic and abiotic stresses) and internal (hormones and other signals) stimuli and integrate them in the regulation of the stomatal pore area. Most abiotic stresses have an impact on stomatal movement either inducing stomatal closures (i.e., Drought, Salinity, High CO2, increase VPD, UV Light, O3, and others) or inducing stomatal opening (i.e., Blue Light, Red Light, Low CO2, Heat, and others). Although the vast majority of available bibliography on stomatal movement regulation are based on experimental systems that address one stimulus at a time, this condition is highly unlikely in nature, since the plants are permanently exposed to multiple stimuli. Therefore, the GCs are continuously monitoring the environmental conditions and integrating those stimuli that induce the stomatal opening with those that induce stomatal closure, in order to achieve a stomatal pore aperture that balances CO2 uptake, H2O economy and leaf temperature. The integration of such a complex convergence of stimuli is achieved by an intricate signaling network with characteristics that resemble a scale free network, in which there are a few very highly connected nodes (called hubs) which represent those signaling components that interconnect different environmental signals. Another feature is the existence of clusters of interconnected components that are linked to the rest of the network through the hubs (Hetherington and Woodward 2003). The degree of complexity and interconnection of the GC signaling network makes it very difficult to discriminate between response paths triggered by a

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Fig. 6.1  Schematic model of the mechanism commanding stomatal movement. Stomata are formed by pairs of specialized cells, called Guard Cells (GCs). Opening inducing stimuli promote the extrusion of H+ through the activation of H+-ATPases, hyperpolarizing the plasma membrane (PM). PM hyperpolarization activates inward rectifying K+ channels (K+in) and anion channels (A−) that drives osmotically active solutes (mostly K+ and Cl− as counterions). This increases the osmotic potential generating water uptake, and increase of GCs volume and the opening of stomatal pore. Conversely, stomatal closure inducing stimuli, inactivates H+-ATPases, and activates Ca2+uptake, producing the depolarization of PM.  The positive shift of the membrane potential inactivates K+in and activates outward rectifying K+ channels (K+out) producing the extrusion of osmolytes accompanied by the Cl− and NO3− anion efflux through slow activated anion channels (SLAC1), favoring water efflux, a reduction of GC volume and the closure of the stomatal pore

particular stimulus, since sooner or later they will end up in one of the several hubs. Moreover, the advances in such an active research field and the evolution of the research techniques is permanently increasing the comprehension of complexity of the network either by adding new signaling components, or by reporting novel interactions between signaling components. One of the new signal molecules recently included in the scenario of the GC signaling network is hydrogen sulfide (H2S). This low molecular weight gas is synthesized enzymatically in members of all kingdoms. H2S and other gases like carbon monoxide (CO) and nitric oxide (NO) that are known as gasotransmitters which

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share a series of characteristics that position them as competent signal molecules, such as (i) freely permeating biological membranes; (ii) being endogenously generated by specific enzymes; (iii) having specific functions at physiologically relevant concentrations; (iv) having donors that can mimic their biological function when applied exogenously; and (v) having specific cellular and molecular targets (Wang 2002). All three gasotransmitters have been reported to participate in a wide variety of physiological processes along the whole plant cycle. Among them, the regulation of stomatal movement has pioneered the role of both NO and H2S in plants (García-­ Mata and Lamattina 2001, 2010; Neill et al. 2002; Lisjak et al. 2010) where they are now recognized to have a key role in the regulation of stomatal movement in response to numerous biotic and abiotic stresses. Although, as expressed above, it is difficult to dissect the paths generated by single stimuli in GCs, the aim of this chapter is to present and updated panorama of the role of H2S in stomatal response to abiotic stresses.

6.2  H  ydrogen Sulfide and Abscisic Acid in Plants Under Drought and Salinity The phytohormone abscisic acid (ABA), sometimes referred as the stress hormone is rapidly synthesized in response to abiotic stress, in particular to drought and salinity, and triggers a fast response along the plant, in particular in the GCs, where it induces stomatal closure or inhibit light induced stomatal opening to reduce stomatal water loss (reviewed by Osakabe et  al. 2014, and Cotelle and Leonhardt 2019). The drought response is originated in the roots, where ABA is synthesized and transported through the xylem to the leaves and ultimately to the GCs (Leung and Giraudat 1998). Although it has been reported that leaves have de novo synthesis of ABA, it has also been shown that the main contribution of ABA in response to the water deficit comes from the roots (Cotelle and Leonhardt 2019). Salt stress is generated by an imbalance in the Na+/K+ ratio produced by the accumulation of salt in the extracellular space that generates an osmotic imbalance that ultimately causes desiccation, and hydric stress-like response including ABA signaling (Munns and Tester 2008; Osakabe et al. 2014). The signaling events trigger in GCs upon ABA reception has kept the attention of plant scientists for the last 3 decades. ABA is considered a master regulator of stomatal movement (Cotelle and Leonhardt 2019). In brief, ABA is transported into the cytosol by a member of the ABC transporters gene family (ABCG40); once in the cytosol ABA binds to the specific PYR/PYL/RCAR (PPR) soluble receptor complex. The binding of ABA to a specific pocket of the PPR receptor generates a conformational change that favors the binding of the negative regulator ABI1 (a member of the PP2C protein phosphatase family) to the ABA-receptor complex, releasing Open Stomata 1 (OST1) kinase (a SNF1-Related Protein Kinases type 2, SnRK2) to trigger several downstream pathways upon autophosphorylation (Cutler

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et al. 2010). The result of these concerted signaling events is the modulation of ion channels, mainly of K+, Ca2+ and Cl−, which determine a decrease in cell turgor and the consequent reduction of the stomatal pore area. After several reports on participation of H2S on plant-pathogen interaction, a couple of independent works introduced the role of H2S in stomatal signaling. These first works, carried out in the GC system, not only positioned H2S in the GC signaling network, but also linked it with those signaling events dependent on ABA and drought (García-Mata and Lamattina 2010; Lisjak et  al. 2010). Despite showing seemingly contrasting results, both works showed H2S as signal molecule acting downstream of ABA. One of those reports linked H2S to the induction of stomatal closure by conecting it to the ABC transporter AtMRP5 (García-Mata and Lamattina 2010), whereas the other showed H2S as an inhibitor of the ABA-dependent stomatal closure, through a reduction of NO endogenous content (Lisjak et  al. 2010). These works were quickly seconded by others that focused on deciphering the role of endogenous H2S in the complex signal network triggered by ABA Thus, genetic works using Arabidopsis thaliana lines deficient in H2S synthesis showed differential expression of different GC specific ion channels, some of which are regulated by ABA (Jin et  al. 2013). Later, the first characterized L-cysteine desulfhydrase enzyme, DES1, which is the main cytosolic source of H2S, was reported to be upregulated by exogenous ABA, and the DES1 mutant lines where shown to be unable to close the stomata in response to ABA, indicating that cytosolic H2S production is involved in ABA dependent stomatal response (Scuffi et al. 2014). This last result was recently confirmed using Arabidopsis double mutant lines of both cytosolic H2S sources LCDES and DES1 (Du et al. 2019), and by in vivo measurement of guard cell H2S production using the fluorescent dye AzMc (Zhang et al. 2019). Existing evidence suggests that, in Arabidopsis, H2S is an early component of the ABA-dependent signaling pathway. Genetic studies carried out with a quadruple mutant of the ABA receptor (pyr1/pyl1/pyl2/pyl4) indicate that H2S acts downstream or independently from the receptor; however, the fact that H2S donors are impaired to induce stomatal closure in abi1 mutants, suggests the H2S might be affecting the binding of ABI1 to ABA-receptor complex (Scuffi et  al. 2014). Nevertheless, it was also reported that H2S production, measured in leaf extracts, was reduced in ABI1 mutants (Jin et al. 2013), hence further work is needed to confirm the putative interaction between ABI1 and H2S and whether H2S is acting upstream or downstream of ABI1. Once ABI1 is recruited by the ABA-PPR, group 3 SnRKs (SnRK2.2, 2.3 and 2.6) are released from ABI1 inhibition and activate several downstream pathways through the phosphorylation of numerous molecular targets such as ion channels (K+ and Cl−), NADPH oxidases, stress response proteins and different transcription factors (Kulik et al. 2011). There are some evidences that suggest that H2S might be modulating GCs group 3 SnRKs activity. Recent data indicates the H2S modulates SLAC1 anion channel activity in an OST1 dependent manner (Wang et al. 2016b) and OST1 is activated by H2S through a posttranslational modification called persulfidation  (Chen et al. 2020). Moreover, SnRK2.2 has been included in a list of

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persulfidated proteins in Arabidopsis (Aroca et al. 2017). On the other hand, H2S inactivates K+in (Papanatsiou et al. 2015), although H2S effect on K+in seems to be mostly independent of ABA, these channels are also phosphorylated by OST1. Therefore, it would be interesting to analyze if the effect of H2S on the K+in is also dependent on OST1. Other downstream targets of SnRKs, are the NADPH oxidases, which are the principal source of ROS production in GCs. As stated above ROS production is involved in the responses to several biotic and abiotic stresses, and it is therefore considered a hub of GC signaling network (Hetherington and Woodward 2003). ABA-dependent ROS production, H2O2 in particular, involves the activation of two isoforms of the Respirative Burst Oxidase Homologs (RBOH) proteins, RBOHD and RBOHF.  ABA-mediated stomatal closure mainly affects RBOHF activity, although RBOHD is also involved, since the closing phenotype of the double mutant rbohF/rbohD is stronger than that of the simple rbohF mutant (Kwak et al. 2003). ABA-dependent activation of RBOHF was reported to involve the phosphorylation of two Ser residues of RBOHF by OST1 (Sirichandra et al. 2009) indicating that ROS production is part of ABA-dependent signaling core in GCs. The evidences, presented above, position H2S as an active component in GC signaling events triggered by ABA, through the interaction with several of the so called hubs, including ROS production, pointed towards a putative role of H2S as a modulator of endogenous ROS levels. As a matter of fact, stomatal closure experiments indicate that RBOHF and D are effectively involved in the induction of stomatal closure by H2S donors (Scuffi et  al. 2018). Moreover, it has been recently demonstrated that H2S activates RBOHD through persulfidation, in response to ABA and that modification is required for ABA-induced stomatal closure (Shen et  al. 2020). However, the fact that RBOHD mutants were impaired to close the stomata in response to H2S suggests that H2S could modulate NADPH oxidase activity in responses triggered by other stimuli than ABA. In line with this result, using Arabidopsis lines expressing the H2O2-specific biosensor, roGFP2-Orp1, it was shown that H2S produces an increase in endogenous levels of H2O2 preferentially in GCs and the neighboring cells (Scuffi et al. 2018). These results were consistent with previous evidence indicating that H2S increases the endogenous hydrogen peroxide concentration in GCs, in a response mediated by extracellular ATP (Wang et al. 2015).

6.3  Hydrogen Sulfide and Light An article from 1978 reported a light-dependent emission of H2S from leaves of several plant species like squash and pumpkin among others (Wilson et al. 1978). Later, this emission was associated to sulfate assimilation processes, where it was demonstrated to be photosynthetic electron transport-dependent (Sekiya et al. 1982). The responses of higher plants to light include de-etiolation, flowering, circadian rhythms control, phototropism, chloroplast relocation, and stomatal movements

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(Chen et  al. 2012). Regarding stomatal movements, it has been known for many years that stomatal conductance is modulated by two photosystems: a blue light-­ dependent one, which increases stomatal conductance and swelling of GC protoplast at low irradiances, and a photosynthetically active radiation (PAR)-dependent one working at higher irradiances (Zeiger and Hepler 1977; Zeiger and Field 1982). Both wavelengths driving photosynthesis, red and blue light, promote stomatal opening to ensure CO2 entry to the mesophyll cells (Fan et al. 2004; Assmann and Jegla 2016). On the other hand, plants are inevitably exposed to UV-B radiation (280–315 nm) that partially passes through the stratospheric ozone layer and reaches the earth’s surface. Many plant species close stomata in response to UV-B (Nogués et al. 1999; He et al. 2011a, b, 2013; Zhu et al. 2014; Li et al. 2017) and some others show a differential response to UV-B depending on the physiological state of GC (Jansen and Van Den Noort 2000; Eisinger et al. 2003). Finally, in the absence of light the stomata remain closed, however, it is still under discussion whether this closed state is due to the lack of light or if dark is a signal per se (Costa et al. 2015).

6.3.1  Blue Light Blue light (BL) (390–500 nm), induces stomatal opening, but this response requires a red-light background indicating the existence of a synergistic effect between both types of light (Shimazaki et al. 2007; Inoue and Kinoshita 2017). In GCs, phototropins 1 and 2 (PHOT1 and PHOT2) are the major BL receptors (Christie 2007; Inoue et al. 2010; Kinoshita and Hayashi 2011). It was shown that phot1 and phot2 single mutants are able to open the stomata in response to both BL under a red light background, however, the stomata of phto1phot2 double mutant remain closed indicating the both BL photoreceptors work simultaneously and redundantly (Kinoshita et  al. 2001). Activation of the plasma membrane H+-ATPase is required for GC membrane hyperpolarization (Fig. 6.1) (Schroeder et al. 1987). Furthermore, starch degradation specifically in GCs is necessary for stomatal opening and occurs during the first 30 min after illumination and is phototropin- and H+-pumping-dependent (Horrer et al. 2016). It has been known for many years that BL induces phosphorylation and activation of plasma membrane H+-ATPase which is essential for stomatal opening (Assmann 1988; Kinoshita and Shimazaki 1999; Shimazaki et al. 2007) and this mechanism is similar in other cell types (Inoue and Kinoshita 2017). On the other hand, once activated, phototropins phosphorylates and activates a cytosol-­ localized kinase BLUS1 (BLUE LIGHT SIGNALING1) at Ser-348 in GCs. This event is essential for H+-ATPase activation and a subsequent binding to 14.3.3 protein (Leonhardt et al. 2004; Hayashi and Kinoshita 2011; Takemiya et al. 2013a; Cotelle and Leonhardt 2016; Yamauchi et al. 2016). Furthermore, two plant-specific proteins called RPT2 (ROOT PHOTOTRO­ PISM2) and NPH3 (NONPHOTOTROPIC HYPOCOTYL3) function as signal

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transducers in phototropin signaling but only RPT2 is involved in stomatal opening (Inada et al. 2004). RPT2 is expressed in several cell types including GCs and form a complex with PHOT1 in vivo (Inada et al. 2004). Another positive regulator of BL-dependent stomatal opening is type 1 protein phosphatase (PP1) which action is also between phototropins and H+-ATPase. PP1 could be a crosstalk point between BL and ABA signaling since PP1 is inhibited by phosphatidic acid (PA) (Takemiya et al. 2006, 2013b; Takemiya and Shimazaki 2010), a second messenger molecule induced by ABA and H2S in GCs and an important inductor of stomatal closure (Jacob et al. 1999; Zhang et al. 2004; Mishra et al. 2006; Distéfano et al. 2012; Scuffi et al. 2018). Down in GC signaling network, BL inhibits S-type anion channels in a phototropin-­dependent manner (Marten et al. 2007). Recently, Hiyama et al. (2017) have characterized two kinases CBC1/2 [CONVERGENCE OF BLUE LIGHT (BL) and CO21/2] which are phosphorylated by PHOT1  in response to BL and inhibit S-type anion channels causing stomatal opening (Hiyama et al. 2017). Furthermore, cryptochromes (CRY) are also BL photoreceptors that mediate BL-responses in animals and plants. In GCs, it is proposed an additive role to phototropins in mediating stomatal opening (Mao et al. 2005; Shimazaki et al. 2007). This event involves the participation of constitutive photomorphogenic 1 (COP1), an E3 ubiquitin ligase, as a negative regulator downstream (Mao et  al. 2005). Experiments conducted in intact leaves suggest that phototropins are directly mediating stomatal conductance, meanwhile the effects of CRY is largely indirect and involves the control of ABA levels (Boccalandro et al. 2012). Finally, [Ca2+]cyt, a second messenger involved in several signaling pathways, is also increased in GCs upon BL illumination and this response is photosynthesisand phototropin-mediated membrane hyperpolarization-dependent (Harada and Shimazaki 2009).

6.3.2  Red Light Red light (RL) induces stomatal opening, but this induction is smaller compared with BL. This response to RL coincides with CO2 assimilation spectra (Sharkey and Raschke 1981). Additionally, RL response is abolished by inhibitors of photosynthetic electron transport suggesting that RL induction of stomatal opening is likely mediated through photosynthesis (Sharkey and Raschke 1981; Tominaga et  al. 2001; Zeiger et al. 2002). However, another scenario is possible where increase in stomatal conductance upon RL irradiation is not dependent of photosynthesis (Baroli et al. 2008). As receptors, Red/far-red light-absorbing phytochromes (PHY) also play a role in the control of stomatal aperture (Chen et al. 2012). There are five PHYs described in Arabidopsis, from PHYA to PHYE (Sharrock and Quail 1989; Clack et al. 1994). PHYB is involved in RL regulation of stomatal opening and its action is in concert with PHYA, and BL receptors CRY and PHOT since multiple mutants combining

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these genes show stronger phenotypes when phyB mutation is present. Similar approaches evidenced that COP1 together with PIFs (phytochrome interacting factors) act downstream of PHYB during stomatal opening (Wang et al. 2010).

6.3.3  UV-B Although high doses of UV-B can cause several damages in plants, UV-B is also a signal for various plant processes (Jenkins 2014). In 2011 the UV-B receptor, UVR8 (UV RESISTANCE LOCUS 8) was characterized (Rizzini et al. 2011). Regarding GCs, stomatal differentiation is one of developmental processes stimulated by UV-B and is UVR8-dependent (Wargent et al. 2009). UV-B-dependent modulation of stomatal closure is still generating some controversies. There are reports indicating that UV-B induces stomatal opening (Jansen and Van Den Noort 2000; Eisinger et al. 2003) while others show induction of stomatal closure (Nogués et al. 1999; He et al. 2011a, b, 2013; Zhu et al. 2014; Li et al. 2017).These discrepancies are fluence rate-dependent and also depend on metabolic-­ state of GC (Jansen and Van Den Noort 2000). Nevertheless, there is more evidence supporting the stomatal closure effect in responses that involve the participation of hormones such ethylene (He et  al. 2011b) and other second messengers like Gα protein, NO, MPKs and RBOH-dependent H2O2 production (He et  al. 2011a, b, 2013; Zhu et al. 2014; Li et al. 2017). Recent studies indicate that upon UV-B radiation H2S induces the accumulation of UV-absorbing compounds maintaining redox homeostasis and alleviates UV-B damage by enhancement antioxidant systems in plant leaves (Li et al. 2016; Rostami et al. 2019). According to the persulfidated proteome recently presented by Aroca et  al. (2017), several members of the light dependent signaling, including the receptors PHOT1, PHOT2, CRY1 and 2 and UVR8 are in the list of persulfidated proteins (Table 6.1). Nevertheless, there are still no works linking H2S to light dependent regulation of stomatal movement. The unique existing evidence is a report in Vicia faba indicating  H2S is required for dark induced stomatal closure, acting downstream of H2O2 production (Ma et al. 2018).

6.4  Stomatal Conductance and CO2 Stomatal GCs sense intercellular CO2 concentration rather than CO2 at the leaf surface and respond differently depending on the level of CO2 (Assmann 1999). CO2 levels in the leaf may vary according to photosynthesis and respiration rates during day/night cycles and also by the increase in atmospheric CO2. Low levels of CO2 stimulate stomatal opening and high CO2 concentrations induce stomatal closure (Assmann 1999; Israelsson et  al. 2006; Young et  al. 2006). Briefly, for stomatal

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Table 6.1  List of proteins participating in guard cell signaling which are persulfidated in basal conditions in Arabidopsis thaliana leaves Protein Pyrabactin resistance 1 (PYR1) PYR1-LIKE 1 (PYL1) SNF1-RELATED PROTEIN KINASE 2.2 (SnRK2.2) ABA DEFICIENT 2 (ABA2) NITRATE REDUCTASE 1 (NIA1) NITRATE REDUCTASE 2 (NIA2) PHOSPHOLIPASE D ALPHA 1 (PLDα1) CALCIUM DEPENDENT PROTEIN KINASE 3 (CPK3) CALCIUM-DEPENDENT PROTEIN KINASE 4 (CPK4) CALCIUM DEPENDENT PROTEIN KINASE 2 (CPK11) CALCIUM DEPENDENT PROTEIN KINASE 21(CPK21) MAP KINASE 4 (MPK4)

Accession number AT4G17870 AT5G46790 AT3G50500

Signaling pathway ABA

AT4G23650

Reference Park et al. (2009) Park et al. (2009) Umezawa et al. (2010) Xie et al. (2006) Bright et al. (2006) Zhang et al. (2004) Mori et al. (2006)

AT4G09570

Zhu et al. (2007)

AT1G35670

Zhu et al. (2007)

AT4G04720

Geiger et al. (2011) Gomi et al. (2005) and Töldsepp et al. (2018) Mori et al. (2006) Wang et al. (2016b) Töldsepp et al. (2018) and Jammes et al. (2009) Hu et al. (2010)

AT1G52340 AT1G77760 AT1G37130 AT3G15730

AT4G01370

ABA/CO2/O3

CALCIUM DEPENDENT PROTEIN KINASE 6 (CPK6)

AT2G17290

ABA/CO2

MITOGEN-ACTIVATED PROTEIN KINASE 12 (MPK12)

AT2G46070

BETA CARBONIC ANHYDRASE 1 (βCA1) BETA CARBONIC ANHYDRASE 4 (βCA4) PHOTOTROPIN 1 (PHOT1) PHOTOTROPIN 2 (PHOT2) ROOT PHOTOTROPISM 2 (RPT2)

AT3G01500

AT3G45780 AT5G58140 AT2G30520

CRYPTOCHROME 1 (CRY1) CRYPTOCHROME 2 (CRY2)

AT4G08920 AT1G04400

CO2

AT1G70410 Blue light

Kinoshita et al. (2001) Inada et al. (2004) Mao et al. (2005) (continued)

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Table 6.1 (continued) Accession number AT5G63860

Signaling pathway UV-B

MITOCHONDRIALNITROGEN FIXATION S (NIFS)-LIKE 1/CYSTEINE DESULFURASE (MtNFS1) ß-CYANOALANINE SYNTHASE (CAS-C1) CYSTEINE SYNTHASE D1 (CYS-D1)

AT5G65720

H2S metabolism

CYSTEINE SYNTHASE D2 (CYS-D2)

AT5G28020

Protein UVB-RESISTANCE 8 (UVR8)

AT3G61440 AT3G04940

CHLOROPLASTIC NIFS-LIKE/ AT1G08490 CYSTEINE DESULFURASE (CpNIFS) D-CYSTEINE DESULFHYDRASE (DCD) AT1G48420 O-ACETYLSERINE (THIOL) LYASE A (OAS-A) O-ACETYLSERINE (THIOL) LYASE B (OAS-B) O-ACETYLSERINE (THIOL) LYASE C (OAS-C) L-CYSTEINE DESULFHYDRASE 1 (DES1) CATALASE 1 (CAT1)

AT4G14880

AT1G20630

CATALASE 2 (CAT2)

AT4G35090

CATALASE 3 (CAT3)

AT1G20620

ASCORBATE PEROXIDASE 1 (APX1)

AT1G07890

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT 1 (GAPC1)

AT3G04120

AT2G43750 AT3G59760 AT5G28030 Redox metabolism

Reference Rizzini et al. (2011) Riemenschneider et al. (2005) Yamaguchi et al. (2000) Yamaguchi et al. (2000) Yamaguchi et al. (2000) Pilon-Smits et al. (2002) Riemenschneider et al. (2005) Wirtz et al. (2004) Wirtz et al. (2004) Wirtz et al. (2004) Álvarez et al. (2010) Böhmer and Schroeder (2011) Jannat et al. (2011a) Jannat et al. (2011b) Aroca et al. (2015) Aroca et al. (2015)

closure, the signaling involves activation of S-type anion and K+out channels and modulation on [Ca2+]cyt (Brearley et al. 1997; Schroeder et al. 2001; Hanstein and Felle 2002; Raschke et al. 2003; Young et al. 2006). Furthermore, long-term exposure to a high CO2 environment causes a decrease in stomatal density with a consequent reduction in stomatal conductance (Gray et al. 2000). Once in solution, CO2 is converted into carbonic acid, bicarbonate and H+ by carbonic anhydrases (CAs).

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Although bicarbonate induces stomatal closure in a H2O2-dependent and SLAC1 activation manner (Kolla et al. 2007; Hu et al. 2010; Xue et al. 2011), there is still no evidence of cytosolic pH changes under elevated CO2 as a way to measure this conversion (Brearley et al. 1997; Kim et al. 2010). In plants there are multiple genes coding for the three types of CAs named α, β, and γ (DiMario et al. 2017). βCA1 localized in chloroplast and βCA4 localized in cytosol are highly expressed in GCs (Leonhardt et al. 2004; Hu et al. 2010) and are considered to act early in stomatal response to CO2, since double mutant ca1ca2 (but not the single mutants) is impaired in stomatal closure in response to high levels of CO2, but still has a  wild type response to blue light and ABA (Hu et al. 2010; Engineer et al. 2016). Interestingly, both βCA1 and βCA4 were also included in the list of persulfidated proteins, under basal conditions (Aroca et al. 2017) (Table 6.1) and, therefore, would be interesting to study the role of this posttranslational modification in response to CO2. HIGH LEAF TEMPERATURE 1 (HT1) has been characterized as another early component of CO2 signaling in GCs. HT1 inhibits OST1 and is, though, considered a negative regulator of CO2-induced stomatal closure preventing the activation of SLAC1 (Tian et al. 2015). Studies made in different ABA signaling-affected mutants submitted to elevated CO2 show that both ABA and CO2 pathways partially overlap (Merilo et al. 2013; Chater et al. 2015). For example, SLAC1 is phosphorylated and activated by other kinases such as Ca2+-dependent protein kinase (CPK) 6 and CPK23 (Wang et al. 2016a). Mitogen-activated protein kinases (MAPKs) are also involved in both pathways, specifically MPK4 and MPK12 (Marten et  al. 2008; Jakobson et al. 2016). It is important to highlight that some of these “common components” are also persulfidated (Table 6.1) (Aroca et al. 2017).

6.5  S  tomatal Conductance and Plant Growth Under Ozone Exposure Ozone (O3) is considered a major troposphere pollutant causing negative effects in plant growth and important reductions in agronomical productivity (Fiscus et  al. 2005; Wilkinson and Davies 2010). O3 enters into leaves through stomata and once in leaf internal air spaces O3 reacts with different components at cell wall or plasma membrane surface generating apoplastic ROS (Fiscus et  al. 2005; Vaultier and Jolivet 2015). As it happens for plant-pathogen interaction, ROS production in response to O3 has a biphasic oxidative burst where the first peak is associated directly with effects of O3 and the second one corresponds to a plant-derivative ROS burst (Schraudner et al. 1998). As a first defense barrier and depending on O3 concentration, plants lower stomatal conductance during the first minutes of exposition and, after 30–40  min, they can recover to pre-exposition values (Moldau et  al. 2011). This type of response occurs also in response to H2S donors but with a different timescale, while stomata close at 90 min and reopen after 120 min (Honda et al. 2015).

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This transient decrease in stomatal conductance in response to O3 also coincides with a dual ROS burst in GCs (Kollist et al. 2007; Vahisalu et al. 2010). It is important to note that at the beginning of O3 exposure, ROS are detected in chloroplast of GCs and later in adjacent cells in a NADPH-dependent manner (Joo et al. 2005; Vahisalu et al. 2010). Comparing with ABA “signalosome”, PYR/RCARs and the PP2Cs ABI1 and ABI2 are also required for O3-induced transient decrease in stomatal conductance indicating that the signaling pathways overlap just like occurs between ABA and CO2 signaling (Kollist et  al. 2007; Vahisalu et  al. 2010; Merilo et  al. 2013). Furthermore, OST1 kinase and anion channel SLAC1 also act in O3-induced stomatal closure (Vahisalu et al. 2008; Vahisalu et al. 2010). Following with ion channels involvement it is also known that O3 prevents stomatal opening through inhibition of K+in channels (Torsethaugen et al. 1999). Other second messengers that participate in O3 response are Ca2+ and NO. O3 induces NO production in GCs which modulates the expression of several defense-­ related genes necessary for a complete response to O3 (Ahlfors et al. 2009). On the other hand, Ca2+concentrations were reported to increase in response to O3 but this was not measured in GCs (Evans et al. 2005). Although some of the signaling hubs that were reported to participate in H2S signaling such as ABI1, ROS and NO are also active in O3 induced stomatal closure, there are no reports of the interaction of these two pathways.

6.6  Conclusion and Perspectives As for other members of the gasotransmitter group, H2S has emerged from “environmental pollutant” to ubiquitous signal molecule. In plants, where it is endogenously synthesized in different subcellular compartments, H2S has been reported to be part of the signaling processes triggered by different biotic and abiotic stresses. In the present chapter we reviewed the data on the participation of H2S in stomatal movement. The compilation of the available bibliography presents H2S as a highly connected component of the GC signaling network (Fig. 6.2). We now face the challenge to unveil the mechanism of action and identify the molecular targets of H2S in vivo. The current knowledge on plant H2S-biology points towards the ability of H2S to modify the activity of target proteins through persulfidation, the posttranslational modification underwent by protein thiols to form persulfides, (Mustafa et al. 2009). A recent proteomic analysis of persulfidated proteins in Arabidopsis thaliana revealed that about 5% of the Arabidopsis proteome is susceptible of persulfidation, including several key components of GC signaling network (Table  6.1). On the other hand, it is known that H2S interacts with other key players like cGMP (Honda et al. 2015) to form a new reactive molecule. Furthermore, at least in animal systems, it was shown that H2S can react directly with NO to form nitroxyl (HNO)- or poly-sulfides (Cortese-Krott et al. 2015) but this process is not shown in plants yet.

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Fig. 6.2  Simplified models of the components of the guard cell signaling networks that participate in abiotic stress induced stomatal closure before (a) and after (b) the emergence of H2S as an active signal molecule in plants. ABA abscisic acid, PPR PYRABACTIN RESISTANCE1

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In short, thinking of the H2S as a gasotransmitter there is still much to be unravel about its mechanism of action but the growing evidence of its participation in signaling processes suggests that it might be considered as a new “hub” in the guard cell signaling.

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Fig. 6.2 (continued) (PYR1)/PYR1-LIKE (PYL1)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR), ABI1 ABSCISIC ACID INSENSITIVE 1, UVR8 UV RESISTANCE LOCUS 8, ROS reactive oxygen species, NO nitric oxide, SnRKs Snf1 (sucrose non-fermenting1)-related protein kinases, SLAC slow activating anion channels, CA carbonic anhydrase, K+in inward rectifying K+ channels. Black filled rectangles: Environmental stresses; Yellow filled circles: ABA-Receptor complex; Full lines: reported interactions; Dotted lines: hypothetical interactions

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Chapter 7

Hydrogen Sulfide and Fruit Ripening Francisco J. Corpas, Salvador González-Gordo, and José M. Palma

Abstract  Fruit ripening is a complex physiological process involving many external modifications affecting shape, color, flavor, and metabolite composition, among others, which are consequence of deeper changes at biochemical, molecular, and cellular level. Hydrogen sulfide (H2S) is a gasotransmitter molecule that is endogenously produced in plant cells by enzymatic processes during sulfur metabolism. At present, H2S is recognized as a new signaling molecule because it has the capacity to affect protein function through a posttranslational modification (PTM) designated persulfidation. The present chapter has the goal to provide an updated comprehensive overview of the H2S metabolism and its implication in the ripening of climacteric and non-climacteric fruits. Moreover, the beneficial effects exerted by the exogenous application of H2S during the ripening period and postharvest storage are also overviewed. Keywords  Antioxidants · Climacteric and non-climacteric fruits · Hydrogen peroxide · Hydrogen sulfide · Persulfidation · Post-translational modification

7.1  Introduction Fruit is the structure of flowering plants which results of the setting and the development of a fertilized ovary that contains the seeds for the future plant propagation. Fleshy fruit ripening is a highly regulated and complex physiological process which involves drastic changes affecting their external appearance as well as their internal metabolism since many biochemical pathways undergo deep adaptations in all F. J. Corpas (*) · S. González-Gordo · J. M. Palma Department of Biochemistry, Cell and Molecular Biology of Plants, Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Estación Experimental del Zaidín (Spanish National Research Council, CSIC), Granada, Spain e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_7

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subcellular compartments, including plastids, mitochondria and peroxisomes (Osorio et al. 2013; Palma et al. 2015; Quinet et al. 2019). As consequence, fruits experiment modifications in flavor, aroma, nutrient composition, hardness, shape, and color, among others (Carrari et  al. 2006; Wahyuni et  al. 2011; Correa et  al. 2018; Wang et al. 2020). In fact, the diversity of fruits is amazing as it can be exemplified by some representative Angiosperm families such as Rosaceae (apples, peaches, strawberries, etc), Solanaceae (tomatoes, peppers, eggplants, etc) or Rutaceae (citrus) which include hundreds of genera and thousands of species with a huge variability of phenotypes (Farinati et al. 2017). At physiological level, fleshy fruits can be divided in two main categories, either climacteric or non-climacteric according to their respiration pattern and their dependency of the ethylene hormone during maturation. Climacteric fruits, such as tomato, banana, and avocado, among others, can be harvested in unripe stage and stored for some period of time, for example at low temperature, and the ripening process will continue because it cannot be stopped once the overproduction of ethylene has been triggered. On the other hand, in non-climacteric fruits such as pepper, citrus, sweet cherry, strawberries, olives or grapes, the ripening does not respond to high ethylene concentrations and it can only occur while they are attached to the parental plant. Therefore, once the plant has been harvested, fruit ripening does not go longer. Besides the involvement or not of ethylene in the process of fruit ripening, there are other biomolecules such as abscisic acid (ABA), auxin (indole-3-acetic acid), brassinosteroids, salicylic acid, melatonin, reactive oxygen species (ROS) or nitric oxide (NO) which have been shown to have the capacity to modulate this process through signaling episodes (Leng et al. 2014; He et al. 2018; Corpas and Palma 2018; Pérez-Llorca et al. 2019). More recently, hydrogen sulfide (H2S) has been revealed to be a new biomolecule which participates in the modulation in the process of fruit ripening (Ziogas et al. 2018; Mukherjee, 2019; Corpas and Palma 2020). Although still scarce, the present chapter has the goal to provide a comprehensive updated overview of the biochemical metabolism of H2S in plant cells and its implications in the fruit ripening and postharvest.

7.2  How H2S Is Endogenously Generated in Plant Cells? At present, it is well recognized that H2S is a key molecule in the metabolism of sulfur in higher plants which is endogenously generated. Based in the available information in the model plant Arabidopsis thaliana, it has been possible to identify so far until six key enzymes which are involved in the metabolism of H2S, although some of them could be composed by several isozymes. These enzymes are located in different subcellular compartments and include: (i) three cytosolic enzymes, L-cysteine desulfhydrase (L-DES), cysteine synthase 1 (OASA1) and L-cysteine desulfhydrase 1(DES1); (ii) two mithocondrial enzymes, D-cysteine desulfhydrase (D-DES) and cyano alanine synthase (CAS); and (iii) the sulfite

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reductase (SiR) located in chloroplasts (Calderwood and Kopriva 2014; Gotor et al. 2015; González-­Gordo et al. 2020a). At cytosolic level, L-DES belongs to the class-V pyridoxal-­ phosphate-­ dependent aminotransferase family and catalyzes the production of H2S from cysteine (Cys) being a key regulator of stomatal movement and closure (García-Mata and Lamattina 2010). On the other hand, the cytosolic OASA1 belongs to the cysteine synthase/cystathionine β-synthase family and is a key enzyme for cysteine biosynthesis and the fixation of inorganic H2S catalyzing the formation of Cys from O-acetylserine and inorganic sulfide. The OASA1 gene expression seems to be induced in leaves, stems and roots by high salt and heavy metal stresses, mediated by ABA (Jia et al. 2016; Li et al. 2018). Finally, L-DES1 is a bifunctional cystathionine γ-lyase/cysteine synthase involved in the Cys homeostasis through the desulfuration of L-Cys, being a key enzyme in the generation of H2S in the cytosol (Laureano-Marín et al. 2014). At mitochondrial level, D-DES belongs to the pyridoxal-5′-phosphate-dependent enzyme family protein and catalyzes the generation of H2S from Cys but using preferentially D-cysteine as substrate (Riemenschneider et  al. 2005). On the other hand, CAS functions as a major β-cyanoalanine synthase and it seems to be involved in the detoxification of cyanide (CN−) that arises from ethylene biosynthesis and maintains a low level of cyanide for proper root hair development (Hatzfeld et al. 2000; Meyer et al. 2003). The chloroplast SiR belongs to the nitrite and sulfite reductase 4Fe-4S domain family and its activity is needed in assimilatory sulfate reduction pathway during both primary and secondary metabolism, and thus it is involved in plant development and growth (Sekine et al. 2007). Figure 7.1 provides an analysis of the metabolic interaction networks, obtained with the program String, of these Arabidopsis enzymes involved in H2S metabolism and located in the different subcellular compartments including chloroplasts, cytosol, and mitochondria. Besides these identified enzymes involved in H2S metabolism, very recently, in peroxisomes from Arabidopsis it has been also reported the presence of H2S with the capacity to modulate the catalase activity, although the enzymatic source of this H2S is still unknown (Corpas et al. 2019a). Based on the available information about these Arabidopsis enzymes involved in the H2S metabolism, Table 7.1 provides an analysis of their orthologue proteins found in tomato (Solanum lycopersicum L.) and pepper (Capsicum annuum L.), which are two representative examples of climacteric and non-climacteric Solanaceae fruits, respectively. Along with all these endogenous enzymatic sources of H2S in plants, it has been started to be considered the involvement of exogenous H2S, which is also known to be part of the atmospheric air as a pollutant (Malone Rubright et al. 2017). In fact, plants have the capacity to interact with the atmospheric H2S in both directions: emission and uptake (Lakkineni et al. 2003; Ausma and De Kok 2019). Thus, the H2S uptake is mainly achieved via stomata and its effects in the endogenous S metabolism depends on its levels because high atmospheric levels are phytotoxic whereas low levels could contribute positively to the plant S requirements (Birke et al. 2015; Fuentes-Lara et al. 2019).

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Fig. 7.1 Protein-protein interaction network of enzymes involved in H2S metabolism in Arabidopsis thaliana. L-cysteine desulfhydrase (L-DES), cysteine synthase 1 (OASA1) and L-cysteine desulfhydrase 1(DES1) are located in the cytosol. D-cysteine desulfhydrase (D-DES) and cyano alanine synthase (CAS) are in mitochondrion, whereas the sulfite reductase (SiR) is in chloroplast. Network nodes represent proteins and colored lines the different types of interaction evidences used in predicting the associations. A green line indicates neighborhood evidence; a blue line indicates co-occurrence evidence; a dark yellow line indicates text-mining evidence; and a black line indicates co-expression evidence

The H2S biochemistry in biological systems is complex and it can generate a battery of related molecules such as hydropersulfides or polysulfides which have relevant functions in the redox homeostasis of the cells (Ono et al. 2014; Álvarez et al. 2017; Fukuto et al. 2018), as well as acting as a signal in root nodule symbiosis in legume (Fukudome et al. 2020). In fact, one of the most relevant physiological functions of the cellular H2S, independently of its source, is that it can function as a signal molecule mainly through a post-translational modification (PTM) called persulfidation. This PTM consists in the oxidation of the thiol group (-SH) present in cysteine residues to the persulfide form (-SSH). Persulfidation can be considered as a mechanism of protein protection because it is reversible compared to a drastic oxidation where the thiol groups are modified to sulfinic (-SO2H) or sulfenic (-SO3H) states which are irreversible processes and usually trigger inhibition of the biological activity (Ono et al. 2014). In higher plants, the number of proteins that undergo persulfidation is increasing (Aroca et al. 2015, 2018; Muñoz-Vargas et al. 2018; Corpas et al. 2019a, b; Gotor et al. 2019; Tao et al. 2020), Thus, it has been found that persulfidation participates in natural physiological processes as well as in the mechanism of response against environmental stresses (Corpas and Palma 2020; Pandey and Gautam 2020; Shen et al. 2020). Table 7.2 shows some plant proteins which have been identified to be persufidated and their functional implications.

Enzyme name L-cysteine desulfhydrase (L-DES) D-cysteine desulfhydrase 2 (D-DES) Sulfite reductase (SiR) Cyanoalanine synthase (CAS) Cysteine synthase 1 (OASA1) L-cysteine desulfhydrase 1 (DES1) 71.95 39.93 33.81

Chloroplast At5g04590 642 Mitochondrion At3g61440 368

At4g14880 322

At5g28030 323

Cytosol

Cytosol

34.33

47.43

Mw AGI code Size (aa) (kD) At3g62130 454 50.69

Arabidopsis thaliana

Mitochondrion At3g26115 427

Cellular location Cytosol 448

5.61 A0A2G2YYZ2 323

5.90 A0A1U8GCP9 325

8.51 A0A2G2YFY5 695 8.71 A0A1U8DSU5 369

8.70 A0A1U8H384

34.45

34.35

78.27 40.07

49.39

Pepper (Capsicum annuum L.) Size Mw pI UniProt ID (aa) (kD) 6.00 A0A1U8DYD9 453 50.30

5.26 Solyc08g014340 386 40.94 5.41

5.94 Solyc09g082060 325 34.24 5.93

9.30 Solyc11g065620 691 77.91 9.29 8.63 Solyc10g012370 351 38.24 8.39

7.56 Solyc01g008900 442 48.60 7.22

Tomato (Solanum lycopersicum L.) Size Mw pI Solyc ID (aa) (kD) pI 6.10 Solyc01g068160 454 50.55 6.64

Table 7.1  Enzymes involved in H2S metabolism in Arabidopsis thaliana and their corresponding orthologues in the non-climacteric pepper (Capsicum annuum L.) and climacteric tomato (Solanum lycopersicum L.) fruits, respectively. Number of amino acids (aa), molecular weight (kD) and theoretical isoelectric point (pI)

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Table 7.2  Plant proteins which have been identified to undergo persulfidation and their function Enzyme Ascorbate peroxidase (APX) Catalase Respiratory burst oxidase homolog protein D (RBOHD) RuBISCO Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Glutamine synthetase (GS) O-acetylserine(thiol)lyase (OAS-TL) L-cysteine desulphydrase (LCD) NADP-isocitrate dehydrogenase (NADP-ICDH) NADP-malic enzyme (NADP-ME) 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6)

Metabolic function Antioxidant in ROS metabolism Antioxidant in ROS metabolism Generation of superoxide radical in ROS metabolism/ Photosynthesis Production of energy in the glycolysis Metabolism of nitrogen Sulfur metabolism

Reference Aroca et al. (2015)

Sulfur metabolism Provide NADPH as a reducing agent Provide NADPH as a reducing agent Ethylene biosynthesis

Chen et al. (2011) Muñoz-Vargas et al. (2018) Muñoz-Vargas et al. (2020) Jia et al. (2018)

Corpas et al. (2019a) and Palma et al. (2020) Shen et al. (2020) Chen et al. (2011) Aroca et al. (2015) Aroca et al. (2015) Chen et al. (2011)

Promote ABA signaling during Chen et al. (2020) stomatal closure

7.3  E  ndogenous H2S Metabolism during Fruit Ripening and Potential Beneficial Effects of the Exogenous H2S Application During Postharvest The available information of endogenous H2S metabolism in plants is relatively limited. One of the best characterized ripening processes, with regard to the biochemical interactions among H2S, ROS and RNS metabolism, has been reported in the non-climacteric sweet pepper (Capsicum annuum L.) fruit during its transition from the immature (green) to the ripe (red) stages. Accumulating data indicate that during ripening, a physiological nitro-oxidative stress (phystress; Palma et  al. 2019) takes place, which is accompanied by an increase of H2S content, but also by a higher cytosolic L- cysteine desulfidrase activity. This process has been found to be characterized by an increase of several stress markers including protein nitration (Chaki et al. 2015), lipid peroxidation (Chu-Puga et al. 2019) and a rise of the proline content (González-Gordo et al. 2019). Moreover, it was reported that the NADPH generating isocitrate dehydrogenase (NADP-ICDH) activity decreased significantly during ripening, being its persulfidation, nitration and S-nitrosation some of the mechanisms involved in its activity regulation (Muñoz-Vargas et al. 2018). In addition, several components of ROS and RNS families were differentially regulated. Thus, whereas the superoxide-generating NADPH oxidase activity and the ascorbate content increased during pepper fruit ripening, other

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parameters decreased such as catalase, ascorbate peroxidase (APX) and S-nitrosoglutathione reductase (GSNOR) activities, as well as the GSH content (Chaki et al. 2015; Rodríguez-Ruiz et al. 2017a, b, 2019; Chu-Puga et al. 2019; González-Gordo et al. 2019; González-Gordo et al. 2020b). More recently it has been demonstrated by in vitro approaches that pepper catalase, a key antioxidant enzyme which keeps under control the cellular H2O2 content, is inhibited by H2S and NO (Corpas et al. 2019a; Rodríguez-Ruiz et al. 2019) which clearly indicates a regulation of the H2O2 metabolism by both H2S and NO.  Figure  7.2 shows a simple model which summarizes all these components involved in the physiological process of sweet pepper ripening characterized by an active nitro-oxidative metabolism. In a recent study in peach fruits, it has been observed that endogenous H2S metabolism is modulated by exogenous NO which triggers (i) lower content of endogenous H2S, Cys, and sulfite; (ii) decreased activity of L-/D-cysteine desulfhydrase, O-acetylserine (thiol)lyase (OAS-TL), and sulfite reductase (SiR); and, (iii) increased activity of β-cyanoalanine synthase (β-CAS) (Geng et al. (2019). Taken together, these data demonstrate a clear regulation of H2S metabolism by NO.

Fig. 7.2  Model of the endogenous hydrogen sulfide (H2S), reactive oxygen species (ROS) and reactive nitrogen species (RNS) metabolism during ripening (green to red) of the non-climacteric sweet pepper (Capsicum annuum L.) fruit. APX ascorbate peroxidase, GSH reduced glutathione, GSNOR nitrosoglutathione reductase, H2S hydrogen sulfide, L-DES L-cysteine desulfidrase

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It should be pointed out that one of the research areas that is being developed concerning the H2S metabolism is its exogenous application to different types of fleshy fruits because, at practical levels, it has been found that this gasotransmitter has the capacity to delay the process of fruit ripening in both climacteric and non-­ climacteric fruits (Hu et al. 2012; Yao et al. 2018; Ziogas et al. 2018). Moreover, it seems that H2S also triggers several biochemical pathways which provide protection during postharvest storage such as delaying senescence, increasing tolerance to cold, alleviating fruit softening and exerting antifungal effects. In many of those cases, it has been shown a modulation of the ROS metabolism where the activity of several antioxidant enzymes such as catalase, superoxide dismutase, and ascorbate peroxidase is up regulated to avoid ROS overproduction under oxidative stress situations such as those which impose senescence or low temperature. Additionally, these antioxidant enzymes could be downregulated allowing an increase of ROS production which is used as a weapon against pathogens (Zhang and Wong 2009; Schmidt et al. 2020). Table 7.3 displays representative examples of climacteric and non-climacteric fruits where the exogenous application of H2S provides diverse beneficial effects.

7.4  Conclusion and Future Perspectives The available information allows confirming that the gasotransmitter H2S is involved in the ripening regulation of fleshy fruit. However, our knowledge of the endogenous H2S metabolism such as how it is generated and its interaction with other biomolecules (phytohormones, NO, melatonin, among others) is still extremely limited being an area which will need future development. Nevertheless, the available experimental data clearly indicate that the exogenous application of H2S exerts beneficial effects on fruit ripening and postharvest being a promising area of research which should be developed considering its biotechnological applications in the horticultural industry. Figure 7.3 displays a simple model where the main implication of H2S in the fruit ripening of both climacteric and non-climacteric fruits is shown, as well as the biochemical mechanisms involved in the defense against some negative effects that undergo during fruit postharvest storage which could be palliated, such as symptoms of softening, chilling damage and fungal infections.

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Table 7.3  Representative examples of the main beneficial effects of the exogenous application of H2S in climacteric and non-climacteric fruits Fruit Climacteric Banana (Musa acuminata, AAA group) Avocado (Persea americana Mill, cv. ‘Hass’) Tomato (Solanum lycopersicum L.) ‘Micro Tom’ Tomato (Lycopersicon esculentum)

H2S donor

Kiwifruit (Actinidia chinensis)

20μM H2S

Kiwifruit (Actinidia deliciosa)

0.5 mM NaHS

Peach [Prunus persica (L.) Batsch] Peach fruits (Prunus persica cv. (L.)

50 mM NaHS Endogenous H2S

Apple (Malus domestica), pear (Pyrus bretschneideri Rehd.) Non-climacteric Grape (Vitis vinifera L. × V. labrusca L. cv. Kyoho) Strawberry (Fragaria × ananassa Duch.) Hawthorn (Crataegus oxyacantha) fruit

0.5 mM NaHS

Orange (Citrus × sinensis) Orange (Citrus sinensis), Mandarin (Citrus reticulata)

100μL L−1 H2S 0.5 mM NaHS

Effects

1 mM NaHS Alleviates fruit softening. Antagonizes ethylene effects

Reference Ge et al. (2017)

200μM NaHS

Protects against frost and day high light Joshi et al. (2020)

0.9 mM NaHS

Postpones ripening and senescence of postharvest tomato fruits by antagonizing the effects of ethylene Triggers ROS generation. Antifungal effects against Aspergillus niger and Penicillium italicum Delays the ripening and senescence. Inhibits ethylene production. Increases antioxidant activities. Regulates cell wall degrading enzyme gene Triggers ROS generation. Antifungal effects against Aspergillus niger and Penicillium italicum Reduces brown rot caused by Monilinia fructicola Exogenous NO decreased endogenous H2S metabolism which affects quality during cold storage Triggers ROS generation. Antifungal effects against Aspergillus niger and Penicillium italicum

0.5 mM NaHS

Yao et al. (2018) and Hu et al. (2019) Fu et al. (2014)

Zhu et al. (2014) and Lin et al. (2020) Fu et al. (2014)

Wu et al. (2018) Geng et al. (2019) Fu et al. (2014)

1 mM NaHS Alleviates postharvest senescence of grape and maintains high fruit quality

Ni et al. (2016)

0.8 mM NaHS 1.5 mM NaHS

Hu et al. (2012) Aghdam et al. (2018)

Prolongs postharvest shelf life and reduces fruit rot disease Confers tolerance to chilling. Triggers H2S accumulation, increases activity of antioxidant enzymes and promotes phenol accumulation Delays senescence Triggers ROS generation. Antifungal effects against Aspergillus niger and Penicillium italicum

Alhassan et al. (2020) Fu et al. (2014)

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Fig. 7.3  Summary of the main effects of the H2S in ripening and postharvest of both climacteric and non-climacteric fruits Acknowledgements  Our research is supported by a European Regional Development Fund cofinanced grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P and PID2019-103924GB-I00), the Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020) (P18-FR-1359) and Junta de Andalucía (group BIO192), Spain.

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Pérez-Llorca M, Muñoz P, Müller M et al (2019) Biosynthesis, metabolism and function of auxin, salicylic acid and melatonin in climacteric and non-climacteric fruits. Front Plant Sci 10:136 Quinet M, Angosto T, Yuste-Lisbona FJ et al (2019) Tomato fruit development and metabolism. Front Plant Sci 10:1554 Riemenschneider A, Wegele R, Schmidt A et  al (2005) Isolation and characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana. FEBS J 272:1291–1304 Rodríguez-Ruiz M, Mateos RM, Codesido V et al (2017a) Characterization of the galactono-­1,4-­ lactone dehydrogenase from pepper fruits and its modulation in the ascorbate biosynthesis. Role of nitric oxide. Redox Biol 12:171–181 Rodríguez-Ruiz M, Mioto P, Palma JM et  al (2017b) S-nitrosoglutathione reductase (GSNOR) activity is down-regulated during pepper (Capsicum annuum L.) fruit ripening. Nitric Oxide 68:51–55 Rodríguez-Ruiz M, González-Gordo S, Cañas A et al (2019) Sweet pepper (Capsicum annuum L.) fruits contain an atypical peroxisomal catalase that is modulated by reactive oxygen and nitrogen species. Antioxidants (Basel) 8:374 Schmidt A, Mächtel R, Ammon A et  al (2020) Reactive oxygen species dosage in Arabidopsis chloroplasts can improve resistance towards Colletotrichum higginsianum by the induction of WRKY33. New Phytol 226:189–204 Sekine K, Fujiwara M, Nakayama M et al (2007) DNA binding and partial nucleoid localization of the chloroplast stromal enzyme ferredoxin:sulfite reductase. FEBS J 274:2054–2069 Shen J, Zhang J, Zhou M et  al (2020) Persulfidation-based modification of cysteine desulfhydrase and the NADPH oxidase RBOHD controls guard cell abscisic acid signaling. Plant Cell 32:1000–1017 Tao C, Tian M, Han Y (2020) Hydrogen sulfide: a multi-tasking signal molecule in the regulation of oxidative stress responses. J Exp Bot 71:eraa093 Wahyuni Y, Ballester AR, Sudarmonowati E et al (2011) Metabolite biodiversity in pepper (capsicum) fruits of thirty-two diverse accessions: variation in health-related compounds and implications for breeding. Phytochemistry 72:1358–1370 Wang R, Angenent GC, Seymour G et al (2020) Revisiting the role of master regulators in tomato ripening. Trends Plant Sci 25:291–301 Wu W, Zhang C, Chen L et al (2018) Inhibition of hydrogen sulfide and hypotaurine on Monilinia fructicola disease in peach fruit. Acta Hortic 1194:257–266 Yao GF, Wei ZZ, Li TT et al (2018) Modulation of enhanced antioxidant activity by hydrogen sulfide antagonization of ethylene in tomato fruit ripening. J Agric Food Chem 66:10380–10387 Zhang X, Wong SM (2009) Hibiscus chlorotic ringspot virus upregulates plant sulfite oxidase transcripts and increases sulfate levels in kenaf (Hibiscus cannabinus L.). J Gen Virol 90:3042–3050 Zhu L, Wang W, Shi J et al (2014) Hydrogen sulfide extends the postharvest life and enhances antioxidant activity of kiwifruit during storage. J Sci Food Agric 94:2699–2704 Ziogas V, Molassiotis A, Fotopoulos V et al (2018) Hydrogen sulfide: a potent tool in postharvest fruit biology and possible mechanism of action. Front Plant Sci 9:1375

Chapter 8

Hydrogen Sulfide Impact on Seed Biology Under Abiotic Stress Emmanuel Baudouin

Abstract  Successful germination is a critical step of plant life cycle that has dramatic impacts on plant performance in natural ecosystems and agrosystems. Paradoxically, seeds can retain viability even after long term exposure to extreme environmental conditions, whereas their germination is possibly modified by only tiny variations of their abiotic environment. Because of the ongoing climate changes that will bring frequent episodes of extreme temperature, rainfall and drought, seed germinating capacity will be dramatically modified, jeopardizing crop yield, plant spreading and survival. Understanding the mechanisms regulating seed germination under abiotic constraints is therefore critical to develop strategies to mitigate the effects of global climate change. During the last decade, the central function of redox processes in the control of seed traits, i.e. dormancy, germination or longevity, has been illustrated in both wild and cultivated species. So far, the investigations have essentially considered the role of reactive oxygen species (ROS), and, to a lesser extent, of nitric oxide (NO), in the regulation of seed biology. The emergence of hydrogen sulfide (H2S) as a new element of the cellular redox balance in plants has recently prompted research on this reactive species in the context of seed biology. This review will present and discuss the possible functions of H2S during seed germination, with an emphasis on the mechanisms through which H2S helps mitigate abiotic stress effects and maintain high germination efficiency under penalizing conditions. Keywords  Seeds · Germination · Hydrogen sulfide · Antioxidants · Abiotic stresses

E. Baudouin (*) Sorbonne Université, Institut de Biologie Paris-Seine, UMR7622 Laboratoire de Biologie du Développement, Paris, France e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_8

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8.1  Introduction The emergence of seeds in Spermatophytes has constituted a major step in the evolution of the Green Lineage, and dramatically improved plant species conservation and dispersion. Indeed, the properties of seeds make them particularly adapted for long-distance spreading and long-lasting survival, two crucial traits for success in complex ecosystems. As plants generally produce huge, but also heterogeneous, seed progeny, seeds allow to evolve diversity and conserve the whole genetic information in temporal and spatial manners, even under changing environmental conditions (Nonogaki 2014). Conversely, the emergence of a new plant generation is directly dependent upon the capacity of seeds to germinate and give rise to a robust plantlet. Beside seed longevity, seed germination efficiency is therefore a key determinant in competitive ecosystems. Since the arrival of agriculture, seeds have also been the pillar of human nutrition and still constitute the primary basis of nutrition. Indeed, improving the production of crop seeds, both quantitatively and qualitatively, is a major economic concern for food industry. Moreover, even when they do not represent the outcome of crop production, seeds generally constitute their income. In this view, seeds are expected to germinate rapidly and homogeneously to give rise to robust seedlings. Improving such seed traits (germination, vigor, and longevity) is therefore highly topical for seed industry and relies on a better understanding of the mechanisms controlling these different responses. The undergoing deterioration of environmental conditions, including global warming, water scarcity and soil pollution, constitutes a major threat for plant development and growth and eventually for the survival of the less-adapted species within the ecosystems. Seed germination, as it constitutes the early stage of plant cycle, is particularly vulnerable and impacted by environmental stresses (Daszkowska-Golec 2011). On the one hand, stresses directly affect the capacity of seeds to germinate and the early survival of the emerging plantlets. On the other hand, the efficiency of germination, that depends on characteristics (desiccation tolerance, stored reserves, dormancy) acquired during seed development on the mother plant, are strongly modulated by the environmental conditions experienced by the mother plant itself. Seed germination therefore represents an original context to investigate the mechanisms underlying stress response in plants, as well as an important model to evaluate stress mitigation strategies. Seed germination is a complex multi-step process that starts with the increase of seed water content upon imbibition and ends up with the protrusion of the embryonic radicle through the protective seed coat tissues (Bewley 1997). In this timeframe, seed metabolism gets reactivated with the sequential resumption of respiration, mRNA translation and gene transcription, and embryonic axis growth operates via cell expansion (Bewley 1997). Nevertheless, mature seeds are frequently released from mother plants in a dormant state, i.e. unable to germinate under favorable conditions because of blockage processes established during seed development and will properly germinate only after dormancy release

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(Finch-Savage and Leubner-Metzger 2006). The switch from dormancy to germination relies on the modification of the balance between abscisic acid (ABA) and gibberellins (GA), the former inhibiting germination when the later participates in its completion. Complex networks controlling ABA and GA metabolism and signaling are therefore at the heart of the regulation of seed germination and integrate environmental cues to trigger germination under appropriate conditions (Vishal and Kumar 2018). During the last decade, key roles played by reactive species derived from oxygen (ROS) or nitrogen (RNS) in the regulation of seed dormancy and germination have been evidenced (for review, Arc et al. 2013; Bailly 2019). Interestingly, these compounds can exert opposite effects functioning as toxic molecules or positive cellular signals, in seeds but also when whole plants are exposed to environmental stresses (Mittler 2017; Bailly 2019). More recently, hydrogen sulfide (H2S) emerged as a new reactive species in plants that shares similar characteristics with ROS/RNS and functions in close interplay with these two classes (Lisjak et  al. 2013; Hancock 2017). The following paragraphs will review the current knowledge on the metabolism and functions of H2S in seeds. A special emphasis will be placed on the role of this molecule during seed germination under abiotic stress.

8.2  Hydrogen Sulfide Metabolism in Seeds As shown in Table 8.1, a series of studies carried out in a range of plant species have evidenced the presence of H2S in seeds (Zhang et al. 2008, 2010b, 2010c; Li et al. 2012; Xie et al. 2014; Baudouin et al. 2016; Chen et al. 2019). These reports indicate that H2S content varies in dry seeds between 0.1 and 1.5 μmol· g−1 DW. Two studies carried out in Arabidopsis thaliana evidenced that H2S content rapidly (within 3 to 6 h) increased upon imbibition leading to a high and steady level of H2S for up to 24–48 h (Baudouin et al. 2016; Chen et al. 2019). Although the profiles of H2S production in these two studies were similar, the amplitude of the increase was different, i.e. 1.25 (Baudouin et al. 2016) and 2.7 folds (Chen et al. 2019). This difference may reflect different experimental procedures for seed germination (15 °C and 22 °C, respectively). Although no detailed kinetics is available for wheat, an accumulation of H2S was also detected 12 h after imbibition (Zhang et al. 2008, 2010c). In contrast to these results, the increase observed in imbibed Jatropha seeds was only slight (1.16 fold) and delayed (after 3 days) (Li et al. 2012). Interestingly, although no increase of H2S content was observed in wheat aleurone layers under control conditions, it was modified upon hormonal treatment, with ABA promoting and GA repressing H2S synthesis (Xie et al. 2014). As a whole, these data support that H2S metabolism is modified during the early steps of seed germination, leading to enhanced H2S concentrations in seeds. Remarkably, seed exposure to abiotic stresses during the time lapse of imbibition led to dramatic modifications of H2S content (Table 8.1). For instance, H2S content was 1.5 fold higher in wheat kernels exposed to copper stress compared to unstressed grains after 12 h imbibition (Zhang

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Table 8.1  Detection of H2S in seeds during imbibition Plant species Arabidopsis thaliana Arabidopsis thaliana Jatropha curcas Triticum aestivum Triticum aestivum

Stress/ treatment None

Imbibition [H2S]max/timea 0-24 h 1.8 μmol·g−1 FW/24 h

None

0-48 h

None None None

0-5d 0.1 μmol·g−1 FW/96 h 0 and 12 h 2.5 μmol·g−1 FW/24 h 0 and 12 h 0.4 μmol·g−1 FW/24 h

4 μmol·g−1 FW/24 h

Arabidopsis High thaliana temperature Triticum aestivum Al

0-48 h

3.5 μmol·g−1 FW/12 h

0-48 h

0.7 μmol·g−1 FW/12 h

Triticum aestivum Cd Triticum aestivum Cu Triticum aestivum Osmotic stress

0-48 h 0-48 h 0-4d

Triticum aestivumb Jatropha curcas

ABA/GA

0-48 h

H2O2

0-5d

Reference Baudouin et al. (2016) Chen et al. (2019) Li et al. (2012) Zhang et al. (2008) Zhang et al. (2010b) Chen et al. (2019)

Zhang et al. (2010b) 0.07 μmol·g−1 DW/0-12 h Huang et al. (2016) 2.5 μmol·g−1 FW/0-12 h Zhang et al. (2008) 3 μmol·g−1 DW/24 h Zhang et al. (2010c) 0.075 μmol·g−1 FW/12 h Xie et al. (2014) ABA 0.16 μmol·g−1 FW/96 h Li et al. (2012)

ABA abscisic acid, GA gibberellins, FW fresh weight, DW dry weight a Highest [H2S] observed in seeds and corresponding time b Experiments realized on isolated aleurone layers

et  al. 2008). A similar increase was also observed in wheat grains submitted to osmotic (Zhang et  al. 2010c) or aluminum stress (Zhang et  al. 2010b) and in Jatrophas curcas seeds exposed to oxidative stress (Li et al. 2012). In these different examples, the increase observed is transient and H2S concentrations progressively decreased back to that of unstressed seeds after 2–4  days of imbibition. In Arabidopsis, the exposure of seeds to high temperature during imbibition did not affect H2S accumulation during the first 12 h, but led to a dramatic decrease thereafter, whereas it remained at highest in unstressed seeds (Chen et  al. 2019). Temperature is a major determinant of seed capacity to germinate and the response to temperature is intimately dependent on seed dormancy status that either restricts (high dormancy) or widen (low dormancy) the temperature window allowing proper germination (Finch-Savage and Leubner-Metzger 2006). Conversely, high temperature induces secondary dormancy in Arabidopsis and inhibits germination (thermoinhibition) (Finch-Savage and Footitt 2017). The different patterns of H2S production reported in seeds imbibed at optimal or high temperature may therefore be a cause or a consequence of different levels of dormancy. Based on these seminal works, H2S appears widely produced in plants seeds during normal germination process and under abiotic constraints. A step forward now requires systematic comparisons of H2S level in (i) a large sampling of distant botanical families/species, (ii)

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covering a variety of seed biology types (orthodox/recalcitrant seeds, primary/secondary dormant with different dormancy types, ecotypes), and (iii) subjected to a panel of abiotic stress conditions (type, intensity, duration, combination). Several metabolic pathways leading to H2S production have been described in plants (Fig. 8.1). On the one hand, H2S is formed as an intermediate during sulfate assimilation by sulfite reductase (SiR), a ferredoxin-dependent enzyme that catalyzes sulfite reduction (Gotor et al. 2019). Then, H2S is essentially used as a substrate by O-acetyl-serine thiol lyase (OAS-TL) to synthesize L-cysteine from O-acetyl-L-serine (OAS) (Wirtz et al. 2004). On the other hand, H2S is generated from L-cysteine by L-cysteine desulfhydrase (L-CDes), together with the formation of pyruvate and ammonia (Romero et  al. 2014). It can also be released from D-cysteine in a similar reaction catalyzed by D-cysteine desulfhydrase (D-CDes) (Riemenschneider et al. 2005). H2S is also formed during the biosynthesis of cyanoalanine from cyanide and cysteine catalyzed by β-cyanoalanine synthase (β-CAS) (García et al. 2010). Finally, OAS-TL possibly catalyzes a reverse reaction, generating H2S and OAS from D-cysteine. As H2S is highly toxic, catabolic pathways also exist that control H2S cellular content. In particular, H2S easily reacts with glutathione disulfide (GSSG) to form glutathione persulfide (GSSH) that is subsequently oxidized to sulfite by the sulfur dioxygenase ETHE1 (Fig. 8.1) (Holdorf et al. 2012; Krüßel et al. 2014). The activities of the enzymes of H2S metabolism have been analyzed in seeds of different species. Early work by Taylorson and Hendricks (1973) evidenced the increase of β-CAS activity in imbibed seeds of pigweed (Amaranthus albus) and

Fig. 8.1  Schematic representation of H2S metabolism in plants. CAS b-cyanoalanine synthase, D-CDes D-cysteine desulfhydrase, L-CDes L-cysteine desulfhydrase, ETHE1 ethylmalonic encephalopathy protein1, GSSH glutathione persulfide, GSSG oxidized glutathione, OAS O-acetylserine, OAS-TL O-acetyl-L-serine(thiol)lyase, SiR sulfite reductase

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lettuce (Lactuca sativa). Whereas cytoplasmic β-CAS activity remained unchanged upon imbibition, increases of mitochondrial β-CAS activity was also observed in imbibed seeds of cocklebur (Xanthium pennsylvanicum), cucumber (Cucumis sativus), barley (Hordeum vulgare) and rice (Oryza sativa) (Hasegawa et  al. 1995). Furthermore, this activity was stimulated by treatment with ethylene in cocklebur seeds (Hasegawa et al. 1995). In A. thaliana, β-CAS activity was rapidly stimulated after imbibition and remained high for 24  h (Baudouin et  al. 2016). Despite the modulation of β-CAS activity during imbibition, it may not be related to the increase of H2S production. Indeed, the main function of β-CAS is the detoxification of cyanide (Machingura et al. 2016). A rapid increase of cyanide production is observed during germination that may participate in dormancy release and promote germination and cyanide burst might be dissipated by β-CAS activation (Oracz et al. 2008). Further investigations are therefore required to evaluate if and to which extent β-CAS activity may participate in the overall increase of H2S content. More convincingly, CDes appear as a major source of H2S in seeds. Both L-CDes and D-CDes activities have been detected in dry Arabidopsis seeds and strongly increased following imbibition (Baudouin et al. 2016; Chen et al. 2019). Strikingly, no increase of H2S content was observed in imbibed mutant seeds for LCD (At3g62130) and DES1 (At5g28030), the major L-CDes, which indicates that these enzymes play crucial role in H2S synthesis in seeds (Baudouin et al. 2016; Chen et al. 2019). In agreement, seeds overexpressing DES1 presented a constitutively high level of H2S (Chen et al. 2019). As the increase of DES1 and LCD enzymatic activities correlated with enhanced transcript levels, it is likely that both activities are at least in part regulated at the transcriptional level (Chen et al. 2019). Finally, high temperature modified DES1 and LCD activities in agreement with the differences in H2S content observed in heat-stressed seeds (Chen et al. 2019). Likewise, ABA and GA treatments modulated LCD activity in a similar way as H2S content in wheat aleurone layers (Xie et al. 2014). It is therefore likely that L-CDes activities account for H2S synthesis during seed germinating in optimal or stressing conditions.

8.3  Hydrogen Sulfide and Germination Capacity Numerous studies have investigated how germination is impacted when H2S content is modulated in seeds (Table 8.2). For this purpose, different strategies can be carried out including treatments with H2S-releasing molecules (NaHS or GYY4137), treatments with H2S scavenger such as hypotaurine (HT) or H2S synthesis inhibitor such as aminooxyacetic acid (AOA) or propargylglycine (PG), and genetic approaches using mutants or overexpressing lines for enzymes of H2S metabolism. These strategies have been widely used to investigate the role of H2S during seed germination under optimal conditions and to examine the capacity of H2S to alleviate abiotic stress effects during this developmental stage.

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Table 8.2  Effect of the modulation of H2S on seed germination Stress/ Plant species treatment Arabidopsis None thaliana Arabidopsis thaliana

None

Jatropha curcas

None

Triticum aestivum Vigna radiata

None

H2S modification Effects NaSH, HT, PG Germination partially inhibited at des1 0.5–1 mM NaSH and by HT/ PG. No phenotype for des1 NaSH, GYY, HT, No effect of NaSH and GYY; AOA, des1, lcd, germination partially inhibited by HT and AOA and in des1/lcd DES1-OE mutants NaSH, AOA Germination stimulated by 0.5–1 mM NaSH and partially inhibited by AOA NaSH No effect up to 1.5 mM NaSH

None

NaSH,

None

NaSH

Medicago sativa Arabidopsis thaliana

Triticum aestivum Triticum aestivum Triticum aestivum Triticum aestivum Medicago sativa Jatropha curcas Vigna radiata

Germination stimulated at 0.5–1 mM NaSH; germination partially inhibited by 10 mM NaSH No effect up to 1 mM NaSH

High NaSH, GYY, HT, Partial alleviation of stress effect temperature AOA, des1, lcd, by NaSH and GYY and in DES1-OE; effect of stress DES1-OE exacerbated by HT and AOA and in des1/lcd mutants Al NaSH Effect of stress reversed at 0.6 mM NaSH Cd NaSH Partial alleviation of stress effect with an optimum at 0.9 mM NaSH Cu NaSH Alleviation of stress effect at 1.4 mM NaSH Osmotic NaSH Partial alleviation of stress effect stress with an optimum at 0.6 mM NaSH Salt stress NaSH Partial alleviation of stress effect with an optimum at 0.1 mM NaSH H2O2 AOA H2O2-promoted germination inhibited by AOA H2O2 AOA, PG, HT No effect on H2O2-promoted germination

Reference Baudouin et al. (2016) Chen et al. (2019)

Li et al. (2012) Zhang et al. (2010b) Li et al. (2015)

Wang et al. (2012) Chen et al. (2019)

Zhang et al. (2010b) Huang et al. (2016) Zhang et al. (2008) Zhang et al. (2010c) Wang et al. (2012) Li et al. (2012)

Li and He 2015

AOA aminoacetic acid, des1 Arabidopsis thaliana L-cysteine desulfhydrase 1 mutant (At5g28030; Salk_103855), DES1-OE Arabidopsis thaliana L-cysteine desulfhydrase 1 overexpressor, GYY GYY4137, HT hypotaurine, lcd Arabidopsis thaliana L-cysteine desulfhydrase (At3g62130; Salk_082099), NaSH sodium hydrosulfide, PG propargylglycine

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Several lines of evidence support a role for H2S produced endogenously during germination under optimal conditions. On the one hand, HT, PG and AOA treatments, that decrease H2S concentration, partially inhibited germination in Arabidopsis and Jatropha curcas (Li et al. 2012; Baudouin et al. 2016; Chen et al. 2019). On the other hand, des1 and lcd mutant seeds also exhibited a lower germination rate compared to WT (Chen et  al. 2019). des1 seed defect was efficiently reverted by the treatment with H2S donor NaSH and was therefore linked to the modification of H2S content. On the other, such phenotype of des1 seeds was hardly observed in freshly harvested dormant seeds, and was dependent on the temperature at which imbibition was performed, which suggests that the physiological state of the seeds and the environmental conditions also modulate the requirement of H2S for germination (Baudouin et al. 2016; Chen et al. 2019). The effects of enhancing H2S content brought more contrasted results. Indeed, no effect of NaSH treatment was observed in Arabidopsis, maize, and wheat (Zhang et al. 2010b; Li et al. 2013; Chen et al. 2019). On the other hand, a stimulation of germination was reported in mung bean, zucchini, and Jatropha curcas (Li et al. 2012; Li and He 2015; Valivand et al. 2019). In addition, toxic effects have also been observed that delay germination and/or lower final germination in several species (Dooley et al. 2013; Baudouin et al. 2016) Based on the numerous examples of stress alleviation in plants fumigated with H2S or treated with H2S donors (for review, Li et al. 2016), the capacity of H2S to mitigate abiotic stress effects during germination has been investigated in a range of seed species/stress combinations (Table 8.2). Improvement of seed germination by NaSH treatment was observed for a broad range of abiotic stresses such as high temperature (Li et al. 2013; Zhou et al. 2018; Chen et al. 2019), metals including Cu (Zhang et al. 2008), Cd (Huang et al. 2016), Al (Zhang et al. 2010b) and Ni (Valivand et al. 2019), and salt and osmotic stress (Zhang et al. 2010c; Wang et al. 2012). H2S treatment appears to mitigate the impact of diverse stress in a same species as illustrated in wheat (Zhang et al. 2010b, c; Huang et al. 2016). Conversely, the effect of high temperature is partially reversed by H2S in different species, i.e. maize and Arabidopsis (Li et al. 2013; Zhou et al. 2018; Chen et al. 2019). These observations suggest that mechanisms underlying H2S effect control generic aspects of plant response to stress conserved among plant species that will be presented in the next paragraph. When different doses of H2S donors have been assayed, an optimal effect has generally been observed at around 0.5 mM NaSH (Zhang et al. 2010c; Wang et al. 2012; Li et al. 2013; Zhou et al. 2018), but it may also be found at concentrations up to 1.5  mM (Zhang et  al. 2010b; Huang et  al. 2016). Interestingly, seed priming with NaHS not only alleviated stress during germination, but it may also ameliorate seedling performance under stress, with important outcome for seed industry and farming (Zanganeh et al. 2018; Valivand et al. 2019).

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8.4  M  olecular Mechanisms Controlled by H2S in Germinating Seeds Although it seems now established that H2S participates in the regulation of seed germination and that the modulation of H2S content is a promising tool for coping with the effects of abiotic stresses during this developmental process, the mechanisms underlying H2S function in seeds are far from being elucidated. In particular, because molecular analyses have been carried out essentially in stressed plants, the mechanisms controlled by H2S during the germination of unstressed seeds can only be speculated by reference to those modulated in seeds germinating under stress. Moreover, evidences are generally correlative and rarely demonstrate a direct effect of H2S, not to speak of the mechanisms involved. The following paragraphs address the most illustrative examples of H2S function described to date, but also more elusive mechanisms that may represent important routes for future investigations..

8.4.1  I nterplay with ROS, Nitric Oxide, and Antioxidant Defense The exposure of plants to environmental stresses generally leads to the elevation of ROS level due to the enhanced ROS formation, in particular H2O2, that are not fully eliminated by the cellular antioxidant machinery. An elevation of H2O2 content has been observed in seeds germinated under penalizing conditions such as excess Cu, Cd, Al or osmotic stresses (Zhang et al. 2008, 2010b, 2010c; Huang et al. 2016). A similar increase in superoxide (O2⦁―) content can also be observed (Zhang et  al. 2010b; Huang et al. 2016). In these experiments, treatments with NaSH strongly lowered H2O2 and O2⦁― accumulation. H2S treatment limited the impact of oxidative stress as it helped restricting lipid peroxidation and maintaining membrane integrity (Zhang et al. 2008, 2010b, c; Wang et al. 2012; Huang et al. 2016). The major effect of NaSH treatment is the stimulation of a range of antioxidant enzymatic activities (superoxide dismutase, catalase and/or ascorbate and guaiacol peroxidases) that account for ROS detoxification (Zhang et  al. 2008, 2010b, c; Wang et  al. 2012; Huang et al. 2016). Such induction might include the transcriptional regulation of the corresponding genes (Wang et  al. 2012). In addition, Arabidopsis isoform of catalase CAT3 and ascorbate peroxidase APX1 have been identified as direct targets for S-sulfhydration, an H2S-based post-translational modification of cysteine residues (Aroca et  al. 2015). Interestingly, the S-sulfhydration of APX1 led to the reversible stimulation of the enzyme activity, therefore providing a direct mechanism of regulation for APX1 by H2S (Aroca et al. 2015). Whether this mechanism is operative in seeds is unknown and no information on the S-sulfhydration status of oxidant/antioxidant enzymes is currently available in seeds. In addition to ROS, connections between H2S and nitric oxide (NO), another reactive species, have been proposed (Wang et al. 2012). The authors evidenced that NaSH treatment enhanced

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NO production in unstressed and salt stressed-imbibed alfalfa seeds. Treatments with the NO scavenger cPTIO impaired the positive effects of NaSH treatment on seed germination and oxidative stress alleviation in salt-stressed seeds (Wang et al. 2012). As mentioned, ROS also have signaling functions and as so, play a critical role in the regulation of seed ability to germinate (Bailly 2019). Indeed, germination can occur only when the H2O2 content in the seed fall into a restricted range referred as the “Oxidative Window”, outside of which seeds remain dormant (too low H2O2 concentration) or undergo rapid ageing (too high H2O2 concentration) (Bailly et al. 2008). Studies carried out in Vigna radiata and Jatropha curcas suggest that H2S could modulate the germination of unstressed seeds through their effect on H2O2 content (Li et al. 2012; Li and He 2015). On the one hand, NaSH treatment increased H2O2 content and NaSH and H2O2 treatments stimulated seed germination. On the other hand, impairing endogenous H2O2 formation abolished the stimulation of germination following NaSH treatment, whereas the stimulation of germination by H2O2 was not affected by H2S depletion. So far, the mechanisms underlying this H2S/H2O2 interplay are unknown, so as its possible physiological relevance for the regulation of seed germination capacity. Although additional investigations are needed to fully establish the relationship between ROS and H2S in germinating seeds and the mechanisms at work, these different examples speak for opposite effects in unstressed and stressed seeds. In the former case, H2S would participate in the lowering of ROS content via stimulation of the antioxidant defense system. In the latter case, it would promote the formation of signaling ROS through mechanisms to be unraveled. Such dual function is reminiscent of that of NO in which it can also promote or decrease ROS accumulation in plants in different physiological contexts (Niu and Liao 2016). In this view, the H2S/ ROS/NO interplay likely constitute a key determinant of seed germination as evidenced in other processes such as stomatal closure (García-Mata and Lamattina 2010).

8.4.2  H2S and Seed Metabolism The transition from dry to germinating seeds is achieved during imbibition and includes the rapid resumption of an active metabolism in a finely orchestrated sequence order (Bewley 1997). At the end of the germination process sensu stricto, the substrates for fueling energetic metabolism and the precursors for macromolecule biosynthesis are provided by the degradation of reserves (starch, triglycerides, proteins) accumulated during seed formation. Therefore, the efficiency of reserve degradation is crucial for seedling establishment before the transition from a heterotrophic to autotrophic metabolism. A series of studies have addressed the effect of H2S treatment on reserve mobilization in unstressed and stressed wheat grains (Zhang et al. 2008, 2010a, b, c; Huang et al. 2016). In this absence of stress, H2S treatment stimulated amylase activities, but not that of esterases (Zhang et al. 2008, 2010b, c; Huang et  al. 2016). Zhang et  al. (2010a) further evidenced the rapid

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stimulation of β-amylase activity by H2S and suggested that this activation might rely on the capacity of H2S to reduce S-S bonds and thereby to release bound β-amylase to a free and active form. Interestingly, an α-amylase-like protein was identified as persulfilated in Arabidopsis which may constitutes another mechanism for the direct regulation of amylase activities by H2S (Aroca et  al. 2017). Under excess Cu, Cd, Al, and osmotic stress, the amylase activities measured in NaSH-­ pretreated grains are always higher which indicates that the maintenance of a high reserve mobilization capacity might explain part of the better germination observed in these conditions (Zhang et al. 2008, 2010b, c; Huang et al. 2016). In contrast, esterase activities were hardly modified by H2S treatment in the absence of stress, but stimulated by H2S under Cu, Cd and Al stress. The stimulation of hydrolytic activities by H2S treatment is also evidenced by an increase of free reducing sugars and contents of most free amino acid (Huang et al. 2016). Although reserve mobilization represents a key process for early seedling growth, it constitutes a late event in the germination program. So far, no information is available on the possible impact of H2S on metabolic process rapidly restarted after imbibition, such as respiration, primary metabolism, translation or transcription, neither under unstressed nor stressed conditions. As many proteins operating in these processes have recently been identified as direct target for H2S (Aroca et al. 2017), it is likely that the elevation of H2S content in seeds should impact these processes.

8.4.3  H  2S and Hormone Signaling in the Regulation of Seed Germination The hormonal balance between ABA and GA content and signaling plays a central role in the decision for a seed to germinate. As ABA is also a key regulator of plant response to abiotic stress, interference, or interplay between H2S signaling and ABA signaling is strongly expected when considering the regulation of seed germination under stressing conditions. The H2S/ABA connection has been clearly evidenced for other processes regulated by ABA such as stomatal closure (García-Mata and Lamattina 2010). Genetic tools have been used to modify H2S formation and analyzed for response to ABA (Baudouin et al. 2016; Li et al. 2018). No difference of ABA sensitivity during germination was observed in Arabidopsis des1 mutant seeds (Baudouin et  al. 2016). In contrast, the overexpression of wheat D-CDes in Arabidopsis led to an increased sensitivity to ABA and a stronger inhibition of seed germination in transformed lines (Li et  al. 2018), which suggests that H2S could function as an intermediate in ABA signaling in seeds. Further investigations using mutants deficient for ABA synthesis or response should provide additional information on the precise relationship between ABA and H2S signaling in seeds. These data contrast with another report recently published by Chen et al. (2019). Indeed, the authors observed that ABA treatment impaired H2S formation, at least partially

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by repressing DES1 and L-CDes gene transcription. These evidence are in good agreement with a role of H2S in promoting seed germination. The study by Chen et al. (2019) also brings a new perspective on the possible interconnection between H2S and ABA signaling. The authors evidenced that H2S was required for seed thermotolerance and that the mechanism at work implied the repression of ABA Insensitive 5 (ABI5) transcription (Chen et al. 2019). ABI5 is a key transcription factor of ABA signaling and a target for multiple post-translational modifications modifying ABA response (Yu et al. 2015). Under high temperature, the transcriptional regulator HY5 is stabilized in the nucleus, interacts with ABI5 promoter, leading to ABI5 transcription and the repression of seed germination (Chen et al. 2019). This is achieved by the partitioning of the E3 ligase COP1 that is preferentially retained in the cytosol and therefore does not participate in HY5 degradation. In the presence of H2S, COP1 is preferentially located in the nucleus, brings HY5 to degradation, leading to the repression of ABI5 transcription and allowing germination (Chen et  al. 2019). Under high temperature, H2S signaling would therefore counteract ABA signaling at the level of ABI5 transcription and the equilibrium between H2S and ABA contents could represent a new balance for seed germination regulation. Whereas the interplay between ABA and H2S becomes increasingly clear, the possible connections between H2S and GA have been poorly addressed. So far, the only system in which this question has been studied is the GA-triggered cell death occurring in cereal aleurone layer occurring at the later stages of grain germination (Fath et al. 2000). In this context, NaSH treatment was found to delay and partially prevent GA-triggered cell death in wheat and barley aleurone layers, suggesting an antagonistic function between H2S and GA (Xie et al. 2014; Zhang et al. 2015). This is further supported by the fact that H2S functioned synergistically with ABA in preventing aleurone cell death (Xie et al. 2014). The alleviation of cell death relied on the capacity of H2S to lower oxidative stress, in particular, by preventing the collapse of the antioxidant defense (Xie et al. 2014; Zhang et al. 2015). Intriguingly, H2S also stimulated α-amylase secretion in barley, a mechanism activated by GA and required for reserve mobilization in the starchy endosperm, suggesting that H2S and GA could also function synergistically (Zhang et  al. 2015). Additional molecular and genetic studies on this system, and more generally on interference between GA and H2S outside of cereal models, are clearly required for supporting whether and understand how these two pathways could be interlinked.

8.5  Concluding Remarks and Open Questions Many studies carried out during the last decade support the strong potential of H2S for stress mitigation during seed germination, and therefore its possible use for translational research in seed science. As the species and stresses that have been analyzed so far remain limited, the coming step will be to evaluate how global H2S effect can be when considering a large range of plant/stress combinations. This is

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particularly topical as the variable physical and physiological properties of seeds might significantly influence H2S availability when exogenous treatments with H2S are to be used, so as the seed sensitivity towards particular stresses. Beyond seed stress physiology, the participation of H2S in the regulation of the germination process itself remains underexplored, although it might bring valuable information on the fundamental mechanisms regulating this process. Moreover, it is still unclear or even unknown if and how H2S takes part in processes such as seed development, seed dormancy establishment and release, seed vigor and/or seed longevity. Future approaches are therefore expected to clarify the ins and outs of H2S in seed biology and require intensive efforts towards the unraveling of the mode of action of this compound at the molecular level in seeds. The use of pharmacological and genetic tools, together with the many molecular markers available for model species and the new probes for H2S detection in leaving tissues, should help address these issues. Noteworthy, seed represents a particularly appropriate playground to investigate H2S interplay with other signaling molecules such as hormones or ROS/ NO. Although the interplay between H2S and ABA evidenced in other physiological contexts is probably also operating in seeds, nothing is known for other hormones such as GA, ethylene, auxins or brassinosteroids that all constitute key regulators of seed biology. Together with information from dedicated studies in seeds, more indirect evidence is probably to come from large scale-approaches that have been recently initiated. For instance, the identification of hundreds of proteins directly modified by persulfidation constitutes a reservoir of potential targets, some of which possibly regulated during germination (Aroca et al. 2017). In addition to proteomic data, the identification of H2S-regulated genes by transcriptomic approaches would help to further unravel the cellular and biochemical processes controlled by H2S signaling in general and seeds in particular. Acknowledgements  This work was supported by Sorbonne Université and the Centre National de la Recherche Scientifique.

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Oracz K, El-Maarouf-Bouteau H, Bogatek R et al (2008) Release of sunflower seed dormancy by cyanide: cross-talk with ethylene signalling pathway. J Exp Bot 59:2241–2251 Riemenschneider A, Wegele R, Schmidt A, Papenbrock J (2005) Isolation and characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana. FEBS J 272:1291–1304 Romero LC, Aroca MÁ, Laureano-Marín AM et al (2014) Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana. Mol Plant 7:264–276 Taylorson RB, Hendricks SB (1973) Promotion of seed germination by cyanide. Plant Physiol 52:23–27 Valivand M, Amooaghaie R, Ahadi A (2019) Seed priming with H2S and Ca2+ trigger signal memory that induces cross-adaptation against nickel stress in zucchini seedlings. Plant Physiol Biochem 143:286–298 Vishal B, Kumar PP (2018) Regulation of seed germination and abiotic stresses by gibberellins and abscisic acid. Front Plant Sci 9:838 Wang Y, Li L, Cui W et al (2012) Hydrogen sulfide enhances alfalfa (Medicago sativa) tolerance against salinity during seed germination by nitric oxide pathway. Plant Soil 351:107–119 Wirtz M, Droux M, Hell R (2004) O-acetylserine (thiol) lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana. J Exp Bot 55:1785–1798 Xie Y, Zhang C, Lai D et al (2014) Hydrogen sulfide delays GA-triggered programmed cell death in wheat aleurone layers by the modulation of glutathione homeostasis and heme oxygenase-1 expression. J Plant Physiol 171:53–62 Yu F, Wu Y, Xie Q (2015) Precise protein post-translational modifications modulate ABI5 activity. Trends Plant Sci 20:569–575 Zanganeh R, Jamei R, Rahmani F (2018) Impacts of seed priming with salicylic acid and sodium hydrosulfide on possible metabolic pathway of two amino acids in maize plant under lead stress. Mol Biol Res Commun 7:83–88 Zhang H, Hu L-Y, Hu K-D et al (2008) Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. J Integr Plant Biol 50:1518–1529 Zhang H, Dou W, Jiang C-X et al (2010a) Hydrogen sulfide stimulates β-amylase activity during early stages of wheat grain germination. Plant Signal Behav 5:1031–1033 Zhang H, Tan Z-Q, Hu L-Y et al (2010b) Hydrogen sulfide alleviates aluminum toxicity in germinating wheat seedlings. J Integr Plant Biol 52:556–567 Zhang H, Wang MJ, Hu LY et al (2010c) Hydrogen sulfide promotes wheat seed germination under osmotic stress. Russ J Plant Physiol 57:532–539 Zhang Y-X, Hu K-D, Lv K et al (2015) The hydrogen sulfide donor NaHS delays programmed cell death in barley aleurone layers by acting as an antioxidant. Oxidative Med Cell Longev 2015:714756 Zhou Z-H, Wang Y, Ye X-Y, Li Z-G (2018) Signaling molecule hydrogen sulfide improves seed germination and seedling growth of maize (Zea mays L.) under high temperature by inducing antioxidant system and osmolyte biosynthesis. Front Plant Sci 9:1288

Chapter 9

Hydrogen Sulfide Signaling in the Defense Response of Plants to Abiotic Stresses Cristiane J. Da-Silva, Ana Cláudia Rodrigues, and Luzia V. Modolo

Abstract  Hydrogen sulfide (H2S) has emerged as a signaling molecule in plants in the late 2000s. Since then, a spectrum of evidence indicates H2S as a key player in plant tolerance to abiotic stresses. This chapter summarizes the production of H2S and its signaling pathways in plants. The main mechanisms of plant defense induced by H2S in response to several abiotic stresses such as high metal availability, high salinity, drought, and extreme temperatures are also highlighted. Finally, the current knowledge on the interplay among H2S, phytohormones, second messengers, and metabolites is also presented. Overall, H2S mitigates the oxidative stress and the damage to organic molecules to maintain seed germination and contribute to plant growth and survival during stressful conditions. Keywords  Hydrogen sulfide · Metal stress · High salinity · Temperature stress · Gene expression · Signaling pathway

9.1  Introduction Plants experience many different types of abiotic stresses including high metal or salt concentrations, drought, and extreme temperatures. Such environmental stresses affect the geographical distribution of plants in nature, limit crop productivity, and threaten food security (IPCC 2019). Plant perception and response to abiotic stress are mediated by phytohormones and signaling molecules, such as nitric oxide (NO), hydrogen peroxide (H2O2), and hydrogen sulfide (H2S) (Zhu 2016; Da-Silva et al. 2019; Khan et al. 2017, 2018, 2020). H2S is a small and lipophilic molecule, which

C. J. Da-Silva · A. C. Rodrigues · L. V. Modolo (*) Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_9

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has long been considered toxic to plants (Li and Lancaster 2013). The studies showing the role of this molecule as a cell signaling in plants began only by the end of the 2000s (Zhang et al. 2008) and now the role of H2S in the tolerance of plants to abiotic stress is widely recognized (Fotopoulos et al. 2013; Li et al. 2016; Da-Silva et al. 2017). Plants that overexpress the genes that encode for enzymes involved in H2S production or those under the effect of exogenous H2S are known to control oxidative stress, maintain proper nutritional status, and assimilate carbon more efficiently (Shen et al. 2013; Mostofa et al. 2015a; Khan et al. 2017, 2018, 2020; Chen et al. 2019). Thus, H2S helps seeds to germinate and also assists plants to grow and produce more even under stress conditions. Most studies show the response of plants to exogenous H2S donor. However, plants are efficient in producing H2S, which allows them to overcome environmental stresses (Lai et al. 2014; Jin et al. 2017; Da-Silva et al. 2017). Four enzymatic systems have been implicated in the production of H2S in plants: (i) L/D-cysteine desulfhydrases, cytoplasmic proteins that convert L-cysteine or D-cysteine to pyruvate with the release of H2S and NH4; (ii) β-cyanoalanine synthase that catalyzes the condensation of L-cysteine to cyanide to produce H2S in mitochondria; (iii) L-cysteine synthase that assists the reversible reaction between L-cysteine and acetate to form O-acetyl-L-serine and H2S in the cytosol, mitochondria, and chloroplasts; (iv) sulfite reductase, a chloroplast enzyme that reduces SO32− to H2S in the presence of ferredoxin (Fig.  9.1) (Li 2015; Da-Silva and Modolo 2018). These enzymes can operate together or separated to produce H2S according to the plant species, developmental stage, and type of stress experienced. For instance, H2S

Fig. 9.1  Sources of hydrogen sulfide (H2S) in plants. L/D-DES L/D-cysteine desulfhydrase, CAS β-cyanoalanine synthase, CS cysteine synthase, SiR sulfite reductase, OAS O-acetyl-L-serine, Fed ferredoxin, CN− cyanide. The size of organelles is merely illustrative and does not represent the actual relative size in plant cells

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content increased in salt-stressed tobacco (Nicotiana tabacum) plants due to increments in the activity of L-cysteine desulfhydrase (LCD), cyanoalanine synthase (CAS), and cysteine synthase (CS) while the activity of sulfite reductase (SiR) remained unaffected under the experimental conditions tested (Da-Silva et al. 2017). In contrast, the endogenous levels of H2S were not affected by the expression of D-cysteine desulfhydrase genes (D-CDS) in Arabidopsis thaliana plants under Cd stress (Jia et al. 2016). In addition, the activity of LCD was not critical for the accumulation of H2S in roots and leaves of Cucumis sativus (cucumber) under high salinity (Jiang et al. 2019). Sulfur-based compounds (e.g. persulfides, polysulfides, and thiosulfate) originated from H2S oxidation also function as signaling substances in plant cells in response to environmental stress (Hancock 2019). Protein persulfidation is the main signaling pathway involving H2S, in which the thiol group (–SH) of cysteine residues is targeted by H2S to form a persulfide group (–SSH). Such post-translational modification can affect the activity of enzymes and subcellular localization as well (Aroca et al. 2018). Cysteine persulfidation also regulates a wide range of biological events, such as carbon metabolism, plant growth and development, and responses to stress. Indeed, it is estimated that 5% of the Arabidopsis protein pool is persulfidated (Aroca et al. 2017). Protein persulfidation seems to occur more often than nitrosylation and this event is believed to be as important as protein phosphorylation (Paul and Snyder 2012). Besides protein persulfidation, H2S can react with NO, hydroxyl radical (•OH), nitrogen dioxide (NO2), superoxide anion (O2•−), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hypochlorite (OCl−). Such reactions not only control the levels of reactive oxygen species (ROS) but also yield species known to modulate several biological processes (Gotor et al. 2019). In fact, the interaction among H2S, H2O2, and NO in plant metabolism has been widely discussed earlier (Da-Silva et al. 2019). Therefore, the current chapter provides the main roles of H2S in plants under abiotic stresses such as metals, high salinity, drought, and extreme temperatures. The crosstalk among H2S, phytohormones, second messengers, and metabolites is also addressed.

9.2  Stress by Metals High metal availability either from natural sources or anthropogenic activities (e.g. mining, sewage irrigation, soil acidification, etc.) is a serious problem in agriculture and natural habitats. The activity and expression of H2S-producing enzymes are reported to increase in plant leaves and roots upon metal stress (Cui et al. 2014; Jia et al. 2016; Yu et al. 2019). The high content of H2S inhibits the absorption, transport, and accumulation of metals in cells in addition to promoting stress tolerance to metallic pollutants (He et al. 2018). Some examples of the effect of H2S on plants challenged with metals are described as follows.

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H2S supply reduces cadmium absorption and accumulation in several plants stressed with cadmium (Cd) (Ali et al. 2014a; Mostofa et al. 2015a; Yu et al. 2019). The overexpression of the H2S-producing enzyme cysteine synthase promoted Cd tolerance in tobacco treated with 100 μM CdCl2 for 7 days (Harada et al. 2001). Those transgenic plants showed higher amounts of phytochelatins in shoots and relatively lower Cd content compared with wild-type plants. The hypothesis raised is that the overexpression of the cysteine synthase gene led to Cd excretion or complexation by phytochelatins. Cis/trans isomerases deficient Arabidopsis (atcsr-2) plants showed lower expression of L/D-cysteine desulfhydrase and thus lower amounts of H2S compared to wild-type plants exposed 30 μM CdCl2 for 3  days. Lower levels of H2S resulted in greater Cd uptake by transgenic plants and less metal ion mobilization into vacuoles (Li et al. 2012a, b). The pre-treatment with the H2S donor NaHS (50 μM) for 6  h provoked an increase of pectin and hemicellulose levels in root cell walls of Brassica napus L. (oilseed rape) exposed to 20 μM CdCl2 for 7 days. Polysaccharides helped root cells to retain Cd and prevented the metal ion translocation to the shoot. Thus, oilseed rape plants treated with H2S and challenged with Cd exhibited lower Cd amounts in leaves and stems, less leaf chlorosis, and higher leaf and root biomass when compared with plants solely exposed to Cd (Yu et al. 2019). Pre-treatment with 50–100 μM NaHS for 6 h inhibited Cd influx into the cytosol of cells of Populus euphratica Oliv. (desert poplar) plants treated with 100 μM CdCl2 for 72 h. Conversely, Cd influx into the vacuole increased due to an antiport Cd/H+. Therefore, the concentration of Cd in the cytosol decreased while that of vacuole increased due to pre-treatment with NaHS. Cd compartmentalization into vacuole in addition to increased antioxidant activity in Cd-exposed cells treated with NaHS lowered the levels of H2O2, lipid hydroperoxide, and decreased programmed cell death (Sun et al. 2013). Similar results were found in Brassica rapa L. (mustard) fumigated with 5 μM NaHS 24 h prior exposure to 5–20 mM CdCl2 for an extra 24 h (Zhang et al. 2015). Accumulation of H2S allowed the control of the levels of ROS increased in Cd stressed plants (Ali et  al. 2014a; Zhang et  al. 2015; Jia et  al. 2016). H2S also decreased the oxidative stress in Oryza sativa L. (rice) plants treated with 100 and 500 μM CdCl2 for 3 days (Mostofa et al. 2015a). In addition to reducing Cd content in leaves and rice roots, 100 μM NaHS enhanced the activity of catalase, superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST), dehydroascorbate reductase (DAR), and methylglyoxal detoxifying enzymes (glyoxalase I and II). The antioxidants ascorbate and glutathione increased in plants under Cd stress whereas the activity of lipoxygenase and content of H2O2, malondialdehyde (MDA), and methylglyoxal decreased in leaves (Mostofa et al. 2015a). The treatment with 200 μM NaHS also enhanced the activity of SOD and guaiacol peroxidase (G-POX) in leaves and roots of oilseed rape upon 15 days of Cd stress (100 and 500 μM CdCl2). The increment in the performance of the antioxidant system resulted in decreases in H2O2, O2•−, •OH, and MDA contents (Ali et al. 2014a). Similarly, ROS and MDA decreased in seedling roots of Medicago sativa L. (alfalfa) exposed to 200 μM CdCl2 for 6 h as a result of

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seedlings pre-treatment with 100 μM NaHS. Under the same conditions, exogenous H2S increased glutathione content and upregulated the transcription of genes that encode for antioxidant enzymes [Cu/Zn-SOD, ascorbate peroxidase 1 (APX1), glutathione synthetase (GS), GR1, GPX] and γ-glutamyl cysteine synthetase (ECS) (Cui et al. 2014). The treatment with 4 μM CdCl2 for 72 h resulted in the accumulation of H2S in mustard roots in the field, which was related to the higher expression of the basic helix-loop-helix transcription factor Br_UPB1s that is involved in the modulation of ROS balance (Lv et al. 2017). ROS generated by cadmium in cells can promote molecular and structural damage in plants under stress, affecting the functioning of molecules and organelles. In shoots, overproduction of ROS due to Cd treatment leads to leaf chlorosis and carotenoids degradation a condition that was reverted by H2S (Ali et al. 2014a; Mostofa et al. 2015a). The exposure of oilseed rape plants to 500 μM CdCl2 or 100–300 μM AlCl3 for 15 days decreased net photosynthetic rate, stomatal conductance, transpiration rate, and internal CO2 concentration. However, the combined treatment of plants with Cd and NaHS or Al and NaHS improved photosynthesis suggesting that H2S mitigated the deleterious effects of Cd or Al in Hordeum vulgare L. (barley) seedlings and oilseed rape plants (Dawood et al. 2012; Ali et al. 2014a; Ali et al. 2015). Notably, the mesophyll cells of Cd-, Al- or Pb-stressed oilseed rape plants presented spongy chloroplasts with dismantled thylakoid membranes and starch grains. Furthermore, mitochondria, Golgi bodies, endoplasmic reticulum, and nucleus seemed to be absent in Cd-stressed oilseed rape plants. The treatment of plants with H2S donor (100 and 200 μM NaHS) reestablished the formation of functional organelles to the normal levels even in the presence of Cd, Al or Pb (Ali et al. 2014a, b, 2015). The root diameter, surface, and volume in oilseed rape plants significantly decreased under Cd stress, while the treatment with NaHS improved root morphology in stressed plants (Ali et al. 2014a). Likewise, H2S donor protected oilseed rape organelles from the negative effects of aluminum (Al) and lead (Pb) (Qian et  al. 2014; Ali et al. 2014b). Damage to root morphology and micromorphology can affect nutrient absorption. Cd stress remarkably enhanced Na absorption but limited the absorption of potassium (K), calcium (Ca), and magnesium (Mg) ions in oilseed rape leaves and roots. On the other hand, the application of NaHS decreased Na concentration and improved the uptake of K, Ca, and Mg. The absorption of the nutrients Ca Mg, Fe, Zn, and Mn was also improved by the treatment of leaves or roots of rice plants with NaHS and challenged with Cd. Oilseed rape plants under Cd treatment presented lower height, shorter stem and root and decreased the number of leaves in comparison to control plants. The treatment with NaHS improved such plant traits (Ali et al. 2014a). The supplementation of nutrient solution with NaHS (100–400 μM) restored the levels of P, Ca, Mg and Fe in barley seedlings challenged with 100 μM AlCl3 for 24 h (Dawood et al. 2012). The concentration of K, Ca, and Mg also decreased in leaves and roots of oilseed rape treated with 300 μM AlCl3, but the application of 300 μM NaHS enhanced the concentrations of these nutrients in cells of both organs

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of Al-stressed plants (Ali et al. 2015). Additionally, rice plant height, fresh weight, and dry weight were higher when Cd-challenged plants were simultaneously treated with H2S (Mostofa et al. 2015a). Equally, the root and shoot length and fresh weight of Brassica oleracea L. (cauliflower) seedlings significantly increased in seedlings under lead toxicity (Chen et al. 2018). Germination, seedling growth, and plant development are negatively affected in the presence of high aluminum (Al) (Zhang et al. 2010a; Dawood et al. 2012; Ali et  al. 2015). H2S was associated with decreased Al absorption by plants and the activation of the antioxidant system (Zhang et al. 2010a; Dawood et al. 2012; Chen et al. 2013; Zhu et al. 2018). Germination of Triticum aestivum L. (wheat) seeds was reduced by 50% in the presence of 30 mM AlCl3 for 48 h whereas radicles were reduced in number and length. The pre-treatment of wheat with 0.3–1.5 mM NaHS for 12 h restored seed germination, likely due to the stimulation of amylases and esterases in the endosperm. The activities of catalase, SOD, APX, and G-POX also increased in wheat seedlings treated with NaHS followed Al stress (Zhang et  al. 2010a). Similar results were noted in barley (Dawood et al. 2012; Chen et al. 2013), oilseed rape (Qian et al. 2014; Ali et al. 2015) and rice (Zhu et al. 2018) plants under Al stress. NaHS at 200 μM increased the expression of ATPases and also citrate efflux in barley under 400 μM AlCl3 for 48 h (Chen et al. 2013). As a result, plants accumulated lower amounts of Al and presented increased root growth. These results corroborate those found for Na+/K+-ATPase activity in barley under 200 μM NaHS and 100 μM AlCl3 for 24 h (Dawood et al. 2012). Most of the Al uptaken by roots is trapped in cell walls and proper alteration of cell wall constituents may alleviate Al toxicity. The pre-treatment of rice seedlings with 2 μM NaHS for 8 h decreased pectin and hemicellulose contents in root cell walls in response to AlCl3 at 50  mM for 24  h (Zhu et  al. 2018). In addition, the expression of the genes SENSITIVE TO ALUMINIUM RHIZOTOXICITY OsSTAR1 and OsSTAR2 increased in the presence of NaHS under high Al. The STAR1-STAR2 complex transports UDP-glucose from cytoplasm to cell wall, which resulted in the lower deposition of Al in such structure (Zhu et al. 2018). H2S also alleviated Al stress by decreasing the expression of OsNRAT1 and increasing the expression of OsALS1 in roots. The protein OsNRAT1 is responsible for Al transport within cells and OsALS1 transports Al from the cytoplasm to the vacuoles. In fact, the induction of OsNRAT1 and OsALS1 genes by NaHS was accompanied by a decrease in Al levels in the cytosol of rice plant cells under Al stress. Pre-treatment with NaHS also increased the expression of OsFRDL4, a gene involved in the efflux of citrate from roots cells, which in turn formed complexes with Al ions and prevented the metal ions from entering into root cells. These data indicate that H2S alleviates Al stress by decreasing the metal content in the apoplast and symplast of rice roots (Zhu et al. 2018). H2S improves the tolerance of lead (Pb)-stressed plants by increasing germination, enhancing growth, restricting lead-induced oxidative stress, and protecting cell structures from metal damage (Ali et al. 2014b, c; Bharwana et al. 2014; Chen et al. 2018). Germination of cauliflower seeds under treatment with 250–500 μM

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Pb(CH3COO)2 for 48 h increased when exposed to 200–300 μM NaHS 12 h prior to the metal stress (Chen et  al. 2018). The H2S donor also increased the content of thiols and glutathione in cauliflower leaves under Pb treatment. It was suggested that H2S was converted to glutathione to form glutathione-Pb complexes or react with ROS (Chen et al. 2018). NaHS at 200 μM stimulated the activity of the antioxidant enzymes SOD, G-POX, catalase, and APX in leaves and roots of Gossypium hirsutum (cotton) exposed to ≥ 50 μM Pb(NO3)2 for 45 days. Thereby, the levels of MDA, electrolyte leakage, and H2O2 production decreased in leaves and roots of Pb-stressed cotton plants (Bharwana et al. 2014). The Pb-triggered oxidative stress was controlled by 100–200 μM NaHS in oilseed rape plants along with 15 days of stress (Ali et al. 2014b). NaHS at 50 or 100 μM improved the photosynthetic performance of cotton plants challenged with 100 μM Pb(NO3)2 for 45 days by increasing the levels of chloroplast pigments (chlorophyll a, b and carotenoids), stomata conductance, transpiration, carbon assimilation, and water-use efficiency in plants of cotton under lead stress [100 μM Pb(NO3)2; 45 days]. The H2S donor also contributed to biomass accumulation in cotton plants under Pb stress (Bharwana et  al. 2014). Oilseed rape plants supplemented with 400 μM Pb(NO3)2 plus NaHS (200 μM) for 15 days increased carbon assimilation, stomatal conductance, transpiration (Ali et al. 2014b). Uptake of N, P, S, Mn, Zn, Fe, and Cu in the leaves and roots of oilseed rape plants under Pb stress was lower, compared to the control, but supplementation of Pb stressed plants with NaHS boosted the uptake of such nutrients (Ali et al. 2014c). Studies on copper (Cu)-stressed plants show that H2S plays a role in restricting metal entry into cells and mitigating oxidative stress (Zhang et al. 2008; Shan et al. 2012). Pre-treatment for 12  h with 0.2–2.6  mM NaHS alleviated the inhibitory effect of 5 mM CuCl2 on the germination of wheat seeds. The increased germination of wheat seeds under Cu plus NaHS treatment may be related to the increased activity of amylase and esterases. In addition, wheat seedlings pre-treated with NaHS had an increment in the activity of SOD and catalase. Indeed, plants treated with NaHS prior to Cu exposure presented the lower activity of lipoxygenase, decreased concentrations of H2O2 and MDA, and higher amounts of free amino acids (Zhang et al. 2008). Pre-treatment for 8 h with NaHS at 0.4 or 1.6 mM also decreased electrolyte leakage and MDA content in wheat seedlings exposed to 100 μM CuSO4; 24 h. The activity of GR, DAR, L-galactono-1,4-lactone dehydrogenase (GalLDH), and γ-glutamyl cysteine synthetase and the content of ascorbate and glutathione (both in the reduced form) were increased in wheat seedlings under relatively high Cu (Shan et al. 2012). The percentage of germinated wheat seeds decreased after treatment with 4 mM Na2CrO4 for 48 h and pre-incubation with 1.2 mM NaHS for 12 h prior to the metal stress rescued seed germination to normal levels (Zhang et  al. 2010b). The H2S donor enhanced the activity of amylase, esterase, SOD, catalase, APX, and G-POX in seeds under Cr toxicity. The lipoxygenase activity and over-production of MDA and H2O2 induced by Cr stress were controlled upon pre-treatment of wheat seeds with the H2S donor (Zhang et al. 2010b). The effect of 50 μM NaHS on the tolerance

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of Arabidopsis plants to treatment with 150 mM K2Cr2O7 for 5 days was investigated (Fang et al. 2016). Cr stress increased H2S content in Arabidopsis and NaHS promoted the increase in the expression of cysteine generation-related genes OASTLa, SAT1, and SAT5 and consequently the accumulation of cysteine. The increase of cysteine levels may result in the accumulation of glutathione as this antioxidant tripeptide contains cysteine in its structure. Actually, NaHS enhanced glutathione content and increased the expression of PCS1/PCS2 and MT2A (related to phytochelatins and metallothioneins synthesis, respectively) in Arabidopsis under Cr stress (Fang et al. 2016). Solanum nigrum L. (blackberry nightshade) plants exposed to 400 μM ZnCl2 for 5 days exhibited an increased amount of H2S and L-cysteine desulfhydrase activity (Liu et  al. 2016). The treatment with 200 μM NaHS 5  days prior to Zn stress decreased the expression of genes related to Zn uptake and homeostasis (ZRT, IRT, NRAMP, MTP, HMA4) and therefore to Zn accumulation in blackberry nightshade root tip roots. The expression of metallothioneins, responsible for Zn transport to vacuoles, and Cu/Zn-SOD, Fe-SOD, CAT2, pAPX was enhanced by NaHS in blackberry nightshade plants. Accordingly, plants exhibited higher leaves and roots fresh weight (Liu et al. 2016). Exposure to 50 μM ZnSO4 for 10 weeks also increased H2S in Capsicum annuum L. (sweet pepper) plants and the treatment with NaHS (200 μM) decreased Zn content in leaves and roots (Kaya et al. 2018). Plants treated with NaHS followed by incubation with Zn were highly hydrated and the leaves accumulated L-proline. Under the same conditions, the activity of catalase, SOD, and G-POX increased whereas the electrolyte leakage and the content of H2O2 and MDA decreased in leaves. The combined treatment with Zn and NaHS increased chlorophyll content and maximal photochemical efficiency and augmented the amounts of P, N, and Fe in leaves and N in roots. Treatment with NaHS also improved the yield of sweet pepper plants and the number of fruits in plants challenged with Zn (Kaya et al. 2018). The pre-treatment with 100 or 200 μM NaHS for 24 h increased the growth of rice seedlings exposed to 100 μM HgCl2 for 3 days (Chen et al. 2017). The use of H2S donor alleviated the Hg stress in plants by increasing the expression of the metallothionein-related gene OsMT-1 as well as the accumulation of non-protein thiols in leaves and roots. Metallothionein and non-protein thiols may prevent the translocation of Hg from root to shoot. The levels of H2O2 and MDA decreased, likely due to a direct antioxidant effect of H2S or increased production of non-­ enzymatic antioxidants as carotenoids since the activity of antioxidant enzymes was found to be decreased (Chen et al. 2017). Incubation of rice plants with 200 μM NiSO4 for 14 days increased the endogenous levels of H2S and supplementation of plants with 100 μM NaHS relieved Ni stress (Rizwan et al. 2019). The treatment with NaHS increased NO3− content and the expression of genes that encode for nitrate reductase, nitrite reductase, glutamate synthase, glutamate oxaloacetate transaminase, glutamine synthetase, and glutamate pyruvate transaminase in leaves. Meanwhile, H2S mitigated ammonium toxicity by decreasing its content in leaves, the activity of glutamate dehydrogenase, and the transcription of genes related to ammonium formation in cells (Rizwan et al.

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2019). NaHS supply restored to the control levels the content of the chlorophyll a, b and carotenoids in Ni stressed plants, a condition that favored plant growth and biomass accumulation when compared with plants solely treated with Ni (Rizwan et al. 2019).

9.3  Salt Stress Salinity is an environmental factor that critically limits plant growth and decreases productivity in semi-arid and arid regions all over the world. Salt stress usually leads to osmotic stress, ion toxicity, severe nutrition disorders, and oxidative stress in plants (Da-Silva and Modolo 2018). H2S plays an important role in the tolerance of plants to salt stress by activating the antioxidant systems, increasing the K+/Na+ ratio, protecting the photosynthetic apparatus, stimulating seed germination, and plant growth, and enhancing the accumulation of osmoregulators (Da-Silva and Modolo 2018). Arabidopsis plants that overexpress cysteine desulfhydrases produced high amounts of H2S and improved tolerance to 150 mM NaCl throughout 21 days of the experiment (Shi et  al. 2015). Conversely, cysteine-desulfhydrases-knockdown plants or plants pre-treated with 100 μM hypotaurine (H2S scavenger) for 7 days presented much lower levels of H2S level and consequently were less tolerant to salt stress. The supplementation of plants with 100 μM NaHS 7 days prior to salt stress inhibited ROS accumulation (H2O2, O2•−), increased the activity of SOD, catalase, G-POX, and GR, and improved the rate of survival in Arabidopsis plants (Shi et al. 2015). Endogenous H2S was also shown to be pivotal to tobacco plants to overcome 300 or 600 mM NaCl for 10 days. The increase of H2S amounts in tobacco plants under salt stress was related to an increment in the activity of the H2S-producing enzymes L/D-cysteine desulfhydrase, cysteine synthase, and β-cyano-L-alanine synthase (Da-Silva et al. 2017). Infiltration of tobacco leaves with 300 μM hypotaurine lowered the activity of catalase, SOD, and APX and decreased the amount of reduced glutathione in cells. Therefore, H2S was responsible for decreasing the contents of H2O2, O2•−, •OH, lipid hydroperoxides, and oxidized proteins (Da-Silva et al. 2017). Pre-incubation of cucumber seeds with 200 μM NaHS for 8 h followed by treatment with 100 mM NaCl for 48 h improved the germination of seeds by enhancing the activity of α/β-amylase in the endosperm (Yu et al. 2013). The height and biomass of salt-stressed seedlings were also improved by the NaHS treatment. The use of an H2S donor increased the activity of catalase, SOD, DAR, G-POX, APX, and GR and diminished the levels of H2O2 and MDA in cucumber hypocotyls and radicles of plants under salt stress. The extract of cucumber hypocotyls and radicles originated from salt-stressed seedlings pre-treated with NaHS efficiently scavenged 2,2-difenil-1-picrylhydrazyl (DPPH) and •OH radicals and caused metal chelation (Yu et al. 2013).

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Treatment of alfalfa seedlings with 175 mM NaCl for 120 h boosted the activity of L-cysteine desulfhydrase and the accumulation of H2S (Lai et al. 2014). In addition, the salt stress was relieved in alfalfa seedlings when seeds were supplemented with 100 μM NaHS 6  h prior to stress. The H2S donor also increased the gene expression and activity of catalase, SOD, GR, DAR, and monodehydroascorbate reductase (MDAR) and stimulated the accumulation of reduced glutathione and ascorbate. Thereby, the content of ROS and lipid hydroperoxides decreased in saltstressed seedlings pre-treated with NaHS, allowing for plant growth and biomass accumulation. The K+ influx was restored by NaHS in salt-stressed seedlings, likely due to the downregulation of SKOR, a gene that encodes for a K+ efflux protein (Lai et al. 2014). SKOR and PM-H+-ATPase were also down-regulated in cucumber roots under salt stress and 15 μM NaHS allowed the plant to keep Na+ and K+ balance during the 7 days of treatment with 200 mM NaCl (Jiang et al. 2019). Lower Na+ and higher K+ levels, coupled with H2S-enhanced antioxidant system decreased electrolyte leakage and the levels of H2O2 and MDA in leaves and roots of cucumber under salt stress. Carbon assimilation, stomatal aperture, pigment content, and photochemical efficiency were also improved in plants simultaneously treated with NaHS and NaCl (Jiang et al. 2019). Salt stress (100  mM NaCl for 48  h) increased the activities of antioxidant enzymes in Zea mays L. (maize) seedlings, but this was not enough to prevent oxidative stress (Shan et al. 2014). The treatment with 600 μM NaHS for 8 h followed by salt stress promoted an abrupt increment in the activity of APX, GR, DAR, γ-glutamyl cysteine synthetase, and GalLDH and accumulation of reduced ascorbate and glutathione. These events lowered the oxidative stress in maize seedlings due to decreased electrolyte leakage and MDA amount (Shan et al. 2014). The oxidative stress and the osmotic stress caused by 300 mM NaCl in leaves of Cynodon dactylon (L.) Pers. (bermudagrass) after 7–14 days was mitigated by 500 μM NaHS (Shi et al. 2013). As observed in other plant species, the H2S donor enhanced the activity of catalase, G-POX and GR and increased the content of glutathione in plants under salt stress. Accumulation of osmoregulators such as L-proline, sucrose, and soluble sugars in bermudagrass plants simultaneously exposed to NaCl and NaHS was reported. Additionally, the content of H2O2, O2•−, and MDA decreased under NaHS treatment, allowing greater survival of plants under salt stress (Shi et  al. 2013). The endogenous levels of H2S increased in Solanum lycopersicum L. (tomato) roots 2  h after the beginning of the treatment with 100  mM NaCl (Da-Silva et al. 2018). Remarkably, the genes CS and CAS that encode for the H2S-­ producer enzymes cysteine synthase and β-cyano-L-alanine synthase, respectively, were already upregulated 1 h after the beginning of plant exposure to NaCl. The activity of catalase and SOD increased while H2O2 and lipid hydroperoxide content decreased in response to simultaneous treatment with 100 mM NaCl and NaHS (25 μM) in comparison to plants treatment with NaCl alone. Hence, endogenous and exogenous H2S contributed to tomato plants overcome NaCl stress (Da-Silva et al. 2018). H2S relieved 4-day salt stress in wheat seedlings by decreasing Na content and Na+/K+ ratio, without affecting K+ levels. The use of 50 μM NaHS for 12 h, together

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with NaCl, resulted in wheat seedlings with higher biomass and higher root and shoot size (compared to plants treated with NaCl only) (Deng et al. 2016). Similarly, the use of 50 μM NaHS 48 h prior to the treatment with 150 mM NaCl for 4 days caused a decrease in Na+ and an increase of the nutrients K, Ca2+, Mg, Fe, Zn, and Mn in roots and leaves of rice plants (Mostofa et al. 2015b). The activity of SOD, catalase, GR, GST, and GPX increased, as well as the content of reduced ascorbate and glutathione. The content of methylglyoxal, a highly cytotoxic compound, decreased in response to an increment in the activity of glyoxalase, an enzyme involved in the methylglyoxal detoxification.

9.4  Water Stress Drought stress has been considered the most devastating environmental stress, causing severe losses to crop worldwide. Plants respond to water-deficit conditions through a series of physiological, cellular, and molecular processes. These responses include stomatal closure, repression of cell growth and photosynthesis, and activation of respiration. H2S increases tolerance to drought stress in plants by restricting oxidative stress, inducing the accumulation of ormoregulators, protecting the photosynthetic apparatus, and controlling stomatal movement (Zhang et al. 2010c; Li et al. 2017; Ding et al. 2018). Indeed, endogenous H2S mediated the transmembrane efflux of K+ from and the influx of Ca2+ and Cl− into guard cells, which lead to stomatal closure in Arabidopsis cultivated in the soil bearing 60% relative humidity (Jin et al. 2017). The effects of H2S on drought-responsive genes were investigated in wheat plants pretreated with 400 μM NaHS for 5 days followed by exposure to 20% polyethyeleneglycol (PEG) 6000 for 3 days (Li et al. 2017). The translocation of Fe to shoot and its accumulation in leaves were more prominent in plants under drought and H2S treatment than in those subjected to drought exclusively. The ribosome biogenesis, protein processing in the endoplasmic reticulum, fatty acid degradation, and cyanoamino acid metabolism were also induced by H2S in leaves of wheat plants under drought. In addition, H2S was involved in plant hormones signal transduction and drought-induced transcription factors (Li et al. 2017). Proteomics analysis identified that H2S regulates the biosynthesis of proteins involved in the carbohydrate metabolism, amino acid metabolism, signaling, photosynthesis, and ascorbate and glutathione metabolism in wheat seedlings (Ding et al. 2018). The use of 50 μM NaHS for 48 h increased the height and decreased lipid peroxidation in wheat seedlings under dehydration (20% PEG 6000 for 4 days). The contents of ascorbate, reduced glutathione, starch, and soluble sugars were also increased in wheat seedlings pre-treated with NaHS and stressed with PEG 6000 (Ding et al. 2018). The expression of genes related to H2S production (LCD, DCD1, NFS1, NFS2, and DES1) was up-regulated, and H2S was produced in Arabidopsis under dehydration stimulated by 0.1 g mL−1 PEG 8000 for 2 h. Pre-treatment with NaHS (3–100

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μM) for 12 h regulated the expression of the drought-responsive genes DREB2A, DREB2B, RD29A, and CBF4. H2S was also involved in the regulation of the drought-associated miRNAs (miR167, miR393, miR396, and miR398) and improved the tolerance of Arabidopsis to drought (Shen et al. 2013). Cysteine desulfhydrase over-expressing Arabidopsis plants and wild-type ones pre-treated with 100 μM NaHS for 7  days showed high H2S levels after dehydration for 6  h. In addition, plants with high production of H2S presented a high survival rate than those knocked down for cysteine desulfhydrase (Shi et al. 2015). Glycine max L. (soybean) plants under drought (devoid of irrigation for 21 days) and sprayed with 50 or 100 μM NaHS showed enhanced activity of catalase and SOD, decreased amounts of MDA, H2O2, and O2•− and low activity of lipoxygenase. Such plants also presented higher chlorophyll levels, higher water content, higher height, and dry mass and displayed a higher survival rate when compared to plants exclusively under drought (Zhang et al. 2010c). Oxidative stress provoked by treatment (7 or 14 days) with 30% PEG 6000 was relieved by 500 μM NaHS treatment in bermudagrass plants. The decrease in ROS content was associated with an increment in the activity of catalase, G-POX, and GR. H2S also increased the content of L-proline, sucrose, and soluble total sugars and the survival rate of plants under PEG 6000 stress (Shi et al. 2013). The endogenous content of H2S and the activity of L/D-cysteine desulfhydrase enzymes were increased in Eruca sativa Mill. (arugula) seedlings after 10  days growing under 30% of field capacity (Khan et al. 2018). In addition, the application of 2 mM NaHS in seedlings under desiccation increased the amounts of L-cysteine, L-proline and glycine betaine and decreased water loss in leaves. The exogenous H2S also alleviated the oxidative stress in arugula seedlings caused by drought. Thus, the electrolyte leakage and content of H2O2, O2•−, and lipid hydroperoxides decreased in seedlings under dehydration. The activity of carbonic anhydrase, a key enzyme of C metabolism, was enhanced in arugula seedlings under dehydration and NaHS treatment (Khan et al. 2018).

9.5  Temperature Stress Temperature tolerance is a species-specific trait that contributes to plants adaptation to their original geographic zones since each species presents a minimum, maximum, and optimum temperature of growth (Hatfield and Prueger 2015). In the face of global warming and climate change (IPCC 2018), temperature stress (heat or cold conditions) is an increased concern for the agricultural sector worldwide. Then, stress-alleviating compounds, such as H2S, show great importance in plants’ thermotolerance. During cold stress, stomata closure limits leaf dehydration. Members of mitogen-­ activated protein kinase (MAPK), such as MPK4, have been reported to help cells to control dehydration (Du et al. 2017). MPK4 is widely distributed in guard cells

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of Arabidopsis plants and its encoding gene exhibited 2.5 times higher expression levels compared to other kinase protein genes in non-stressed plants fumigated with 50 μM NaHS. Pre-treatment with 50 μM NaHS for 30 min provoked inhibition by 30% of stomata opening in wild type plants under cold stress (4 °C) than it did in cold-stressed plants with no H2S fumigation. Pre-treatment of mp4k mutant plants with NaHS did not cause the expected response, indicating that H2S inhibited stomatal aperture in Arabidopsis through the MPK4 signaling cascade (Du et al. 2017). H2S also alleviated cold stress symptoms via reinforcement of enzymatic and non-­ enzymatic antioxidant systems. Low temperatures may cause chilling injury by increasing ROS production and cell membrane damages, which compromise the quality and shelf-life of many horticultural products (Luo et  al. 2015; Liu et  al. 2019). Fumigation with 0,5  mM NaHS reduced the chilling injury symptoms in Musa spp., AAA group (banana) fruits stored at 7 °C for 7 or 14 days, and kept at 20 °C for 2, 4 or 6 days for ripening. NaHS treatment increased by 33% of the total phenolic compounds in banana fruits submitted to chilling temperatures for 7 or 14  days. NaHS also increased the ability to scavenge DPPH radicals and ferric reducing antioxidant potential (from 7% to 15%) in plants submitted to cold stress for 7 or 14 days (Luo et al. 2015). Increased content of soluble sugar is also another way of plants to cope with cold stress as a strategy of osmotic adaptation. NaHS (500 μM) led to sugar accumulation in bermudagrass within 7 days, but not after 14 days of cold stress (Shi et al. 2013). MDA content decreased by 19% (compared to the control) in all treatments in which plants were pre-treated with H2S donor followed by 7 °C stress, except for fruits ripening for 6 days after 14 days of chilling temperatures (Luo et al. 2015). Likewise, MDA content diminished 30 and 26% in bermudagrass after 500 μM NaHS plus 4 °C treatment for 7 or 14 days, respectively, when compared to control. Peroxidase, catalase, and GR activities increased in plants under cold plus 500 μM NaHS treatment for 7 and 14 days (Shi et al. 2013). The activity of catalase and GR also increased in banana fruits under conditions similar to those imposed on bermudagrass plants (7  °C plus 0.5  mM NaHS for 0–14 days) (Luo et al. 2015). Over-expression of genes related to H2S biosynthesis in Arabidopsis controlled ROS production in plants as a result of an enhancement of the activity of antioxidant enzymes. In contrast, knockdown plants, minimally controlled ROS production in cold stressed plants (Shi et al. 2015). The mutant plants provide a better understanding of in vivo roles of H2S as the treatment of plants with exogenous H2S may have limitations to address changes in plant metabolism. Arabidopsis plants knocked down for genes that encode for L/D-­cysteine desulfhydrase (lcd and dcdI) fail to properly express genes related to the plant response to cold stress. Conversely, over-expression of H2S-encoding genes increased the expression of cold stress-related genes in Arabidopsis. Thus, the expression of certain genes related to the tolerance of plants to multiple (a)biotic stresses is modulated by H2S (Shi et al. 2015). Similarly, LCD and DES expression in leaves of four cucumber genotypes increased upon plant maintenance at 4  °C.  Cold-exposed cucumber plants treated with hypotaurine exhibited lower expression of genes related to the biosynthesis of cucurbitacin C while those plants were supplied with H2S presented higher expression of such genes and consequently

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accumulation of cucurbitacin C when exposed to 4  oC. It is hypothesized that H2S modulates gene expression by sulfhydrating transcription factors, but more in vivo evidence is needed to demonstrate it (Liu et al. 2019). The H2S-triggered biosynthesis of secondary metabolites is another way of plants coping with cold stress. Plants respond differently to heat stress depending on the developmental stage. Exogenous H2S provided as 0.5  mM NaHS increased the germination of maize seeds by 37.5% at 39  °C (Zhou et  al. 2018). MDA also decreased in Fragaria x ananassa cv. ‘Camarosa’ (strawberry) plants whose leaves were treated with 100 μM NaHS after exposure of plants to 42 °C for 8 h. NaHS application also alleviated the injuries caused to photosystem II by heat stress as it increased photochemical efficiency in strawberry leaves (Christou et  al. 2014). Likewise, exposure of the woody species Populus trichocarpa (black poplar) with 50 μM NaHS followed by incubation at 35 °C for 3 days yielded phytochemical efficiency compared to that of non-­stressed plants (Cheng et al. 2018). The tolerance of black poplar to heat stress appears to be mediated by S-nitrosoglutathione reductase (GSNOR) that indirectly affects S-nitrosylation processes in cells via the depletion of the major endogenous NO donor S-nitrosoglutathione. H2S is believed to act upstream of GSNOR because H2S was found to induce GSNOR activity (Cheng et al. 2018). Maize seeds under high temperature successfully germinated upon treatment with NaHS (Li et al. 2018a).

9.6  I nterplay Among H2S, Plant Hormones, and Secondary Messengers This section intends to exclusively address the interplay among H2S, hormones and second messengers as the crosstalk among H2S, H2O2 and NO has already been widely discussed recently (Da-Silva et al. 2019). Salicylic acid (SA) is a small phenolic hormone that contributes to plant tolerance to several stresses. Like H2S, SA protects plants by boosting the antioxidant system. SA-induced heat tolerance in maize seedlings was improved by supplying seedlings with 0.5 mM NaHS (Li et al. 2015). Survival percentage increased from 70% (SA treatment) to 85% (NaHS plus SA) in maize seedlings. SA at 0.5 mM increased L-DES activity indicating that SA and H2S interact with each other and that H2S may act downstream of SA in plants under heat stress (Li et  al. 2015). Crosstalk between SA and H2S was also observed in Arabidopsis seedlings under 100 μM Cd for 72  h. SA at 0.5  mM induced L-DES activity and, therefore, the accumulation of H2S in cells (Qiao et al. 2015). High temperatures also up-regulate the expression of genes related to the production of abscisic acid (ABA), which in turn suppresses the germination of seeds at supra-optimal temperatures. The increase of seed germination under relatively high temperature (32 °C) was 10 times higher in Arabidopsis that over-expressed DES1-ox compared to wild-type Col. The germination of seeds of knocked down

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plants for DES and LCD enzymes (des1 and lcd plants) was considerably lower than that of wild-type plants. The increment in germination under heat stress was also accompanied by a 20% decrease in transcriptional levels of ABI5 in DES1-ox compared to Col or des1 and lcd plants. ABI5 is a transcription factor that works in ABA signaling and induces seed dormancy (Chen et  al. 2019). The factor ELONGATE HYPOCOTYLE 5 (HY5) that induces the expression of ABI5 by binding to the ABI5 promoter and mediating ABA signaling and the CONSTITUTIVE PHOTOMORPHOGENESIS (COP1), a ring finger E3 ligase that triggers the degradation of nuclear HY5 in the absence of light were also investigated (Chen et al. 2019). Under heat stress, seeds accumulated H2S that led to the accumulation of COP1 in the nucleus of radicle cells. COP1 degraded HY5, which decreased the transcription of ABI5 and consequently allowed Arabidopsis seeds to germinate even at high temperatures (Chen et al. 2019). The enhancement of ABA levels in response to drought stress decreased the turgor of guard cells. This was a result of the increasing outcome of K+ from inside the guard cells through the action of ion-­channels. Leaves of 4-week-old wild type and lcd/des 1 mutant Arabidopsis plants were submitted to 20 μM ABA for 3 h. ABA decreased stomata opening in both genotypes, an inhibition that was much less prominent in lcd/des 1 mutant plants (Du et al. 2019). Similar results were found in Arabidopsis defective for LCD proteins solely. Increasing ABA concentrations had a smaller effect on stomatal closure in lcd plants in comparison to wild type ones. On the other hand, ABA-deficient mutant Arabidopsis failed to respond to 80 μM NaHS treatment under the same treatment conditions. These results suggest an overly complex interplay between H2S and ABA in stomatal movement under drought stress. Additionally, lcd mutants showed lower levels of TPC1 (Ca2+ channel-related gene) expression whereas the expression of ACA9 and ACA11 (Ca2+-ATPase) and CAX1 (Ca2+-H+ antiporter gene) were comparatively higher (Jin et al. 2013). The involvement of MPK4 in the stomatal movement was also investigated using mpk4 mutant Arabidopsis plants (Du et al. 2019). ABA at 20 μM increased stomata closure but the number of open stomata in mpk4 stomata was 25% higher than that of wild type plants. Stomata aperture in mpk4 was totally inhibited by treatment with an H2S donor. Therefore, MPK4 has an important role in the control of stomatal closure by H2S mediation during drought stress, and that the ABA signaling pathway is closely related to the H2S-­MPK4 cascade (Du et  al. 2019). However, further studies are needed to deepen into the understanding of the influence of other MAP kinase members and Ca2+ in stomatal movement in response to H2S signaling during drought. In Setaria italica (foxtail millet), 1 μM methyl jasmonate (MeJA) increased the endogenous amount of H2S in seedlings under Cd stress; the MeJA-induced Cd tolerance was decreased by H2S scavenger (Tian et al. 2017). The effect of H2S on drought stress has also been addressed through the RNA-­ Seq perspective to identify candidate genes that contribute to H2S-dependant drought tolerance in wheat plants (Li et al. 2017). Eight hormones were found to participate in wheat tolerance to drought, in which ABA is one of the key regulators of stomata closure. ABA signaling initiates when ABA binds to pyrabactin resistance (PYR) and PYR-like (PYL) receptors. Thus, type 2C protein phosphatases

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(PP2Cs), which are negative regulators of ABA signaling, are repressed. The signaling process undergoes the activation of SNF1-related kinases (SnRK2 kinases). NaHS induced the expression of PYR/PYL and suppressed the one of PP2C genes in wheat under drought stress. H2S also interacts with auxin probably by regulating the hormone’s homeostasis and the polar auxin transport. Gibberellin (GA) signaling is disrupted by H2S that represses the expression of GA insensitive dwarf1 (GID1), a gene that encodes for a GA receptor. The expression of the pathogenrelated gene PR-1 was up-regulated by H2S and the expression of PR1 is known to be induced by SA. H2S also induced the expression of genes related to cytokinin metabolism [histidine phosphotransfer proteins (AHPs) and response regulators (A-ARRs)] in plants under drought. The F-box protein coronatine insensitive1 (COI1) is crucial in the jasmonic acid (JA) signaling by promoting the degradation of the jasmonate-ZIM domain (JAZ) repressor proteins. H2S induced COI1 in wheat under drought stress (Li et  al. 2017). Ethylene is detected by the receptor ETR, whose downstream genes were differently expressed in response to exogenous H2S in plants under drought. Finally, H2S interfered with the brassinosteroid signaling pathway by downregulating the hormone’s receptor and related targets (Li et al. 2017). Secondary messengers can also interact with H2S to help plants cope with abiotic stress. These secondary messengers can regulate transcription, translation, and post-­ translation processes and influence plant metabolism and defense system of tissues far from the stressing agent. Calcium (Ca2+) is one of the most notable secondary messengers in plant cells (Valivand et al. 2019). Ca2+ concentration fluctuates in the cytosol during plant stress and differences in cytosolic concentrations are mediated by Ca2+ channels, pumps, and carriers located in plasma membranes and tonoplasts (Fang et al. 2014). Such process is known as the Ca2+ signature and is decoded by Ca2+ sensors such as calmodulins (CaMs), CaM like proteins, and Ca2+-dependent protein kinases (CDPKs) (Fang et al. 2017). The interaction of Ca2+-CaM2 complex with the transcription factor TGA3 induces the transcription of LCD, which culminated in the increase of endogenous H2S in Arabidopsis exposed to Cr6+. The Cr stress did not increase TGA3 expression in Arabidopsis but increased the transcriptional and translational levels of CaM2, which accordingly enhanced the levels of Ca2+/CaM2/TGA3 complex (Fang et  al. 2017). Pre-treatment of 10-day-old Cucurbita pepo (zucchini) cv. Courgette d’Italie plants with 100 μM NaHS plus 15 mM Ca2+ followed by Ni stress [50 mg L−1 Ni(NO3)2] showed higher height and biomass accumulation compared to Ni-exposed plants (Valivand et al. 2019). Ca2+ channel blocker, Ca2+ chelator, or CaM antagonist revoked the effect of NaHS, suggesting that H2S acts upstream of Ca2+ in tolerance of zucchini plants to Ni. Incubation of roots with Ca2+ increased H2S in roots and leaves, while simultaneous treatment with NaHS and Ca2+ increased the expression of CaM by 43% when compared to Ca2+-treated plants (Valivand et al. 2019). Supplementation of heat-stressed tobacco cells suspension with Ca2+ enhanced the production of H2S and therefore thermotolerance whereas EGTA, a Ca2+ chelator, abolished this effect (Li et  al. 2012a, b). Alternatively, an H2S scavenger blocked the growth of Ni-stressed zucchini plants that was stimulated by Ca2+. Thus, H2S may act downstream of Ca2+ for

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Ni stress tolerance (Valivand et al. 2019). Treatment of zucchini with roots H2S led leaves to respond to Ni stressed plants. Ca2+ inhibitors reversed such responses while Ca2+ applied in roots resulted in higher accumulation of H2S in leaves. The same relation was recorded in foxtail millet, in which the endogenous H2S increased upon treatment of plants with 20 mM CaCl and H2S levels dropped in the presence of a Ca2+ chelator (Fang et al. 2014). Regardless of H2S acting up- or downstream of Ca2+, evidence suggests the role of the gasotransmitter and the cation as potent long-­ distance messengers (Valivand et al. 2019). The interaction of H2S with secondary metabolites is also documented. Mustard seedlings treated with 10 μM CdCl2 and 20 μM eugenol increased the endogenous levels of H2S by 50.7% when compared to plants solely treated with Cd. Eugenol is the main constituent of essential oils from clove trees. The increase in H2S levels stimulated the accumulation of reduced glutathione and therefore Cd tolerance in mustard seedlings (Hu et al. 2018). The use of an H2S scavenger compromised by 25% of the thermotolerance promoted by methylglyoxal in maize seedlings when compared to seedlings treated with 50 μM methylglyoxal plus 500 μM NaHS. Methylglyoxal, a byproduct of glycolysis and photosynthesis (reduced form of pyruvate), is a reactive carbonyl species long been considered a toxic compound. The role of methylglyoxal in stress signaling is still unclear, but it was shown to induce twice as much as the activity of LCD in maize when compared to control. Thus, methylglyoxal seems to act upstream of H2S in the tolerance of maize seedlings to heat stress (Li et al. 2018b).

9.7  Conclusions The stress tolerance mechanisms in plants can be activated by H2S through changing the function and subcellular localization of target proteins via persulfidation. Under controlled conditions, the increase in H2S production or the exogenous supply of this molecule are known to improve the plant performance under extreme environmental conditions such as high salinity, heavy metals, drought, and low or high temperatures. Currently, the evaluation of the efficiency of plants that overexpress genes related to H2S synthesis is of paramount importance to better understand the role of H2S in plant response to environmental stresses. Field conditions associated with the use of nanotechnology to efficiently deliver H2S to plant cells will be valuable to expand our knowledge on H2S-mediated plant responses to environmental changes. Such approaches will assist the development of even more tolerant plants in a moment of growing demand for food and projections of global climate change. Acknowledgements  Authors are thankful to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001 for financing part of the research mentioned in this chapter.

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Chapter 10

A Transcriptomic and Proteomic View of Hydrogen Sulfide Signaling in Plant Abiotic Stress Susana González-Morales, Raúl Carlos López-Sánchez, Antonio Juárez-­ Maldonado, Armando Robledo-Olivo, and Adalberto Benavides-Mendoza

Abstract  Plants often endure biotic and abiotic stress. To survive and reproduce, plants not only need to grow and develop but also need to tolerate and continuously adjust to environmental stress. Hydrogen sulfide (H2S) is a gasotransmitter with a signaling role in plants, which is endogenously generated in plant cells. Furthermore, H2S is related to some physiological processes such as germination, root development, and fruit ripening. Both endogenous H2S and exogenous applications of H2S donors can trigger different defense responses against stress in plants. The most studied mechanism is the stimulation of the antioxidant system, which efficiently removes excessive reactive oxygen species (ROS) and maintains redox homeostasis in plants. Furthermore, H2S has an essential role in protein regulation through the persulfidation process in post-translational modifications. At the proteomic and transcriptomic level, H2S can regulate proteins and genes and generate changes in metabolism, specifically in signal transduction, as well as in the defense response.

S. González-Morales CONACYT-Universidad Autónoma Agraria Antonio Narro, Saltillo, Mexico R. C. López-Sánchez Centro de Estudios de Biotecnología Vegetal, Facultad de Ciencias Agropecuarias, Universidad de Granma, Bayamo, Cuba A. Juárez-Maldonado Departamento de Botánica, Universidad Autónoma Agraria Antonio Narro, Saltillo, Mexico A. Robledo-Olivo Departamento de Ciencia y Tecnología de Alimentos, Universidad Autónoma Agraria Antonio Narro, Saltillo, Mexico A. Benavides-Mendoza (*) Departamento de Horticultura, Universidad Autónoma Agraria Antonio Narro, Saltillo, Mexico e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_10

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However, the different mechanisms of H2S signaling network in plants are still not completely clear. This review approaches the effect of H2S at the proteome and transcriptome level and its interaction with other molecules in plants under abiotic stress such as drought, salinity, extreme temperatures, and heavy metals. Keywords  Hydrogen sulfide · Antioxidant metabolism · Photosynthesis · Osmolyte biosynthesis · Stomatal opening · Postharvest

10.1  Introduction Sulfur (S) is an essential mineral nutrient that plays a crucial role in the structure and function of proteins, the cell redox state, and plant responses to biotic and abiotic stress (Gläser et al. 2014). Sulfate (SO42−) is the primary source of S for plants and represents the most stable form of S in the soil. Plants absorb SO42− from the soil solution through SO42− transporters located in the root cells (Boldrin et al. 2016). Plants must assimilate SO42−, an oxidized form of S, and convert it to reduced forms, metabolically useful for the biosynthesis of a wide range of compounds, including cysteine (Cys) and glutathione (GSH) (Jez 2019). As a first step in the assimilation of S, ATP-sulfurylase (ATP-S) catalyzes the activation of SO42− and adenosine-5′-phosphosulfate, which is subsequently reduced to sulfide (S2−) and incorporated into Cys. In turn, Cys acts as a precursor or donor of reduced S for a range of S compounds such as methionine (Met), GSH, homo-GSH (h-GSH), and phytochelatins (PC) (Anjum et  al. 2015). A wide variety of metabolites in which S is involved have been identified; these metabolites are derived from various metabolic pathways and have diverse functions ranging from proteogenic amino acids (Cys, Met), hormone derivatives (sulfojasmonate and sulfated brassinosteroids), antioxidants (GSH), secondary metabolites (sulfoflavonoids) and signaling molecules (phosphonucleotides and H2S) (Gigolashvili and Kopriva 2014). Hydrogen sulfide (H2S) is among the gaseous molecules that function as signaling agents, along with nitric oxide (NO) and carbon monoxide (CO) (Li 2013; Calderwood and Kopriva 2014; Hancock and Whiteman 2014; Yamasaki and Cohen 2016; Corpas et al. 2019a). H2S is an inorganic, flammable gas, soluble in water, with a characteristic odor of decomposing organic material. It has been historically considered as a pollutant and a toxic gas for life, however, in the last decade, a considerable amount of evidence has indicated its function as a gasotransmitter (Aroca et  al. 2018). H2S plays vital roles in post-translational protein modification, cell signaling, and physiological processes such as germination, fruit ripening, and root development (Paul and Roychoudhury 2020; Corpas and Palma 2020). H2S can serve as an alternative source of S for plants, which can be significant under anaerobic conditions in waterlogged soils (Calderwood and Kopriva 2014). Due to its gaseous nature, H2S can diffuse to different parts of cells and modify the redox state by supplying S to them (Pandey and Gautam 2020) or by induction of antioxidant

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enzymes (Shi et al. 2013). Furthermore, due to its highly lipophilic nature, H2S can quickly diffuse through the lipid bilayer of cell membranes and organelles (Cuevasanta et al. 2017). Although the signaling molecule H2S was once believed to be a phytotoxic contaminant, it is now recognized that plants have H2S-generating enzymes. These enzymes include desulfhydrases, specifically cytosolic L and D Cys desulfhydrases, and the mitochondrial enzymes D-Cys desulfhydrase and cyanoalanine synthase and chloroplastic sulfite reductase (Lisjak et al. 2013). Regarding the removal of H2S, the enzyme O-acetylserine (thiol) lyase (OASTL) seems to fulfill a dual function: on the one hand, the removal and detoxification of excess H2S; but also, the assimilation of H2S that enters the stomata in the form of reduced sulfur compounds such as Cys or GSH on the other (Youssefian et al. 1993). Catalase (CAT) and superoxide dismutase (SOD) can also metabolize H2S to transform it into polysulfides, and the latter is thought to be an ancient mechanism to detoxify H2S (Olson et al. 2018); however, it was also reported that H2S, as well as NO and reactive oxygen species (ROS)/reactive nitrogen species (RNS), can modulate catalase activity by inhibition (Corpas et al. 2019a; Palma et al. 2020). Various physiological processes of plants, and responses to abiotic stress are regulated by H2S (Fotopoulos et al. 2015). H2S confers tolerance to abiotic stress by regulating the expression of genes related to stress responses (Pandey and Gautam 2020), the metabolism of ROS, and the improvement in metabolic homeostasis (He et al. 2018). H2S has been shown to mitigate the harmful effects of salinity, heavy metals, drought, alkalinity, hypoxia, and high temperatures by improving the activity of antioxidant enzymes (Yu et al. 2013; Yang et al. 2016; Montesinos-Pereira et al. 2018; Kaya et al. 2020a). The stress-alleviating effect of H2S is associated with the increases in the activity of ROS detoxification enzymes involved in the ascorbate-­ glutathione (AsA-GSH) cycle, such as ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and one of the critical enzymes in the synthesis of GSH such as glutamylcysteine synthetase (γ-ECS) (Shan et al. 2014). Furthermore, it has been reported that H2S can improve resistance to drought through transcriptional regulation associated with microRNAs (miRNAs) in Arabidopsis (Shen et al. 2013). This chapter aims to show the signaling network where H2S intervenes in the process of tolerance to abiotic stress from the transcriptional and proteomic point of view.

10.2  P  articipation of H2S, Polysulfides, and Reactive Sulfur Species in Stress Signaling Plant cell metabolism involves a complex network of signaling molecules that facilitate communication between different metabolic pathways, their regulation, and responses under specific environmental and physiological conditions (Corpas et al. 2019b). Being a gaseous and small molecule, H2S can cross cell

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membranes and reach intracellular targets without the need for carriers or transporters and can spread rapidly (Shivaraj et al. 2020). Regulation of H2S concentration is a crucial factor; H2S at an adequate level plays a protective role in plants under environmental stress, while in high concentration, it leads to H2S toxicity by inhibiting the mitochondrial electron transport chain (Fakhari et al. 2019). Reactive Cys residues in the proteome are essential components of redox signaling pathways; reversible changes in the oxidation state of cysteines allow them to function as redox switches in multiple signaling pathways (Kabil et al. 2014). Signaling in cells involving reactive compounds is well established. ROS and NO are influential in controlling a range of physiological responses in plants, and H2S is an important signaling molecule (Hancock and Whiteman 2016) that interacts with ROS and NO signaling agents during stress acclimatization processes. Abiotic stress causes nutrient imbalance, oxidative stress, osmotic stress, membrane damage, and protein denaturation. However, endogenously synthesized or exogenously applied, H2S can alleviate these damages by inducing processes related to membrane repair and reconstruction, activation of the antioxidant system and the cellular detoxification system, the regulation of ionic and nutrient balance, synthesis of heat shock proteins (HSPs) and synthesis of osmolytes (Singh et al. 2019). H2S interacts with the ROS-mediated oxidative stress response network at multiple levels, including regulating ROS processing systems by transcriptional or post-­translational modifications. H2S-ROS crosstalk also involves other factors such as NO, affecting critical cellular processes such as autophagy (Chen et  al. 2020a). H2S has a similar function and is coupled to NO in plant response to stress and stomatal regulation (Singh et al. 2015). An interaction between H2S, NO, and abscisic acid (ABA) in stomatal movements have also been described (Li et  al. 2016). Regarding the H2S-ABA interaction to trigger the closure of stomata and facilitate adaptation to abiotic stress, ABA has been reported to induce the production of H2S catalyzed by L-cysteine ​​desulfhydrase 1 (DES1) in guard cells, and that H2S too, in turn positively regulates ABA signaling through persulfidation of 1(OST1)/SNF1 related to protein kinases 2.6 (SnRK2.6) (Chen et al. 2020b). H2S actively participates in ethylene-induced stomatal closure regulation and interacts with H2O2 to regulate the Na+/H+ antiporter under salinity stress (Hou et al. 2013a; Li et al. 2014a). H2S has been shown to regulate the activity of the vacuolar ATPase (V-ATPase) in cadmium (Cd)-stressed cucumber roots, while H2O2 is responsible for the decrease in enzyme activity (Kabała et  al. 2019). Changes in V-ATPase activity induced by both markers are unrelated to the expression of the V-ATPase genes, suggesting that H2S and H2O2 affect enzyme activity at the post-translational level, for example, through a well-known mechanism that involves the formation of disulfide regulatory bonds (Kabała et al. 2019).

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Interaction of H2S with phytohormones, such as gibberellins and salicylic acid (SA) in plant development processes and during stress responses has already been studied (Liu et al. 2020a). For example, H2S has been found to modulate root development in Arabidopsis, modifying the expression of actin-binding proteins (ABPs), which regulate the location of PIN proteins responsible for auxin transport (Jia et al. 2015). On the other hand, H2S can delay gibberellic acid (GA)-mediated programmed cell death (PCD) in wheat aleurone layers by modulating GSH homeostasis and heme oxygenase-1 (HO1) expression (Xie et  al. 2014). It can even react directly with lipid radicals to prevent lipid oxidation or activate a signaling pathway to indirectly amplify the activation of antioxidant enzymes and decrease oxidative stress (Amooaghaie et al. 2017). One proposed mechanism for H2S signaling is protein persulfidation, that is, the posttranslational conversion of cysteinyl thiolates (Cys-S -) to persulfides (CysS-S -). However, relatively weak reactivity of H2S to oxidized thiols, such as disulfides, the low concentration of disulfides in the reducing medium of the cell, and the low level of H2S in the stress-free cellular state raise doubts about the possibility of persulfide formation by the reaction between an oxidized thiol and a sulfide anion or a reduced thiol and oxidized H2S (Mishanina et al. 2015). Greiner et al. (2013) observed that H2S does not directly cause the protein thiol oxidation process, but rather that the polysulfides formed in H2S solutions are directly responsible for the process. H2Sx polysulfides are Reactive Sulfur Species (RSS) like H2S2, H2S3, H2S4, and H2S5 (Kharma et al. 2019). The RSS are characterized as S-containing redox-active molecules that are capable, under physiological conditions, of oxidizing biomolecules. RSS are considered key molecules in signaling (Gruhlke and Slusarenko 2012). In addition to the polysulfides mentioned above, among the RSS are the thiyl radical (RS•), disulfide (RSSR), disulfide-S monoxide (thiosulphinate) [RS (O) SR’], disulfide dioxide-S (thiosulfonate) [RS (O) 2SR’], sulfenic acid (RSOH), and sulfinic acid (RSO2H). The reaction of ROS and RNS with thiols, producing RSS, can be seen as an initial step in detoxification of ROS and RNS (Diaz-Vivancos et  al. 2015; García et  al. 2019). Because the resulting generation of RSS depends on the initial oxidation of the thiol and the reaction with other sulfur-containing molecules, they can be considered “second generation RSS.” Therefore, a fundamental RSS division from a biological point of view can be drawn from their biosynthetic pathway and genesis mechanism. While first-generation RSS are already present in the cell in stress-free plants, second-generation RSS are produced enzymatically in the cell or by the action of oxidants, e.g., ROS and/or RNS after injury or oxidative stress. An example of a first-generation RSS would be allyl cysteine (alliin) sulfoxide in garlic, which is the precursor for allicin synthesis. In contrast, sulfenic acid and allicin products arising after injury through the alliinase enzyme action (Zheng et  al. 2016; Mukherjee 2019) are second-generation RSS.  The cellular impact of allicin exemplifies the effects of RSS on metabolism: alkyl-mercapto glutathione (GSSA), the initial

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product of the reaction between GSH and allicin, is a potent antioxidant with a significant effect on thiols (Gruhlke and Slusarenko 2012).

10.3  T  he H2S Signaling Network Seen Through Transcriptomics and Proteomics Proteins are directly connected to cell function and reflect the complex molecular and physiological mechanisms in plants. As far as is known, the molecular mechanism by which H2S exerts its action involves an oxidative post-translational modification of Cys residues to form a persulfidated thiol motif, a process called protein persulfidation (Aroca et al. 2017; Filipovic et al. 2018). Persulfidation is also known as sulfhydration (an inaccurate term that implies hydration); instead, persulfidation involves sulfuration (addition of sulfur atom) (Filipovic et al. 2018). This process increases the reactivity of modified Cys due to the higher nucleophilicity of persulfide compared to the thiol group (Paul and Snyder 2012). Protein persulfidation has also been shown to be mediated by NO, which provides strengthening against over-­ oxidation (Aroca et al. 2018). The persulfidation process is ubiquitous and modifies many proteins, a much higher amount than those modified by ROS and RNS (Ida et  al. 2014). Post-­ translational modifications (PTM) of proteins by persulfidation have been shown to change biological functions, enzyme activity, structures, and subcellular locations (Shibuya et al. 2013). According to Aroca et al. (2017), a large number of persulfidated proteins are in chloroplasts, where most of the H2S production occurs during the S assimilation process and where ROS are also produced. Then the persulfidation of proteins using H2S can serve as a mechanism to increase the antioxidant capacity of thiol sinks (Filipovic and Jovanović 2017). Protein polysulfidation, another kind of redox-dependent regulatory mechanism, occurs when proteins and enzymes have cysteine polysulfides, including CysSSH at their specific Cys residues [cysteine persulfide/polysulfides [CysSSH/CysS–(S)n –H)] (Kasamatsu et al. 2016). Protein polysulfidation can be catalyzed by cysteinyl-tRNA synthetases (CARSs), and it seems independent of H2S oxidation and independent of the reaction with NO. Protein polysulfidation occurs during the translation process in mammals, bacteria, and mitochondria (Akaike et al. 2017).

10.3.1  H2S and the Plant-Stress Proteome H2S is a gaseous signaling molecule that has been shown to improve drought resistance in plants. The pretreatment with sodium hydrosulfide (NaHS, an H2S donor) significantly increases plant height, and the leaf relative water content of seedlings

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under drought stress. H2S improves CAT, SOD, and peroxidase (POD) activities in leaves under drought; this gaseous mediator can alleviate damage in plants induced by different abiotic stresses via improvement of antioxidant systems (Christou et al. 2013). In another study in wheat seedlings under drought stress, H2S was found to significantly regulate the level of 120 proteins (63 decreased and 57 increased). These proteins were related to photosynthesis, carbohydrate metabolism, signal transduction, stress, protein synthesis, and secondary metabolism (Ding et al. 2018). In Eruca sativa plants under dehydration stress, H2S reduced oxidative stress and increased the activities of antioxidant enzymes (SOD, POD, and CAT) and the activation of carbonic anhydrase activity, and OASTL (involved in the biosynthesis of Cys), L-cysteine desulfhydrase (LCD), and D-cysteine desulfhydrase (DCD) activity (synthesizes more H2S from accumulated Cys) also occurred (Khan et al. 2018). In wheat plants, the effects of H2S in drought stress were studied by Kolupaev et al. (2019a). H2S treatment increased the activity of SOD and prevented a stress-induced decrease in the activities of CAT and guaiacol peroxidase (GPO) in the leaves. The above prevents the drought-induced accumulation of H2S and lipid peroxidation. The involvement of H2S in plant responses to multiple abiotic stress has been well studied. Shi et al. (2013) studied the effect of H2S application in Bermudagrass under salt, osmotic, and cold stress. Exogenous application of H2S confers tolerance to abiotic stress, which is evidenced by decreased electrolyte leakage and increased survival rate under stress conditions. H2S alleviates ROS burst and cell damage induced by stress, by increasing antioxidant enzymes (CAT, POD, and GR). Salt stress is one of the most critical environmental factors limiting plant growth, and H2S can play a role in plant responses to this stress. Wei et al. (2019) studied the effect of salt stress in Malus hupehensis Rehd. var. pingyiensis treated with H2S. The application of H2S reduced the NaCl-induced inhibition of root elongation, decreased H2O2 content, and enhanced SOD, GPO, and CAT activity in the mitochondria compared to the stress condition; this protected plants against salt stress by decreasing ROS accumulation and by regulating membrane stability and antioxidant system. Similarly, in Arabidopsis thaliana plants under salt stress, the treatment with H2S increases salt resistance (Yastreb et al. 2020), which was expressed in a decrease in oxidative damage, a reduction in water deficit, and preservation of photosynthetic pigments. H2S prevented the stress-induced decrease in the activity of antioxidant enzymes (SOD and CAT) and contributed to the increase in GPO activity. The proteomic analysis of Kandelia obovata plants under salinity showed modulation by H2S in the expression of 37 differentially expressed proteins (Liu et al. 2020b). The proteins primarily enriched were related to photosynthesis, primary metabolism, stress response, and hormone biosynthesis. H2S increased photosynthetic electron transfer, chlorophyll biosynthesis, and carbon fixation in K. obovata leaves under salt stress (Liu et al. 2020b). H2S confers tolerance to alkalinity stress in Brassica oleracea (Montesinos-Pereira et  al. 2018). The application of H2S improves the antioxidant response, inducing SOD activity, and improving processes involved in

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GSH homeostasis, boosting the reduced GSH content and the operation of critical enzymes in GSH synthesis and the AsA-GSH cycle. H2S improves the germination of seeds and the survival percentage of maize seedlings under heat stress (Li et al. 2013). The above result from the improvement in the activity of Δ1-pyrroline-5-carboxylate synthetase and lower proline dehydrogenase (PDH) activity, which in turn induced accumulation of endogenous proline in maize seedlings, followed by mitigated accumulation of malondialdehyde (MDA) and increased survival percentage of maize seedlings under heat stress (Li et al. 2013). Tolerance to abiotic stress is also related to the increase of the antioxidant system that neutralizes ROS. Depending on the prevalent stress, the response of the plant’s antioxidant system to H2S is different. The application of H2S in wheat under heat stress increased SOD, CAT, APX, and GPO activity (Yang et al. 2016; Kolupaev et al. 2017). Under chilling stress, H2S increases the wheat’s SOD and GPO activity (Kolupaev et al. 2019b). In maize under hypoxia, H2S increases CAT, SOD, APX activity, and GSH content (Peng et al. 2016). Similarly, the application of H2S in maize under heat induces antioxidant system (APX, GR, GPO, SOD, and CAT activities) and osmolyte biosynthesis (Δ1-pyrroline-5-carboxylate synthetase, ornithine aminotransferase, betaine aldehyde dehydrogenase, and trehalose −6-­phosphate phosphatase activities) (Zhou et al. 2018). To minimize the detrimental effects of heavy metal exposure and their accumulation, plants have evolved detoxification mechanisms (Yadav 2010). Plants experience oxidative stress upon exposure to heavy metals that lead to cellular damage. Tolerance to Cd stress in plants with the application of H2S inhibited the ROS burst by inducing antioxidant activity (SOD, CAT, APX, POD) (Huang et al. 2016; Jia et al. 2016; Fu et al. 2019; Kaya et al. 2020b). Tolerance to Cd in cucumber plants is also correlated with the increase of H2S content by desulfhydrase activity and the protection of V-ATPase, responsible for generating a proton gradient across the tonoplast (Kabała et al. 2019). Among the heavy metals (HMs), lead (Pb) is a widespread ecosystem pollutant and affects food security and public health. The effect of H2S in cauliflower under Pb stress was the increase of seed germination and seedling growth along with the reduction of ROS and MDA by the increment of antioxidant enzyme (Chen et al. 2017). The same effect is reported in Brassica napus L. (Ali et al. 2014). Pb tolerance is also related to the increase of nitrate reductase activity; this enzyme is involved in the assimilation of nitrate into amino acids and is sensitive to HMs toxicity, including Pb (Zanganeh et al. 2019). Tolerance to HMs like Al, Cu, Hg, Cr, and Zn and excess of nitrate in plants with H2S application is also related to the increase of the antioxidant enzyme activities (Zhang et al. 2010; Dai et al. 2016; Chen et al. 2017; Kaya et al. 2018; Zhu et al. 2018; Qi et al. 2019; Ahmad et al. 2020). Nickel tolerance in rice plants with H2S application is related to the increase in nitrate content, with more activity of biosynthesizing enzymes (nitrate reductase, nitrite reductase, glutamate synthase, glutamate oxaloacetate transaminase, glutamine synthetase, and glutamate pyruvate transaminase), indicating a positive role of H2S on nitrogen metabolism (Rizwan et al. 2019).

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The post-harvest life of vegetables and fruits is significantly affected by H2S. In broccoli plants, H2S increases the LCD and DCD activities, which enhances endogenous H2S and activates key enzymes involved in energy metabolism, including ATPases, cytochrome c oxidase (CCO), succinate dehydrogenase (SDH), glucokinase, fructokinase, glucose-6-phosphate dehydrogenase (G6PDH) and 6-­phosphogluconate dehydrogenase (6PGDH). The above suggests that H2S can effectively inhibit the yellowing and maintain an adequate metabolic activity, prolonging the shelf life of post-harvest broccoli (Li et al. 2017a). H2S also alleviates post-harvest senescence of grape by modulating the antioxidant defenses (CAT and APX) and reducing MDA and ROS (Ni et al. 2016). Photosynthesis is the key metabolic process, and H2S has a vital role in modulating photosynthesis in Spinacia oleracea plants (Chen et al. 2011). The activity of ribulose-1,5-bisphosphate carboxylase (RuBisCO) and the protein expression of the RuBisCO large subunit (RuBisCO LSU) were enhanced by H2S (Chen et al. 2011). Chen et  al. 2014a reported that a set of proteins are involved in H2S-regulated metabolism or signaling pathways in Spinacia oleracea. The authors found that 65 proteins were up-regulated, whereas 27 were down-regulated. These proteins were functionally divided into 9 groups, including energy production and photosynthesis, cell rescue, development, cell defense, metabolism, protein synthesis and folding, and cellular signal transduction. Proteins regulated by H2S and their functions are described below, using the results of 35 studies in which H2S was applied to the plants under abiotic stress (Table 10.1).

10.3.2  H2S and the Plant-Stress Transcriptome Land plants are continuously exposed to multiple abiotic stress factors like drought, heat, and salinity. In A. thaliana plants, drought induces more H2S production by Cys degradation by inducing the activity of LCD and DCD enzymes. Drought associated genes DREB2, RD29A, and CBF4, showed expression patterns related to LCD and DCD. Treatment with H2S showed a higher survival rate and displayed a significant reduction in the stomatal aperture size (Jin et al. 2011). H2S regulates the ion channels related to stomatal closure in plant response to drought stress (Jin et al. 2013). In A. thaliana plants, the relationship of ABA and H2S with mutants was studied. H2S may be an important link in stomatal regulation by ABA via ion channels, affecting the expression of ABA receptor candidates; on the other hand, ABA itself influences H2S production. In A. thaliana plants, H2S can improve drought resistance through regulating drought-associated miRNAs (Shen et  al. 2013). Drought usually induces plant growth inhibition and oxidative damage. D1 protein in the PSII reaction center is the most sensitive target of PS II damage. H2S alleviated drought-induced PS II damage owing to a higher level of transcription of the gene PsbA and fast D1 protein turnover (Li et al. 2015). H2S increases plant height

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Table 10.1  Proteins regulated by the application of H2S in different plant species H2S chemical form 500 μM NaHS 0.5 mM NaHS

Species Triticum aestivum T. aestivum

Drought

2 mM NaHS

Eruca sativa

Dehydration

T. aestivum

CAT, POD, GR

0.1–0.5 mM NaHS 500 μM NaHS

SOD, CAT, GPO

0.05 mM NaHS

SOD, CAT, GPO

50 μM NaHS

APX, CSD2, PDX1, HSP, GS2, GS1:1, PGK, TPI, AsA-GSH SOD, γ-ECS, APX, GR, glyoxalase I and II, GPO

200 μM NaHS

Proteins SOD, CAT, POD W5A5Z6, W5A2Y8, W5BBW7, W5IAG4, W5F3S8, W5EDB0, W5H6J0, W5BQ07, C1K737, W5ENK5, P69448, W5HCI7, W5H2V3, W5E2N8, W5DYM3 OAS-TL, LCD, DCD, CA, SOD, POD, CAT SOD, CAT, POD

0.5 mM NaHS

Cynodon dactylon Malus hupehensis Arabidopsis thaliana Kandelia obovata Brassica oleracea

Stress type Drought

Reference Ma et al. (2016) Ding et al. (2018)

Khan et al. (2018) Drought Kolupaev et al. (2019a) Salinity, osmotic Shi et al. and cold (2013) Salinity Wei et al. (2019) Salinity Yastreb et al. (2020) Salinity Liu et al. (2020b) Alkalinity

P5CS, ProDH SOD, CAT, APX

NaHS Zea mays 0–1.5 mM NaHS T. aestivum

Heat Heat

SOD, CAT, GPO

100 μM NaHS

T. aestivum

Heat

Z. mays

Heat

T. aestivum

Chilling

APX, GR, GPO, SOD, 0.5 mM NaHS CAT, P5CS, OAT, BADH CAT, GPO 0.1 and 0.5 mM NaHS CAT, SOD, APX, GSH 0.02–1.0 mM NaHS SOD, CAT 50 μM NaHS

Z. mays

Hypoxia

A. thaliana

POD, CAT, APX, LOX

0.9 mM NaHS

T. aestivum

SOD, CAT, APX

200 μM NaHS

Hordeum vulgare

Cadmium toxicity Cadmium toxicity Cadmium toxicity

Montesinos-­ Pereira et al. (2018) Li et al. (2013) Yang et al. (2016) Kolupaev et al. (2017) Zhou et al. (2018) Kolupaev et al. (2019b) Peng et al. (2016) Jia et al. (2016) Huang et al. (2016) Fu et al. (2019) (continued)

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Table 10.1 (continued) Proteins V-ATPase, NADPH oxidase, L-CD, D-CD SOD, CAT, POD

H2S chemical form 100 μM NaHS 0.20 mM NaHS

SOD, CAT, POD, APX, GR

0.2 mM NaHS

NR

0.5 mM NaHS and salicylic acid 100, 200 μM NaHS 0.3–1.5 mM NaHS 2 μM NaHS

POD, APX, CAT, GR GPO, SOD, APX, CAT SOD, APX, CAT, POD APX, SOD, GPO, GR SOD, CAT SOD, CAT, POD SOD, CAT, POD, APX Nitrate reductase, nitrite reductase, glutamate synthase, glutamate oxaloacetate transaminase, glutamine synthetase, glutamate pyruvate transaminase SOD, CAT, POD, APX

Species Cucumis sativus T. aestivum Brassica oleracea var. botrytis Z. mays

Brassica napus L. T. aestivum Oryza sativa

0.4, 0.8, 1.2 mM T. aestivum NaHS O. sativa 100, 200 μM NaHS 0.2 mM NaHS Capsicum annuum 100 μM NaHS C. sativus

Stress type Cadmium toxicity Cadmium toxicity Lead toxicity

Reference Kabała et al. (2019) Kaya et al. (2020a) Chen et al. (2018)

Lead toxicity

Zanganeh et al. (2019)

Lead toxicity

Ali et al. (2014) Aluminum Zhang et al. toxicity (2010) Zhu et al. Aluminum toxicity (2018) Copper toxicity Dai et al. (2016) Mercury toxicity Chen et al. (2017) Zinc toxicity Kaya et al. (2018) Nitrate toxicity Qi et al. (2019) Nickel toxicity Rizwan et al. (2019)

100 μM NaHS

O. sativa

200 μM NaHS

B. oleracea var. botrytis B. oleracea L. var. Italica

Chromium toxicity Without stress (post-harvest)

Ahmad et al. (2020) Li et al. (2017b)

Vitis vinifera L. × V. labrusca L.

Without stress (post-harvest)

Ni et al. (2016)

0.8 mM NaHS LCD, DCD, ATPase, CCO, SDH, glucokinase, fructokinase, G6PDH, 6PGDH APX, CAT 1 mM NaHS

(continued)

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Table 10.1 (continued) Proteins RuBISCO, RuBISCO LSU 65 protein up-regulated, 27 protein down-regulated

H2S chemical form 100 μM NaHS 100 μM NaHS

Species Spinacia oleracea S. oleracea

Stress type Without stress (photosynthesis) Without stress

Reference Chen et al. (2011) Chen et al. (2014a)

SOD superoxide dismutase, CAT catalase, POD peroxidase, W5A5Z6 alpha-glucosidase, W5A2Y8 starch synthase isoform IV, W5BBW7 D-3-phosphoglycerate dehydrogenase, chloroplastic, W5IAG4 caffeoyl-CoA O-methyltransferase 2, W5F3S8 phosphatidylinositol-glycan biosynthesis class X protein, W5EDB0 PHD finger protein, W5H6J0 xyloglucan endotransglucosylase/hydrolase protein 22, W5BQ07 urease accessory protein G, C1K737 multiprotein bridging factor 1, W5ENK5 chlorophyll binding proteins, P69448 chlorophyll binding proteins, W5HCI7 chlorophyll degradation protein, W5H2V3 RuBISCO large subunit binding protein, W5E2N8 chloroplastic, W5DYM3 ATP-dependent Clp protease ATP-binding subunit, OAS-TL O-acetylserine (thiol) lyase, LCD L-cysteine desulfhydrase, DCD D-cysteine desulfhydrase, CA carbonic anhydrase, GR glutathione reductase, GPO guaiacol peroxidase, APX ascorbic acid peroxidase, CSD2 copper/zinc superoxide dismutase, PDX1 pancreatic and duodenal homeobox 1, HSP heat-shock protein, GS2 glutamine synthetase 2, GS1:1 glutamine synthetase 1, PGK phosphoglycerate kinase, TPI triosephosphate isomerase, AsA-GSH ascorbate–glutathione, γ-ECS gluatmylcysteine synthetase, P5CS Δ1-pyrroline-5-carboxylate synthetase, ProDH proline dehydrogenase, OAT ornithine aminotransferase, BADH betaine aldehyde dehydrogenase, GSH glutathione, LOX lipoxygenase, V-ATPase vacuolar H+-ATPase, NADPH oxidase nicotinamide adenine dinucleotide phosphate oxidase, L-CD L-cysteine desulfhydrase, D-CD D-cysteine desulfhydrase, NR nitrate reductase, CCO cytochrome C oxidase, SDH succinate dehydrogenase, G6PDH glucose-6-phosphate dehydrogenase, 6PGDH 6-phospho- gluconate dehydrogenase, RuBISCO ribulose-1,5-bisphosphate carboxylase, RuBISCO LSU ribulose-1,5-bisphosphate carboxylase large subunit, NaHS sodium hydrosulfide

and the leaf relative water content of seedlings under drought stress by incrementing of the expression levels of ABA biosynthesis and ABA reactivation genes in leaves, whereas the expression levels of ABA biosynthesis and ABA catabolism genes were up-regulated in roots (Ma et al. 2016). To elucidate the regulatory mechanisms of H2S on drought tolerance in wheat plants, Li et al. (2017c) analyzed a transcriptome. The authors found that 7552 transcripts exhibited differential relative expression. H2S alleviated drought damage, probably modifying the transport systems, plant hormone signal transduction, protein processing pathway, fatty acids, and amino acid metabolism. Mitogen-activated protein kinase (MAPK) is an important signaling molecule that links the growth and developmental signals and environmental stimuli to cellular responses. MPK4 is important downstream of H2S in the drought stress response and stomatal movement, and that the H2S-MPK4 cascade is involved in ABA-mediated stomatal movement to regulate the drought stress (Du et al. 2019). NOSH is a novel donor as it can donate NO and H2S simultaneously to plants. The effect of this molecule in drought-­ stressed Medicago sativa plants was the acclimation to the stress and the differential regulation of multiple defense-related transcripts, including antioxidant enzymes (Antoniou et al. 2020).

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Salinity is a major environmental stress that imposes both ionic toxicity and osmotic stress on plants, which leads to nutritional disorders, oxidative damage, and the resultant limitation of plant growth and crop yield. H2S rescues the NaCl-­ induced inhibition of seed germination and seedling growth due to the stimulation of antioxidant metabolism and the reestablishment of ion homeostasis. APX1, APX-2, Cu/Zn-SOD gene expression and enzyme activities were modified by H2S (Wang et al. 2012). H2S promoted the level of gene expression and the phosphorylation level of plasma membrane H+-ATPase (AHA3, AHA4); additionally, H2S changes the level of Na+/H+ antiporter protein (Li et al. 2014a). H2S had significant rescuing effects on 150 mM NaCl-induced inhibition of plant growth. H2S increased endogenous NO production and controlled the K+/Na+ balance by decreasing the net K+ efflux, increasing the transcription of HvAKT1 that codifies an inward-­rectifying potassium channel, and the transcription of HvHAK4 that encodes a high-affinity K+ uptake protein. H2S and NO promoted the transcription of PM H+-ATPase (HvHA1) and Na+/H+ antiporter (HvSOS1), responsible for maintaining a lower level of Na+ content in the cytoplasm. H2S and NO modulated Na+ entering into the vacuoles with up-regulation of the transcription of vacuolar Na+/H+ antiporter (NHE1), vacuolar Na+/H+ antiporter (HvVNHX2), and H+-ATPase subunit β (HvVHA-β) (Chen et al. 2015). H2S contributes to increasing plasma membrane H+-ATPase activity in short-term salt stress and low temperature treated plants by stimulating the expression of several genes encoding isoforms of the plasma membrane proton pump (CsHA2, CsH4, CsH8, CsH9, and CsHA10) (Janicka et al. 2018). When NaCl stresses plants, they can limit their growth due to osmotic imbalance and Na+ poisoning. However, the H2S application has been shown to maintain Na+/ K+ homeostasis in cucumber plants through transcriptional regulation of PM H+ATPase, SOS1, and SKOR expression (Jiang et al. 2019). H2S alleviates salt stress in wheat seedlings not only by strengthening antioxidant defense systems but by coordinating signal transduction pathways related to the stress response at a transcriptional level, up-regulating expression levels of genes related to the antioxidant system, SOS pathway, and MAPK pathway, as well as the transcription factor dehydration-­responsive element-binding gene (Ding et al. 2019). H2S and phospholipase Dα1 regulated the antioxidant enzyme system under osmotic stress in A. thaliana plants. The ROS level, electrolyte leakage, and MDA are decreased by NaHS application under osmotic stress, demonstrating that H2S maintains membrane integrity (Zhao et al. 2020). H2S up-regulates the expression level of MAPK, and both regulate the expression levels of the cold-responsive genes inducer of CBF expression 1 (ICE1), C-repeat-binding factors (CBF3), cold-responsive 15A (COR15A), and cold-­ responsive 15B (COR15B). Also, H2S regulates stomatal movement in response to cold stress (Du et  al. 2017). H2S is involved in chilling stress response in grape plants and modifies superoxide anion radical content, MDA content, the relative permeability of cell membrane and SOD activity, as well as expression of stress-­ responsive genes VvICE1 and VvCBF3; gene expression changes were in good agreement with the profiles of H2S accumulation and corresponding H2S synthetase

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activity (LCD) (Fu et al. 2013). Indole-acetic acid (IAA) acts as a downstream signaling molecule involved in H2S-induced chilling tolerance in cucumber seedlings. H2S enhances flavin monooxygenase (FMO) activity and the relative expression of FMO-like proteins (YUCCA2), which in turn elevated endogenous IAA levels. H2S-induced IAA production accompanied by an increase in chilling tolerance, as shown by the decrease in stress-induced electrolyte leakage and ROS accumulation and increase in gene expressions and enzyme activities of photosynthesis (Zhang et al. 2020). H2S treatment in seedlings under chilling stress increases the endogenous content of the GSH. mRNA levels and activities of the key photosynthetic enzymes (ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), transketolase (TK), sedoheptulose 1,7-biphosphatase (SBPase), and fructose-bisphosphate aldolase (FBAase), also, GSH-associated genes (GST Tau, MAAI, APX, GR, GS, and MDHAR) are significantly up-regulated (Liu et  al. 2020c). H2S upregulated abiotic and biotic stress-related genes and inhibited ROS accumulation. MIR393-mediated auxin signaling, including MIR393a/b and their target genes (TIR1, AFB1, AFB2, and AFB3) are transcriptionally regulated by H2S and link with the induction of antibacterial resistance by H2S (Shi et al. 2015). Higher tolerance to HMs like Cd, Zn, and excess of nitrate in plants with H2S application, is related to increased antioxidant gene expression (Cui et al. 2014; Liu et al. 2016a; Guo et al. 2018; Liang et al. 2018). H2S enhanced antioxidant enzyme activity leading to a decline in ROS accumulation and lipid peroxidation. H2S plays a protective role in cucumber seedlings under nitrate stress, and MAPK/NO signaling (CsNMAPK transcript level up-regulated) are involved in the process by regulating antioxidant enzyme activities (Qi et al. 2019). Cd stress induces the expression of H2S synthase-encoding genes LCD, DCD1, and DES1 (Zhang et al. 2015). H2S and Cys cycle plays a key role in plant responses to Cd stress. H2S promotes the expression of Cys synthesis-related genes SAT1 and OASA1, which leads to endogenous Cys accumulation. H2S weakened Cd toxicity by inducing the metallothioneins (MTs) gene expression (Jia et al. 2016). H2S and proline cooperate to alleviate Cd-damage in foxtail millet. H2S markedly exacerbate Cd-induced alterations in proline content, the activities of proline-5-carboxylate reductase (P5CR) and PDH, and the transcript levels of P5CR and PDH (Tian et al. 2016). Ca2+ and H2S induced by chromium stress activate the heavy-metal chelators synthesis-related genes, such as metallothioneins (MT3) and phytochelatins (PCS1) (Fang et al. 2014). H2S has been shown to regulate expression levels of the OASTLa, SAT1, and SAT5 genes related to Cys generation. H2S can regulate the expression of genes associated with Cys metabolism to modulate chromium stress-mediated Cys accumulation in Arabidopsis (Fang et al. 2016). H2S mitigates aluminum-induced root inhibition and the absorption of Al in roots. H2S elevates the secretion of citrate and reverse the negative effects of Al on gene expression of citrate transporter HvAACT1, cytosolic APX, HvAPX1, and protein level of plasma membrane H+-ATPase (Chen et al. 2013). SO2 donor pretreatment increase endogenous H2S accumulation and the antioxidant capability and decrease endogenous Al content in wheat grains to alleviate Al stress, also

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decreased the expression of Al-responsive genes TaWali1, TaWali2, TaWali3, TaWali5, TaWali6, and TaALMT1 in radicles exposed to Al stress (Zhu et al. 2015). Calcium (Ca2+) and H2S induce adaptation to nickel stress through enhancing the AsA-GSH cycle, redox homeostasis, and expression of PCs genes in seedlings (Valivand et al. 2019). H2S mitigates the inhibition in plant root elongation under boron (B) stress by inducing changes in the expression level of genes encoding pectin methylesterase (CsPME) and expansin (CsExp). Increases in PME and expansin activities may underlie the inhibition of root elongation by toxic B (Wang et al. 2010). Selenium (Se) toxicity causes the inhibition of endogenous H2S synthesis in root tips. Se (IV) stress reduces the expression of LCD and DCD homologs in the roots of B. rapa. The increase in endogenous H2S by applying NaHS could notably alleviate the induction of Se (IV)-induced ROS through inducing oxidative damage and subsequent cell death in root tips (Chen et al. 2014b). H2S delays the post-harvest senescence of broccoli, and higher levels of carotenoids, anthocyanin, and ascorbate were maintained with a concomitant decrease in MDA, H2O2, and the superoxide anion. H2S elevates activities of GPO, APX, CAT, and GR and diminishes lipoxygenase, polyphenol oxidase, phenylalanine ammonia-­lyase, and protease activities also, and H2S reduces the expression of the chlorophyll degradation related genes BoSGR, BoCLH2, BoPaO, BoRCCR, as well as cysteine protease BoCP1 and lipoxygenase gene BoLOX1 (Li et al. 2014b). In another post-­harvest example (Ge et al. 2017), H2S antagonized the impact of ethylene by negatively regulating the expression of banana ripening-related genes MaACS1, MaACS2, MaACO1, and macta pectate lyase (MaPL), resulting in a decrease in the rate of ripening and senescence of the fruit. H2S pretreatment could maintain a better appearance and nutritional quality than ethylene treatment alone and prolonged the storage period of post-harvest tomato fruits. H2S attenuates the expression of beta-amylase encoding gene BAM3, UDP-glycosyltransferase encoding genes, ethylene-­ responsive transcription factor ERF003 and DOF22 (Yao et al. 2020). H2S triggers the upregulation of target genes responsible for HO-1/CO-induced adventitious root formation, including CsDNAJ-1 and CsCDPK1/5. The heme oxygenase-­1/carbon monoxide (HO-1/CO) acts as a downstream signal system in the auxin-induced pathway leading to cucumber adventitious root formation (Lin et al. 2012). The D−/L-CDes-generating H2S is involved in the regulation of ethylene-­ induced stomatal closure in A. thaliana. LCD genes are expressed in guard cells, and the level of expression depends on the stimuli that promote the opening or closing of stomata (Hou et al. 2013b). H2S is involved in the ABA signaling network in stomatal guard cells. DES1 gene for LCD is required for ABA-dependent NO production and stomatal closure (Scuffi et al. 2014). H2S treatment causes the modulation of the HO-1 gene. H2S delays programmed cell death in GA-treated wheat aleurone cells modulating GSH homeostasis and HO-1 gene expression (Xie et al. 2014). Genes regulated by H2S and their functions are described below, using the results of 42 studies in which H2S were applied to the plants under abiotic stress (Table 10.2).

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Table 10.2  Genes differentially expressed by the application of H2S in different plant species

Genes DREB2, RD29A, CBF4 ABA receptors in the guard cells MIR167a, MIR167c, MIR167d, MIR393a, MIR396a, ARF8, TIR1, AFB2, AFB3, GRF1, GRF2, GRF3, CSD1, CSD2 PsbA

H2S chemical form 80 μM NaHS 80 μM NaHS 50 μM NaHS

0.4 mM NaHS 500 μM NaHS

Species Arabidopsis thaliana A. thaliana

Drought

A. thaliana

Drought

Triticum aestivum T. aestivum

Drought

TaZEP, TaNECD, TaAAO, TaSDR, Ta8’-OH1, Ta8’-OH2, TaGLU1 TaGLU4 TaRCAR, TaCHLH T. aestivum Traes_2BL_A0786494A, Traes_2DL_ 0.4 mM NaHS AFAFDC9F2, Traes_3AL_7199364DD, Traes_4BL_7C507ED43, Traes_4DS_ DBAA2CC451, Traes_5BS_ AB86BB5DE, Traes_6DS_F3077393C, TRAES3BF019300240CFD_g, TRAES3BF020200120CFD_g, TRAES3BF098900070CFD_g MPK4 5 μM NaHS A. thaliana NR, GST17, FeSOD, APX, PIP, Cu/Zn SOD APX1, APX-2, cu/Zn-SOD

AHA3, AHA4 HvAKT1, HvHAK4, HvHA1, HvSOS1, NHE1, HvVNHX2, HvVHA-β CsHA2, CsH4, CsH8, CsH9, CsHA10

PM HC-ATPase, SOS1, SKOR

TaGR, TaDHAR, TaGS, TaSOS1, TaSOS2, TaSOS3, TaMPK1, TaMPK4, TaDREB2 SOD, POD, CAT

100 μM NOSH or NOSH-A 100 μM NaHS 500 μM NaHS 50–100 μM NaHS 100 μM NaHS 5,10, 15, 20 mM NaHS 0.5 mM NaHS 150 μM NaHS

Stress type Drought

Drought

Reference Jin et al. (2011) Jin et al. (2013) Shen et al. (2013)

Li et al. (2015) Ma et al. (2016)

Drought

Li et al. (2017c)

Drought

Du et al. (2019) Antoniou et al. (2020) Wang et al. (2012) Li et al. (2014a) Chen et al. (2015) Janicka et al. (2018) Jiang et al. (2019)

Medicago sativa

Drought

M. sativa

Salinity

A. thaliana

Salinity

Hordeum vulgare Cucumis sativus

Salinity

C. sativus

Salinity and low temperature Salinity

T. aestivum

Salinity

Ding et al. (2019)

A. thaliana

Osmotic

Wu et al. (2018) (continued)

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Table 10.2 (continued)

Genes MPK4, ICE1, CBF3, COR15A, COR15B VvICE1, VvCBF3

H2S chemical form Species 5 μM NaHS A. thaliana

Stress type Cold

NaHS

Vitis vinifera

Chilling

FMO, YUCCA2, rbcS, rbcL, FBA, TK, ICE, CBF1, COR

1.0 mM NaHS

C. sativus

Chilling

Rubisco, TK, SBPase, FBA, RBOH, GST Tau, MAAI, APX, GR, GS, MDHAR MIR393, MIR393a/b, TIR1, AFB1, AFB2, AFB3 APX2, CAT1, CAT2, SOD, HSP70, TFT6, GSH1, LOXD, ACS6 SOD, POD, CAT, APX

1.0 mm NaHS

C. sativus

Chilling

NaHS

Abiotic and biotic stress Solanum Nitrate lycopersicum toxicity S. Nitrate lycopersicum toxicity

CsNMAPK LCD, DCD1, DES1

LCD, DES1, SAT1, OASA1, MTs, PCs P5CR, PDH Cu-SOD, Zn-SOD, APX1, GPO MT3, PCS1, LCD, DCD2, DES, TPC1, MRP5, ACA9, CaM, CBL, CDPK, CIPK1, CIPK2 OASTLa, SAT1, SAT5, PCS1, PCS2, MT2A HvAACT1, HvAPX1 TaWali1, TaWali2, TaWali3, TaWali5, TaWali6, TaALMT1 CDPK, PCs

CsPME, CsExp

100 μM NaHS 100 μM NaHS

Chromium toxicity

Shi et al. (2015) Guo et al. (2018) Liang et al. (2018) Qi et al. (2019) Zhang et al. (2015) Jia et al. (2016) Tian et al. (2016) Cui et al. (2014) Fang et al. (2014)

Chromium toxicity Aluminum toxicity Aluminum toxicity

Fang et al. (2016) Chen et al. (2013) Zhu et al. (2015)

Cucurbita pepo

Nickel toxicity

Valivand et al. (2019)

C. sativus

Boron toxicity

Wang et al. (2010)

A. thaliana

100 μM C. sativus NaHS 5 μM NaHS Brassica. rapa L. ssp. pekinensis 50 μM A. thaliana NaHS NaHS Setaria italica L. 100–500 μM M. sativa NaHS 50 μmol/L S. italica NaHS 50 μM NaHS 50–800 μM NaHS 1.2 mmol/L NaHSO3 and Na2SO3 100 μM NaHS and 200 μM hypotaurine 200 μM NaHS

Reference Du et al. (2017) Fu et al. (2013) Zhang et al. (2020) Liu et al. (2020c)

A. thaliana H. vulgare T. aestivum

Nitrate toxicity Cd toxicity

Cd toxicity Cd toxicity Cd toxicity

(continued)

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Table 10.2 (continued)

Genes MT, CAT2

LCD, DCD BoSGR, BoCLH2, BoPaO, BoRCCR, BoCP1, BoLOX1

MaACS1, MaACS2, MaACO1, MaPL

BAM3, ERFoo3, DOF22

CsDNAJ-1, CsCDPK1/5

LCD

DES1

HO-1

H2S chemical form 50, 100, 200, 400 μM NaHS 0–2.0 mM NaHS NaHS

Species Solanum nigrum L.

Stress type Reference Zinc toxicity Liu et al. (2016b)

Selenium toxicity Without Brassica oleracea var. stress (post-harvest italica senescence) 1 mM NaHS Musa spp. Without stress (postharvest) Without 0.90 mM S. NaHS lycopersicum stress (post(fruit) harvest) 0.1–100 μM C. sativus Without NaHS stress (root induction) H2S-­ A. thaliana Without stress producing (stomata mutants movement) H2S-­ A. thaliana Without stress producing (stomata mutants movement) 0.1–100 μM T. aestivum Without NaHS stress (programmed cell death) B. rapa

Chen et al. (2014b) Li et al. (2014b)

Ge et al. (2017)

Yao et al. (2020)

Lin et al. (2012) Hou et al. (2013b)

Scuffi et al. (2014) Xie et al. (2014)

10.4  Conclusion The manipulation or promotion of H2S metabolism in plants is a promising tool to induce tolerance to several types of abiotic stresses. In the ambit of proteomics and transcriptomics, the most studied mechanism by which H2S promotes stress tolerance is the activation and promotion of the antioxidant system. However, stress tolerance is also related to H2S biosynthesis, persulfidation, regulation of photosynthesis, carbon and nitrogen metabolism, signal transduction, protein synthesis, synthesis of secondary metabolites, osmolyte biosynthesis, stomatal opening, nutrient transport, osmotic balance, and programmed cell death. Also, the application of H2S in post-harvest can be beneficial because it produces a decrease in ripening and an improvement in the nutraceutical quality of fruits.

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Chapter 11

Cysteine and Hydrogen Sulfide: A Complementary Association for Plant Acclimation to Abiotic Stress M. Nasir Khan , Manzer H. Siddiqui , Mazen A. AlSolami, Riyadh A. Basahi, Zahid H. Siddiqui, and Saud Alamri

Abstract  Plants are always in a state of fighting against detrimental effects imposed by environmental stresses. Plants counter these adverse conditions through their defense system comprised of a well-orchestrated network of proteins, enzymes, hormones, metabolites and signaling molecules. Exposure of plants to these abiotic stresses usually lead to the induction of plants’ defense system through a network of signaling molecules. Hydrogen sulfide (H2S) is considered as an important signaling molecule and is involved in the protection of plants against various abiotic stresses such as drought, salinity, metal, chilling, cold, heat, UV radiations etc. Cysteine (Cys) serves as a precursor molecule for the biosynthesis of H2S by Cys desulfhydrases. However, plants synthesize Cys in a reaction catalyzed by O-acetylserine(thiol) lyase, which also synthesizes H2S from Cys in a reverse reaction. Cys not only serves as a precursor of H2S but also the primary organic compound containing reduced sulfur and acts as sulfur donor for biosynthesis of various biomolecules and defense compounds. Directly or indirectly, Cys alleviates abiotic stresses in plants through affecting the functioning of various cellular processes and molecules. These include antioxidant defense system, redox homeostasis, glutathione, phytochelatins, metallothioneins etc. The present chapter is focused on the role of Cys and its allied molecules and products in the mechanisms responsible for plant acclimation to environmental stresses. In the light of available information, biosynthesis of Cys and H2S and their mode of action during plant adaptive responses is also discussed. M. N. Khan (*) · M. A. AlSolami · R. A. Basahi Department of Biology, College of Haql, University of Tabuk, Tabuk, Saudi Arabia e-mail: [email protected] M. H. Siddiqui · S. Alamri Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia Z. H. Siddiqui Department of Biology, Faculty of Science, University of Tabuk, Tabuk, Saudi Arabia e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_11

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Keywords  Cysteine · Glutathione · Cysteine desulfhydrase · Cyanide · O-acetylserine(thiol)lyase · Phytochelatins · Redox homeostasis

11.1  Introduction Normal growth and development of a plant is determined by an array of factors ranging from environmental to nutritional and hormonal but there exists a set of signaling molecules which play vital role in growth, development, and survival of the plants. Among these, hydrogen sulfide (H2S) has been established as a significant signaling molecule after its detection in plants (Wilson et al. 1987; Sekiya et al. 1980; Rennenberg et al. 1990). Early studies on the impact of H2S in plant growth were carried out by McCallan et al. (1936), and Benedict and Breen (1955) who observed injurious effects of H2S on plants. Later, Rodriguez-Kabana et al. (1965) and Thompson and Kats (1978) observed that H2S caused significant stimulation in growth of the plants. In recent years, significant number of evidences showed that H2S is involved in various plant processes such as stomatal movement, flower senescence, seed germination, root morphogenesis, and photosynthesis (Carlos and Lorenzo 2010; Zhang et al. 2011; Li et al. 2012a; Lisjak et al. 2010). In addition, H2S plays vital role in the mitigation of adverse effects of various abiotic stresses such as drought, heat, salinity, heavy metals, dark, and cold (Li et al. 2013, 2012b, 2015; Christou et al. 2013, 2014; Jin et al. 2013; Khan et al. 2017a, b, 2018, 2020; Wei et al. 2017; Geng et al. 2019), and also functions as a potent antioxidant (Li et al. 2012c) and signaling molecule (Corpas et al. 2019; Corpas 2019; Calderwood and Kopriva 2014; Hancock and Whiteman 2014). In the last decade various studies have been carried out to explore, the role of H2S in plants as a signaling molecule and its interaction with other signaling molecules such as nitric oxide (NO), carbon monoxide (CO), and reactive oxygen species (ROS) (Khan et al. 2017a, b, 2020; Kabała et al. 2019; Geng et al. 2019; Kaya et al. 2019). However, it has yet to be established how H2S acts, the pathway(s) in which it might be involved and how and in what sequence H2S works in association with other signaling molecules (Hancock and Whiteman 2018; Corpas et al. 2019; Corpas 2019). Sulfate is regarded as the most abundant form of sulfur in nature that is absorbed by the plants. In plants, sulfate is reduced and is assimilated into Cys, the primary organic compound containing reduced sulfur (Takahashi et  al. 2011). Cysteine (Cys) serves as a reduced sulfur donor molecule in the biosynthesis of various biomolecules and defense compounds. In addition to its role as amino acid, Cys also functions as a precursor molecule for a variety of sulfur containing metabolites such as methionine, vitamins, cofactors, Fe–S clusters (Van Hoewyk et al. 2008; Romero et  al. 2014) and many other defense compounds (Rausch and Wachter 2005). In plants, Cys serves as a precursor molecule for the synthesis of antioxidant

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glutathione (GSH), the key regulator of cellular redox homeostasis (Noctor et al. 2012) which is synthesized in two ATP dependent reactions (Fig. 11.1) (Cruz et al. 2010). Cys plays pivotal role in plant growth (James et al. 2019; Khan et al. 2020), and detoxification of ROS generated during abiotic stresses (Takahashi et al. 2011; Noctor et  al. 2012; Cao et  al. 2014). However, high reactivity of Cys makes it a highly phytotoxic molecule when its concentration overpasses a certain threshold. The maintenance of Cys levels, at least 10 times lower than GSH, is essential for normal functioning of cellular system under nonstress conditions (Meyer et  al. 2001; Krueger et  al. 2009). Therefore, Cys homeostasis must be maintained via degradation of excess Cys to some non-toxic and usable products. One such degradation product is H2S which is synthesized by desulfurylation of Cys by Cys desulfhydrase (CDes). Therefore, Cys plays central role in the biosynthesis of H2S in plants and thus uninterrupted supply of Cys is vital for H2S-mediated cellular processes. The present chapter focusses the interdependence of H2S and Cys and their involvement in the regulation of plant defense system in response to various environmental stresses.

Fig. 11.1  Biosynthesis of cysteine and hydrogen sulfide and their involvement in the protection mechanisms of plants against abiotic stress-induced impairments. APR adenosine 5′-phosphosulfate (APS) reductase, ATPS ATP sulfurylase, GS glutathione synthetase, GSH reduced glutathione, GSSG oxidized glutathione, H2S hydrogen sulfide, HM heavy metal, LCD/DCD L/D–cysteine desulfhydrase, MT metallothionein, OAS O-acetylserine, OASTL O-acetylserine(thiol) lyase, PC phytochelatin, PCS phytochelatin synthase, ROS reactive oxygen species, SAT serine acetyltransferase, SiR sulfite reductase, β-CAS β-cyanoalanine synthase, γ-EC γ-glutamylcysteine, γ-ECS γ-utamylcysteine synthetase

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11.2  Homeostasis of Cys and H2S 11.2.1  Regulation of Cys Homeostasis Biosynthesis of Cys requires O-acetylserine (OAS) and sulfide as precursor molecules which are provided by carbon, nitrogen, and sulfur assimilation pathways (Koprivova and Kopriva 2014). Nitrogen component of Cys is produced through nitrate assimilation pathway. Nitrate reductase is the first enzyme of nitrate assimilation pathway which reduces nitrate to nitrite. The enzyme nitrite reductase reduces nitrite to ammonium which enters to the glutamate synthetase–glutamine oxoglutarate aminotransferase (GS–GOGAT) cycle to produce glutamate and glutamine (Krapp 2015). Assimilation of nitrogen and its incorporation into the carbon backbone results in the amino acid serine which on acetylation, by the enzyme serine acetyltransferase (SAT), produces OAS. The sulfur component of Cys is provided through sulfate assimilation pathway. Plant roots absorb sulfur in the form of sulfate which is transported to the shoot by sulfate transporters (Takahashi et al. 2012) and produces adenosine 5′-phosphosulfate (APS) by the enzyme ATP sulfurylase. There exist two pathways using APS, one leads to the generation of reduced sulfur compounds (sulfite and sulfide), the other results in the synthesis of sulfated compounds. The dominant pathway, that occurs in plastid, leads to reduced sulfur compounds. The APS is reduced by APS reductase (APR) to produce sulfite using glutathione (GSH) as electron donor, whereas reduction of sulfite by sulfite reductase (SiR) produces sulfide. The enzyme O-acetylserine(thiol)lyase (OASTL) facilitates incorporation of sulfide into OAS to produce Cys (Fig.  11.1) (Takahashi et  al. 2011; Rennenberg and Herschbach 2014), the primary form of reduced organic sulfur, which serves as a reservoir of sulfur for successive cellular metabolic reactions (Pivato et al. 2014). As mentioned in the preceding paragraph, SAT generates OAS from serine, while OASTL catalyzes the formation of Cys from OAS and sulfide. These two enzymes (SAT and OASTL) interact and form hetero-oligomeric Cys synthase complex that regulates Cys synthesis (Feldman-Salit et al. 2012). The SAT is the rate limiting enzyme of Cys biosynthesis (Wirtz and Hell 2006); the activity of SAT is exclusively observed in association with OASTL in Cys synthase complex, whereas the complex formation inactivates OASTL (Wirtz and Hell 2006). The Cys-synthase complex modifies the kinetic properties of SAT; as a result it synthesizes OAS more efficiently. On the contrary, OASTL in its free form synthesizes Cys more competently (Wirtz and Hell 2006). The regulation of Cys synthesis by Cys-synthase complex depends on the relative amounts of sulfide and OAS (Jez and Dey 2013). Sulfide stabilizes the complex to form OAS and Cys, but when OAS is in excess, the complex dissociates to reduce OAS production (Gotor et al. 2015). In Arabidopsis thaliana, five SAT (Howarth et al. 2003) and nine OASTL genes (Wirtz et al. 2004) have been identified. The SAT and OASTL isoforms have been reported to form specific complexes. In cytosol, SAT5 and OASTL A form Cys synthase complex, in chloroplasts the complex is constituted by SAT1 and OASTL

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B, and in mitochondria Cys synthase complex is formed by SAT3 and OASTL C, that results in OAS and Cys synthesis in all three compartments (Heeg et al. 2008; Watanabe et al. 2008a; Birke et al. 2013). In addition, SAT 2 and SAT 4 have also been reported in cytosol but with a different amino acid sequence and much lower expression level than the other SATs (Kawashima et al. 2005). Moreover, isoforms of SAT and OASTL demonstrate exchangeability of sulfide, OAS and Cys between the compartments (Heeg et al. 2008, Watanabe et al. 2008a, Lee et al. 2014). Studies using knockout mutants showed that OASTL C is an important regulator of SAT3 activity in mitochondria which chiefly supplies OAS, whereas OASTL A is the main contributor of Cys in cytosol (Wirtz et al. 2012; Birke et al. 2013), and chloroplast is the main source of sulfide (Birke et al. 2013). The enzyme OASTL belongs to the superfamily of β-substituting alanine synthases (Hatzfeld et  al. 2000; Watanabe et  al. 2008a, b). In A. thaliana, a large diversity of OASTLs has been reported and the true OASTLs and OASTL-like proteins are debated (Jost et  al. 2000; Álvarez et al. 2010; Bermudez et al. 2010). However, authentic OASTLs are defined by their property to interact with SAT that can be verified using different approaches (Bonner et  al. 2005; Heeg et  al. 2008). The OASTLs are considered authentic encoded by OAS-A1 (At4g14880), OAS-B (At2g43750), and OAS-C (At3g59760) and are located in the cytosol, plastids, and mitochondria of Arabidopsis cells, respectively (Wirtz et al. 2004). In addition, there exists another isoform of OASTL which is encoded by ATCYS-C1 (At3g61440), and functions as a β-cyanoalanine synthase (β-CAS) which catalyzes the formation of β-cyanoalanine from cyanide and Cys in mitochondria. On the other hand, CS-LIKE (At5g28030) proteins in the cytosol were observed to cleave Cys to form sulfide, ammonia and pyruvate and is recognized as a novel L-Cys desulfhydrase (DES1) (Hatzfeld et al. 2000; Álvarez et al. 2010). Furthermore, CS26 (At3g03630, Bsas5;1) functions as a S-sulfocysteine synthase accepting thiosulfate instead of sulfide as donor of reduced sulfur for incorporation into OAS (Bermudez et  al. 2010). On the basis of Genevestigator microarray database (www.genevestigator.com) and various studies it can be concluded that the three OASTL isoforms OAS-A1, OAS-B, and OAS-C and the β-cyanoalanine synthase isoform ATCYSC1 are the most highly expressed in Arabidopsis cells. The remaining OASTL-like proteins located in the cytosol [encoded by ATCYS-D1 (At3g04940), ATCYS-D2 (At5g28020), and CS-LIKE (At5g28030)] and in the plastid [encoded by CS26 (At3g03630)] are expressed at much lower levels and have not been characterized enzymatically (Álvarez et al. 2010). It is well documented that H2S and Cys are crucial for the survival of plants under adverse environmental conditions but their increased concentration beyond certain level can be phytotoxic and lower level of H2S and Cys can also lead to weak protection of plans against various stresses. Therefore, maintenance of H2S and Cys homeostasis is equally important for adaptation of plants under adverse environmental conditions. Cytosolic Cys homeostasis is precisely regulated by the action of various Cys-degrading enzymes in different plant species (Papenbrock et al. 2007). As explained above, de novo synthesis of Cys is chiefly carried out in the cytosol by the action of OAS-A1, whereas degradation of Cys also takes place in the cytosol by

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the action of L-Cys desulfhydrases (DESs; EC 4.4.1.1), also designated as DES1, which was previously known as CS-LIKE (At5g28030) (Álvarez et al. 2010). The enzyme DES1 catalyzes the formation of H2S, ammonia and pyruvate from Cys in a stoichiometric ratio of 1:1:1. The role of DES1 was further confirmed by knockout des 1 mutant of Arabidopsis that showed increased Cys levels (Álvarez et al. 2010). However, in the leaves of des 1 mutant of Arabidopsis, endogenous H2S concentration was only 30% less than the quantified amount in the wild type (Álvarez et al. 2012). On the contrary, oas-a1 knockout mutants that are deficient in the most abundant form of cytosolic OASTL show decreased Cys levels (Lopez-Martin et  al. 2008a; Álvarez et al. 2010). Therefore, contrasting activities of DES1 and OAS-A1 maintain cytosolic Cys homeostasis (López-Martín et al. 2008b; Gotor et al. 2010; Álvarez et al. 2010). There are two additional important enzymes in Cys metabolism, CASC1, a β-CAS and SCS, a S-sulfocysteine synthase. The enzyme CASC1 catalyzes the conversion of Cys and cyanide to H2S and β-cyanoalanine (Hatzfeld et al. 2000; Yamaguchi et al. 2000), and SCS catalyzes the incorporation of thiosulfate (instead of sulfide like an authentic OASTL) to OAS to form S-sulfocysteine (Gotor and Romero 2013).

11.2.2  Regulation of H2S Homeostasis In plants, synthesis of H2S takes place through enzymatic pathways which include CDes [(L-cysteine desulfhydrase, LCD; EC 4.4.1.1 and D-cysteine desulfhydrase, DCD; EC 4.4.1.15)], SiR, β-CAS, and cysteine synthase (CS) (Rausch and Wachter 2005). In plants, LCD degrades L-Cys to produce H2S, ammonia, and pyruvate in the cytosol, nucleus, and mitochondria, whereas DCD degrades D-Cys to generate H2S in mitochondria. In chloroplast, SiR reduces sulfite to produce H2S using ferredoxin as electron donor. Cytoplasm and mitochondria of plant cells also use β-CAS to convert cyanide and L-Cys into cyanuric acid and H2S. The enzyme CS, also known as OASTL, synthesizes H2S from Cys in the cytosol, chloroplast, and mitochondria. This reversible reaction produces Cys from H2S and OAS and thus plays significant role in maintaining H2S homeostasis. In recent years, it has been established that L-Cys desulfhydrase 1 (DES1) is the only enzyme involved in the generation of H2S from Cys in plant cytosol (Romero et al. 2013; Aroca et al. 2017a). DES1 catalyzes the desulfuration of Cys to H2S, ammonia, and pyruvate (Gotor et al. 2010; Alvarez et al. 2012) and this H2S plays vital role in signaling (Romero et al. 2013; Gotor et al. 2015). Chloroplast is the main source of sulfide produced from sulfite reduction through sulfate assimilation pathway (Takahashi et al. 2011). Sulfide is believed to runoff into the cytosol from other cell organelles, however, sulfide in these organelles is present in ionized form (H+ and SH−) which cannot be transported across the membrane into the cytosol (Kabil and Banerjee 2010). Romero et  al. (2013) concluded that cytosolic DES1 induces production of H2S that is utilized by the plants for signaling purpose.

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11.3  I nvolvement of H2S and Cys in Plant Adaptive Responses to Abiotic Stresses Perception of stress stimulus at cellular level leads to a downstream signaling cascade. This signaling cascade is executed by a network of chemical compounds and signaling molecules which induce the defense system of the plants at cellular, sub-­ cellular and molecular levels to respond against abiotic stress-induced impairments. Among these, H2S has been reported to play significant role in the protection of plants against various environmental stresses through activating defense system of plants and/or by acting as a signaling molecule against abiotic stresses such as salinity (Wang et al. 2012), high temperature (Li et al. 2013), osmotic (Khan et al. 2017a, b), drought (Jin et al. 2018; Khan et al. 2018), cold (Geng et al. 2019), and heavy metals (Khan et al. 2020). A significant number of studies showed that exposure of plants to abiotic stresses triggers endogenous accumulation of H2S (Ma et  al. 2015a, b; Chen et  al. 2011; Khan et al. 2017a, b, 2018, 2020). Lai et al. (2014), reported that increasing concentration of NaCl gradually caused the induction of total LCD activity and the increase of endogenous H2S production. Increased activities of LCD, DCD, and OASTL enzymes coupled with improved endogenous levels of H2S were also reported under osmotic (Khan et  al. 2017a, b) and drought stresses (Khan et  al. 2018). Similar increase in H2S synthesizing enzymes was also reported under low temperature (Fu et al. 2013; Du et al. 2017), high temperature Chen et al. (2016a), altitude gradient (Ma et al. 2013), UV-B radiation (Li et al. 2016), and hypoxia (Cheng et al. 2013). Elevation of H2S levels was reported to be strongly associated with the abiotic stress-induced expression of H2S-synthesising enzymes-encoding genes (Jin et al. 2011; Fang et al. 2014; Zhang et al. 2015; Jia et al. 2016). Moreover, it was reported that the DES1 defective mutant (des1) was more sensitive to drought and displayed accelerated leaf senescence, while the leaves of over-expression mutant (OE-DES1) showed increased H2S production rate, H2S content and contained adequate chlorophyll levels, accompanied by improved drought resistance (Jin et al. 2018). Effect of exogenous application of H2S donors on the plants under different abiotic stresses has also been investigated and it was observed that H2S signaling can also be triggered by exogenously applied H2S. Garcia-Mata and Lamattina (2010), reported that treatment with the H2S donors sodium hydrosulfide (NaHS) or GYY 4137 increased relative water content and protected the plants against drought stress. Jin et al. (2011), reported that treatment with NaHS stimulates the expression of drought associated genes and displayed a higher survival rate and significant reduction in the size of the stomatal aperture that improved drought resistance in Arabidopsis. Li et al. (2012b) observed that application of NaHS to tobacco suspension cultured cells improved heat tolerance and alleviated decrease in the vitality of cells, and increase in electrolyte leakage and accumulation of malondialdehyde content, a product of lipid peroxidation. Under salinity and non-ionic osmotic stress, leaves of strawberry seedlings, pre-treated with NaHS exhibited enhanced H2S synthesis (Christou et  al. 2013). Cheng et  al. (2013), observed that root tip death

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induced by hypoxia was strongly enhanced by inhibition of the key enzymes responsible for endogenous H2S, whereas, exogenous H2S pretreatment significantly alleviated hypoxia-induced root tip death in pea seedlings and, therefore, enhanced the tolerance of the plant to hypoxic stress. Strawberry plants, pre-treated with NaHS, exhibited a significant increase in H2S content after 1  h exposure to heat stress (Christou et al. 2014). Transgenic A. thaliana overexpressing CDes and NaHS pre-­ treated plants exhibited higher endogenous H2S level and improved abiotic stress tolerance and biotic stress resistance, while CDes knockdown mutants and hypotaurine (H2S scavenger) pre-treated plants showed lower endogenous H2S level and decreased stress resistance (Shi et al. 2014). Enhanced accumulation of H2S under abiotic stress has been found to be connected with the enhanced expression of Cys-­ synthesizing genes and Cys content (Jia et al. 2016; Khan et al. 2018, 2020). Khan et al. (2018), in Eruca sativa, reported that exogenous application of NaHS induced the synthesis of Cys and H2S by enhancing the activity of OASTL, LCD, and DCD enzymes and improved the activities of antioxidant enzymes and accumulation of osmolytes that resulted in the alleviation of dehydration stress. Exogenous application of NaHS also enhanced endogenous H2S content that can increase cold stress resistance through MPK4  in Arabidopsis, resulting in a better adaptability to the environment (Du et al. 2017).

11.4  Mode of Action of H2S and Cys Under Abiotic Stresses On the basis of available information, as discussed in the preceding paragraphs, it is evident that abiotic stress and exogenous H2S activate H2S synthesizing enzymes that induce endogenous synthesis of H2S which in turn protects the plants against abiotic stress induced impairments. In plants, Cys acts as a substrate for H2S synthesis and an H2S-Cys cycle plays vital role in plant responses to environmental stresses. In the forthcoming paragraphs an attempt is made to shed light on the operational mechanism of H2S and Cys in the protection of plants against various abiotic stresses.

11.4.1  M  ode of Action of H2S in Abiotic Stress Tolerance of Plants Role of H2S in the alleviation of various abiotic stresses have already been discussed. However, it is quite imperative to explain here the mode of action by which H2S provides protection to the plants. To respond to abiotic stresses plants are fitted with various defense systems such as enzymatic and non-enzymatic antioxidant defense system which counter the generation of and scavenge ROS. Plants use cellular redox homeostasis for maintaining interne milieu of the cell under stress

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conditions. Plants counter osmotic stress and maintain normal hydration level of cells under stress conditions by synthesizing organic osmolytes such as trehalose, glycerol, inositols, sorbitols, proline, glycine betaine, taurine etc. Furthermore, under hypoxic conditions plants alter their metabolism and switch over from carbohydrate metabolism to fermentation (Banti et al. 2013). Plants counter metal stress by synthesizing metal-chelates, organic acids and polyphosphates that cause restriction and sequestration of toxic metals either in apoplasm or symplasm (Khan et al. 2020). However, protection of plants against any stress requires timely and precise activation of theses defense systems before the onset of stress-induced damage. This activation of defense system is facilitated by a network of signaling molecules which transmit the stress stimulus to the defense machinery which gets activated and provides protection to the plants from the stress. Role of H2S as signaling molecule has been well established and H2S is considered as third gasotransmitter after nitric oxide (NO) and carbon monoxide. However, for H2S to be considered as signaling molecule it needs a system for its generation at the right time and right place that would require activation of enzyme (s) which is able to make a signal. And also, there should be a system which could remove the molecule after fulfilling its signaling function, so that further signaling could be blocked (Hancock 2010; Hancock and Whiteman 2014). The H2S qualifies these criteria as it is synthesized by desulfhydrases in plants (Álvarez et  al. 2010) and there also exists a dedicated system of removal of H2S by the enzyme OASTL.  Moreover, in a signal transduction pathway, soon after its generation a signaling molecule needs to get the message moved to the next component. Once the compound has arrived at the place of its action it would need to be recognized for what it is, and therefore be able to relay a unique message, and to elicit a unique response. During the course of this signaling cascade, H2S interacts with several other signaling molecules such as hydrogen peroxide (H2O2), NO, reactive nitrogen species (RNS), abscisic acid (ABA), ethylene etc. that assist H2S in moving to its destination (Hancok and Whiteman 2016). Therefore, it indicates that H2S does not work alone but as part of a team comprised of various other molecules. 11.4.1.1  Interaction of H2S with Other Signaling Molecules Interaction of H2S and NO has been studied by various laboratories (Khan et  al. 2017a, b, 2020; Corpas et al. 2019). Khan et al. (2017a, b) observed that NO and H2S together markedly improve the activities of antioxidant enzymes and caused additional accumulation of osmolytes that collectively resulted in the protection of plants against osmotic stress-induced oxidative damage (Fig. 11.1). H2S also promoted stomatal closure (Zhang et  al. 2010) by inducing NO-mediated 8-nitro-­ cGMP/8-mercapto-cGMP synthesis, which triggers stomatal closure (Honda et al. 2015), stomatal opening (Lisjak et al. 2010, 2011) and upregulates the transcript levels of genes involved in the ascorbic acid  – glutathione (AsA-GSH) cycle (Christou et al. 2013). Various researchers concluded that H2S acts downstream of NO in the signaling cascade. For example, in maize, exogenous NO induced heat

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tolerance via a mechanism that appears to be mediated by an increase in L-DES activity, which is involved in H2S production (Li and Lancaster 2013). Khan et al. (2017a, b), reported that protection of wheat seedlings from osmotic stress was mediated by NO induced activation of the enzymes involved in H2S biosynthesis. In another study on Vigna radiata, Khan et al. (2020) reported that H2S acts downstream of NO but the interaction of the two is mediated by calcium under cadmium stress. In addition, H2S also interacts with ABA in response to stress stimulus (Ma et al. 2016). Exogenous H2S is found to induce stomatal closure, while scavenging of H2S partially blocks ABA-dependent stomatal closure, indicating the protective role of H2S in plants against drought stress (Garcia-Mata and Lamattina 2010). Moreover, H2S-induced stomatal closure can be reversed by cPTIO (an NO-specific scavenger), also confirming that the function of H2S in stomatal closure is mediated by NO. A study carried out by Scuffi et al. (2014), indicates that ABA fails to induce stomatal closure in isolated epidermal strips of des1 mutants, demonstrating that DES1 was required for ABA-dependent stomatal closure. It is not only NO and ABA, but H2S also facilitates the maintenance of cellular redox homeostasis (Lai et al. 2014; Da-Silva et al. (2017). Da-Silva et al. (2017) observed that salt stress induced the biosynthesis of H2S with a concomitant increase in the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX)] and GSSG content, whereas a reduction in GSH content and stomatal conductance was observed. It has been shown that H2S induced antioxidant enzyme activities and protected plants from drought stress (Ma et al. 2016), osmotic stress and dehydration stress (Khan et al. 2017a, b, 2018). Cheng et  al. (2013), reported that H2S pretreatment alleviated hypoxia-induced root tip death from ROS damage by inhibiting ethylene production. Khan et  al. (2018) reported that exogenous application of H2S protected Eruca sativa plants against dehydration stress by enhancing the Cys content, activities of carbonic anhydrase and antioxidant enzymes, and by accumulation of osmolytes (proline and glycine betaine). Treatment of tomato plants with NO and H2S donors has been shown to alleviate oxidative damage caused by exposure to NaCl (Da-Silva et al. 2018). Hydrogen sulfide not only controls but also interacts with ROS/RNS. Presence of high cellular NO content allows its interaction with superoxide (O2·–) to generate peroxynitrite rather than dismutating to H2O2 (Reiter 2006). Peroxynitrite can react with H2S (Carballal et al. 2011) and new signals are generated. Similarly, reaction of H2S with NO generates nitrosothiols (Whiteman et al. 2006). Effect of H2S has also been observed on the enzymes involved in the synthesis of ROS and NO. Li et  al. (2014), in Arabidopsis roots, reported that H2S increased ROS generation through the alteration of activities of NADPH oxidase and glucose-6-phosphate dehydrogenase (G6PDH). H2S-mediated generation of H2O2 and NO is associated with H2S-induced induction of respiratory burst oxidase homologue (Rboh) types D/F and nitrate reductase activity (Scuffi et al. 2014, 2018), respectively. Therefore, it is clear that H2S has the capacity to modulate the generation of ROS and NO in the cells.

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11.4.1.2  H2S and Persulfidation Interaction of H2S with other signaling molecules is described above, but the molecular mechanism through which H2S exerts its action is not yet fully understood. However, the primary signaling mechanism of H2S occurs through post-­translational modification of Cys residues on target proteins through a process known as persulfidation (Mustafa et al. 2009; Aroca et al. 2015). In this process, reactive Cys residues on target proteins are modified via conversion of the thiol group (-SH) into a persulfide group (-SSH). Persulfidation is believed to play crucial role in the protective mechanisms against ROS and RNS. Recently, Aroca et al. (2017a) reported the presence of persulfidation-modified Cys residues in 106 proteins from Arabidopsis leaf extracts. Proteins modified by persulfidation show functional changes in enzyme activities and therefore, modified Cys is capable of interacting with several proteins and exhibit greater reactivity due to the increased nucleophilicity of persulfide compared with the thiol group (Paul and Snyder 2012; Aroca et  al. 2015, 2017b). Recently, several low molecular weight persulfides have been identified such as, Cys-persulfide (CysSSH), GSH persulfide (GSSH) and their persulfurated species Cys-SSnH and GSSnH that have been recognized as effective redox regulators (Kasamatsu et al. 2016; Kimura et al. 2017). Aroca et al. (2015), observed the effect of sulfhydration (persulfidation) on the activity of enzymes. They reported that NaHS significantly decreased the activity of glutamine synthetase (GS), whereas activity of ascorbate peroxidase (APX), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were enhanced as a result of persulfidation. Recently, the enzyme cysteinyl-tRNA synthetases (CARSs), using L-cysteine as substrate involved in persulfidation, has been identified in prokaryotic and mammalian system. The Cys polysulfides bound to tRNA are incorporated into polypeptides that are synthesized de novo in the ribosomes, suggesting that these enzymes are the principal Cys persulfide synthases in vivo (Akaike et al. 2017). Under oxidative stress, persulfidated proteins react with ROS/RNS and form an adduct (RSSO3H) that may be restored by thioredoxin to free thiol (Filipovic and Jovanović 2017). The APX1 is inactivated by the oxidation of Cys32, while persulfidation by H2S increases the activity of the enzyme (Aroca et  al. 2015). During oxidative stress, an increased persulfidation has been reported and speculated that persulfidation is the protective mechanism against oxidative damage of proteins (Aroca et al. 2018). Moreover, several proteins associated with the ABA-dependent regulation of stomatal movement are modified by persulfidation and S-nitrosylation (Aroca et al. 2017a). Therefore, it can be concluded that a crosstalk exists between NO and H2S mediated by S-nitrosylation and persulfidation, respectively. Based on proteomic approaches, persulfidation also modifies several transcription factors and chromatin modifiers such as histones, acetyltransferases, and methyltransferases (Sen et  al. 2012; Aroca et  al. 2017a; Yang 2015). This modification affects their specificity to DNA and their binding affinity, resulting in distinct transcriptional responses. Therefore, H2S also exhibits its signaling role, through persulfidation, in epigenetic regulation of chromatin by histone modification, and chromatin structure alteration.

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11.4.2  M  ode of Action of Cys in Abiotic Stress Tolerance of Plants In plants, Cys has been reported to play significant role in enhancing tolerance of plants to abiotic stresses (Takahashi et  al. 2011; Lopez-Martin et  al. 2008b; Dominguez-Solis et al. 2004). These studies also get consistence from other works which observed an increase in free Cys concentration in response to various abiotic stresses (Ruiz and Blumwald 2020; Khan et al. 2018, 2020). However, a concomitant increase in GSH was also recorded together with Cys which indicates that there is a need of Cys for the biosynthesis of sulfur-rich compounds having anti stress activity (Zagorchev et al. 2012). Although, Cys serves as a potent chelator of heavy metals ions, but Cys-metal ion complexes induce the Fenton reaction leading to the production of hydroxyl radical (˙OH), and free Cys may also get oxidized to various by-products (Bashir et al. 2012). Therefore, accumulation of free Cys may lead to the loss of sulfur, which adversely affects the anti-stress mechanisms of the plant. Hence, synthesis of Cys should lead to its conversion or modification to such compounds which could assist the plants in combating stresses. Such a need is fulfilled when Cys acts as a sulfide donor and plays decisive role in the synthesis of various sulfur containing compounds such as glutathione (GSH), pytochelatins (PCs), metallothioneins (MTs) etc. which assist in enhancing the stress tolerance of plants (Takahashi et al. 2011; Dominguez-Solis et al. 2004). 11.4.2.1  Cys and Glutathione in the Cellular Redox Homeostasis Glutathione (GSH) is involved in the protection of plants against oxidative stress (Grill et al. 2001; Tausz et al. 2003), as it functions as a reductant in the enzymatic detoxification of ROS in the AsA-GSH cycle. Cys acts as a precursor for the synthesis of GSH, a low-molecular weight thiol (Fig. 11.1), which serves as a pool for reduced sulfur. GSH is a tripeptide of γ-glutamate (γ-Glu), Cys and glycine (Gly) which exists in reduced (GSH) and oxidized disulfide form (GSSG). The potential of glutathione as a protectant is associated with the pool size of GSH, its redox state (GSH/GSSG) and the activity of glutathione reductase (GR). Abiotic and biotic stress tolerance in plants is chiefly governed by GSH.  Several stress conditions induce biosynthesis of GSH, and its accumulation compensates for decrease in the capability of other antioxidants. For instance, oxidative stress induced catalase inhibition (May and Leaver 1993) and catalase deficiency (Willekens et al. 1997; Queval et al. 2009) induces GSH synthesis. In addition, the genes encoding the enzymes involved in GSH biosynthesis are also affected by various stresses (Schäfer et al. 1997, 1998; Xiang and Oliver 1998). Biosynthesis of GSH takes place in a two-step ATP-dependent reaction (Fig.  11.1). In the first step, γ-glutamylcysteine (γ-EC) is synthesized from L-glutamate and L-Cys by the enzyme γ-glutamylcysteine synthetase (γ-ECS; encoded by GSH1 gene) (Hell and Bergmann 1990), while the second step yields

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GSH by adding glycine to the C-terminal end of γ-EC by the enzyme glutathione synthetase (GS; encoded by GSH2 gene) (Meister 1988). Similar to any metabolic product, the rate of GSH synthesis is also determined by the supply of the substrates and/or modulation of the enzymes activities involved in the pathway. Concentration of Cys actively controls the rate of GSH synthesis as confirmed by the exogenous supply of Cys which resulted in increased GSH content (Noctor et al. 1996, 1997; Zechmann et al. 2008). In addition, overexpression of γ-ECS, the first enzyme of the GSH synthesis pathway, results in enhanced γ-EC and GSH content without depletion of Cys pool (Arisi et al. 1997). Although, no clear mechanisms are available behind the reason of increasing γ-ECS activity and/or Cys concentration that trigger accumulation of GSH, few studies reveal that induction of GSH1, encoding γ-ECS, and/or GSH2, encoding GS, occurred in response to abiotic and biotic stresses (Xiang and Oliver 1998; Parisy et al. 2007). Concentration of Cys actively influences the rate of GSH synthesis. It has been observed that exogenously applied Cys enhances tissue GSH content (Strohm et  al. 1995; Noctor et  al. 1996, 1997). Moreover, synthesis of GSH is controlled by the synthesis of Cys and in turn Cys synthesis may be customized to its demand for the synthesis of GSH (Noctor et al. 2012). This control is chiefly regulated by the activity of γ-ECS that might act mainly to modify the rate of GSH production to the defensive needs of the plant (Schneider and Bergmann 1995). It has been observed that overexpression of γ-ECS results in enhanced level of γ-EC and GSH without compromising the Cys pool which exhibits that Cys can be made available to support ongoing γ-EC and GSH synthesis (Noctor et al. 1996, 1998; Arisi et al. 1997). As mentioned in the preceding pages, APR may limit sulphate reduction and SAT may limit the biosynthesis of OAS, the precursor of Cys. Overexpression of γ-ECS enhances in vitro activities of APR and SAT that may lead to improved Cys supply for GSH biosynthesis (Noctor et al. 2012). Therefore, it is evident that Cys is the key player in maintaining GSH pool for the normal functioning of AsA-GSH cycle in maintaining redox homeostasis. In addition, higher level of GSH is also maintained by overexpression of GR in the chloroplast (Foyer et al. 1995). Furthermore, oxidized form GSSG is more readily degraded than the GSH (Foyer et al. 1995), thus it strongly implicates chloroplastic GR activity as a factor that influences GSH levels through its regeneration from GSSG (Noctor et al. 2012). Several plant species contain alternate GSH analogs, in which glycine is replaced by β-alanine, serine or glutamate (Klapheck et  al. 1995; Matamoros et  al. 1999; Moran et  al. 2000; Galant et  al. 2011). Among these GSH analogs, homo-GSH (h-GSH; γ-Glu-Cys-β-Ala) has been detected along with GSH in many legumes (Moran et  al. 2000). Hydroxymethyl–GSH (γ-Glu-Cys-Ser) has been reported in grasses (Klapheck et  al. 1994). Moreover, GSH-like peptides having glutamate (Glu) instead of Gly (γ-Glu-Cys-Glu) have been reported in maize under cadmium stress (Meuwly et al. 1995). Of these, h-GSH is well known GSH analog which has been reported in fourteen different legumes (reviewed by Galant et  al. 2011). However, GSH analogs were not reported in all assayed tissue types of broad bean and lupine (Matamoros et al. 1999), while cowpea and pea have h-GSH in roots and

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nodules, but not in leaves (Moran et al. 2000). The concentration of GSH and h-GSH also vary in different parts of the same plant species, for instance soybean leaves contain 50–200-fold more h-GSH, whereas seeds contain 130-times more h-GSH than GSH (Matamoros et al. 1999). The concentration varied in roots and root nodules of soybean which were reported to have 80-fold and ∼4-fold more h-GSH than the GSH, respectively (Matamoros et al. 1999). Although, the exact role of h-GSH is vague, but possibly h-GSH replaces GSH and plays important role as a dominant cellular redox-buffer during nitrogen fixation and is required for proper development of nodules (Frendo et al. 2005). It is evident that Cys is the key player in maintaining GSH pool for the normal functioning of AsA-GSH cycle in redox homeostasis and thus maintains interne milieu of the cell under stress conditions (Foyer and Noctor 2011). GSH plays important role in scavenging ROS, the by-products of aerobic metabolism, via AsA-­ GSH cycle keeping ROS level under control (Noctor and Foyer 1998; Khan et al. 2017a, b, 2020). The AsA-GSH cycle is operated by four enzymes i.e., APX, GR, monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) and involves two metabolites (AsA and GSH). The cycle starts with the scavenging of H2O2 by the APX using AsA as electron donor and terminates with the regeneration of GSH from GSSG with the help of enzyme GR using NADPH.  Therefore, maintenance of AsA and GSH pools is vital for the normal functioning of AsA-GSH cycle. In this cycle, AsA is oxidized to the univalent monodehydroascorbate (MDHA) which gets reduced rapidly to regenerate AsA by reduced ferredoxin. In addition, MDHAR rapidly reduces MDHA to AsA using NADPH.  When MDHA is not reduced it gets rapidly disproportionated to divalently oxidized dehydroascorbate (DHA) which gets reduced to AsA by DHAR (Foyer and Halliwell 1976). AsA and GSH scavenge ROS either directly or indirectly by serving as electron donors (Noctor et al. 2012). Under nonstress conditions leaves typically maintain 20:1 ratio of GSH:GSSG (Mhamdi et al. 2010), this ratio varies greatly across the tissues with higher value in the cytosol and lower in the vacuole (Meyer et  al. 2007; Queval et  al. 2011). However, excessive accumulation of ROS leads to an increased load on AsA-GSH cycle that may quickly shift GSH/GSSG towards a slightly more oxidized value. However, the acclimation reaction induces ROS scavenging capacity of the AsA-­ GSH cycle along with the induction of the antioxidant levels and activities of associated enzymes than the non-stressed values leading to restored GSH/GSSG redox balance. On the other hand, under weak or slow acclimatory responses oxidative stress eventually depletes the antioxidative system and causes an imbalance between oxidative load and scavenging that leads to the degradation (Tausz et  al. 2004). Leipner et al. (1999) observed that chilling tolerance of maize cultivars was probably correlated with increasing levels of the GSH and GR activity under stress conditions. Later, Kocsy et al. (2001) reported that the substances such as herbicides, that increase GSH biosynthesis, lead to increased chilling tolerance, while the inhibition of GSH biosynthesis reduced chilling tolerance (Kocsy et al. 2000). Higher concentration of GSH in chilled maize plants were the result of induction of key enzymes of GSH synthesis, as well as sulphate reduction, which also increases Cys levels

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(Kopriva et al. 2001). GSH is also involved in the detoxification of heavy metals and acts as a precursor of phytochelatins, which form a complex with metals that is then transported into the vacuole and further metabolized (Cobbett and Goldsbrough 2002; Rea et al. 2004; Noctor et al. 2012; Labrou et al. 2015). GSH is also a substrate for glutathione S-transferases, which catalyzes the conjugation of GSH with xenobiotics such as herbicides (Marrs 1996). 11.4.2.2  Cys and Phytochelatins Phytochelatins (PCs), are Cys rich peptides that play significant role in the protection of plants from metal toxicity through metal chelation (Fig. 11.1). The structure of PCs is symbolized as γ-glutamate (Glu)-cysteine (Cys) dipeptide followed by a glycine (Gly) [(γ-Glu-Cys)n-Gly]. Presence of PCs have been reported in different groups of plants including gymnosperms, angiosperms, and bryophytes (Wόjcik and Tukiendorf 2004; Wόjcik et al. 2005). However, in some plants, the C-terminal Gly can be replaced by serine as (γ-Glu-Cys)n-Ser, glutamine as (γ-Glu-Cys)n-Gln, glutamate as (γ-Glu-Cys)n-Glu and alanine as (γ-Glu-Cys)n-β-Ala (Kanaujia 2017). The enzyme phytochelatin synthase (PCS) uses GSH as substrate to synthesize PCs in the cytosol that can bind heavy metals and thus reduces metal toxicity in the plant cell (Cobbett and Goldsbrough 2002; Clemens 2006) through sequestering PC-metal complexes in the vacuole (Cobbett and Goldsbrough 2002). Presence of metals such as nickel (Ni), copper (Cu), zinc (Zn), silver (Ag), mercury (Hg), lead (Pb) and arsenic (As) have been shown to activate PCS-encoding gene and thus cadmium (Cd) is one of the most effective activators for PCs (Ha et al. 1999; Manara 2012; Vatamaniuk et al. 2001; Clemens and Peršoh 2009). The N-terminal of the enzyme PCS has been reported to contain the active site and four highly conserved Cys residues that appear to play an important role in heavy-metal-­induced PC-catalysis (Anjum et al. 2014); whereas the variable C-terminal site could bind the heavy metals via conserved Cys residues and translocate them to the catalytic N-terminal domain (Vestergaard et al. 2008). Overexpression of PCS genes has been reported to improve metal-metalloids tolerance in different plant species including A. thaliana (Vatamaniuk et al. 1999), Triticum aestivum (Clemens et al. 1999), Allium sativum (Zhang et al. 2005), and Brassica juncea (Heiss et al. 2003). Brunetti et al. (2011) observed that at relatively low Cd concentrations, tobacco plants over-expressing PC biosynthetic gene AtPCS1 is more tolerant to Cd than the wild type. Whereas, at higher Cd concentration, Arabidopsis seedlings over-expressing AtPCS1 are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Song et al. (2014), concluded that PCs play significant role in the homeostasis of essential metals and detoxification of non-essential toxic metal(loid)s in plants. Some other Cd-inducible genes were also found to be involved in PCs-dependent pathway such as HsfA4a (Shim et al. 2009), HMT1 (Huang et al. 2012), PAD2–1 and VTC2–1 (Koffler et al. 2014), and ZAT6 (Chen et al. 2016b). Chen et al. (2015), reported that overexpression of gene MAN3, which encodes an endo-β-mannanase

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enhanced Cd accumulation and tolerance through enhancing PCs synthesis. Brunetti et  al. (2015), observed that an ABC-type transporter AtABCC3 was involved in PC-mediated Cd-tolerance pathway. Song et al. (2017) observed that exposure of Arabidopsis plants to Cd overexpressed ferrochelatase-1 gene (AtFC1) that improved the generation of GSH and PCs, and accumulated more Cd than wild-type. In addition, overexpression of GSH and PCs synthesizing enzymes (γ-ECS and PCS, respectively), resulted in higher levels of GSH and PCs leading to more efficient metal sequestration (Zagorchev et  al. 2013; Song et  al. 2012). However, several studies also exhibit contradictory results. Overexpression of γ-ECS in Arabidopsis (Xiang et al. 2001) and tomato (Goldsbrough 1998) did not enhance resistance to Cd stress, despite increased levels of GSH and PCs. Also, the expression of bacterial γ-ECS in Arabidopsis did not enhance Cd tolerance and even caused Cd sensitivity (Li et al. 2005). Moreover, overexpression of AtPCS1 in Arabidopsis led to hypersensitivity to Cd despite enhanced PC production (Lee et  al. 2003a, b; Li et  al. 2004). However, pos-transcriptional regulation of PCS activity is well established (Cobbett 2000), therefore, PCs contribute to alleviate metal and/or Cd toxicity (Casarrubia et al. 2020). 11.4.2.3  Cys and Metallothioneins In addition to PCs, Cys also contributes to the synthesis of another group of metal-­ binding proteins known as metallothioneins (MTs). The enzymatic synthesis of PCs distinguishes them from MTs, which are gene encoded polypeptides, produced through mRNA translation. MTs are characterized as low molecular weight (4–8 kDa) Cys-rich peptides (Kagi 1993) that can bind a variety of metals through the thiol groups of their Cys residues by mercaptide bonds (Cobbett and Goldsbrough 2002; Blindauer and Leszczyszyn 2010). Since their discovery as Cd-binding proteins in horse kidneys, the corresponding functions of MTs such as detoxification of metals have been found throughout the plant kingdom as well as in prokaryotes (Cobbett and Goldsbrough 2002; Cobbett 2003; Roosens et  al. 2005; Freisinger 2008). The first MT was identified in wheat by Lane et  al. (1987). Since then, numerous MT genes have been isolated from different plant species. MT proteins typically contain two metal-binding, Cys-rich domains and are classified based on the arrangement of Cys residues (Cherian and Chan 1993). The plant MTs are represented by the family 15 and most plant MT genes are grouped into four types viz. MT1, MT2, MT3, and MT4 (Robinson et al. 1993; Rauser 1995, 1999; Cobbett and Goldsbrough 2002). Although, all the four MTs function as metal chelators (Guo et al. 2008), they are also involved in various other processes including fruit ripening (Moyle et al. 2005), root development, embryo germination (Yuan et al. 2008), suberization (Mir et al. 2004), response to multiple abiotic stresses (Li et al. 2015), regulation of cell growth and proliferation, DNA damage repair, and scavenging of ROS (Cherian and Kang 2006; Kumar et al. 2012; Ansarypour and Shahpiri 2017). The distribution of four MTs also vary with different plant organs. Leaves and roots predominantly contain MT1 genes, whereas MT2 genes are expressed primarily in

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leaves, stems, and developing seed (Zhou and Goldsbrough 1995; Murphy et  al. 1997; Guo et al. 2003; Waters et al. 2005). MT3 genes are expressed in leaves or in ripening fruits (Ledger and Gardner 1994), and the expression of MT4 genes are reported in seed as well as in reproductive organs and vegetative tissues (Chyan et al. 2005; Leszczyszyn et al. 2013). Although, many reports have indicated the roles of MTs in the sequestration and homeostasis of metals and ROS scavenging, the mechanisms through which MTs mediate these functions remain unclear.

11.5  Conclusions As mentioned in the preceding pages, H2S, GSH, PCs, MTs play crucial role in combating various abiotic stresses. However, the source material for all these is provided by Cys. The Cys acts as a pool for reduced sulfur which is required for the biosynthesis of various biomolecules and defense compounds. Cys not only acts as a precursor for H2S, but also takes part in the detoxification of ROS generated during abiotic stresses, maintenance of redox homeostasis, and detoxification of metals. On the contrary, high reactivity of Cys makes it a highly phytotoxic molecule when its concentration overpasses a certain threshold. Therefore, continuous supply of Cys as well as removal of excess Cys is vital for normal functioning of the defense system and for avoiding Cys toxicity, respectively. Hence, Cys homeostasis must be maintained via its synthesis and degradation. Synthesis of Cys is carried out by the action of OASTL enzyme and degradation of excess Cys takes place by the action of CDes which produce H2S. Persulfidation plays crucial roles in H2S-indued protective mechanisms against oxidative stress. On the basis of available information, it can be concluded that Cys is the key player behind the mechanisms involving H2S in combating abiotic stress-induced damage in plants, and Cys and H2S homeostasis is vital for the normal functioning of the defense system. However, further investigations are required to unravel the underlying mechanisms having new signaling roles of Cys and H2S in the acclimation of plants to abiotic stresses.

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Chapter 12

Hydrogen Sulfide and Posttranslational Modification of Proteins: A Defense Strategy Against Abiotic Stress Dengjing Huang, Changxia Li, Chunlei Wang, and Weibiao Liao

Abstract  Hydrogen sulfide (H2S), as a signaling gasotransmitter, has been reported to be involved in the regulation of diverse biological processes. Nevertheless, the underlying H2S-regulated mechanisms in plant biological functions are poorly understood. A new way of post-translational modification of proteins, named persulfidation, was found and used to explain the core mechanism of H2S action. Persulfidation results in the modification of cysteine residues on target proteins, via conversion of the thiol group into a persulfide group. Persulfidated proteins exhibit functional changes in enzyme activities and therefore, modified cysteine can interact with several other proteins and expresses greater reactivity due to the increased nucleophilicity of persulfide compared with the thiol group. Persulfidation is believed to play crucial role in the protective mechanisms through affecting antioxidant system, autophagy, and stomatal closure. In the present chapter the importance of persulfidation in H2S-mediated plant adaptive responses to various abiotic stresses and methods for the detection of protein persulfidation are presented. Also, the crosstalk of H2S, NO, and ROS has been analyzed, especially in relation to posttranslational modification of proteins. Keywords  Abiotic stresses · Autophagy · Hydrogen sulfide · Persulfidation · Post-translational modification · S-nitrosylation

D. Huang · C. Li · C. Wang · W. Liao (*) College of Horticulture, Gansu Agricultural University, Lanzhou, People’s Republic of China e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1_12

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12.1  Introduction Recently, hydrogen sulfide (H2S) has emerged as an important signaling molecule with many physiological functions in both animals and plants (Vandiver and Snyder 2012; Gotor et al. 2019). The first descriptions of H2S roles in plants are dated to the 1960s and the molecule is now accepted as the third gasotransmitter alongside of nitric oxide (NO) and carbon monoxide (CO). H2S has been shown to be completely or partially involved in a wide range of physiological processes including seed germination, root organogenesis, photosynthesis, stomatal movement, fruit ripening, as well as in leaf, flower and fruit senescence (Dooley et al. 2013; Fang et al. 2014; Álvarez et al. 2012; Huo et al. 2018; Ziogas et al. 2018). H2S has also been shown to participate in the regulation of adverse conditions such as metal stress (Mostofa et al. 2015; Qian et al. 2014; Amooaghaie et al. 2017), drought and heat tolerance (Ma et al. 2016; Li et al. 2014), dehydration stress (Khan et al. 2018), and salt stress (Lai et al. 2014). Endogenous H2S production was observed in various plants in the 1970s, while the production mechanism was only illustrated in recent decades. The production of H2S mainly derives from cysteine metabolism. In plant cells, several enzymes involved in cysteine metabolism present in subcellular compartments including the cytosol, mitochondria and chloroplasts are available for the production of H2S. These enzymes include L-cysteine desulfhydrase (LCD), previously known as Cys synthase-­like, and cysteine synthase (CS) in the cytosol; D-cysteine desulfhydrase (DCD) and cyano alanine synthase (CAS) in mitochondria; and sulfite reductase (SiR) in the chloroplast (Filipovic and Jovanovic 2017; Calderwood and Kopriva 2014). In the cytosol, H2S can be released from cysteine by the action of DCD and LCD, with the accompanying formation of ammonia and pyruvate. Mitochondria can also be a source of H2S which is generated during the detoxification of cyanide by the action of the CAS, which catalyzes the formation of β-cyanoalanine (Hatzfeld et al. 2000; Yamaguchi et al. 2000). H2S production occurs mainly via the photosynthetic sulfate-assimilation pathway in chloroplasts in the reaction catalyzed by the SiR (Takahashi et al. 2011). The endogenous production of H2S has been shown to be induced in response to various abiotic stress conditions. For example, Liu et  al. (2019) showed that the content and production rate of H2S were significantly enhanced in cucumber leaves exposed to low temperature (4 °C) for 12 h, and treatment of low temperature also increased the expression of genes (CsaLCD, CsaDES1, and CsaDES2) encoding the enzymes involved in H2S generation. The process of H2S-generation causes Cys depletion. In Arabidopsis, both H2S and Cys can be activated by chromium (Cr6+) stress, leading to increase in the contents of H2S and Cys. The results suggested that 1 mmol/L concentration of Cys was consumed by Cr6+ stress-induced H2S generation, and this H2S-generation-caused Cys depletion did not affect the Cr6+ stress-­ mediated Cys increase (Fang et  al. 2016). Enhanced endogenous H2S level in response to Cr6+ was again explored by Fang et al. (2017). They provided evidence for the regulatory mechanism of H2S production in response to Cr6+ stress, which

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was achieved through the calcium/calmodulin 2-mediated pathway, involving the transcription factor TGA3 (Fang et  al. 2017). Lai et  al. (2014) reported that salt stress increased the activity of LCD-associated endogenous H2S production in seedlings of Medicago sativa. Ma et al. (2016) reported that endogenous H2S content of wheat was increased in response to drought stress. Du et al. (2019) also reported that drought stress increased H2S production rate, H2S content and expression levels of LCD and DES1 genes encoding H2S-synthesizing enzymes. Additionally, elevating endogenous H2S level was observed during the response of metal stresses, such as cadmium (Cd), aluminum and lead (Mostofa et  al. 2015; Qian et  al. 2014; Amooaghaie et al. 2017). Plenty of evidence are available that support H2S can interact with other signaling molecules and modificate their signals. There is increasing interest in the interaction of H2S with plant hormones such as abscisic acid (ABA), gibberellic acid (GA), and ethylene (Scuffi et  al. 2014; Xie et  al. 2014; Jia et  al. 2018). Further recent evidence suggests that H2S also plays a critical role in the NO signaling pathways (Li et al. 2013). In addition, studies in several species have documented the involvement of NO and hormones such as ethylene and ABA in the regulation of plant H2S production (Li et al. 2013; Scuffi et al. 2014; Jia et al. 2018). For example, ethylene increased H2S content of tomato leaves under normal and stress conditions (Jia et al. 2018). The H2S production level of ABA-treated guard cells in Arabidopsis was increased, suggesting that ABA-activated DCD synthesized H2S in those special cells (Zhang et  al. 2020). Similar results have been reported by Scuffi et  al. (2014). Pretreatment with sodium nitroprusside (SNP, a NO donor) increased the DCD activity and H2S content in maize coleoptiles and roots (Li et  al. 2013). Additionally, some transcription factors also positively regulate H2S production. For instance, an Arabidopsis bZIP transcription factor, TGA3, promotes H2S production by regulating LCD expression (Fang et al. 2017). They further confirmed that TGA3 binding affinity to ‘TGACG’ cis-acting elements in the LCD promoter enhances LCD expression in a calcium/calmodulin 2-dependent way (Fang et al. 2017). Although increasing studies highlight the importance of H2S as a signaling molecule, its primary mechanism of action is poorly comprehended. A new post-­ translational modification (PTM) of proteins, named persulfidation, previously known as S-sulfhydration, was found and used to explain the underlying mechanism of H2S action. Persulfidation is the process in which cysteine residues on target proteins are modified via conversion of the thiol group (-SH) into a persulfide group (-SSH). Persulfidated proteins were involved in the regulation of important biological processes, such as carbon metabolism, plant responses to abiotic stresses, plant growth and development, and RNA translation (Aroca et  al. 2017a). The role of protein persulfidation in other important biological processes such as antioxidant system, autophagy and stomatal closure has also been described (Laureano-Marin et  al. 2016). Therefore, persulfidation plays a key role not only in regulating the activity of modified proteins but it may also regulate cellular localization of proteins. Within this context, we focus our discussion on the detection methods of

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protein persulfidation, target protein of persulfidation and role of persulfidation in the mechanism of H2S action.

12.2  Protein Persulfidation and Detection Methods in Plants In recent years, it has been widely accepted that the main mechanism by which H2S has been proposed to regulate a huge number of the processes is the formation of persulfides on specific protein cysteine residues (Mustafa et al. 2009; Aroca et al. 2015). It is the third mechanism of action of H2S based on chemical reactivity that the modification of proteins by the oxidation of cysteine residues to form the corresponding persulfides (therefore, called persulfidation). Several detection methods have been developed in recent years based on the nucleophilic characteristic of persulfide groups. Conversely, due to their instability and similarity to thiol groups, the development of a specific method for persulfide detection is challenging. Herein, these detection methods have been summarized, including further explanations of the reactions and procedures. Spectroscopy is an indirect detection of protein persulfides based on their reaction with 1-fluoro-2,4-dinitrobenzene to form mixed disulfides (Sawahata and Neal 1982). Subsequently, treatment with 1,4-dithiothreitol (DTT) releases 2,4-­dinitrobenzenethiol (DNBT), which absorbs at 408  nm under alkaline (1  M NaOH) conditions. The protein persulfide concentration can be estimated via using an extinction coefficient of 13,800 M−1 cm−1 for DNBT. Mass spectrometry (MS) can be used for direct detection of the protein persulfides (Mustafa et  al. 2009). However, the relative instability of persulfides limits their direct detection using MS analysis. To solve these problems, persulfidated proteins can be blocked with agents such as N-ethylmaleimide or iodoacetamide, thus stabilizing the persulfide modification (Francoleon et  al. 2011; Pan and Carroll 2013). Then, the presence of persulfide in proteins can be analyzed by MS. Some proteomic analysis methods of persulfides have only recently started to emerge. The modified biotin switch method (mBSM) was first used for proteomic analysis of persulfides. Unlike thiols, persulfides would not react with the electrophilic thiol-blocking reagent, S-methyl methanethiosulfonate (MMTS; Mustafa et al. 2009). On that basis, the Snyder’s group proposed that persulfides could be detected in biological samples by treating samples with S-methyl methanethiosulfonate to alkylate free thiols and then with an activated disulfide probe [N-(6(biotinamide) hexyl)-3′-(2′-pyridyldithio)-propionamide, biotin-HPDP] that would react with persulfides forming thiopyridone and tagging the proteins with biotin, which can later be detected. Another method for persulfide detection involves blocking free thiols and persulfides with Cy5-maleimide and subsequently reducing the R − S − S − maleimideCy5 adduct, which results in the loss of fluorescence. The loss of red fluorescence is detected following separation of proteins by gel electrophoresis. While relatively simple, the limitation of this method is that it is based on the absence of a signal

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associated with persulfides rather than on a positive signal, which can be coupled to MS for proteomic analysis. Furthermore, since maleimides are known to react with amines, extensive labeling can give high backgrounds obscuring changes in signal intensity when persulfides are present at low concentrations. In addition, several reactive compounds have been recently reported and used in animal systems for the detection of persulfides. These are (i) the fluorescent Cy5-­ maleimide (Sen et al. 2012), (ii) maleimide-PEG 2-biotin (Dóka et al. 2016), and (iii) iodoacetyl-PEG2-biotin (Gao et al. 2015), among others. However, most methods hitherto described have shown weaknesses since they lack specificity. Based on the methods previously described a new approach showing higher specificity to detect persulfidated proteins was recently reported and named as the tag-switch method. It employs methylsulfonyl benzothiazole (MSBT) to block both thiols and persulfide groups; then, a nucleophilic attack by the cyanoacetate-based reagent CN-biotin is performed labeling only the persulfide groups, which are purified with streptavidin conjugates and analyzed by Western blot, or directly by liquid chromatography coupled to mass spectrometry (LC-MS/MS).

12.3  Protein Persulfidation and H2S in Plants Protein persulfidation is a post translational modification of protein cysteine residues via conversion of the -SH group into a -SSH group. The target proteins of persulfidation, their functions and effects have been recently deciphered. In animal system, it is reported that persulfidation can either activate or inactivate enzymes. For example, glyceraldehyde-3-phosphate dehydrogenase and Parkin E3 ligase are activated and PTP1B is inactivated through persulfidation (Mustafa et  al. 2009; Vandiver et al. 2013; Krishnan et al. 2011). In addition, protein-protein interactions are modified by persulfidation. In mice, interaction of Kelch-like ECH-associated protein1 (Keap1) and Nuclear factor (erythroid-derived2)-like2 (Nrf2) are negatively regulated via persulfidation (Yang et al. 2008). H2S-producing associated proteins improve after H2S release (Mustafa et al. 2009). In plant system, few studies have revealed and analyzed the target proteins of persulfidation and their function. Table  12.1 shows a list of plant proteins which have been observed to undergo persulfidation, and how their protein function is modulated. Ascorbate peroxidase (APX), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6) and so on have been identified as target proteins of persulfidation. Persulfidation plays a key role in regulating the activity of modified proteins. The activities of some modified proteins are upregulated. H2S positively regulates the leaf photosynthesis of Spinacia oleracea by increasing the quantity and activity of RuBisCO and enhancing photosynthetic electron transfer through persulfidation modification (Chen et al. 2011). In sulfur metabolism, O-acetylserine(thiol)lyase (OAS-TL) and LCD were also modified by persulfidation resulting in increasing activities (Chen et al. 2011).

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Table 12.1  Summary of persulfidated proteins and their functions and effects in plant Enzyme Carbon metabolism-ribulose bisphosphate carboxylase/ oxygenase (RuBisCO) O-acetylserine(thiol)lyase (OAS-TL) L-cysteine desulfhydrase (LCD)

Function Photosynthesis

Effect Activity upregulated

Sulfur metabolism

Activity upregulated

Reference Chen et al. (2011)

Chen et al. (2011) Sulfur metabolism Activity upregulated Chen et al. (2011) Ascorbate peroxidase (APX) Antioxidant Activity upregulated Aroca et al. (2015) Activity upregulated, Aroca et al. Glyceraldehyde 3-phosphate Energy production in (2015, dehydrogenase (GAPDH) the glycolysis; cellular enhancing nuclear 2017a) localization metabolism Csa5G156220 Secondary metabolism Activity upregulated Liu et al. (2019) Csa5g157230 Secondary metabolism Activity upregulated Liu et al. (2019) Generation of Activity upregulated Shen et al. Respiratory burst oxidase homolog protein D (RBOHD) superoxide radical (2020) Ethylene biosynthesis Activity Jia et al. 1-aminocyclopropane-1-­ carboxylic acid oxidase (ACO) downregulated (2018) Aroca et al. Glutamine synthetase (GS) Metabolism of nitrogen Activity downregulated (2015) Provides NADPH as a Activity Muñoz-­ NADP-malic enzyme (NADP-ME) reducing agent downregulated Vargas et al. (2020) Catalase (CAT) Antioxidant Activity Corpas et al. downregulated (2019a) NADP-isocitrate dehydrogenase Provides NADPH as a Activity Muñoz-­ (NADP-ICDH) reducing agent downregulated Vargas et al. (2018) Inhibits actin Li et al. Actin Involved in organelle polymerization (2018) movement, in cell division and expansion Chen et al. SNF1-RELATED PROTEIN Promote ABA Promotes ABA-­ (2020a) KINASE2.6 (SnRK2.6) signaling induced stomatal closure

Aroca et al. (2015) identified 106 persulfidated proteins using mBSM and liquid chromatography-tandem mass spectrometry analysis. They further confirmed that APX and GAPDH were upregulated through persulfidation (Aroca et  al. 2015). Then, Aroca and colleagues indicated that persulfidated proteins were mainly involved in primary metabolic pathways such as the tricarboxylic acid cycle, glycolysis, and the Calvin cycle (Aroca et al. 2017a). Very recently, a gel-shift assay revealed increased levels of persulfidation of two bHLH transcription factors (Csa5G156220 and Csa5g157230) in cucumber. These transcription factors may enhance their binding activities towards the promoter of a key gene involved in the

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synthesis of cucurbitacin C, which is an important secondary metabolite for defense against adverse environmental stress (Liu et al. 2019). H2S regulates persulfidation of the NADPH oxidase-like respiratory burst oxidase homolog protein D (RBOHD) at Cys825 and Cys890, enhancing its activity (Shen et al. 2020). In contrast, the activities of persulfidation-induced specific proteins are down-regulated. Persulfidation is somehow similar to S-nitrosylation. Wang et al. (2015) revealed that S-nitrosylation inhibited SnRK2.6 activity. It is reported that 1-­aminocyclopro pane-­1-carboxylic acid oxidase 1 and 2 (ACO1 and ACO2) activities were also inhibited by persulfidation at cysteine 60 (Jia et  al. 2018). The activity of persulfidation-­induced glutathione synthetase is down-regulated (Aroca et al. 2015). NADP-malic enzyme (NADP-ME) could be modulated by persulfidation during pepper fruit ripening, leading to decrease in its activity (Muñoz-Vargas et al. 2020). With the persulfidation of catalase (CAT), the activity of this enzyme declined significantly (Corpas et  al. 2019a). NADP-isocitrate dehydrogenase (NADP-ICDH) was identified as a target of persulfidation in animal cells, although its impact on this activity has not been determined. Muñoz-Vargas et  al. (2018) indicated that NADP-ICDH activity was inhibited during sweet pepper (Capsicum annuum L.) fruit ripening. Besides, persulfidation may also regulate nuclear localization of proteins, polymerization, stomatal closure and other processes with significant consequences in plant systems. For example, persulfidation has been reported to enhance the nuclear localization of GAPDH (Aroca et al. 2017a). It is reported that persulfidation inhibited actin polymerization. Overaccumulation of H2S caused the depolymerization of actin and inhibited root hair growth (Li et al. 2018). H2S persulfidates SnRK2.6 to promote ABA signaling and ABA-induced stomatal closure (Chen et al. 2020a). Besides, some current studies suggest the mutual influence of H2S and protein persulfidation in plants. Sen et al. (2012) showed that increased expression of H2S-­ producing enzyme cystathionine β-synthase, besides producing H2S, induces persulfidation of Cys38 in the p65 subunit of nuclear factor kB (NF-kB), suggesting that H2S positively regulates the persulfidation of target proteins. In contrast, H2S production was also induced by persulfidation of some proteins related to H2S production. For instance, H2S-producing protein (DES1) was activated by H2S via persulfidation at Cys44 and Cys205 in the presence of ABA, which led to the transient overproduction of H2S in guard cells (Shen et al. 2020). From the proteome persulfidation data, Aroca et al. (2017b) indicated that the SnRK2.6 protein has two persulfidated residues (Cys131 and Cys137). In in vitro assays, SnRK2.6 activity was enhanced by H2S through persulfidation of Cys131 and Cys137. Then, Chen et al. (2020a) found that ABA positively regulates the production of H2S by activating DES1 in guard cells, and ABA signaling in turn was also positively mediated by H2S via persulfidation of Open Stomata 1 (OST1)/ SnRK2.6. Jia et al. (2018) showed that H2S positively induces persulfidation of ACO1 and ACO2 in tomato in a dose-­ dependent manner by using mBSM, suggesting that H2S regulates ethylene biosynthesis. Hence, H2S plays vital role in protein persulfidation which in turn mediates H2S production at the same time.

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12.4  P  rotein Persulfidation in Plant Adaptive Responses to Abiotic Stress 12.4.1  Antioxidant Defense System The induction and accumulation of reactive oxygen species (ROS) is considered as the consensus occurrence in plants during the environmental stress. Plants survive in the harsh conditions by maintaining the balance between ROS production and elimination. H2S as the simplest thiol found in cells can interact with ROS antagonistically and synergistically in response to stresses at multiple levels. For example, hydrogen peroxide (H2O2) works together with H2S in the resistance against the polyamine-mediated UV-B radiation in white clover (Li et  al. 2016). Mei et  al. (2017) combined pharmacological, anatomical, and molecular approaches systematically to study the positive effects of NADPH oxidase-derived H2O2 in H2S-­ induced lateral root formation. As proof, the translation levels of cell cycle regulatory genes including SlCYCA2;1, SlCYCA3;1, SlCDKA1, and SlKRP2 were compared with or without the presence of H2O2 scavenger. Above expression changes were consistent with the phenotypes of H2O2 resulted in tomato seedlings (Mei et  al. 2017). While in Medicago sativa, the Cd2+ toxicity was aggravated by the LCD/ DCD-mediated H2S mainly by manipulating reduced (homo)glutathione (hGSH) and ROS homeostasis. When pretreated with sodium hydrosulfide (NaHS), the decrease of hGSH level caused by Cd2+ in alfalfa seedling roots was downregulated. Further research suggests that H2S synthetic inhibitor DL-propargylglycine increased ROS abundance, but this was rescued by GSH. Considering the function of GSH in eliminating excess ROS through ascorbate-glutathione pathway or the ROS scavenging by superoxide dismutase, it is not hard to understand the effect of H2S on Cd2+-induced oxidative stress. The question about how H2S increases glutathione contents was answered by Chen et al. (2020b), who demonstrated that H2S combines with O-acetylserine to form a key GSH substrate, cysteine. Moreover, post-translational redox modification like persulfidation could increase the activity of glutamate cysteine ligase (Aroca et al. 2017a), which is one of the key enzymes in GSH formation (Noctor et al. 2012). H2S also increases the activities of antioxidant enzymes including APX, dehydroascorbate reductase (DHAR), glutathione reductase (GR), and CAT, thereby antagonizes oxidative stress. Except for scavenging ROS by enzymes or by GSH pathway, H2S also reacts with the oxidative forms of protein cysteine to form persulfides (Filipovic et al. 2018). In Arabidopsis, 57 antioxidant enzymes/proteins including APX, CAT1, CAT2 and CAT3, DHAR1, DHAR2, DHAR3, GR1, and GR2, could be persulfidated (Aroca et al. 2015, 2017a). NaHS inactivates glutathione synthesis, while activates APX and GAPDH. These changes were reversed by the addition of DTT, an inhibitor of disulfide bond formation. Cys32 was the detected persulfidation site of cytosolic APX1 (cAPX1) which enhanced its catalytic activity (Aroca et al. 2015). Under glyphosate-triggered oxidative stress, the activity of photorespiratory H2O2-generating glycolate oxidase and that of CAT were inhibited by NaHS. Persulfidation of different forms of APX and

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CAT in Arabidopsis peroxisomes inhibits the activity, and this was further verified to be consistent in both bovine liver and pepper fruit (Corpas et al. 2019b). Therefore, H2S plays critical roles in the adaption of plant to various oxidative stresses, especially through persulfidation.

12.4.2  Autophagy Autophagy (‘self-eating’), a major system for vacuole/lysosome to degrade cytoplasmic components, is involved in the digestion of cell contents, thereby recycling the necessary nutrients or the degradation of toxic and damaged components, maintaining normal cellular activities. In the plants, there are two types of autophagy: microautophagy and macroautophagy (Klionsky and Ohsumi 1999). In microautophagy, invaginated vacuolar membrane directly engulfs the cytoplasmic components (including individual proteins, protein aggregates, and organelles). While macroautophagy is a relative multi-stage process, during which bulk cytosolic constituents and organelles are firstly sequestered into double-membrane autophagosome and then delivered into the vacuolar lumen for degradation through the fusion of autophagosome outer membrane and vacuolar membrane (Aubert et  al. 1996; Herman et al. 1981; Robinson et al. 1998; Swanson et al. 1998; Toyooka et al. 2001; Bassham 2007). Autophagy occurs at basal levels under favorable growth conditions and is extremely important in the regulation of plant development (Inoue et al. 2006; Sláviková et al. 2005). However, more attentions are paid on the beneficial effect of autophagy under stressful conditions, such as oxidative stress, starvation (Chung et  al. 2009; Guiboileau et  al. 2013), saline, drought, heat stress, osmotic stress and sugar excess (Avin-Wittenberg, 2019; Janse van Rensburg et al. 2019; Han et al. 2011; Yu and Xie 2017; Zhuang et al. 2015). In plants, autophagy is associated with survival and longevity, however, the exact role of autophagy as a death-­ promoting or life-promoting process is unclear. Although some related explorations have been reported, for instance, constitutively stressed 1 (COST1), a plant-specific DUF641 family protein whose mutant showed defect in development and strong drought resistance phenotype, negatively regulates autophagy, and whose aggregation or degradation under drought activates autophagy, thereby withstanding drought stress (Bao et al. 2020). Usually, proteins or genes involved in autophagy (termed ATG) have been identified to be required in the development of autophagosome. Studies have shown that different ATG operate specific functions in autophagy process. For example, ATG1, ATG11, and ATG13 respond for induction of the phagophore, ATG5, ATG12, and ATG9 for transport of lipids for membrane enlargement and ATG4, ATG8, ATG3, ATG7 for phagophore expansion and closure (Marshall and Vierstra 2018; Doelling et al. 2002; Hanaoka et al. 2002). Till now, H2S has been extensively reported to enhance plant tolerance or defense against varies stresses. Little is known, however, about the molecular mechanisms underlying H2S regulating autophagy in plants. In plants, DES1 is the only protein which is identified to be involved in the degradation of cysteine leading to the

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synthesis of H2S in Arabidopsis cytosol (Álvarez et al. 2010). In des1 mesophyll protoplasts, the senescence-associated vacuoles were detected, and DES1 deficiency resulted in the accumulation and lipidation of autophagy close-related protein ATG8. Phenotype of Arabidopsis leaf in T-DNA insertion mutants des1–1 and des1–2 exhibits premature leaf senescence, and this was reversed by the addition of H2S (Álvarez et al. 2010). In addition, the accumulation and lipidation of ATG8 was also recovered by H2S in both des1 mutants and carbon-starved wild type Arabidopsis plants. These results indicate that H2S generated by DES1 in plant cytosol negatively regulates autophagy, thereby rescuing the premature leaf senescence induced by autophagy (Álvarez et al. 2012). Similar results were obtained in the nitrogen-­ deprived Arabidopsis and the subsequent experiments demonstrated that the mechanism of sulfide repressing autophagy is independent of ROS which induced autophagy (Laureano-Marín et al. 2016). To further explore the mechanism of H2S in autophagy regulation, Laureano-Marín et al. (2020) detected the autophagy process induced by ABA-triggered persulfidation in Arabidopsis through comparative and quantitative proteomic analysis. The results demonstrated that the autophagy mediated by the ATG cysteine protease AtATG4a was persulfidated, especially in Cys170, and this caused conformational changes and intramolecular rearrangements of the catalytic site and inactivation of AtATG4a activity. Thus, one of the mechanisms about H2S that negatively regulates autophagy is due to the specific persulfidation of ATG4 at Cys170 (Laureano-Marín et al. 2020).

12.4.3  Stomatal Closure Plants develop a variety of defense mechanisms to adapt and survive in the hostile environment. For instance, regulating opening and closing of stomata to limit water loss or importing carbon dioxide to enhance photosynthesis (Schroeder et al. 2001a). Drought is one of the most serious stresses to restrict plant growth and development as well as yield (Fedoroff et al. 2010; Geiger et al. 2009; Zhu 2002, 2016). Stomatal movement is one of the typical stress responses of plants under drought stress. Therefore, it is important to explore the stomatal movement in plant response to environmental stimuli. ABA causes stomatal opening through reducing the turgor and volume of the guard cells (Schroeder et al. 2001b; Pandey et al. 2007). Similarly, H2S is also revealed to induce stomatal opening. Lisjak et al. (2010) found that both NaHS and GYY4137 (a slow releasing H2S donor) causes stomatal opening in the light. Subsequent experiment demonstrates that H2S leads to the efflux of K+ and influx of Ca2+ and Cl−, thus regulating stomatal movement (Jin et al. 2017). Recently, H2S has been discovered to coordinate with ABA signaling in the modulation of stomatal apertures (Scuffi et al. 2014; Lisjak et al. 2010). This can be further proved in ABA-associated mutants (aba3 and abi1), in which the H2S-induced stomatal closure was impaired (Du et al. 2019), and DES1 knockout mutant whose ABA-­ induced stomatal closure was nullified (Scuffi et al. 2014). DES1, as the first and most characterized enzyme for H2S generation in plants, is widely used to study the

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mechanism of H2S action in the regulation of stomatal closure. Although the coupling of the DES1/H2S signaling pathways to stomatal movement has not been thoroughly elucidated, some research results have been achieved. Zhang et al. (2020) deduced that DES1 mediates ABA-dependent stomatal closure through the LONG HYPOCOTYL1 signaling pathway. Moreover, DES1-produced H2S also interacts with other second messengers like MAP kinase and ROS to manipulate the switch of opening or closing of stomata (Du et al. 2019; Gudesblat et al. 2007; Shen et al. 2020). Taking ROS as an example, the H2S produced by DES resulted in the persulfidation of RBOH D and F isoforms in Arabidopsis, thereby affecting its activity to generate ROS and mediating stomata movement. The persulfidation of protein is also an important way in which H2S participates in ABA signaling to jointly regulate stomatal movement. Firstly, H2S endogenous producer DES1 can be persulfidated under the stimulation of ABA. Mass spectrometric analysis suggested that DES1 was persulfidated at Cys44 and Cys205 in a redox-dependent manner. This posttranslational modification enhanced the activity of DES1 and promoted H2S accumulation (Shen et  al. 2020). Whereafter, as the increase of H2S concentration, RBOHD was driven to be persulfidated at Cys825 and Cys890, thereby improving the ability of RBOHD to generate ROS. The function of protein persulfidation has also been proved physiologically (Shen et  al. 2020). Moreover, the production of H2S catalyzed by DES1 is monitored to be induced by ABA. Further research found that H2S in guard cells positively regulates the activity of OST1/SnRK2.6 through persulfidation at its Cys131 and Cys137 (Chen et al. 2020a). Bimolecular fluorescence complementation analysis and results on pull-down experiment also showed that H2S strengthened the interaction between SnRK2.6 and ABA response element-binding factor 2. Physiologically, SnRK2.6 persulfidation regulates ABA-induced Ca2+ signaling in guard cells thereby affecting stomatal movement (Chen et  al. 2020a). Except for ABA, H2S and ETH act synergistically on stomatal closure in similar mechanism of ABA. What differs is that the persulfidated proteins in ETH pathway are ACOs at Cys60, this giving rise to the inhibition of ACO activity (Shen et al. 2020). Taken together, these observations imply that H2S plays a crucial role in the regulation of stomatal movement in plant response to abiotic stresses, especially through the specific and reversible redox-based persulfidation modifications.

12.5  T  he Crosstalk of H2S with Other Signaling Molecules and Protein Persulfidation Emerging data in recent years suggest that H2S may be a signaling molecule in plants as important as NO and H2O2 (Corpas et al. 2019b; Deng et al. 2019; Gotor et al. 2019). The forthcoming paragraphs are dedicated to the crosstalk of H2S with other signaling molecules in relation to persulfidation.

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12.5.1  Crosstalk of H2S and NO in Relation to Persulfidation Like other signaling molecules, NO and H2S, due to their low molecular weight, can cross membranes and reacts with different compounds and carry out their functions in plant cells. More important, in spite of their toxicity, NO and H2S are also important signaling molecules when below the toxic threshold. In plant, NO regulates a series of physiological processes from seeds germination to postharvest ripening and senescence, as well as the response to abiotic and biotic factors (Fancy et al. 2017; Besson-Bard et al. 2008). As happened with NO that was seen to participate in defense against adverse environmental conditions, H2S seems to be also involved in similar ways (synergistically or antagonistically) in these processes. Up to date, although the upstream and downstream relationship between NO and H2S are still confusing, reports about their collaboration on resistance to various abiotic stresses are springing up (Wang et al. 2012; Li et al. 2013; Amooaghaie et al. 2017; Khan et al. 2017, 2020). High temperature (HT) stress leads to the accumulation of ROS and reactive nitrogen species (RNS), which results in leaf damage. Intriguingly, the transcriptional level and enzyme activity of S-nitrosoglutathione reductase (GSNOR) were increased, which strengthens the capability to scavenge excessive ROS and RNS, thereby improving poplar resistance to HT stress (Cheng et  al. 2018). Once the biosynthesis of H2S was suppressed, the positive effect of GSNOR on HT stress was reversed. H2S and NO also alleviate wheat cobalt toxicity by modulating the activity of ROS scavenging enzymes including APX, superoxide dismutase (SOD), peroxidase (POX), as well as the ascorbate level, etc. (Ozfidan-­ Konakci et al. 2020). Apart from transcriptional levels and crosstalk with other regulators, protein PTMs of cysteine residues is another functional way for H2S and NO to perform their physiological functions. Protein thiols are crucial to enzymatic reactions that need the involvement of cysteine (Richau et al. 2012). H2S and NO reversibly combine to protein cysteine residues to induce persulfidation (Aroca et  al. 2017a) or S-nitrosation (Corpas et al. 2019a; Astier et al. 2012), which regulate both the structure and activity of susceptible enzymes. Aroca et al. (2018) compared the PTM proteins in Arabidopsis and found that 2330 proteins underwent persulfidation and 929 underwent S-nitrosation, and 612 proteins could be modified by both. Thus, they speculated that persulfidation is more important than S-nitrosation in Arabidopsis leaves. Although few studies on the analysis of both PTMs have been reported, some results have been obtained in oxidation-reduction related enzymes including APX, CAT, GAPC and RBOHD/F (Fig. 12.1). cAPX, an important enzyme to modulate plant H2O2 content, was indicated to be S-nitrosated during both heat shock or H2O2 treatment in tobacco suspension cells (de Pinto et al. 2013). Additionally, Yang et al. (2015) reported that APX1 was S-nitrosated at Cys32, enhancing its capacity of H2O2 scavenging, leading to the increased resistance to oxidative stress. Similar to S-nitrosation, APX persulfidation also improved its activity (Aroca et  al. 2015).

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Fig. 12.1  The interaction of H2S, nitric oxide (NO) and reactive oxygen species (ROS) during protein persulfidation. Trx thioredoxin, APX ascorbate peroxidase, CAT catalase, GAPC glyceraldehyde-­3-phosphate dehydrogenase, RBOHD NADPH oxidase-like respiratory burst oxidase homolog protein D, Trx thioredoxin

Another antioxidant enzyme CAT which exclusively locates in peroxisomes also undergoes both PTMs (Palma et al. 2020). Differently, CAT activity was inhibited by NO and H2S through S-nitrosation and persulfidation, respectively (Clark et al. 2000; Aroca et al. 2015). Different PTMs also result in opposite regulation in some cases, like GAPC, whose activity could be inhibited by H2O2 directly, showing positive regulation in persulfidation (Aroca et al. 2015), while negative one in S-nitrosation (Lindermayr et al. 2010; Sell et al. 2008). The different PTMs in above enzymes means that PTM at cysteine thiols is an important way for H2S and NO to synergistically regulate plant stress responses. In ABA-induced stomatal movement, excessive accumulation of H2S leads to RBOHD persulfidation at Cys890, which increases its ability to generate ROS, thus promoting stomatal closure and enhancing tolerance to drought stress (Shen et al. 2020). Inversely, S-nitrosation of RBOHD at the same site also enhances the immunity of Arabidopsis (Yun et al. 2011).

12.5.2  Crosstalk of H2S and ROS in Relation to Persulfidation The accumulation of ROS under various stresses is a common phenomenon in plants. There is a battery of enzymes that facilitate the ROS scavenging in plant cells. These include CAT, APX, SOD and thioredoxin (Trx) etc., as well as other ROS-scavenging systems. However, in spite of the presence of such an antioxidant system, a higher accumulation of ROS under stress conditions has been observed that can lead to survival threat. In such situation H2S has been observed to play

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decisive role in combating excess ROS generation. A growing number of researches have evidenced that H2S cooperates with ROS to regulate plant response to adverse conditions antagonistically and synergistically (Scuffi et  al. 2018; Li et  al. 2016, 2019). H2S and its ionic forms (HS− and S2−) cannot react with protein thiols directly, but it can react with sulfenic acid like oxidized cysteine residues to form persulfide (Filipovic et al. 2018; Benchoam et al. 2019). Oxidative conditions can increase the level of persulfidation in H2O2-treated cells (Cuevasanta et al. 2015; Wedmann et al. 2016). As mentioned previously, sustainable H2S accumulation drives persulfidation of the RBOHD, which increases its ability to generate ROS and lead to stomatal closure (Shen et al. 2020). Continuous ROS accumulation under stress contributes to protein sulfenylation, by which ROS reacts with persulfidated proteins to form perthiosulfenic acids (R-SSOHs) (Heppner et al. 2018). The sulfenylated proteins can be further oxidized by ROS into sulfinic (-SO2H) or sulfonic (-SO3H) acid, which may irreversibly render the proteins functionally inactive (Paulsen and Carroll 2013) (Fig. 12.1). Compared to sulfenylation, persulfidation may be more helpful for plants to resist oxidative stress, because of the reversible reduction of thioredoxin which prevent the formation of irreversible thiol modification (Wedmann et al. 2016; Ren et al. 2017). The results of Aroca et al. (2017a) and Huang et al. (2019) showed that there are many proteins sensitive to both the above modifications. They mainly function in primary metabolic pathways, amino acid biosynthesis and protein synthesis. Even though it’s hard to figure out the detailed relationships between ROS, NO and H2S in plant stress response, a great number of data evidence that they do jointly play important roles in resisting the hardship of environmental conditions.

12.6  Conclusions and Future Perspectives The available information suggests that H2S modifies structure and function of various proteins through persulfidation. Moreover, interaction of H2S with other signaling molecules including ABA, NO, and ROS plays decisive role in plant adaptive responses to abiotic stress through persulfidation. Although H2S-induced persulfidation operates through the modification of cysteine residues of target proteins, in some targets the modified cysteine is the active site, while in others it is not the case. Therefore, the specificity of cysteine residues needs to be elucidated. Also, understanding the molecular basis of crosstalk of H2S with other signaling molecules and their sequence of action during protein PTMs will unravel the underlying mechanisms involved in the tolerance to plants to abiotic stresses. The study on H2S and protein PTMs in plants is growing. Advanced molecular approaches and omics technologies should be adopted to systematically analyze the impact of H2S on plant tolerance to adverse environmental conditions, especially in the aspects of PTMs.

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Acknowledgements  This work was supported by the National Natural Science Foundation of China (Nos. 32072559, 31860568, 31560563 and 31160398); the Research Fund of Higher Education of Gansu, China (No. 2018C-14); the Natural Science Foundation of Gansu Province, China (Nos. 1606RJZA073, 1606RJZA077 and 1606RJYA252).

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Index

A Abiotic stresses, v, vi, 5, 6, 8, 13–24, 31–50, 61, 63, 67, 73–81, 88, 90, 92, 99–101, 123–135, 139–155, 161–178, 187–203, 215–228 Abscisic acid (ABA), v, 3, 6–8, 15, 18, 19, 32, 38, 45, 49, 61, 68, 74, 77, 81, 90–92, 94, 96, 98–101, 110, 111, 114, 125, 126, 128, 133–135, 152–154, 164, 169, 172, 175, 176, 195, 196, 217, 220, 221, 224, 225, 228 Adenosine 5'-phosphosulfate (APS), 63, 189, 190 Alfalfa, 5, 17, 20, 38, 62, 132, 142, 148, 222 Aluminum, 5, 21, 126, 143, 144, 171, 177, 217 Antioxidant defense systems, 16, 132, 173, 194, 222–223 Antioxidant enzymes, 7, 17, 18, 20, 21, 23, 35, 42, 44–49, 62, 64, 66, 77, 115–117, 131, 143, 145, 146, 148, 151, 162–163, 165, 167, 168, 172–174, 194–196, 222, 227 APS reductase (APR), 189, 190, 199 Ascorbate-glutathione cycle, 7, 62, 65–67, 80 Ascorbate peroxidase (APX), 5, 15–21, 23, 35–39, 42, 44, 46, 49, 67, 77, 78, 80, 97, 114–116, 131, 143–145, 147, 148, 163, 168–172, 174–177, 197, 200, 219, 220, 222, 226, 227 Autophagosome, 4, 223 Autophagy, 4, 5, 164, 217, 223–224 Auxins, 19, 49, 61, 74, 110, 135, 154, 165, 174

B β-cyanoalanine synthase (β-CAS), 3, 115, 191 Biotin switch method, 218 Brassica napus, 5, 67, 142, 168, 171 Brassica oleracea, 37, 39, 144, 167, 170, 171, 178 Brassinosteroids, 68, 110, 135, 154, 162 C Cadmium, 5, 19, 39, 60, 142, 143, 164, 170, 171, 196, 199, 201, 217 Calcium, v, 2, 20, 31–50, 62, 65, 74, 75, 79, 96, 100–101, 143, 154, 175, 196, 217 Calmodulins, 23, 154, 217 Capsicum annuum, 67, 111, 113–115, 146, 171, 221 Carbonic anhydrases, 3, 33, 96, 97, 150, 167, 172, 196 Carotenoids, 18, 22, 143, 145–147, 175 Catalases, 5, 7, 16, 35–37, 41, 62, 77, 78, 97, 111, 114–116, 131, 142, 144–151, 163, 171, 196, 198, 220, 221, 227 Chilling, 5–7, 21, 22, 35, 79, 116, 117, 151, 168, 170, 173, 174, 177, 200 Chlorpromazine, 40 Cobalt, 60, 226 Cold stresses, 5, 22, 34, 79, 150–152, 167, 173, 194 Copper, 5, 19, 60, 80, 125, 145, 171, 172, 201 Cucumis sativus, 37, 128, 141, 170, 171, 176–178

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 M. N. Khan et al. (eds.), Hydrogen Sulfide and Plant Acclimation to Abiotic Stresses, Plant in Challenging Environments 1, https://doi.org/10.1007/978-3-030-73678-1

235

Index

236 Cyanuric acid, 62, 192 Cysteine desulfhydrase, 7, 65, 147, 150, 189 Cysteines, v, 15, 16, 20, 62, 65, 75, 76, 79, 97, 111, 112, 114, 127, 131, 141, 143, 145, 146, 148, 162, 164–166, 175, 187–203, 216–219, 221–224, 226–228 Cysteine synthase, 3, 15, 16, 33, 62, 63, 75, 97, 110–113, 140–142, 147, 148, 192, 216 D Dehydroascorbate reductase (DHAR), 18, 21, 38, 67, 77, 142, 163, 200, 222 Diphenyleneiodonium, 45 Drought, v, vi, 5–7, 13, 17–19, 36, 38, 41, 44, 45, 61, 62, 74–77, 88, 90–92, 139, 141, 149, 150, 153–155, 163, 166, 167, 169, 170, 172, 176, 188, 193, 196, 216, 217, 223, 224, 227 E EGTA, 40–42, 154 Electrolyte leakage, 17, 21, 65, 66, 79, 145, 146, 148, 150, 167, 173, 193 Ethylene, 7, 8, 16, 40, 49, 61, 68, 74, 95, 110, 111, 114, 117, 128, 135, 154, 175, 195, 196, 217, 220, 221

H H+-ATPase, 5, 18, 21, 88, 89, 93, 94 Heat shock proteins (HSPs), 6, 23, 34, 36, 78, 164 Heat stress, 23, 40, 50, 78, 79, 152, 153, 155, 168, 194, 223 Heavy metal stresses, 5, 19–21, 59–68, 79–80, 111 High temperature (HT), 23, 35–38, 49, 78, 126, 128–130, 134, 152, 193, 226 Homo-GSH (h-GSH), 162, 199, 200 Hordeum vulgare, 128, 143, 170, 176, 177 Hydrogen sulfide signaling, 139–155 Hydroxyl, 14, 42, 141, 198 Hydroxylamine, 46 Hypotaurine, 44, 64, 128, 129, 147, 151, 177, 194 Hypoxia, 46, 80, 163, 168, 170, 193, 194 J Jasmonic acid (JA), 45, 49, 154

F Fumigation, 18, 35, 151

L Lanthanum chloride, 41 L-arginine, 46 Lipid peroxidation, 5, 17–19, 35, 77, 78, 114, 131, 149, 167, 174, 193 Low temperatures, 6, 22, 34–36, 46, 74, 78, 110, 116, 151, 173, 176, 193, 216

G Gasotransmitters, 2, 6, 32, 46, 48, 89, 90, 99, 101, 116, 155, 162, 195, 216 Germination, vi, 4, 8, 16, 17, 20, 21, 23, 37, 39, 40, 44, 61, 66, 67, 77, 124–126, 128–135, 144, 145, 147, 152, 153, 162, 168, 173, 188, 202, 216, 226 Glutathione, 15, 17, 36, 38, 62, 65–67, 77, 78, 80, 115, 127, 142, 143, 145–149, 155, 162, 165, 172, 189, 190, 195, 198–201, 221, 222 Glutathione reductase (GR), 15, 18, 21, 23, 35, 36, 38, 39, 42, 66, 67, 77, 78, 80, 142, 145, 147–151, 163, 167, 168, 170–172, 174, 175, 177, 198–200, 222 Glycerol, 75, 195 Glycolate oxidase, 43, 222

M Macroautophagy, 223 Malondialdehyde (MDA), 18–23, 35, 40, 65–67, 77, 79, 80, 142, 145–148, 150–152, 168, 169, 173, 175, 193 Medicago, 4, 19, 37, 78, 129, 142, 172, 176, 217, 222 Metallothioneins (MTs), 21, 66, 67, 80, 146, 174, 198, 202–203 Methionine, 162, 188 Microautophagy, 223 Mitogen-activated protein kinase (MAPK), 22, 96, 150, 172–174 Monodehydroascorbate reductase (MDHAR), 21, 36, 38, 67, 148, 174, 177, 200

Index

237

N NADPH oxidases, 7, 41, 43–45, 47–49, 91, 92, 114, 170, 172, 196 Nicotiana tabacum, 36, 41, 141 Nitrite reductase, 46, 146, 168, 171, 190

Proline dehydrogenase (PDH), 23, 35, 65, 79, 168, 174, 177 Pyrroline-5-carboxylate, 6, 35, 75 Δ¹-pyrroline-5-carboxylate synthetase (P5CS), 75, 79, 170, 172

O O-acetylserine, 22, 33, 63, 97, 111, 115, 127, 163, 172, 189, 190, 222 O-acetylserine(thiol)lyase (OASTL), 33, 114, 189, 190, 219, 220 Oryza sativa, 67, 128, 142, 171 Osmolytes, vi, 6, 7, 14, 23, 37, 61, 74–81, 89, 164, 168, 178, 194–196 Osmotic stresses, 5, 17, 18, 36, 38, 45, 47, 61, 74–76, 78, 79, 81, 126, 129–131, 133, 147, 148, 164, 173, 193, 195, 196, 223 Ozone, 93, 98–99

R Reactive nitrogen species (RNS), v, 2, 5, 114, 115, 125, 163, 165, 166, 195–197, 226 Reactive oxygen species (ROS), 2, 5–7, 14, 15, 18, 20–22, 31–50, 62–67, 74, 76–80, 92, 98–101, 110, 114–117, 125, 131–132, 135, 141–143, 145, 147, 148, 150, 151, 163–169, 173–175, 188, 189, 194, 196–198, 200, 202, 203, 222, 224–228 Relative water content (RWC), 17–20, 37, 77, 166, 172, 193 Respiratory burst oxidase homolog protein D (RBOHD), 7, 92, 114, 220, 221, 225–228 Rubisco, 4, 16, 114, 169, 172, 174, 177, 219, 220

P Pectin methylesterase (PME), 5, 21, 64, 175 Peroxidases, 15, 35–39, 43, 44, 78, 131, 142, 151, 167, 171, 172, 196, 226 Peroxisomes, 43, 44, 46, 110, 111, 223, 227 Peroxynitrite, 16, 46, 62, 141, 196 Persulfidation, v, vi, 4–7, 16, 22, 24, 32, 44, 46, 47, 49, 64, 67, 68, 91, 92, 99, 112, 114, 135, 141, 155, 164–166, 178, 197, 203, 217–228 Persulfides, v, 44, 99, 112, 127, 141, 165, 166, 197, 217–219, 222, 228 3-phosphate dehydrogenase, 16, 114, 197, 219, 220 Phosphatidic acid (PA), 43, 94 Photosynthesis, 4, 7, 8, 14, 16, 18–21, 61, 65, 77, 80, 88, 93–95, 114, 143, 149, 155, 167, 169, 171, 174, 178, 188, 216, 219, 220, 224 Phototropins, 93, 94, 96 Phytochelatins, 62, 65, 66, 79, 80, 142, 146, 162, 174, 189, 201–202 Polyamines, 14, 19, 37, 43, 44, 46, 77, 78 Post translational modification (PTM), 112, 166, 217, 219, 226, 227 Programmed cell death (PCD), 64, 142, 165, 175, 178 Proline, 6, 15, 18, 21–23, 34–38, 65, 74, 75, 77–79, 114, 168, 172, 174, 195, 196

S Salicylic acid (SA), 2, 32, 49, 50, 61, 65, 74, 110, 152, 154, 165, 171 Salinity, v, vi, 5, 13, 17, 38, 39, 61, 74, 75, 88, 90–92, 141, 147, 155, 163, 164, 167, 169, 170, 173, 176, 188, 193 Salt stresses, 5, 17–18, 37–39, 45, 62, 74, 75, 77–78, 90, 129, 147–149, 167, 173, 196, 216, 217 Serine acetyltransferase (SAT), 189–191, 199 S-nitrosoglutathione reductase, 23, 115, 152, 226 S-nitrosylation, 4, 152, 197 Sodium hydrosulfide (NaHS), 4, 7, 14, 17–23, 35–42, 47, 48, 62, 64–67, 76–80, 117, 128–130, 142–155, 166, 170–173, 175–178, 193, 194, 197, 222, 224 Sodium nitroprusside (SNP), 20, 48, 217 Solanum lycopersicum, 111, 113, 117, 148, 177, 178 Solanum nigrum, 21, 67, 146, 178

238 Spermidine, 44, 77 Spinacia oleracea, 4, 19, 36, 38, 169, 172, 219 S-sulfhydration, 65, 131, 217 Sugars, 4, 23, 34–37, 75, 77, 79, 133, 148–151, 223 Sulfite reductase (SiR), 3, 15, 16, 33, 62, 63, 75, 76, 110–113, 115, 127, 140, 141, 163, 189, 190, 216 Superoxide, 14, 36, 42, 62, 65, 114, 131, 141, 173, 175, 196, 220

Index Superoxide dismutase (SOD), 5, 15, 17, 19, 20, 22, 35–39, 42, 44, 49, 62, 78, 80, 116, 131, 142, 144–150, 163, 167, 168, 170–173, 176, 177, 196, 222, 226, 227 T Thermotolerance, 23, 134, 150, 154, 155 Trifluorpromazine, 40 Triticum aestivum, 36, 37, 126, 129, 144, 170, 171, 176–178, 201