# REDOX HOMEOSTASIS MANAGERS IN PLANTS UNDER ENVIRONMENTAL STRESSES

EDITED BY: Nafees A. Khan, Naser A. Anjum, Adriano Sofo, Rene Kizek and Margarete Baier PUBLISHED IN: Frontiers in Environmental Science

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ISSN 1664-8714 ISBN 978-2-88919-878-8 DOI 10.3389/978-2-88919-878-8

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## **REDOX HOMEOSTASIS MANAGERS IN PLANTS UNDER ENVIRONMENTAL STRESSES**

Topic Editors:

**Nafees A. Khan,** Aligarh Muslim University, India **Naser A. Anjum,** University of Aveiro, Portugal **Adriano Sofo,** University of Basilicata, Italy **Rene Kizek,** University of Veterinary and Pharmaceutical Sciences, Czech Republic **Margarete Baier,** Free University of Berlin, Germany

Spring forest with hornbeam trees and saw grasses in the locality of Baba, Czech Republic. Personal photography of the Co-Editor Prof. Rene Kizek, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.

The production of cellular oxidants such as reactive oxygen species (ROS) is an inevitable consequence of redox cascades of aerobic metabolism in plants. This milieu is further aggravated by a myriad of adverse environmental conditions that plants, owing to their sessile life-style, have to cope with during their life cycle. Adverse conditions prevent plants reaching their full genetic potential in terms of growth and productivity mainly as a result of accelerated ROS generation-accrued redox imbalances and halted cellular metabolism. In order to sustain ROSaccrued consequences, plants tend to manage a fine homeostasis between the generation and antioxidants-mediated metabolisms of ROS and its reaction products.

Well-known for their involvement in the regulation of several non-stress-related processes, redox related components such as proteinaceous thiol members such as thioredoxin, glutaredoxin, and peroxiredoxin proteins, and key soluble redox-compounds namely ascorbate (AsA) and glutathione (GSH) are also listed as efficient managers of cellular redox homeostasis in plants. The management of the cellular redox homeostasis is also contributed by electron carriers and energy metabolism mediators such as non-phosphorylated (NAD+) and the phosphorylated (NADP+) coenzyme forms and their redox couples DHA/AsA, GSSG/GSH, NAD+/NADH and NADP+/NADPH. Moreover, intracellular concentrations of these cellular redox homeostasis managers in plant cells fluctuate with the external environments and mediate dynamic signaling in pant stress responses.

This research topic aims to exemplify new information on how redox homeostasis managers are modulated by environmental cues and what potential strategies are useful for improving cellular concentrations of major redox homeostasis managers. Additionally, it also aims to provide readers detailed updates on specific topics, and to highlight so far unexplored aspects in the current context.

**Citation:** Khan, N. A., Anjum, N. A., Sofo, A., Kizek, R., Baier, M., eds. (2016). Redox Homeostasis Managers in Plants under Environmental Stresses. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-878-8

# Table of Contents



Anja Steffen-Heins and Bianka Steffens

## Editorial: Redox Homeostasis Managers in Plants under Environmental Stresses

Naser A. Anjum<sup>1</sup> \*, Nafees A. Khan<sup>2</sup> \*, Adriano Sofo<sup>3</sup> \*, Margarete Baier <sup>4</sup> \* and Rene Kizek <sup>5</sup> \*

<sup>1</sup> Centre for Environmental and Marine Studies and Department of Chemistry, University of Aveiro, Aveiro, Portugal, <sup>2</sup> Department of Botany, Aligarh Muslim University, Aligarh, India, <sup>3</sup> School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, Potenza, Italy, <sup>4</sup> Plant Physiology, Dahlem Centre of Plant Sciences, Institute of Biology, Freie Universität Berlin, Berlin, Germany, <sup>5</sup> Department of Human Toxicology and Pharmacology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

Keywords: redox homeostasis, plant life, oxidative stress, environmental stress, redox couples

#### Edited by:

Bruno Silva Nunes, University of Porto, Portugal

#### Reviewed by:

Miguel Machado Santos, University of Porto, Portugal Glória Catarina Pinto, University of Aveiro, Portugal

#### \*Correspondence:

Naser A. Anjum anjum@ua.pt; Nafees A. Khan naf9@lycos.com; Adriano Sofo adriano.sofo@unibas.it; Margarete Baier margarete.baier@fu-berlin.de; Rene Kizek kizek@sci.muni.cz

#### Specialty section:

This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science

> Received: 18 March 2016 Accepted: 28 April 2016 Published: 13 May 2016

#### Citation:

Anjum NA, Khan NA, Sofo A, Baier M and Kizek R (2016) Editorial: Redox Homeostasis Managers in Plants under Environmental Stresses. Front. Environ. Sci. 4:35. doi: 10.3389/fenvs.2016.00035 **Redox Homeostasis Managers in Plants under Environmental Stresses**

**The Editorial on the Research Topic**

Environmental stresses, grouped broadly into abiotic (physical environment e.g., drought, temperature regimes, UV-B radiation, salinity, and metals/metalloids) and biotic (e.g., pathogen, herbivore) significantly modulate the survival, reproduction, and productivity of plants/crops (Redondo-Gómez, 2013). In particular, environmental stresses caused by climate change, such as drought, high salinity, and low and high temperatures are predicted to become more severe and widespread (Osakabe et al., 2013). At cellular level, the sustenance of plant life under stressful environment is controlled by homeostasis in the usual redox reactions. Redox reactions can contribute various reactive oxygen species (ROS). One-, two-, and three-electron reduction of O<sup>2</sup> or excitation of triplet oxygen (3O2) can occur and cause the formation of superoxide radical (O•− 2 ) or hydroperoxyl radical (HO• 2 ), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen (1O2), respectively. As integral signaling molecules, ROS regulate growth and development of plants, and also modulate their responses to biotic and/or abiotic stimuli (Baxter et al., 2014). However, beyond their steady-state cellular concentrations, ROS and their reaction products can modulate plant stress responses and/or severely impair the cellular redox homeostasis (Dietz, 2003; Oelze et al., 2008; Baxter et al., 2014; Juszczak et al.). Nevertheless, extreme environmental conditions are inevitable for plants and can excessively over-reduce or overoxidize cellular environment. Previous conditions are likely to cause an imbalance in the generation and metabolism of ROS (and their reaction products), loss in the cellular redox homeostasis and finally the arrest in the cellular metabolism (Foyer and Noctor, 2009, 2012).

Plants are equipped with several strategies to efficiently metabolize and tightly regulate the steady-state levels of cellular ROS (and its reaction products), and manage cellular redox homeostasis at its optimum. The list of major cellular redox homeostasis managers includes redoxrelated components such as proteinaceous thiol members such as thioredoxin, glutaredoxin, and peroxiredoxin proteins, and key soluble redox-compounds such as ascorbate (AsA) and glutathione (GSH). Electron carriers and energy metabolism mediators such as non-phosphorylated (NAD+) and the phosphorylated (NADP+) coenzyme forms and their redox couples DHA/AsA, GSSG/GSH, NAD+/NADH, and NADP+/NADPH also contribute to cellular redox homeostasis (Schafer and Buettner, 2001; Anjum et al., 2010, 2012a,b; Foyer and Noctor, 2011, 2012; Suzuki et al., 2012; Giordano, 2013). The cellular redox homeostasis has also been regarded as the major "integrator" of information from metabolism and the plant–environment relationship (Foyer and Noctor, 2009). Hence, unveiling insights into the role and underlying mechanisms of redox homeostasis in plants under environmental stresses has been the major focus of current plant research (Schafer and Buettner, 2001; Foyer and Noctor, 2009, 2011, 2012; Anjum et al., 2010, 2012a,b; Suzuki et al., 2012; Giordano, 2013).

The present volume "Redox Homeostasis Managers in Plants under Environmental Stresses" updates the readers on the subject, and discusses research progress and current understanding on the subject. Among the significant 17 article types, original research reports exemplified new information on how redox homeostasis managers are modulated by environmental cues and what potential strategies are useful for improving cellular concentrations of major redox homeostasis managers. Additionally, detailed updates to specific topics and so far unexplored aspects were highlighted by focused review/mini-review articles.

Cellular redox homeostasis is impacted by abiotic factors that can cause elevations in ROS (and their reaction products) at varying levels in the major energy organelles, such as chloroplast and mitochondria (Das et al.). Soil salinity is a serious threat to crop productivity worldwide that causes oxidative stress through imposing ion toxicity, osmotic stress, and metabolic imbalance (Adem et al., 2014; Abd Elgawad et al.). Notably, salinity stress significantly impacts electron flow in the electron transport chain in these organelles, disturbs the status of adenine (ATP) and pyridine nucleotides (NADH, NADPH), and eventually leads to elevation in cellular ROS and lipid peroxidation (LPO; Srivastava et al.). However, the extent of salinity-impact on ATP, NADH, NADPH, cellular ROS, and LPO can be higher in glycophytes (such as Brassica juncea; salt-sensitive) when compared with halophytes (such as Sesuvium portulacastrum; salt-tolerant; Srivastava et al.). Redox active compounds, AsA and GSH can play a significant role in the protection of plants against a number of abiotic stresses including temperature (low/chilling and high) and drought (Anjum et al.; Awasthi et al.; Das and Roychoudhury; Lukatkin et al.). Notably, the cellular level of AsA and GSH can differ in Vigna radiata during its ontogeny under drought exposure (Anjum et al.). In addition, sulfur nutrition was revealed as a potential strategy for the management of improved cellular pools of these redox active compounds. The role of polyamines and brassinosteroids for the maintenance of the cellular redox homeostasis in plants exposed to major abiotic stresses was critically discussed by Saha et al. and Vardhini and Anjum, respectively. Montero-Palmero et al. showed that plant endogenous factors like ethylene can modulate the early oxidative stress induced by mercury (Hg). Monoterpenoid indole alkaloids and phenols can be used as a defense tool against stress factors and can also benefit plants depleted in GSH (Vera-Reyes et al.). Dehydrogenases involved in the regeneration of NADPH such as glucose-6-phosphate dehydrogenase (G6PDH), 6 phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME), and NADP-isocitrate dehydrogenase (NADP-ICDH) can support the protection of plants against nitro-oxidative stress induced by adverse environmental conditions (Corpas and Barroso). Information is scanty in the literature on the mechanisms involved in the control of each mitochondrial enzyme at the post-translational level (Millar et al., 2011; Nunes-Nesi et al., 2013). To this end, Yoshida and Hisabori evidenced that oxidation can inactivate mitochondrial isocitrate dehydrogenase; whereas, the later can be reactivated by thioredoxin-dependent reduction in Arabidopsis.

Insights into the role of redox active compounds AsA and GSH, proteinaceous thiol members such as thioredoxins, peroxiredoxins, and glutaredoxins, electron carriers and energy metabolism mediators phosphorylated (NADP) and nonphosphorylated (NAD+) coenzyme in the ROS-metabolism and the maintenance of redox homeostasis in abiotic stressedplants were also critically discussed (Kapoor et al.). Notably, the enzymes of the AsA-GSH pathway, a key part of the network of reactions involving enzymes and metabolites with redox properties can have various subcellular isoforms, differ from each other (with respect to their spatial and temporal expression), and can also be differentially regulated by stress types (Anjum et al., 2010, 2012a,b; Gill and Tuteja, 2010). The knowledge gap available on the major mechanisms underlying the regulation of major isoforms of the AsA-GSH pathway enzymes was provided by Pandey et al. These authors provided major insights into the gene families of the AsA-GSH pathway, and also described their roles in the management of cellular redox homeostasis in plants exposed to abiotic and biotic stress conditions. Carvalho et al. elaborated the information available on the main mechanisms underlying plant tolerance to stresses (abiotic and biotic) via phenolic compounds (such as simple flavonoids like anthocyanins). Furthermore, it has recently been demonstrated that condensed proanthocyanidins (tannins) are solubilized into the vacuole or linked to cell wall polysaccharides and largely control the nutraceutical properties of the grape berry and its derivatives such as wine (Tenore et al., 2011; De Nisco et al., 2013).

An improved cellular redox homeostasis and plant-tolerance to environmental challenges are also achieved by employing several seed/plant-priming strategies (Tanou et al.; Bhanuprakash and Yogeesha, 2016). Notably, extreme abiotic and biotic stresses can severely impact or kill the organisms; whereas, low stress levels can exhibit priming effects and benefit stressed-plants (Hadacek and Bachmann). In an attempt to understand this idiosyncratic phenomenon, Hadacek and Bachmann explored the phenomenon of life from a more chemical perspective, elaborated insights into chemical structure diversity and recapitulated the basic reaction chemistry of low-molecularweight metabolites (LMWMs). Additionally, contributions of LMWMs to a homeodynamic systems chemistry of living organisms were also dissected. Non-metabolized and/or elevated levels of ROS (and its reaction products) are the major violators of cellular redox homeostasis in stressed plants. However, the science of cellular redox homeostasis is lagging behind due to a major problem related with the quantification ROS and also with the identification of their short lifetime. Thus, the technique of electron paramagnetic resonance (EPR) spectroscopy could be a panacea to the said issue and can allow disentangling the origin of specific ROS and transient alterations in their cellular levels (Steffen-Heins and Steffens).

Herein, research reports discussing "cellular redox homeostasis in plants" in context with stresses caused by climatic changes (such as drought and salinity) and toxic chemicals (such as Hg) were least explored. Also, report is scanty on the "cellular redox homeostasis in plants" vs. biotic factors. However, contributions gathered herein concluded that the "cellular redox homeostasis in plants" is central to the plant stress defense, and the future investigations in this area can help in dissecting more insights into plant responses to environmental stresses. Recent advances in the subject were nicely presented and elaborated, and also identified and listed important open questions and challenges in the article types. Further, this research topic advocates to intensify and integrate biochemical, physiological and molecular-genetic studies on various aspects of the "cellular redox homeostasis in plants." Contributions included in this e-book can be useful for budding scientists working on the subject, and can also encourage further dialog, research and development on the "cellular redox homeostasis in plants."

### AUTHOR CONTRIBUTIONS

NAA prepared the first draft of this "Editorial." All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

### REFERENCES


### FUNDING

NAA gratefully acknowledges the partial financial supports received from FCT (Government of Portugal) through contracts (SFRH/BPD/64690/2009 and SFRH/BPD/84671/2012), the Aveiro University Research Institute/CESAM (UID/AMB /50017/2013), and "COMPETE" through Project n.◦ FCOMP-01-0124-FEDER-02800 (FCT PTDC/AGR-PRO/40 91/2012).

### ACKNOWLEDGEMENTS

We are thankful to contributors for their significant contributions, and also to the reviewers who provided their timely evaluation of the manuscripts. We are also grateful to Prof. Donat Häder for providing us an opportunity to work on this exciting research topic. Timely help rendered by the Front. Environ. Sci. editorial office team is also appreciable that made our efforts successful in bringing out the present treatise on "Redox Homeostasis Managers in Plants under Environmental Stresses."


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Anjum, Khan, Sofo, Baier and Kizek. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Oxidative environment and redox homeostasis in plants: dissecting out significant contribution of major cellular organelles

#### *Priyanka Das 1, Kamlesh K. Nutan1, Sneh L. Singla-Pareek2 and Ashwani Pareek1 \**

*<sup>1</sup> Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India <sup>2</sup> Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India*

#### *Edited by:*

*Naser A. Anjum, University of Aveiro, Portugal*

#### *Reviewed by:*

*Naser A. Anjum, University of Aveiro, Portugal Brahma B. Panda, Berhampur University, India Yehia ElTemsah, Cairo University, Egypt*

#### *\*Correspondence:*

*Ashwani Pareek, Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, Room No. 401, New Delhi 110067, India e-mail: ashwanip@mail.jnu.ac.in*

Plant cells are often exposed to oxidative cellular environments which result in the generation of toxic reactive oxygen species (ROS). In order to detoxify the harmful ROS, plants have evolved various strategies including their scavenging and antioxidant machinery. Plant cells contain many enzymatic and non-enzymatic antioxidants which aid in removing the toxic oxygen molecules. Various antioxidant molecules localized within different cellular compartments play crucial role(s) during this process, which includes both redox-signaling and redox-homeostasis. The present review gives an overview of cellular oxidative environment, redox signaling operative within a cell and contributions of major cellular organelles toward maintaining the redox homeostasis. Additionally, the importance of various antioxidant enzymes working in an orchestrated and coordinated manner within a cell, to protect it from stress injury has been presented. We also present the state-of-the-art where transgenic approach has been used to improve stress tolerance in model and crop species by engineering one or more than one of these components of the ROS scavenging machinery.

**Keywords: redox homeostasis, reactive oxygen species, ascorbate peroxidase, catalase, superoxide dismutase, abiotic stress**

### **INTRODUCTION**

Plants are obligate aerobic organisms like animals and they require oxygen for mitochondrial energy production. Furthermore, plants can deal with much higher concentration of oxygen as the green tissues of plants continuously produce oxygen through the process of photosynthesis during day time. In plants, the green leaves contain 2.5 fold higher oxygen concentration than the non-green parts like root. In both green and non-green parts, the oxygen concentration is much higher than the oxygen concentration found in animal cells (Vanderkooi et al., 1991). Plant tissues experience wide oxygen fluctuations under abiotic stress conditions, making the surroundings strongly hypoxic (Bailey-Serres and Voesenek, 2008). Plant seeds also experience huge oxygen variations. When green young seeds are photosynthetically dynamic, the light-dark reaction generates large and quick variations in the internal oxygen concentrations of *Brassica napus*. The variation ranges from strong hyperoxia (*>*700μM in day) to severe hypoxia (*<*1mM in night). Similar situations have also been observed in many other species (Borisjuk and Rolletschesk, 2008).

As a natural result of the oxygen metabolism, plants continuously produce reactive oxygen molecules/species (ROS) like superoxides and peroxides (Panda et al., 2013; Kangasjarvi and Kangasjarvi, 2014; Vainonen and Kangasjarvi, 2014). Although, high concentration of these ROS has negative effect on plants, specific concentrations of ROS play vital roles in cell signaling. Continual exposure to ROS creates an oxidative environment which affects the redox balance of the cell. Alterations in redox state in intracellular region also have a major consequence on cell functions as various cellular signaling pathways regulating cell division and stress reaction systems are sensitive to redox situation (Chiu and Dawes, 2012). Severe redox situation often leads to senescence and death of the cell and ultimately the organism.

Antioxidants with low molecular weight like ascorbate, tocopherol and glutathione, are redox buffers which act as enzyme cofactors and play crucial roles in defense, cell proliferation to aging and death (Tokunaga et al., 2005). Antioxidants supply necessary information on redox state of the cell, and they control the expression of the genes linked with abiotic and biotic stresses to increase stress defense. Maintaining the level of these ROS at a balanced state is always crucial for plants and for this purpose, plants have adopted various cellular mechanisms. Growing facts suggest models for redox homeostasis where the antioxidant-ROS communications play as a metabolic interface for signals derived from metabolism and from the environment. Present topic talks about the roles of various cellular organelles in maintaining the redox homeostasis in plant cells and ultimately helping toward abiotic stress tolerance in plant.

### **OXIDATIVE ENVIRONMENT, ANTIOXIDANT INTERACTIONS AND REDOX SIGNALING IN PLANT CELL**

Oxidative environment is generated when ROS is produced by a specific or by combination of multiple stresses (Thorpe et al., 2004). Process of generation of oxygen in cell has been mentioned or reviewed by many researchers (Aung-Htut et al., 2012; Kumar et al., 2012; Khanna-Chopra et al., 2013; Ghosh et al., 2014). The first product of specialized water producing reactions catalyzed by oxidases is superoxide and from superoxide, other ROS are produced subsequently. Singlet oxygen is also produced while capturing of light and process of photochemistry is going on. Numerous enzymatic processes generate superoxide (O− 2 ) or hyderogen peroxide (H2O2). Most of the cellular compartments (chloroplast, mitochondria, peroxisome, and cytoplasm) in higher plants participate in the generation of ROS inside the cell (**Figure 1**).

Abiotic stresses like drought, salinity, low temperature or high temperature often limit the CO2 fixation and reduce the generation of NADP+ through Calvin cycle. Therefore, over-reduction of the photosynthetic electron transport chain (ETC) is occurred which generates superoxide radicals and singlet oxygen in the chloroplasts (Li and Jin, 2007). To avoid the over-reduction of the ETC under stress conditions, higher plants modified the pathway of photorespiration to regenerate NADP+ (Shao et al., 2006). H2O2 is generated in the peroxisomes as a by-product of photorespiratory pathway (Foyer and Noctor, 2005).

To control the production of the highly toxic ROS, higher plants possess enzymatic and non-enzymatic antioxidant defense systems that help in scavenging of ROS and protection of plant cells from oxidative damage (Foyer and Noctor, 2005). High accumulation of non-enzymatic ROS scavengers, and different biochemical properties, different localization and differential inducibility at the transcript or protein level of antioxidant enzymes provide the antioxidant systems, a very flexible unit that can control ROS accumulation temporally and spatially (Foyer and Noctor, 2005; Shao et al., 2006). The antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), ascorbate peroxidase (APX), GR and monodehydroascorbate reductase (MDAR) play major role in scavenging the toxic ROS inside the plant cell along with the non-enzymatic ROS scavengers like ascorbic acids and reduced glutathione (**Figure 1**).

Plants have evolved inbuilt mechanism to sense, transduce, and translate ROS signals into appropriate cellular responses. This particular process requires the existence of redox-sensitive proteins that can take part both in oxidation and reduction reactions and may regulate the switching-on or -off depending upon the cellular redox state (Shao et al., 2006). The redoxsensitive proteins are directly or indirectly oxidized by ROS via the ubiquitous redox-sensitive molecules, such as thioredoxins (Trxs) or glutathione (Nakashima and Yamaguchi-Shinozaki, 2006). The cellular metabolism under oxidative stress is directly modulated by redox-sensitive metabolic enzymes, but the redoxsensitive signaling proteins complete their action via downstream

signaling components, such as phosphatases, kinases and transcription factors (Foyer and Noctor, 2005; Li and Jin, 2007). Molecular mechanisms of redox-sensitive regulation of protein have also been explained for plants and other living organisms (Cvetkovska et al., 2005; Foyer and Noctor, 2005). ROS mediated signaling involves hetero-trimeric G-proteins and MAP kinase regulated protein phosphorylation and protein Tyr phosphatases (Pfannschmidt et al., 2003; Foyer and Noctor, 2005; Kiffin et al., 2006). Mitogen-activated protein kinase (MAPK) cascades are mainly engaged by eukaryotes which have got much concentration for research since long years. The minimal signal transduction unit is considered to have a stimulus-activatable MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), a MAP kinase (MAPK) and their downstream targets. A chronological phosphorylation-activation process begins which transmit the signal from the MAPKKK to the target, which may be a transcription factor (TF) whose activity and localization is influenced by phosphorylation. The proportions of phosphorylation activation and transmission indicate that MAPKKKs can be activated by particular stimuli and the signaling pathways may congregate at the MAPKK level of the cascade. A single MAPKK could then phosphorylate several MAPKs. The signaling through MAPKKKs and MAPKKs could continue through other mechanisms as well besides phosphorylation of their direct downstream targets (**Figure 2**). This occurs with the *Arabidopsis* MAPKKK, MEKK1, which may phosphorylate the WRKY53 TF and additionally, bind to its promoter which functions as an activator for transcription (Miao et al., 2007). Salinity and cold reactive MEKK1-MKK1/2-MPK4/6 signaling cascade (Teige et al., 2004), which appears to have a bi-directional communication with ROS: the MEKK1 protein have been reported to be stimulated and stabilize by H2O2 and also the MAPK components -MPK4 and MPK6 have been found to be activated by ROS and various abiotic stresses (Teige et al., 2004).

In higher plants, the biochemical and structural basis of kinase pathway activation by ROS is yet to be established, but thiol oxidation probably has a key contribution here (Yabuta et al., 2004; Foyer and Noctor, 2005). Stromal ferredoxin-thioredoxin system is the well-known redox signal transduction system in plants which functions during photosynthetic metabolism of carbon. Signal transmission occupies disulfide-thiol alteration in target enzymes (Yabuta et al., 2004). Increasing authentication shows that plant hormones are situated downstream of the ROS signal. Induction in accumulation of stress hormone, such as salicylic acid and ethylene, is caused by H2O2 (Kiffin et al., 2006). Plant hormones are not only placed downstream to the ROS signal, ROS also play a role as secondary messengers in many hormone signaling pathways (Kwon et al., 2006). It indicates that backward or forward interactions may possibly occur between different hormones and ROS (Rio et al., 2006; Terman and Brunk, 2006).

### **REDOX HOMEOSTASIS IN PLANTS**

Concurrent occurrence of both reduced and oxidized forms of electron transporters is required for competent flux through electron transport cascades in plants. This condition is known as redox poising and it involves an uninterrupted change of electrons to oxygen molecule from diverse sites in the respiratory and photosynthetic electron transport chains. The reactive character

**FIGURE 2 | Schematic depiction of cellular ROS sensing and signaling mechanisms through MAP kinase signaling pathway.** Intracellular ROS can also influence the ROS-induced mitogen-activated protein kinase (MAPK) signaling pathway through inhibition of MAPK phosphatases (PPases) or downstream transcription factors. Whereas, MAP kinases regulate gene expression by altering transcription factor activity through phosphorylation of serine and threonine residues, ROS regulation occurs by oxidation of cysteine residues.

of these ROS means not only that their increasing concentration should be controlled but also that they are capable to play as signaling molecules. The level of accretion of ROS is determined by the antioxidative system which enables cells to preserve the cellular components in an active state for metabolism.

Similar to many other aerobic animals, plants preserve most cytoplasmic thiols in the reduced (2SH) condition, as the low thiol disulfide redox potential imposed by millimolar concentrations of glutathione is the thiol buffer. Nevertheless, plant cells produce high concentrations of ascorbate, an added hydrophilic redox buffer which gives strong defense against oxidative stress. Redox homeostasis is directed by the large pools of these antioxidants which maintain the level of reductants and oxidants in a balanced state. Tochopherols (Vitamin E) are important liposoluble redox buffers produced by the plants. Although, tochopherol is known as a major singlet oxygen scavenger, it also can efficiently scavenge other ROS (Foyer and Noctor, 2005). Furthermore, as the tocopherol redox couple has an additional constructive midpoint potential than the ascorbate pool, it further amplifies the range of efficient superoxide scavenging. The capacity of the glutathione, ascorbate and tocopherol pools, to play as redox buffers in plant cells, is one of their significant characteristics.

ROS signaling pathways are made achievable by homeostatic regulation accomplished by antioxidant redox buffering. As the antioxidants constantly process ROS, they decide the duration and the specificity of the signal of ROS. Plant cells usually handle the high rate of generation of ROS in a very careful way. Even though, cellular oxidation is significant in all biotic and abiotic stress reactions, the level and physiological consequence of oxidative injury is arguable. For instance, plants with low cytosolic APX and CAT activities show less severe stress indications than the plants which require either one of these enzymes (Rizhsky et al., 2002). It has also been established that cell death mediated by singlet oxygen is not a direct consequence of damage *per se* but somewhat is genetically programmed through the EXECUTOR1 pathway (Wagner et al., 2004). Moreover, plants adapt very well to depletion of antioxidants by signaled induction of other defense systems such as: tocopherol-deficient Arabidopsis *vte* mutant seedlings have high amounts of lipid peroxides, but the mature plants show slightly abnormal phenotype (Kanwischer et al., 2005). Furthermore, it has been well-established that most of the cellular organelles play important roles to maintain the redox homeostasis in the plant cell (Andreev, 2012; Ferrández et al., 2012; Lázaro et al., 2013). Following section describes the contribution of major cell organelles toward maintaining cell redox homeostasis under oxidative environment.

### **INVOLVEMENT OF MAJOR CELLULAR ORGANELLES IN MAINTAINING REDOX HOMEOSTASIS IN PLANT CELL CONTRIBUTION OF CHLOROPLAST**

Dithiol-disulphide exchange based post translational alteration comprises a fast and reversible mechanism of regulation in a cell. Thus, it allows the competent adaptation of metabolism to the ever-changing environmental conditions. Trxs with a pair of cysteine residues at their active site act an important role in disulphide reduction of protein by using NADPH as reducing agent (Jacquot et al., 2009). This reaction is catalyzed by NADPHdependent thioredoxin reductase (NTR). All the living organisms (including Bacteria, animals, and plants) possess two component NTR/Trx redox systems (Meyer et al., 2005). However, plant chloroplasts have an intricate set of particular Trxs, which additionally utilize a chloroplast specific ferredoxin-dependent thioredoxin reductase (FTR), unlike the other heterotrophic organisms. Hence, rather than the NADPH, the redox regulation of chloroplast is mainly dependent on photosynthetic electron transport chain-reduced ferredoxin in the presence of light. It has been reported that a unique NTR with a Trx domain at its C-terminus (named as NTRC) is utilized in oxygenic photosynthetic organisms and is localized in chloroplasts (Serrato et al., 2004). NTRC is capable of reducing disulphides of the target proteins by using NADPH and hence, it performs as NTR/Trs system in a single polypeptide (Pérez-Ruiz and Cejudo, 2009). After discovery of these results, a new picture appeared according to which both NADPH and ferredoxin (FD) can be used for maintaining the chloroplast redox homeostasis (Spínola et al., 2008). At night, reduced FD become limiting and NADPH produced from the sugar play as a major source of reducing power and thus, NTRC play an essential role for maintaining the redox homeostasis. It has also been reported that non-green plastids also have the components of FTR/Trx system which suggests that the redox regulation is also occurring in the non-photosynthesizing plant parts (Balmer et al., 2006). The damage in the vital regulatory enzymes of starch synthesis i.e., ADP-glucose pyrophosphorylase, in the NTRC knock out mutant indicated that NTRC might play important role in the redox homeostasis of non-green tissues. The expression analysis of NTRC further showed that it is found in both green chloroplasts and non-green plastids and it could regulate the redox homeostasis in the green and non-green plant parts (Kirchsteiger et al., 2012). Taking together all the recent findings, it can be concluded that redox regulation is an important function of all the plastids (including green and non-green plastids). However, in chloroplasts this depends on light or sugar and in non-green plastids it depends entirely on the NADPH which is generated from the metabolism of sucrose by the oxidative pentose phosphate pathway (**Figure 3**).

### **CONTRIBUTION OF MITOCHONDRIA**

Mitochondria also play important role in plant cell redox homeostasis. In the photosynthetic cells, the power house mitochondria are the second key organelle after chloroplasts. Mitochondria have a great contribution toward redox homeostasis during the oxidative reactions operating in mitochondria and peroxisome in the light. Plant mitochondria have specific ETC components which functions in photorespiration process. In leaves, oxygenic photosynthesis certify that mitochondria function in a carbohydrate and oxygen loaded environment. This specific cellular environmental condition ensures the existence of mitochondrial redox signaling and homeostasis. Malate and pyruvate are imported to mitochondria and subsequently oxidized to produce ATP. Another essential function of mitochondria is metabolism of compounds like glutamate and other amino acids, and production of precursors for biosynthetic processes (Ishizaki et al., 2005). In these processes, the expression of the necessary proteins

depends upon the developmental stage of the plant and type of the cell. Tricarboxylic acid (TCA) cycle is common to all plant mitochondria but, here, the TCA cycle operates depending on tissue type or environmental factors. Here, APX functions to dissipate electrons without generation of ATP and thus, prevent the formation of ROS during over reduction of the mitochondrial ETC (Vanlerberghe and McIntosh, 1992). Interestingly, APX is a target of redox-modification via the mitochondrial thioredoxin system (Gray et al., 2004). Induction of APX transcription is caused by abiotic stress factors such as low temperature (Vanlerberghe and McIntosh, 1992). By using male sterile mutant tobacco, the role of mitochondria in cellular homeostasis has been shown (Dutilleul et al., 2003). These mutant plants do not have the functional complex I, which is a key complex required for maintaining the redox homeostasis in cell (Noctor et al., 2004). It has also been reported that knockout plants lacking type II peroxiredoxin F of mitochondria possess a strong phenotype, particularly under stress and when APX is inhibited (Finkemeier et al., 2005). Ultimately, interruption of the TCA cycle by decreasing the quantity of mitochondrial MDH (malate dehdrogenase) had remarkable effect on photosynthesis and plant growth (Nunes-Nesi et al., 2005).

### **CONTRIBUTION OF PEROXISOME**

Peroxisome is contributing majorly in maintaining cellular redox homeostasis by having the key enzyme CAT inside the peroxisomal boundary. CAT depletes the peroxisomal H2O2 generated through photorespiratory glycolate oxidase pathway and maintains redox homeostasis of the cell. Plants deficient in CAT have always accumulated high levels of H2O2. It has been reported that *cat2* mutants grown at relatively low light, possess increased diaminobenzidine staining (Bueso et al., 2007). It has also been reported that *cat2* and *cat2:cat3* knockout plants contains two folds increase in extractable H2O2 (Hu et al., 2010). The CATlacking tobacco plants are also more sensitive to diseases as they are not altered in their protein, which is related to pathogenesis, but the tobacco leaves show bleaching due to H2O2 accumulation in peroxisomes (Chamnongpol et al., 1998). It has also been reported that young leaves are less susceptible than the older leaves, in *Cat1* deficient tobacco plants, upon high light exposure (Willekens et al., 1997). Remarkably, double antisense plants deficient in both APX and CAT showed decreased photosynthesis. The reduction of photosynthetic activity is regarded as an approach to avoid the formation of ROS (Rizhsky et al., 2002). Tobacco mutants with increased CAT activity confirmed higher photosynthesis rates under photorespiratory situations than the control, probably because these plants are more tolerant to O2 inhibition of photosynthesis (Zelitch, 1990). Willekens et al. (1997) also reported that *Cat1* deficient tobacco plants were unable to maintain ascorbate, particularly glutathione pools in the reduced state when exposed to elevated light conditions. Therefore, peroxisomal localized CAT is an essential enzyme for protecting ascorbate and glutathione pools from oxidation. Additionally, Willekens et al. (1997) also reported that glutathione are the major sulfohydryl component in plants cells, for maintaining the redox homeostasis in light stressed cells. Brisson et al. (1998) have reported that increase in CAT activity reduces the photorespiratory loss of CO2.

### **CONTRIBUTION OF VACUOLE**

It has been known that the antioxidant system in the vacuolar compartment is comprised of various components of enzymatic and non-enzymatic origin. Apart from the cell wall, Class III peroxidases (POX) are also localized inside the vacuoles and play significant role to quench ROS inside the vacuole, where the secondary metabolites accumulate. Although, the exact function of vacuolar POX is not known, few recent reports show that the vacuolar POX control the level of H2O2 in photosynthesizing plant cells at the time of oxidation of some vacuolar phenolic substrates with H2O2 as an electron acceptor (Costa et al., 2008; Brunetti et al., 2011). The presence of POX in the vacuole and the apoplast is a feature of these subcellular compartments known to gather the major part of secondary metabolites which serves as peroxidase substrates (Idanheimo et al., 2014). It has been reported that vacuoles can generate ROS by a mechanism comparable to that in the plasmalemma-apoplast system. This mechanism is

supported by operation of the tonoplast located NADPH oxidase and the vacuolar or tonoplast-surface located superoxide dismutase. These data were acquired from proteomic analysis of the tonoplast membrane proteins and biochemical recognition of the enzymes (Shi et al., 2007; Whiteman et al., 2008; Pradedova et al., 2011). However, the convincing and direct experimental confirmation for functioning of such enzymes in the vacuolar compartment is not yet reported. The presence of superoxide producing NADPH oxidase in membranes of animal phagocytes and lysosomes cannot be taken as enough evidence for the presence of a similar enzyme in the tonoplast of plant cell. The schematic representation of mechanism of ROS quenching involving vacuolar enzymes is shown in **Figure 4**.

### **CONTRIBUTION OF CELL WALL AND PLASMA MEMBRANE**

Apart from the major cell organelles, cell wall also plays crucial role in maintaining redox balance in the cell. Similar to the other organelles, oxidative burst also occurs in the plant cell wall where, molecular oxygen is reduced to O− <sup>2</sup> and then undergoes spontaneous dismutation at a higher rate at acidic pH (O'Brien et al., 2012). Class III POX present in the cell wall are able to oxidize NADH and catalyze the formation of O− <sup>2</sup> . The cell wall oxidases catalyze the oxidation of NADH to NAD+, which in turn reduces oxygen to superoxide. This superoxide consequently dismutated to produce H2O2 andO2 (Bhattachrjee, 2005; O'Brien et al., 2012). Additionally, amine oxidases and oxalate oxidases have been proposed to generate H2O2 in the apoplast (Munné-Bosch et al., 2013). NADPH oxidase present in cell membrane is another source of H2O2 for oxidative burst (O'Brien et al., 2012). Aluminum, a soil pollutant, is also responsible for oxidative burst through activating the cell wall-NADH peroxidase and/or plasma membrane-associated NADPH oxidase (Achary et al., 2012). However, it is evident that presence of SOD in the cell wall is responsible for the efficient scavenging of the O− <sup>2</sup> (Apel and Hirt, 2004). It has also been reported that along with class III POX, APX is also present in cell wall and plasma membrane which is responsible for depletion of H2O2 and helps in maintaining cellular redox homeostasis (Apel and Hirt, 2004; O'Brien et al., 2012).

### **CROSS TALK AMONG CELLULAR ORGANELLES**

The peroxisomal extension, named peroxules, can expand over the chloroplastic exterior and curl around it, in a very quick manner and connect with other peroxisomes (Sinclair et al., 2009). Morphology of peroxisome can modify under stress situations which induce a quick key between spherical motile organelles with extensive tubular-beaded shape with extended peroxules (Sinclair et al., 2009). Stromules are stroma-filled tubules present in chloroplasts, consisting of thin extensions of the stroma (Hanson and Sattarzadeh, 2008) and these can often join together and have been shown to enter into channels of the nucleus (Kwok and Hanson, 2004). Chloroplasts, peroxisome and mitochondria have high rates of ROS metabolism which vary with the changing environmental conditions. Close interactions between chloroplast, peroxisomes and mitochondria could enhance cellular metabolic synchronization under stress situations and contribute to plant stress acceptance/tolerance (Rivero et al., 2009). Furthermore, increase of mitochondria and peroxisomes at the diffusion/penetration site of a fungus has been shown which probably occur for detoxification of the ROS at the infected site of the fungus *Erysiphe cichoracearum* (Koh et al., 2005). Form the above studies, it is quite convincing to state that that the cellular organellar crosstalk play significant role in cell signaling, avoiding stress situation and maintaining the cell redox homeostasis.

### **DEVELOPMENT OF TRANSGENIC PLANTS TOLERANT TO ABIOTIC STRESS BY ENHANCING ROS DEFENSE MECHANISMS**

In past, researchers have developed several transgenic plants by manipulating various genes involved in enzymatic and nonenzymatic ROS scavenging mechanisms which have shown increased tolerance to abiotic stresses (**Table 1**).

Over-expression of genes encoding ROS-scavenging enzymes such as SOD (Prashanth et al., 2008), CAT (Al-Taweel et al., 2007), APX (Kim et al., 2008), MDAR (Etrayeb et al., 2007), DHAR (Ushimaru et al., 2006), GR (Kornyeyev et al., 2003) and GPX (Gaber et al., 2006) in various plants isolated from same or different organisms were shown to possess higher tolerance to one or more abiotic stresses by minimizing the oxidative damage. Complete neutralization of ROS molecules involves more than one enzymes localized in same or different sub cellular compartments of cell. Transgenic Cassava (*Manihot esculenta* Crantz) has also shown the increased level of other important ROS scavenging enzymes such as MDR, DHAR, and GR.

Similarly, overexpression of critical enzymes involved in the biosynthetic pathway of antioxidants play a significant role in combating different abiotic stresses. Overexpression of P5CS (Yamada et al., 2005; Vendruscolo et al., 2007), a key enzyme for proline biosynthesis leads to increased tolerance to drought in transgenic plants. Liu et al. (2008) generated tobacco transgenic


**Table 1 | Representative reports for raising transgenic plants by overexpressing enzymes involved in ROS scavenging, which show improved tolerance to various abiotic stresses.**

plants by overexpressing VTE1 gene, encoding tocopherol cyclase (VTE1), an important enzyme involved in tocopherol biosynthesis. They have showed that the VTE1 overexpressing plants have higher tolerance to drought. Increased accumulation of another important antioxidant -ascorbic acid in AtERF98 TF overexpressing transgenic arabidopsis, showed increased tolerance to salinity (Zhang et al., 2012).

Apart from ROS-scavenging enzymes and non-enzymatic antioxidants, over-expressing ROS-responsive signaling and regulatory genes also responsible for stress tolerance in plants. The regulatory genes which regulate a large set of genes involved in acclimation mechanisms, including ROS-scavenging enzymes proved beneficial in enhancing tolerance to abiotic stresses such as drought, salinity, oxidative, cold and heavy metal stress. In Arabidopsis, over-expression of mitogen-activated kinase kinase 1 (MKK1) enhanced the activity of MAPK cascade, which is also activated by ROS (Teige et al., 2004; Wrzaczek et al., 2013) leads to increased tolerance to abiotic stresses by controlling stress-associated ROS levels under abiotic stress (Xing et al., 2008). Likewise, over-expression of transcription factors (*Zat12* or *JERF3, Zat10*) control the expression of various ROSscavenging genes encoding enzymes showed higher tolerance to salt, drought or osmotic stresses (Sakamoto et al., 2004; Davletova et al., 2005). Rai et al. (2013) have reported that overexpression of AtDREB1A/CBF3 of Arabidopsis under the control of stress inducible promoter (rd29A) in tomato (cv. Kashi Vishesh) showed higher accumulation of ROS scavenging enzymes and antioxidants with greater tolerance to drought-induced oxidative stress.

It has been established that the transgenic plants produced through gene pyramiding or co-expression of several antioxidant genes could able to give better stress tolerance than the plants overexpressing a single antioxidant gene (**Table 2**). It has been reported that co-expression of Mn-SOD and APX could able enhance multiple abiotic stress tolerance in *Nicotiana tabacum*. Co-expression of maize ZmCu/ZnSOD and ZmCAT


**Table 2 | Representative reports for raising transgenic plants by co-expressing enzymes involved in ROS scavenging, which show improved tolerance to various abiotic stresses.**

showed higher photosynthetic efficiency and salinity tolerance ability of transgenic cabbage (*Brassica campestris* L.) better than the independent ZmCu/ZnSOD and ZmCAT transgenic plant (Tseng et al., 2007). Likewise, co-expression of MeAPX2 and MeCu/ZnSOD in cassava (*Manihot esculenta* Crantz) showed higher tolerance to MV mediated H2O2 stress as well as two fold tolerance to chilling stress as compare to the wild type plants (Xu et al., 2014).

### **CONCLUSION AND FUTURE PROSPECTS**

Normally, ROS are generated by metabolic activity of the plants and act as signaling molecules for activating plant metabolic pathway. However, under environmental stresses, generation of ROS increase in different compartments of the cell such as chloroplast, peroxisomes and mitochondria. Higher accumulation of ROS leads to oxidative stress in plant causing damage to the cell membranes (lipid peroxidation) and biomolecules like nucleic acid, protein and lipid by oxidative damage. To combat the harmful effect of increased ROS accumulation, plants are equipped with effective ROS scavenging mechanisms. Plants have evolved two types of scavenging tools (i) scavenging enzymes such as SOD, CAT, MDAR, dehydroascorbate reductase (DHAR), GR and glutathione peroxidase (GP) and (ii) antioxidant molecules like ascorbic acid, α-tocopherols, glutathione, proline, flavonoids and carotenoids. ROS are key signaling molecules interacting with each other and with other cellular antioxidant systems to maintain proper balance between various cellular metabolic pathways, which get disrupted under unfavorable environments. Therefore, it is not the ROS, but their concentration in cell which decides their good or bad effect on plant. A lot of information about the ROS generation, role of free radicals in intra cellular communication and their effective scavenging have been accessible, but there are gaps in our understanding of complete ROS scavenging and signaling pathway. Future research in this area will be useful for designing the strategy to achieve the potential yield under unfavorable environments. Although, through transgenic technology of ROS scavenging components, abiotic biotic stress tolerance in various crop plants has been improved to some extent, this needs to be improved further in future by gene pyramiding to achieve the near potential yield of crops under rapidly changing climate.

### **ACKNOWLEDGMENTS**

The authors gratefully acknowledge financial support from University Grant Commission (through resource network program to JNU and Dr D. S. Kothari fellowship to PD) and Department of Biotechnology, Government of India.

### **REFERENCES**


seedling tolerance to salinity and osmotic stresses. *Plant Cell Rep.* 29, 917–926. doi: 10.1007/s00299-010-0878-9


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 29 November 2014; accepted: 30 December 2014; published online: 15 January 2015.*

*Citation: Das P, Nutan KK, Singla-Pareek SL and Pareek A (2015) Oxidative environment and redox homeostasis in plants: dissecting out significant contribution of major cellular organelles. Front. Environ. Sci. 2:70. doi: 10.3389/fenvs.2014.00070*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2015 Das, Nutan, Singla-Pareek and Pareek. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

Edited by: *Naser A. Anjum, University of Aveiro, Portugal*

Reviewed by:

*Naser A. Anjum, University of Aveiro, Portugal Girdhar Kumar Pandey, University of Delhi, India Polavarapu Bilhan Kavi Kishor, Osmania University, India*

#### \*Correspondence:

*Ashish K. Srivastava, Plant Stress Physiology and Biotechnology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India ashishbarc@gmail.com*

#### †Present Address:

*Sudhakar Srivastava, Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi, India; Vinayak H. Lokhande, Department of Botany, Shri Shiv Chhatrapati College of Art, Commerce and Science, Pune, India*

#### Specialty section:

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science*

> Received: *30 December 2014* Accepted: *28 February 2015* Published: *23 March 2015*

#### Citation:

*Srivastava AK, Srivastava S, Lokhande VH, D'Souza SF and Suprasanna P (2015) Salt stress reveals differential antioxidant and energetics responses in glycophyte (Brassica juncea L.) and halophyte (Sesuvium portulacastrum L.). Front. Environ. Sci. 3:19. doi: 10.3389/fenvs.2015.00019*

## Salt stress reveals differential antioxidant and energetics responses in glycophyte (Brassica juncea L.) and halophyte (Sesuvium portulacastrum L.)

Ashish K. Srivastava\*, Sudhakar Srivastava † , Vinayak H. Lokhande † , Stanislaus F. D'Souza and Penna Suprasanna

*Plant Stress Physiology and Biotechnology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India*

Salt stress, considered as one of the major environmental factors, decreases crop productivity world-wide and hence, investigations are being made to understand the cellular basis of salt tolerance in plants. In our earlier studies, maintenance of redox homeostasis and energetics were found as key determinants of salt tolerance in a halophyte *Sesuvium portulacastrum* (high salt accumulator). The redox homeostasis is defined as integrated ratio of different redox couples present inside the cell. In recent years, it has also been proposed as general stress response regulator in plants, bacteria as well as animals. In view of this, present study was performed to compare responses of redox state and energetics of *S. portulacastrum* with a glycophyte *Brassica juncea* (low salt accumulator). The data revealed activation of antioxidant defense in *S. portulacastrum* which either avoided or delayed the accumulation of different reactive oxygen species (ROS). In contrast, due to the lack of co-ordination, although the non-enzymatic antioxidants were increased, significant oxidative damage was seen in *B. juncea*. Further, the decreased NADPH oxidase activity suggested that basal redox signaling was also affected in *B. juncea*. In order to correlate these changes with chloroplastic and mitochondrial electron transport chain, NADP/NADPH and NAD/NADH ratios were measured. The NADP/NADPH ratio suggested that the process of photosynthesis was minimally affected in *S. portulacastrum* which might have contributed to its lower level of ROS under salt stress. The comparatively lower NAD/NADH and ATP/ADP ratios in *S. portulacastrum* as compared to *B. juncea* indicated the active and better utilization of energy generated to support different processes associated with salt tolerance. Thus, the findings suggest that co-ordinated regulation of antioxidant defense to avoid oxidative damage and proper utilization of energy are the key determinants of salt-tolerance in plants.

Keywords: antioxidants, ascorbate, energetics, glutathione, redox state, reactive oxygen species

### Introduction

A major challenge toward world agriculture involves production of 70% more food crop for an additional 2.3 billion people by 2050 (FAO, 2009). Salinity is a major stress limiting the increase in the demand for food crops. More than 20% of cultivated land worldwide (∼ about 45 hectares) is affected by salt stress and the amount is increasing day by day. Plants on the basis of adaptive evolution can be classified roughly into two major types: the halophytes (that can withstand salinity) and glycophytes (that cannot withstand salinity and eventually die). Majority of crop species belong to this second category. Thus salinity is considered as one of the most important stresses that hamper crop productivity worldwide (Gupta and Huang, 2014). During salinity stress, availability of atmospheric CO<sup>2</sup> is reduced because of an increased stomatal closure in order to avoid water loss via transpiration and hence, consumption of NADPH by the Calvin cycle is decreased. Due to the over-reduction of electron transport chain, electrons get misleaded from oxygen to form reactive oxygen species (ROSs) like superoxide radicals (O2-) and hydrogen peroxide (H2O2). Additionally, to meet the increased energy demand of the cell which can support different defense processes such as enhanced antioxidant capacity, osmolytes biosynthesis, ion transport and vacuolar sequestration, the activity of mitochondrial electron transport is also increased which further contributes to the generation of ROSs (Munns and Tester, 2008). Plants have complex antioxidant defense mechanisms including superoxide dismutase (SOD) and the ascorbate (ASC)-glutathione (GSH) cycle (Mittler, 2002). SOD constitutes the first line of defense converting O•− 2 to hydrogen peroxide (H2O2), which is further reduced to water and oxygen by ascorbate peroxidase (APX) and catalase (CAT). APX uses two molecules of ASC to reduce H2O<sup>2</sup> to water, with the concomitant generation of two molecules of monodehydroascorbate (MDHA), whereas CAT does not need any reductant for action against H2O2. Regeneration of ASC from MDHA occurs in sequential steps and utilizes GSH. This results in generation of oxidized glutathione (GSSG), which is in turn, re-reduced to GSH by NADPH, a reaction catalyzed by glutathione reductase (GR). Ascorbate and GSH both can accumulate in millimolar concentrations in cells and function as molecular antioxidants, in addition to serving various other roles, reacting directly with various ROS (Noctor and Foyer, 1998).

Among different experimental approaches adopted to understand plant responses to salt stress, one of the strategies is to compare halophytes and glycophytes. In this context, Thellungiella salsuginea is widely used as halophytic model and its responses are compared with its close relative Arabidopsis thaliana. The lipidomic analysis has revealed that remodeling of plastidic lipids is important for maintaining the integrity and fluidity of plastidic membranes which contributes to PEG-induced osmotic tolerance of T. salsuginea (Yu and Li, 2014). A proteomics study has been done to identify novel proteins associated with high salt tolerance of T. salsuginea (Vera-Estrella et al., 2014). The physiological responses of T. salsuginea and A. thaliana have also been studied to understand the mechanism of boron exclusion and tolerance in plants (Lamdan et al., 2012). The species of Eutrema such as Eutrema parvulum and E. salsugineum have been contrasted with A. thaliana to understand the regulatory role of genes associated with aldehyde dehydrogenase gene superfamily (Hou and Bartels, 2015). Using Mesembryanthemum crystallinum (halophyte) and Brassica juncea, the mechanism of nickel accumulation and tolerance has been studied (Amari et al., 2014). The halophyte Cakile maritima has been compared with Brassica juncea to understand cadmium accumulation and tolerance (Taamalli et al., 2014). In the same line, Sesuvium portulacastrum is a mangrove associate which is known for its high capacity to accumulate salt. Its physiological, redox, and energetics behavior toward salt stress has been reported (Lokhande et al., 2011). In the present study, these responses were compared with that of B. juncea which is a glycophyte and low salt accumulator. The findings confirmed the significance of co-ordinated antioxidant responses and effective utilization of energy as important determinants of salt tolerance in plants.

### Materials and Methods

### Plant Material and Treatment Conditions

The seeds of Indian mustard (B. juncea cv. TM- 2) were surface sterilized with 30% ethanol for 3 min and then washed thoroughly to remove any traces of ethanol. They were then allowed to germinate in plastic pots containing sand: soil (1:1) mixture at 25◦C under light providing 115µmol photons m−<sup>2</sup> s −1 illumination, with 12 h photoperiod. After 6-days of germination, water was changed with ½strength Murashige and Skoog's (MS) medium (Murashige and Skoog, 1962). After 15-days of germination, plants having secondary leaves were used for salt treatment. The Sesuvium—MH clone (Lokhande et al., 2010) was used for the present study. Four nodal sectors (∼4.0 cm) containing single pre-existing axillary bud with two opposite leaves were planted in the plastic pots containing sand: soil (1:1) mixture. The 45 day-old plants were used for the present study. For salt treatment, B. juncea was subjected to 250 mM NaCl, while S. portulacastrum was given two different NaCl concentrations (250 or 1000 mM). All the salt solutions were prepared in ½strength MS medium. An independent set with no NaCl was maintained as control. At 2, 4, and 8-days after treatment, secondary leaves were harvested and stored at -80◦C until further analyses. The NaCl concentrations were selected on the basis of preliminary experiments to assess plant tolerance in terms of growth to various NaCl concentrations (100–300 mM for B. juncea and 200–1000 mM for S. portulacastrum).

### Assay of Reactive Oxygen Species (Superoxide radicals, Hydrogen Peroxide) and the Level of Malondialdehyde

For the estimation of hydrogen peroxide (H2O2) levels, plant samples were homogenized in 0.5% (w/v) trichloroacetic acid (TCA) in an ice bath and centrifuged at 14,000 × g for 15 min at 4 ◦C. For H2O<sup>2</sup> determination, 0.5 ml of supernatant was mixed with 0.5 ml 100 mM potassium phosphate buffer (pH 7.0) and 1 ml of freshly prepared 1 M potassium iodide. Reaction was allowed to develop for 1 h in dark and absorbance was measured at 390 nm (Alexieva et al., 2001). The amount of H2O<sup>2</sup>

was calculated from a standard curve prepared using the known concentrations of H2O2. The rate of superoxide radicals (O•− 2 ) production was measured following the method of Chaitanya and Naithani (1994). About 500 mg of fresh plant samples were homogenized under N<sup>2</sup> atmosphere in cold (0-4◦C) in 100 mM sodium phosphate buffer (pH 7.2) containing 10 mM sodium azide to inhibit SOD activity. After centrifugation at 22,000 × g at 4◦C for 20 min, the level of O•− <sup>2</sup> was measured in the supernatant by its capacity to reduce NBT (extinction coefficient; ε = 12.8 mM−<sup>1</sup> cm−<sup>1</sup> ). The reaction mixture (1 ml) contained 100 mM sodium phosphate buffer (pH 7.8), 0.05% (w/v) NBT (Nitro blue tetrazolium chloride), 10 mM sodium azide and 0.2 ml of extract. Absorbance was measured at 580 nm at 0 and 60 min. The level of O•− 2 is expressed as an increase in absorbance per min per gram fresh weight. Lipid peroxidation was determined by estimation of the malondialdehyde (MDA) content following Heath and Packer (1968) as described previously (Srivastava et al., 2006).

### Assay of NADPH Oxidase Activity

Plants were homogenized in 20 mM HEPES [4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid; pH 7.0] containing 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 2.5% polyvinylpyrrolidone (PVP) under chilled conditions. Homogenate was squeezed through four layers of cold cheese cloth and centrifuged at 12,000 × g for 15 min at 4◦C. Protein content of supernatant was measured following the protocol of Lowry et al. (1951). NADPH-dependent O •− 2 generation was measured using NBT as an electron acceptor, whose reduction was monitored at 530 nm. Monoformazan concentrations (and therefore O•− 2 concentrations) were calculated using ε of 12.8 mM−<sup>1</sup> cm−<sup>1</sup> . The reaction mixture consisted of Tris buffer (50 mM Tris-HCI, pH 7.4), 5 mM NBT, 1 mM MgCl2, 1 mM CaCl2, 5 mM NADPH and a suitable aliquot of enzyme extract. The selective reduction of NBT by O•− <sup>2</sup> was calculated from the difference in the NBT reduction rate in the presence and absence of SOD (50–100 units ml−<sup>1</sup> ; Sigma, USA). No NBT reduction with NADPH was observed in the absence of protein fractions.

### Assays of Enzymes of Antioxidant System

For the assay of antioxidant enzymes, plant samples (500 mg) were homogenized in 100 mM chilled potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 1 mM PMSF and 1% PVP (soluble, MW 3,60,000) at 4◦C. Homogenate was squeezed through four layers of cold cheese cloth and the extract thus obtained was centrifuged at 15,000 g for 15 min at 4◦C. For all enzyme assays, respective sample and reagent blanks were run in duplicate. The activity of SOD (EC 1.15.1.1) was assayed following the method of Beauchamp and Fridovich (1971). The reaction mixture for SOD activity assay contained 40 mM phosphate buffer (pH 7.8), 13 mM methionine, 75µM NBT, 2µM riboflavin, 0.1 mM EDTA and a suitable aliquot of enzyme extract. After the reaction under light for 15 min, the absorbance was taken at 560 nm. One unit of activity is the amount of protein required to inhibit 50% initial reduction of NBT under light. For measurement of the CAT (EC 1.11.1.6) activity, extraction was done in the buffer containing 50 mM Tris–HCl (pH 7.0), 0.1 mM EDTA, 1 mM PMSF and 0.3 g g−<sup>1</sup> fw PVP. Activity was measured by the method of Aebi (1974). The reaction mixture comprised of 50 mM sodium phosphate buffer (pH 7.0), 20 mM H2O<sup>2</sup> and suitable aliquot of enzyme. Decrease in the absorbance was taken at 240 nm (molar extinction coefficient of H2O<sup>2</sup> was 0.04 cm<sup>2</sup> µmol−<sup>1</sup> ). Enzyme activity was expressed as units mg−<sup>1</sup> protein. The activity of APX (EC 1.11.1.11) was measured by estimating the rate of ascorbate oxidation (extinction coefficient 2.8 mM−<sup>1</sup> cm−<sup>1</sup> ). The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 0.1 mM H2O2, 0.5 mM sodium ascorbate, 0.1 mM EDTA and suitable aliquot of enzyme. The change in absorbance was monitored at 290 nm (Nakano and

FIGURE 1 | Time dependent modulation of superoxide radicals (A), hydrogen peroxide (B) and malondialdehyde (C) in NaCl-exposed Sesuvium portulacastrum and Brassica juncea. All values represent the mean of three replicates. Different letters indicate significantly different values at a particular duration (DMRT, *P* = 0.01).

Asada, 1981) and enzyme activity was expressed as units mg−<sup>1</sup> protein. For the estimation of the GR (EC 1.6.4.2) activity plant material was extracted in 0.1 M potassium phosphate buffer (pH 7.5) containing 0.5 mM EDTA. Activity was assayed by following the method of Smith et al. (1988). The reaction was started by adding following in order; 1.0 ml of 0.2 M potassium phosphate buffer (pH 7.5) containing 1 mM EDTA, 500µl 3 mM 5,5′ dithiobis (2-nitrobenzoic acid) in 0.01 M phosphate buffer (pH 7.5), 250µl H2O, 100µl 2 mM NADPH, 50µl enzyme extract, and 100µl 20 mM GSSG. The increase in absorbance was monitored for 5 min at 412 nm. The rate of enzyme activity was calculated using standard curve prepared by known amounts of GR (Sigma, USA). Activity of enzyme was expressed as units mg−<sup>1</sup> protein.

### Estimation of Redox Couples (GSH/GSSG and ASC/DHA Ratio)

The level of reduced (GSH) and oxidized (GSSG) glutathione was determined fluorometrically using o–phthaldialdehyde (OPT) as fluorophore (Hissin and Hilf, 1976). The level of total, reduced and oxidized ascorbate (ASC) contents in plants was measured following the protocol of Gillespie and Ainsworth (2007). Plant samples (50 mg) were homogenized in 1 ml 6% trichloroacetic acid (TCA) under chilled conditions and centrifuged at 13,000 × g for 5 min at 4◦C. To 200µl of sample 100µl 75 mM phosphate buffer (pH 7.0) was added. In total ASC, 100µl DTT (dithiothreitol; 10 mM) was added and incubated for 10 min at room temperature to reduce the pool of oxidized ASC. Then, 100µl NEM (N-ethylmaleimide; 0.5%) was added to remove excess DTT. For reaction, 500µl 10% TCA, 400µl 43% orthophosphoric acid, 400µl 4% 2,2′ -bipyridyl, and 200µl 3% FeCl<sup>3</sup> were added to all tubes. After incubation at 37◦C for 1 h, absorbance was measured at 525 nm. The level of dehydroascorbate (DHA) was calculated by subtracting ASC values from total ASC.

### Determination of Adenine and Pyridine Nucleotides

The analysis of adenine and pyridine nucleotides was performed by High Performance Liquid Chromatography (HPLC) as described previously by Srivastava et al. (2011). In brief, samples (100 mg) were subjected to either acid extraction using 0.6 M perchloric acid (for the measurement of ATP, ADP, NADP, and NAD) or alkaline extraction using 0.5 M potassium hydroxide (for the measurement of NADPH and NADH). The extract was centrifuged at 14,000 × g at 4◦C for 10 min followed by neutralization with either 0.5 M KOH or 1 M KH2PO4, respectively and re-centrifuged at 14,000 × g at 4◦C for 10 min to remove the precipitate. Supernatant was filtered using 0.22µm syringe filters and used for the HPLC injection. The mobile phase consisted of 0.1 M KH2PO<sup>4</sup> solution at pH 6.0 (Buffer A) and a 0.1 M KH2PO<sup>4</sup> solution at pH 6.0, containing 10% (v/v) of CH3OH (Buffer B). The chromatographic conditions were as follows: 8 min at 100% of buffer A, 7 min at up to 25% of buffer B, 2.5 min at up to 90% of buffer B, 2.5 min at up to 100% of buffer B, held for 7 min at 100% B, 5 min at up to 100% buffer A and held for 8 min at 100% buffer B to restore the initial condition. The flow rate was 1 mL/min and detection was performed at 254 nm (Waters 996, PDA detector). Separation was performed on a 10µm C18 analytical column (250 × 4.6 mm) equipped with a guard column. The peaks were identified using the standard samples. The analytical recovery was tested by adding a known amount of standard compound prior to extraction and recovery was found to be 94 to 100% for different compounds. The data were analyzed using Empower software.

### Statistical Analysis

The experiments were carried out in a randomized block design. One–Way analysis of variance (ANOVA) was done on all the data to confirm the variability of data and validity of results and Duncan's multiple range test (DMRT) was performed to determine the significant difference between treatments using the statistical software SPSS 10.0.

### Results

### Effect of Salinity Stress on the Level of Reactive Oxygen Species and Lipid Peroxidation

In S. portulacastrum, no significant change in MDA level was observed at any salt concentration and duration. The increase in ROS levels was also limited to longer duration. At 8 d, H2O<sup>2</sup> level was increased under both 250 and 1000 mM NaCl treatment; while O•− 2 level was increased only under 1000 mM NaCl treatment as compared to that of control (**Figures 1A–C**). By contrast, in B. juncea, time-dependent increase in MDA level was observed (**Figure 1C**) and ROS levels also increased significantly. The maximum increase of 2.5- and 4.8-fold in O•− 2 and H2O<sup>2</sup> levels were observed at 8 d after treatment, as compared to that of control (**Figures 1A–B**).

### Effect of Salinity Stress on the Activity of NADPH Oxidase

The activity of NADPH oxidase was not affected significantly in S. portulacastrum except for the 22% increase at 1000 mM NaCl after 2 d treatment. In B. juncea, NADPH oxidase activity declined significantly at all the durations as compared to that of control (**Figure 2**).

### Effect of Salinity Stress on the Activities of Antioxidant Enzymes

In S. portulacastrum, dose-dependent increase in SOD activity was observed until 4 d, with the maximum of 30% under 1000 mM NaCl as compared to that of control. At 2 and 8 d after treatment, SOD activity was almost unchanged, except at 1000 mM at 2 d where it was increased by 20% as compared to that of control (**Figure 3A**). Unlike SOD, CAT activity was increased at all the durations, except at 8 d where the increase was observed only at 1000 mM NaCl concentration (**Figure 3B**). The APX activity increased at both NaCl concentrations; however, maximum of 2.8-fold was observed under 250 mM at 2 d after treatment as compared to that of control. At 4 and 8 d, comparable increase in APX activity was observed under both 250 and 1000 mM NaCl as compared to that of control (**Figure 3C**). The GR activity was almost unchanged at both NaCl concentrations and all durations (**Figure 3D**). In B. juncea, except for CAT, activities of SOD, APX, and GR decreased at all the durations (**Figures 3A–D**).

### Effect of Salinity Stress on GSH and ASA Pools and their Redox Couples (GSH/GSSG and ASC/DHA Ratio)

In S. portulacastrum, GSH level was unaltered until 4 d; however; at 8 d it showed significant reduction with the maximum

of 40% under 1000 mM NaCl as compared to that of control (**Figure 4A**). The GSH/GSSG ratio showed an increase in comparison to control until 4 d at 250 mM NaCl but was at par to control at 1000 mM NaCl. At 8 d, GSH/GSSG ratio was decreased

with the maximum of 66% under 1000 mM NaCl as compared to that of control (**Figure 4B**). In B. juncea, GSH level increased beyond 2 d and at 8 d, it was 2.5-fold higher than that of control. The GSH/GSSG ratio was at par with control till 4 d and at 8 d, it was 1.8-fold higher than that of control.

Under NaCl stress, a distinct increase in ASC level was observed in both S. portulacastrum and B. juncea, however, the level of increase was comparatively higher in B. juncea (**Figure 4C**). The ASC/DHA ratio was unchanged in S. portulacastrum at 250 mM NaCl; however, at 1000 mM, it significantly increased. In contrast, B. juncea showed a significant decrease of ASC/DHA ratio at all the durations (**Figure 4D**).

### Effect of Salinity Stress on the Level of Adenylate and Pyridine Nucleotides

In S. portulacastrum, ATP level was declined at both 250 and 1000 mM NaCl (Supplementary Figure 1A), hence, ATP/ADP ratio declined significantly (**Figure 5A**). Similar profile was also observed for NADP/NADPH and NAD/NADH ratios. The maximum decrease in these ratios was observed under 1000 mM NaCl at 8 d after treatment (**Figures 5B,C**). In B. juncea, ATP/ADP ratio remained at par with control, except at 4 d at which it was increased by 1.5-fold as compared to that of control (**Figure 5A**). The NADP and NADPH levels although increased (Supplementary Figures 2, 3), NADP/NADPH ratio was reduced with the maximum of 60% at 8 d after treatment as compared to that of control (**Figure 5B**). In contrast, the NAD/NADH ratio was increased beyond 2 d of stress treatment (**Figure 5C**).

### Discussion

The salinity stress in plants is known to drastically reduce its growth and productivity (Gupta and Huang, 2014). Thus, in order to maintain the crop productivity in saline affected regions, it is essential to understand the mechanism of salt toxicity in plants. Generally, most crop plants are glycophytes and can withstand salt concentration in the range of 50–250 mM; however, there are specific type of plants referred as halophytes which can tolerate upto 1 M salt concentration. In an earlier study, physiological responses of a halophyte, S. portulacastrum have been reported and redox homeostasis and energetics were found to be the key determinants of salt tolerance (Lokhande et al., 2011). To further strengthen this hypothesis, in the present work, similar responses were studied in B. juncea (Indian mustard – a glycophyte) and compared with that of S. portulacastrum.

In the presence of salt stress, series of events leading to perturbation of cellular metabolism are: less water availability, stomata closure, altered gaseous exchange, inhibition of photosynthesis, effect on electron flow in ETC in chloroplast and mitochondria, increase in the production of ROS and disturbed status of adenine (ATP) and pyridine nucleotides (NADH, NADPH). Thus, the oxidative damage and altered energetics are mainly responsible for affecting the general metabolism and plant growth under salt stress (Srivastava et al., 2011). In S. portulacastrum, the increase in ROS levels was seen only at longer duration; however, in B. juncea, the increase in ROS and MDA levels were seen as early

as 2 d after treatment (**Figures 1A–C**). This clearly suggested that S. portulacastrum has better ability to avoid or delay the oxidative damage under salt stress conditions. In recent years, significant progress has been made with respect to ROS function in plants and they are now not seen as negative by-product of oxidative metabolism but they are known to serve as important signaling mediators under stress (Gilroy et al., 2014). The ROS based signaling is termed as "redox signaling" and is important for growth and survival of the plants under normal as well as salt stress conditions (Srivastava and Suprasanna, in press). There are many pro-oxidant enzymes in plants which are responsible for the ROS formation. NADPH oxidase is one such pro-oxidant which is an important regulator of calcium signaling and downstream signal transduction associated with salt tolerance (Marino et al., 2012; Drerup et al., 2013). The NADPH oxidase activity in S. portulacastrum was unaffected while in B. juncea, it decreased significantly (**Figure 2**). This indicates that apart from oxidative damage, the basal ROS signaling is also disturbed in glycophytes under salt stress. Thus, differential ability of halophytes and glycophytes to perceive stress seems to be responsible for differential stress response. The NADPH oxidase might be one of the possible candidates responsible for this; however, this needs to be investigated further.

Significant research has been conducted to establish that antioxidant defense is important for maintaining proper plant growth under different abiotic stress conditions, especially halophytes (Boestfleisch et al., 2014; Canalejo et al., 2014; Yildiztugay et al., 2014). In the same line, to see how the stress imposition and oxidative stress in two plant species (glycophytes and halophyte) affected their antioxidant metabolism and vice versa, activities of enzymatic antioxidant and levels of non-enzymatic antioxidants were measured. The enzymatic antioxidants showed a significant decline except CAT in B. juncea, whereas in S. portulacastrum, there was either a increase or no change (**Figures 3A–D**). Apart from suggesting the better ability of S. portulacastrum to activate its antioxidant defense under stress, this also indicated that halophytic enzymes are comparably more robust and stable than glycophytic enzymes. In recent years, various efforts have been made where antioxidant gene from a halophyte has been used to make transgenic glycophytes with higher salt tolerance. For instance, cytosolic copper/zinc SOD from a mangrove plant Avicennia marina has been used to increase the abiotic stress tolerance in rice (Prashanth et al., 2008). The peroxisomal APX from halophyte Salicornia brachiata was used to confer salt and drought tolerance in tobacco (Singh et al., 2014). Among molecular antioxidants, ASC and GSH are involved in the functioning of ASC-GSH cycle and can also independently function as antioxidants. Hence, maintenance of ASC/DHA and GSH/GSSG ratio is necessary to allow antioxidant functions to operate properly. In B. juncea, the GSH/GSSG and ASC/DHA ratio was either maintained or decreased which indicated plant's inability to stimulate their ASC and GSH regenerating systems as observed in the case of GR. Thus, despite an increase in absolute ASC and GSH, ROS levels continued to increase significantly. On the contrary in S. portulacastrum, both ASC/DHA and GSH/GSSG showed increasing trend; except for the decrease in GSH/GSSG ratio at 1000 mM NaCl which signifies the active utilization of GSH to avoid the oxidative damage at such a higher salt concentration.

Apart from the lack of co-ordinated activation of antioxidant defense, the over-reduction of electron transport chain (ETC) is also an important contributor of oxidative damage under stress. The over-reduced ETC in chloroplasts and mitochondria apparently result in significant decline in ratios of NADP/NADPH and NAD/NADH, respectively (Munns and Tester, 2008). In the present study, both the plants demonstrated the decreasing trend in NADP/NADPH ratio; however the extent of decrease was higher in B. juncea than S. portulacastrum. This suggests two possibilities, first: either the photosynthesis was not affected to a significant level in S. portulacastrum; second: there might be the activation of alternative salvage pathway to utilize reducing equivalents, such as photorespiration. Although, comparison of glycophyte Arabidopsis and its halophytic relative Thellungiella showed that photosynthesis and gas exchange in halophyte is minimally affected under salt conditions (Stepien and Johnson, 2009), the contribution of salvage pathway cannot be completely ignored, however this needs further investigation. To see the effects on plant energetic, the levels of adenylate (ATP, ADP) and pyridine nucleotides (NAD, NADH) were measured. In B. juncea, NaCl stress increases the NAD/NADH ratio which signifies the effect on energy metabolism. However, the ATP/ADP ratio remained unchanged which suggests that ATP formed is not actively utilized. By contrast, in S. portulacastrum, significant consumption of ATP and NAD was observed which led to the decline in ATP/ADP and NAD/NADH ratios. Thus, apart from the ATP and NAD generation, their proper channelization or consumption is also important to activate

### References


different mechanisms responsible for imparting salt tolerance in plants.

In summary, two plant systems (S. portulacastrum – a halophyte and B. juncea – a glycophyte) differing in their salttolerance ability have been compared. The findings reveal better antioxidant defense to avoid oxidative damage and proper energy consumption as key determinants of salinity tolerance in these two contrasting plants. The differential response of NADPH oxidase is also seen which needs to be investigated further in the context of stress perception and downstream signaling.

### Supplementary Material

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fenvs. 2015.00019/abstract


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Srivastava, Srivastava, Lokhande, D'Souza and Suprasanna. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Temperature stress and redox homeostasis in agricultural crops

### *Rashmi Awasthi , Kalpna Bhandari and Harsh Nayyar\**

*Department of Botany, Panjab University, Chandigarh, India*

#### *Edited by:*

*Naser A. Anjum, University of Aveiro, Portugal*

#### *Reviewed by:*

*Naser A. Anjum, University of Aveiro, Portugal Aryadeep Roychoudhury, St. Xavier's College, India Yogesh Abrol, Bhagalpur University, India*

#### *\*Correspondence:*

*Harsh Nayyar, Department of Botany, Panjab University, Chandigarh 160014, India e-mail: harshnayyar@hotmail.com* Plants are exposed to a wide range of environmental conditions and one of the major forces that shape the structure and function of plants are temperature stresses, which include low and high temperature stresses and considered as major abiotic stresses for crop plants. Due to global climate change, temperature stress is becoming the major area of concern for the researchers worldwide. The reactions of plants to these stresses are complex and have devastating effects on plant metabolism, disrupting cellular homeostasis and uncoupling major physiological and biochemical processes. Temperature stresses disrupt photosynthesis and increase photorespiration thereby altering the normal homeostasis of plant cells. The constancy of temperature, among different metabolic equilibria present in plant cells, depends to a certain extent on a homeostatically regulated ratio of redox components, which are present virtually in all plant cells. Several pathways, which are present in plant cells, enable correct equilibrium of the plant cellular redox state and balance fluctuations in plant cells caused by changes in environment due to stressful conditions. In temperature stresses, high temperature stress is considered to be one of the major abiotic stresses for restricting crop production worldwide. The responses of plants to heat stress vary with extent of temperature increase, its duration and the type of plant. On other hand, low temperature as major environmental factor often affects plant growth and crop productivity and leads to substantial crop loses. A direct result of stress-induced cellular changes is overproduction of reactive oxygen species (ROS) in plants which are produced in such a way that they are confined to a small area and also in specific pattern in biological responses. ROS (superoxide; O− <sup>2</sup> , hydroxyl radicals; OH−, alkoxyl radicals and non-radicals like hydrogen peroxide; H2O2 and singlet oxygen; 1O2) are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates which ultimately results in oxidative stress. ROS may also serve as signaling molecules in mediating important signal transduction pathways that coordinate an astonishing range of diverse plant processes under temperature stress. To counter temperature induced oxidative stress, plants upregulate a variety of enzymatic and non-enzymatic antioxidants which are also information-rich redox buffers and important components of redox signaling that interact with biomembrane-related compartments. They provide essential information on cellular redox state, and regulate gene expression associated with stress responses to optimize defense and survival, stress acclimation and tolerance. The work done by various researchers has explored a direct link between ROS scavenging and plant tolerance under temperature extremes in various crops which include legumes, cereals, oil crops and vegetables. There is ample need to develop temperature tolerance in crop plants by exploring suitable strategies to manage oxidative stress and maintain cellular redox state. Here, we summarize the studies linking ROS and temperature stress in plants, their generation and site of production, role of ROS as messengers as well as inducers of oxidative damage and strategies for the development of temperature stress tolerance involving redox homeostasis in various agricultural crops.

**Keywords: temperature stress, oxidative damage, legumes, cereals, homeostasis**

### **INTRODUCTION**

Plants are constantly subjected to different environmental conditions, which cause alterations in their metabolism in order to maintain a steady-state balance between energy generation and consumption and also in their redox state (Suzuki et al., 2011). Several environmental conditions result in stress in plants to adversely affect the metabolism, growth and development and may even lead to death under long-term exposures (Boguszewska and Zagdanska, 2012). Various abiotic stresses include drought, salt, low/high temperature, flooding and anaerobic conditions, which limit crop growth and productivity (Lawlor and Cornic, 2002). Among all the stresses, temperature stresses (cold or heat) can have devastating effects on plant growth and metabolism, also leading to alterations in redox state of the plant cell which is one of the important consequences of the fluctuating environment conditions (Suzuki and Mittler, 2006; Suzuki et al., 2011; Bita and Gerats, 2013). A delicate balance exists between multiple pathways residing in different organelles of plant cells, known as cellular homeostasis (Kocsy et al., 2013). This coordination between different organelles may be disrupted during temperature stresses due to variation in temperature optimum in different pathways within cells (Hasanuzzaman et al., 2013a). The constancy of temperature, among different metabolic equilibria present in plant cells, depends to a certain extent on a homeostatically-regulated ratio of redox components, which are present virtually in all plant cells (Suzuki et al., 2011). Several pathways, which are present in plant cells enable correct equilibrium of the plant cellular redox state and balance fluctuations in plant cells caused by changes in environment due to stressful conditions which are otherwise sensitive to changes in environmental conditions, especially temperature stresses (Foyer and Noctor, 2005, 2012; Suzuki et al., 2011). Plant Redox changes result in modification or induction of various physiological and biochemical processes through regulatory networks including ROS and antioxidants by reprogramming transcriptome which include the set of all RNA molecules, proteome including all proteins expressed by genome and metabolome such as metabolic intermediates, hormones and other signaling molecules etc. (Foyer and Noctor, 2009). Furthermore, reactions of plants to temperature stresses are complex and have adverse effects on plant metabolism by disrupting cellular homeostasis and uncoupling major physiological and biochemical processes (Hasanuzzaman et al., 2013a; Hemantaranjan et al., 2014). These stresses alter the normal homeostasis of plant cells by disrupting photosynthesis and increasing photorespiration (Noctor et al., 2007). A direct result of stress-induced cellular changes is overproduction of reactive oxygen species (ROS) in plants which are produced in such a way that they are confined to a small area and also in specific pattern in biological responses. The production of ROS is an inevitable consequence of aerobic metabolism during stressful conditions (Bhattacharjee, 2012). ROS are highly reactive and toxic, affecting various cellular functions in plant cells through damage to nucleic acids, protein oxidation, and lipid peroxidation, eventually resulting in cell death (**Figure 1**) (Bhattacharjee, 2005; Amirsadeghi et al., 2006; Suzuki et al., 2011; Tuteja et al., 2012). ROS toxicity due to stresses is considered to be one of the major causes of low crop productivity worldwide (Vadez et al., 2012).

ROS system consists of both free radicals including superoxide (O− <sup>2</sup> ), hydroxyl radicals (OH−), alkoxyl radicals and non-radicals like hydrogen peroxide (H2O2) and singlet oxygen (1O2) (Gill and Tuteja, 2010). During stress conditions, these species are always formed by the leakage of electrons from the electron transport activities of chloroplasts, mitochondria, and plasma membranes or also as a by-product of various metabolic pathways localized in different cellular compartments (Del Rio et al., 2006; Gill and Tuteja, 2010; Sharma et al., 2012; **Figure 2**). Depending upon their concentrations, ROS play dual role as both deleterious and beneficial species in plants (Kotchoni and Gachomo, 2006). At low/moderate concentrations, ROS act as second messengers in various intercellular signaling pathways that mediate many responses in plants, thus regulating cellular redox state whereas at higher concentrations they have detrimental effects on plant growth (Mittler, 2002; Torres et al., 2002; Yan et al., 2007; Miller et al., 2008; Sharma et al., 2012). Plants have various metabolic and developmental processes which are regulated by cross-talk between ROS and hormones (Kocsy et al., 2013). ROS can activate the synthesis of many plant hormones such as brassinosteroids, ethylene, jasmonate and salicylic acid (Ahmad et al., 2010). In contrast, some hormones such as auxins, ABA, salicylic acid can also result in ROS generation (**Figure 3**). The redox state of the cell may be affected by plant hormones through transcriptional stimulation of genes coding for molecules involved in redox system (Laskowski et al., 2002). Various metabolic and developmental processes which involve interaction between ROS and hormones in plants include stomatal closure (Yan et al., 2007; Neill et al., 2008), programmed cell death (Bethke and Jones, 2001), gravitropism (Jung et al., 2001), control of root apical meristem organization (Jiang and Feldman, 2003) and acquisition of tolerance to both biotic and abiotic stresses (Torres et al., 2002; Miller et al., 2008).

These ROS are continuously reduced/scavenged by plant antioxidative defense systems which maintain them at certain steady-state levels under stressful conditions (Tuteja et al., 2012). An efficient anti-oxidative system comprising of the nonenzymatic as well as enzymatic antioxidants is involved in scavenging or detoxification of excess ROS (Noctor et al., 2007; Sharma et al., 2012). Various enzymatic antioxidants comprise of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), enzymes of ascorbate-glutahione (AsA-GSH) cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) (Noctor and Foyer, 1998; Foyer and Noctor, 2003) whereas Non-enzymatic antioxidants include phenolics, ascorbate (AsA), glutathione (GSH), carotenoids, and tocopherols (Apel and Hirt, 2004; Gill and Tuteja, 2010). Increased activities of many antioxidant enzymes have been observed in plants to combat oxidative stress induced by various environmental stresses and also to maintain cellular homeostasis (Blokhina et al., 2003; Almeselmani et al., 2006). Maintenance of a high antioxidant capacity to scavenge the toxic ROS has been linked to increase in tolerance of plants to these environmental stresses (Suzuki et al., 2011; Hasanuzzaman et al., 2013a). Transgenic lines with altered levels of antioxidants have been developed for improving stress-induced oxidative stress tolerance in various crop plants (Chen et al., 2010; Hasanuzzaman et al., 2013b). Transgenics developed with concurrent expression of multiple antioxidant enzymes are found to have more tolerance to multiple environmental stresses compared to those transformed with one or two genes (Suzuki et al., 2011; Sharma et al., 2012).

### **TEMPERATURE STRESSES**

Temperature stress is becoming a major area of concern for plant scientists due to climate change, affecting crop production worldwide (Hasanuzzaman et al., 2013b). Every plant species has optimum temperature limits for its growth and development and abnormal temperatures have devastating effects on plant growth and metabolism (Yadav, 2010; Suzuki et al., 2011; Hasanuzzaman et al., 2012a, 2013b; Kumar et al., 2013a,b). According to global climate change scenarios, high temperature stress is considered as a critical factor for plant growth and productivity and the plant responses to high temperature vary with the extent of temperature increase, its duration and type of plant (Mittler, 2006; Wahid et al., 2007; Hasanuzzaman et al., 2012a). High temperature may adversely affect vital physiological processes like photosynthesis, respiration, water relations and membrane stability and also modulate levels of hormones, primary and secondary metabolites (Hemantaranjan et al., 2014). Furthermore, for the duration of plant ontogeny, enhanced expression of a variety of heat shock and stress-related proteins and production of ROS constitute the major plant responses to heat stress (Saidi et al., 2011; Hasanuzzaman et al., 2013a; Hemantaranjan et al., 2014). Higher ROS concentrations are associated with lipid peroxidation; mainly cellular membranes are particularly susceptible to oxidative damage (Sharkey, 2005; Suzuki and Mittler, 2006). In addition, acquired thermotolerance, i.e., the ability of plants to develop heat tolerance was shown to be mediated in plants by enhancing cellular mechanisms that prevent oxidative damage under high temperature conditions in crops (Larkindale and Huang, 2004; Suzuki and Mittler, 2006). According to various studies, different types of signal transduction pathways and defense mechanisms due to heat stress are involved in sensing of ROS and helpful in providing thermotolerance to crop plants (**Figure 4**; Apel and Hirt, 2004; Kreslavski et al., 2012; Hasanuzzaman et al., 2013b; Miura and Furumoto, 2013). In contrast, low temperature stress or cold stress is another factor that often affects plant growth and productivity and leads to substantial crop losses (Croser et al., 2003; Yadav et al., 2004; Beck et al., 2007; Yadav, 2010; Sanghera et al., 2011; Miura and Furumoto, 2013). Cold stress or low temperature, which includes both chilling stress (*<*20◦C) and freezing stress (*<*0◦C) is one of the most significant abiotic stresses of agricultural plants, affecting plant development and yield and consequently reducing crop production (Lang et al., 2005; Thakur et al., 2010). It results in micro-organelle disruption, phase transition in cell membrane lipids and generation of ROS (Kim et al., 2013). It also induces cascades of alterations in metabolic pathways which

stress conditions organelles with highly oxidizing metabolic activities such as mitochondria, chloroplast and peroxisomes are major sites of ROS production (Mittler et al., 2004). In mitochondria, ROS production is likely to occur in complex I and Q zone (Blokhnia and Fagerstedt, 2006). In the chloroplast, during photosynthesis, energy from sunlight is captured and transferred to photoystem I (PS I) and photoystem II (PS II). O-− <sup>2</sup> , which is produced mainly by electron leakage from Fe-S centers of PS I or reduced ferredoxin (Fd) is then converted to H2O2 by SOD (Gechev et al., 2006). Under excess light conditions, PS II is able to generate 1O2 by energy transfer from the triplet state chlorophyll (Asada, 2006). In peroxisomes, ROS is produced mainly during photorespiration and also during β-oxidation ribulose-1,5-bisphosphate (RuBP) by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), can also use O2 to oxygenate ribulose-1,5-bisphosphate. Under abiotic stress conditions, which impair CO2 fixation in the chloroplast, the oxygenase activity of RuBisCO increases and the glycolate that is produced moves from the chloroplast to peroxisomes, where it is oxidized by glycolate oxidase (GO) forming H2O2 (Takahashi and Murata, 2008). In peroxisomes, H2O2 can also be formed directly from O2 by enzyme systems such as xanthine oxidase (XO) coupled to SOD (Mhamdi et al., 2010). Plasma membrane-bound NADPH oxidases as well as cell-wall associated peroxidases are the main sources of O-− <sup>2</sup> and H2O2 producing apoplastic enzymes (Mhamdi et al., 2010).

include changes in membrane fatty acid composition, activity of antioxidant enzymes, gene regulation and changes in redox state (Shahandashti et al., 2014). According to various reports, mechanisms governing the temperature response in higher plants are being extensively probed to improve the cold tolerance in agricultural crops (Chinnusamy et al., 2007; Thakur et al., 2010). Various cellular changes, which are induced by either high temperature or low temperature lead to the overproduction of toxic compounds, especially ROS that result in oxidative stress (Mittler, 2002). ROS have toxic potential effects as they can induce protein oxidation, DNA damage, lipid peroxidation of membranes (malondialdehyde content) and destruction of pigments (Apel and Hirt, 2004; Xu et al., 2006; Hasanuzzaman et al., 2012a), however plants have evolved variety of responses to extreme temperatures that help in minimizing damages and provide cellular homeostasis (Kotak et al., 2007). Direct link exists between ROS scavenging and plant stress tolerance under temperature stress conditions which is often related to enhanced activities of antioxidative defense enzymes that confers stress tolerance to either high temperature or low temperature stress (Huang and Guo, 2005; Almeselmani et al., 2009).

### **TEMPERATURE STRESSES AND OXIDATIVE DAMAGE IN CROPS**

### **LEGUMES**

Grain legumes are important food commodities and also the essential components of crop rotation throughout the world (McDonald and Paulsen, 1997). High temperature stress is

**redox environment.** Various spatial and temporal changes in the levels of ROS and hormones at various organizational levels are important in operation of regulatory network. The concentration-dependent effects of ROS and their

including both activation and inhibition of certain signaling pathways. This model shows the interactions between various hormones and ROS. ABA, abscisic acid; JA, jasmonate; ROS, reactive oxygen species; SA, salicylic acid; NO, nitric oxide.

predicted to occur more frequently further affecting various aspects of leguminous crops (Hatfield et al., 2011; Ibrahim, 2011; Reddy et al., 2012). ROS produced during extreme temperature conditions have been demonstrated to cause oxidative damage leading to cellular injury in legumes (Apel and Hirt, 2004). In *Phaseolus vulgaris*, increased H2O2 content was observed at 46– 48◦C, which further led to lipid peroxidation in membranes and accumulation of malondialdehyde (MDA) (Nagesh and Devaraj, 2008; Kumar et al., 2011). In chickpea (*Cicer arietinum*), at 40/30◦C (day/night) temperatures under controlled conditions, symptoms of heat stress arise in the form of chlorosis of leaves, membrane damage and loss of viability of tissues. The damage to the plants becomes intensive at 45/35◦C (Kumar et al., 2011) that was attributed to increased oxidative damage as lipid peroxidation and H2O2 content, which was relatively greater in heat-sensitive genotypes, especially at 40/30 and 45/35◦C. According to Kaushal et al. (2011), oxidative damage, measured as lipid peroxidation and hydrogen peroxide concentration, increased with heat stress (45/40◦C), pertinently lipid peroxidation was found to increase to a greater extent indicating membrane injury. Also, decrease in the activities of enzymatic antioxidants (SOD, CAT, APX, GR) was observed, which was due to their denaturation at higher temperature, i.e., 45/40◦C. When compared with other grain legumes such as pigeonpea, groundnut and soybean, chickpea was the most sensitive in terms of oxidative damage, membrane thermostability and PSII function (Srinivasan et al., 1999). In another legume, Mungbean (*Vigna radiata* L), which is a summer-season crop, the seedlings exposed to higher temperature of 50◦C for 2 h (lethal temperature) as well as pretreated with 40◦C for 1 h, were analyzed for MDA content and antioxidative enzymes. The results showed that the growth in lethal temperature was extremely poor which improved when pre-treatment of 40◦C was applied before 50◦C. The content of MDA in seedlings treated with lethal temperature was highest at any harvest, which reduced when seedlings were pre-treated with 40◦C prior to lethal stress (Mansoor and Naqvi, 2013). These observations were attributed to heat acclimation, which improved the antioxidant defense. In soybean, heat stress enhanced membrane permeability and electrolyte leakage as a result of oxidative damage, which in turn reduced the ability of the plasma membrane to retain solutes and water (Lin et al., 1984). In another related study, increased membrane lipid peroxidation due to heat stress was noticed which aggravated the membrane injury in soybean (*Glycine max*) (Tan et al., 2011). Also, the crop exposed to day/night temperature of 38/28◦C for 14 days at flowering stage showed damage to chloroplast and thylakoids membranes (Tan et al., 2011). Heat induced

to activate calcium channels, which induces Ca2<sup>+</sup> influx (Saidi et al., 2009), thus the MAPK cascade leading to gene expression. Secondary signals like ROS, H2O2, NO, and ABA lead to stress-tolerance (Hua, 2009; Mishkind

oxide; PLD, Phospholipase D; PIPK, Phosphadidylinositol-4,5-biphosphate kinase; PA, Phosphatidic acid; IP3, D-myo-inositol-1,4,5-triphosphate; DAG, Diacylglycerol.

membrane damage has been reported in broad bean (Hamada, 2001) and soybean (Djanaguiraman et al., 2011). ROS arising out from heat stress were implicated as primary agents causing oxidative injury in all these studies.

Many economically important legumes are sensitive to temperature below 15◦C (Ouellet, 2007). Stressful low temperatures lead to disruption of respiration by affecting respiratory rate which may at first increase in response to chilling (Kaur et al., 2008) but on continued exposure, it decreases (Munro et al., 2004) or plants may resort to some alternative respiratory pathway as found in case of mungbean (*Vigna radiata*) and pea leaves (Gonzalez-Meler et al., 1999). Besides these implications, other harmful effects of low temperature reported are loss of membrane fluidity and rigidification (Vigh et al., 2007; Jewell et al., 2010), generation of ROS (Wang et al., 2009a,b; Turan and Ekmekci, 2011). At metabolic levels, chilling stress negatively affects photosynthesis as described in pea (*Pisum sativum*; Guilioni et al., 1997), mungbean (*Vigna radiate;* Gonzalez-Meler et al., 1999), beans (*Phaseolus vulgaris;* Tsonev et al., 2003), chickpea (*Cicer arietinum;* Nayyar et al., 2005b; Berger et al., 2006), pigeon pea (*Cajanus cajan*; Sandhu et al., 2007), faba beans (Torres et al., 2011), soybean (Ohnishi et al., 2010; Board and Kahlon, 2012). The loss of membrane integrity is the primary damage of chilling temperatures due to oxidative stress, which results in the production of H2O2 and MDA content due to lipid peroxidation (Nayyar and Chander, 2004; Tambussi et al., 2004; Nayyar et al., 2005a,b,c,d). In mungbean, exposure of plants to low temperature showed damage to PSII, further reducing photochemical efficiency due to photoinhibition and damage to chloroplast (Saleh, 2007). It also resulted in swelling of plastids and accumulation of lipid drops, ultimately leading to disorganization of entire plastid (Ishikawa, 1996). In mungbean, 5 days old seedlings subjected to stressful low temperature (4◦C for 2 days) showed irreversible chilling injury as evident from increased electrolyte leakage contents due to membrane damage (Chang et al., 2001). Chillinginflicted membrane damage was also reported in broad bean (Hamada, 2001). Chickpea is a chilling- sensitive crop and its productivity is adversely affected by chilling temperatures as chilling stress is the principal cause for crop reduction in chickpea (Nayyar et al., 2005b). Increased electrolyte leakage was reported in chickpea under cold stress (5/13◦C mean min. and max. temperature), thereby indicating altered membrane permeability, structural disintegration and membrane injury in chickpea (Croser et al., 2003; Nayyar et al., 2005a). For chickpea, same results were observed in various studies conducted by different researchers (Bakht et al., 2006; Turan and Ekmekci, 2011; Shahandashti et al., 2014). The decrease in photosynthetic capacity was observed in soybean (*Glycine max*), which was partly due to chilling-associated oxidative damage to chloroplast components. Also, the lipid peroxidation and oxidative damage to thylakoid proteins were observed in leaves of soybean exposed to chilling stress under light (Tambussi et al., 2004). In another study of *Glycine max*, a much larger reduction was observed in the speed of germination of radical length at chilling temperature, which was probably due to decrease in activity of numerous enzymes involved in degradation of seed storage reserves, transport of degradation products and their metabolism in the embryonic roots. This decrease in enzymatic activity was resulted due to generation of ROS induced by chilling stress (Borowski and Michalek, 2014).

### **CEREALS**

High temperature stress is considered as a key stress factor with high potential impact on crop yield of cereals (Hasanuzzaman et al., 2013a). On the other hand, long term exposure of cereals to low temperature showed reduction in photochemical efficiency of PSII due to photoinhibition and damage to chloroplast (Kratsch and Wise, 2000; Hasanuzzaman et al., 2013a). One of the major consequences of high temperature stress in cereals is oxidative damage caused by imbalance of metabolic processes such as photosynthesis and respiration either by increasing the ROS or by decreasing the oxygen radical scavenging ability in the cell (Mittler, 2002; Wormuth et al., 2007; Barnabas et al., 2008). High temperature stress leads to the peroxidation of membrane lipids leading to the production of malondialdehyde (MDA), which is a good indicator of free radical damage to cell membranes (Hasanuzzaman et al., 2013a,b). Heat-stress-induced membrane peroxidation and aggravated membrane injury was observed in wheat (Savicka and Skute, 2010), rice and maize (Kumar et al., 2012c) and sorghum (Tan et al., 2011). High temperature stress in sorghum (*Hordeum vulgare*) resulted in lipid peroxidation of membranes to cause membrane injury. Membrane damage and MDA content increased by 110 and 75%, respectively which was due to increased H2O2 and O<sup>−</sup> <sup>2</sup> content (Mohammed and Tarpley, 2010). High temperature stress decreased antioxidant enzyme activities and increased oxidant production in sorghum (Djanaguiraman et al., 2010). In this study, SOD, CAT and POX activities were decreased during heat stress (22, 15, and 25% lower than control plants) and the inhibition of all antioxidant enzymes in heat-stressed plants relative to control plants indicated inactivation of all antioxidant enzymes by heat stress. In wheat seedlings, gradual increase in H2O2 content was observed (0.5, 0.58, 0.78, and 1.1μmol g−<sup>1</sup> FW) in response to different heat shock treatments of 22, 30, 35, and 40◦C for the time period of 2 h (Kumar et al., 2012a). Oxidative damage due to ROS production during long term exposure to high temperature led to changes in MDA content and O− <sup>2</sup> production which were observed at two growth stages, i.e., early stages (4-dayold) and late stages (7-day-old) of wheat (*Triticum aestivum)* seedlings development (Savicka and Skute, 2010; Cossani and Reynolds, 2012). In another study on wheat, increased MDA concentration was observed in first leaf of wheat seedlings during high temperature stress conditions, which is due to the increased production of superoxide radical (O− <sup>2</sup> ) (Bohnert et al., 2006). According to Kumar et al. (2012c), high temperature of 40/35◦C (day/night temperature) resulted in 1.8-fold and 1.2- to 1.3-fold increase of MDA content in rice and maize genotypes, respectively over the control treatment. A further increase of MDA content was observed at 45/40◦C, in both the crops, where an increase of 2.2- to 2.4-fold was noticed in rice genotypes compared to 1.7-fold increase in maize genotypes. With rise in temperature to 45/40◦C, oxidative damage increased further in rice genotypes (Kumar et al., 2012c; Theocharis et al., 2012; Yang et al., 2012).

Cold stress, especially the chilling stress in cereal crops, is one major form of stress which affects the crop growth and yield (Hasanuzzaman et al., 2013a). Cold stress-induced tissue dehydration further leads to membrane disintegration, reduced growth and development of plants in maize which was due to the accumulation of MDA content as a result of lipid peroxidation in membranes (Farooq et al., 2009; Yadav, 2010). According to Yordanova and Popova (2007), exposure of wheat plants to low temperature (3◦C) for 48 and 72 h resulted in decreased levels of chlorophyll, CO2 assimilation, transpirations rates and photosynthesis due to the reduced activities of ATP synthase, which further restricted RuBisCo regeneration and limited photophosphorylation (Allen and Ort, 2001). Physiobiochemical responses to cold stress in tetraploid and hexaploid wheat were studied where, the elevated levels of electrolyte leakage index, H2O2 and MDA content were observed in stressed plants (Nejadsadeghi et al., 2014).

According to some previous reports, oxidative stress as a result of chilling stress has been observed in some other crops also (Turan and Ekmekci, 2011). Cold stress adversely affected membrane properties and enzymatic activities leading to plant and tissue necrosis, as observed in banana (*Musa* spp.) (Chinnusamy et al., 2007). Some other crops, which are chilling-sensitive and have been studied for the adverse effects on growth and development include Coffee plant (*Coffea Arabica*; Alonso et al., 1997), tomato (*Lycopersicum esculentum*; Starck et al., 2000) and its wild varieties, potato (*Solanum* spp.; Svensson et al., 2002), Citrus plant (Hara et al., 2003), muskmelons (*Cucumis melo;* Wang et al., 2004), cotton (*Gossipium hirusutum*; Zhao et al., 2012), and sugarcane (*Saccharum officinarum* L; Badea and Basu, 2009; Thakur et al., 2010; Aghaee et al., 2011; Anjum et al., 2011; Zhu et al., 2013).

Some other crops, where damage due to ROS in response to heat stress has been reported are *Gossipium hirsutum* (Crafts-Brandner and Law, 2000; Snider et al., 2009), *Lycopersicon esculentum* (Willits and Peet, 2001; Rivero et al., 2004; Wahid et al., 2007), *Nicotiana tabacum* (Wang et al., 2006; Tan et al., 2011), *Malus domestica* (Ma et al., 2008), *Brassica juncea* (Rani et al., 2013; Wilson et al., 2014), and *Cucurbita* sp. (Ara et al., 2013).

### **REDOX HOMEOSTASIS IN TEMPERATURE-STRESSED CROPS HIGH TEMPERATURE STRESS**

Plants tend to combat ROS production by inducing an antioxidant system consisting of enzymatic and non-enzymatic components under extreme temperature conditions as their defense system and also maintain their redox homeostasis (Sairam and Tyagi, 2004; Wahid et al., 2007; Hasanuzzaman et al., 2013a) (**Figure 5**). Various studies on plants are available which indicate tolerance to temperature stress with an increase in antioxidants (Gill and Tuteja, 2010; Kaushal et al., 2011; Kumar et al., 2011, 2012b, 2013a; Hasanuzzaman et al., 2012a).

(Suzuki and Mittler, 2006). During stress, ROS overproduction can pose a threat to plant cells, and many stress conditions can enhance the expression of ROS-scavenging enzymes (De Gara et al., 2010). ROS are actively produced by cells (e.g., by NADPH oxidase in membranes) in

both the perpetuation of their production and their scavenging by enzymes such as CAT, catalase; APX, ascorbate peroxidase; GPX, guaiacol peroxidase; SOD, superoxide dismutase, and GR, glutathione reductase (Alscher et al., 2002; Suzuki et al., 2011).

Though all the reports indicate to up-regulation of similar types of enzymatic and non-enzymatic antioxidants, their degree and type of expression varies depending upon the plant type, duration and intensity of the stress. Nagesh and Devaraj (2008) observed increased activities of glutathione reductase (GR), peroxidase (POX) and ascorbic acid content in *Phaseolus vulgaris* plants during high temperature stress. Increased levels of sugars, proline, glutathione and ascorbate and activities of peroxidase (POX), glutathione reductase (GR) and ascorbate peroxidase (APX) were observed in lablab (*Dolichos lablab*) seedlings (D'Souza and Devaraj, 2013). In lentil, Chakraborty and Pradhan (2011) observed initial increase in CAT, APX, and SOD activities as temperature increased from 20 to 50◦C before declining at 50◦C. Likewise, in chickpea, the oxidative stress assessed by measuring the activity of enzymatic antioxidants such as CAT, SOD, APX, and GR elevated in plants grown at 40/35◦C but decreased at 45/40◦C (Kaushal et al., 2011). To cope up the oxidative stress, increased levels of antioxidants were observed at 40/30◦C, which decreased markedly at 45/35◦C suggesting their impairment. Heat-tolerant genotypes possessed greater activities of ascorbate peroxidase (APX) and glutathione reductase (GR), which possibly influenced the heat tolerance (Kumar et al., 2011). Seedlings of soybean (*Glycine max*) exposed to high temperature at 45◦C showed increased activities of peroxidases (POX), glutathione reductase (GR) and ascorbate peroxidase (APX) (D'Souza, 2013); similar findings have been observed in sorghum (Djanaguiraman et al., 2010). The activity of SOD, APX, CAT, GR, and POX increased significantly at all stages of growth in wheat cultivar C306 (heat-toelrant) while the PBW 343 (heat-sensitive) genotype showed a significant reduction in CAT, GR, and POX activities in response to high temperature stress in wheat (Almeselmani et al., 2009). Thermotolerance acquired in a set of wheat (*Triticum aestivum*) genotypes was correlated with higher activities of antioxidants such as catalase and superoxide dismutase, higher ascorbic acid concentration and less oxidative damage (Sairam et al., 2000; Almeselmani et al., 2006). A study conducted on wheat by Baldawi et al. (2007) showed heat tolerance to be associated with higher activities of SOD, APX, GR, GST, and CAT. In an another study conducted by Kumar et al. (2012c), comparative responses of *Oryza sativa* and *Zea mays* revealed the higher expression of enzymatic and non-enzymatic antioxidants. In enzymatic oxidants CAT, APX, and GR were found to be significantly higher in *Zea mays* compared to *Oryza sativa* while no variations existed for superoxide dismutase at the highest temperature applied (45/40◦C), whereas the nonenzymatic antioxidants (AsA and GSH) were also maintained significantly at greater levels at 45/40◦C in maize than in *Oryza sativa* genotypes. Therefore, *Zea mays* genotypes were able to retain their growth under heat stress partly due to their superior ability to cope up with oxidative damage by heat stress compared to *Oryza sativa* genotypes as suggested by these findings. The relative sensitivity of these plant groups to heat stress may also be reflected from the observation that *Zea mays* and *Oryza sativa* belong to C4 and C3 plant groups, respectively (Kumar et al., 2012c). Pearl millet plantlets showed significant increase in SOD, CAT and peroxidase activities during heat stress (Tikhomirova, 1985). In a similar fashion, exposure of a thermo-tolerant (BPR5426) and thermo-sensitive (NPJ119) Indian mustard (*Brassica juncea*) genotype to high temperature (45◦C) revealed higher SOD, CAT, APX and GR activities in tolerant genotypes (Rani et al., 2013). Under heat stress conditions, activity of antioxidant enzymes such as SOD, APX, POX, CAT increased, while H2O2 and MDA decreased, which increased shoot weight in tomato (Ogweno et al., 2008). According to these various studies, maintaining the redox state is vital to tolerate mild heat stress while severe stress, even for short periods, impairs this ability. Therefore, understanding of the expression of antioxidants in heat-stressed plants of various crops may be a significant step toward improving redox state and heat tolerance in crop plants.

### **LOW TEMPERATURE STRESS**

Low temperature stress was shown to enhance the transcript, protein, and activity of different ROS scavenging enzymes of antioxidative machinery which is linked to acquisition of stress tolerance (Saito et al., 2001; Posmyk et al., 2005; Morsy et al., 2007; Janska et al., 2009; **Figure 5**). Higher cold tolerance was observed in plants having enhanced activities of anti-oxidative enzymes in chickpea (Kumar et al., 2011). The chilling experiments carried out by Wang et al. (2009b) on alfalfa (*Medicago sativa*) genotypes with different chilling sensitivities showed that the chilling tolerant-genotypes had high anti-oxidative activity over the chilling-sensitive ones. The pod walls in chickpea exposed to cold stress upregulated the anti-oxidative enzymes to protect pods and developing seeds from chilling injury (Kaur et al., 2008). Cold acclimation in chickpea imparted cold tolerance at 2 and 4◦C, which was attributed to enhanced activities of SOD, APX, GR, and POX (Turan and Ekmekci, 2011). CAT, SOD and GR represent first lines of antioxidant defense which prevent formation of more toxic ROS and play essential role in cellular H2O2 signaling in chickpea (Shahandashti et al., 2014). In a subsequent study in chickpea, Turan and Ekmekci (2011) exposed the chickpea cultivars to chilling treatment and reported the enhanced activities of PSII and anti-oxidative enzymes in acclimated plants. Higher activities of CAT, APX and GR were found in pod walls of tolerant genotypes of chickpea which led to increased translocation of GSH from pod wall to seeds and contribute to ROS scavenging and tolerance to pod wall against low temperature stress (Kaur et al., 2009). Soybean seedlings exposed to very low temperature treatments (1◦C) resulted in increased activities of anti-oxidative enzymes (Posmyk et al., 2001, 2005; Borowski and Michalek, 2014). The tolerant genotypes of some cereals growing under cold stress showed higher expression of antioxidants implicating their role in governing the cold tolerance. In chillingtolerant winter rye leaves, the contents of ascorbic acid and α-tocopherol were found to be increased appreciable than the sensitive genotype. Three antioxidant enzymes were studied in two wheat cultivars, winter wheat and spring wheat, under low temperature stress conditions. The levels of endogenous peroxides were strongly increased in spring cultivar and to lesser extent in winter wheat (Apostolova et al., 2008) at low temperature (Streb and Feierabend, 1999). In rice, higher activities of antioxidant enzymes (CAT, SOD, APX) and higher AsA content was recorded which possibly provided cold tolerance (Huang and Guo, 2005; Guo et al., 2006). Antioxidant enzymes have significant importance in providing chilling tolerance in cold-stressed *Zea mays* where levels of APX, MDHAR, DHAR, GR and SOD were found to be elevated (Hasanuzzaman et al., 2013a). There are some examples of other crops such as *Coffea* sp. (Hasanuzzaman et al., 2013a), tomato (Zhao et al., 2009), Cucumber (Yang et al., 2011), grapes (Wang and Li, 2006), *Medicago sativa* (Ibrahim and Bafeel, 2008) where cold tolerance has been reported to be linked to upregulation of various antioxidants.

### **PLANT ACCLIMATION TO TEMPERATURE STRESSES AND REDOX HOMEOSTASIS**

### **HIGH TEMPERATURE STRESS**

Plants acclimate rapidly to different environmental conditions and manifest different mechanisms for surviving under extreme temperature conditions, together with long-term evolutionary adaptations at morphological and phonological level, involving changes in membrane lipid compositions, leaf orientation and transpirational cooling or short-term avoidance or acclimation mechanisms (Wahid et al., 2007; Larkindale and Vierling, 2008; Bita and Gerats, 2013). The acclimation of plants to moderately high temperature plays an important role in inducing plant tolerance to subsequent lethal high temperatures (He et al., 2003). Under high temperature conditions, many crop plants undergo early maturation, which is strongly related to decreased yield and may occur as a result of involvement of escape mechanism (Adams et al., 2001). Among general heat acclimation mechanisms involving various stress proteins, osmo-protectants, antioxidant enzymes, ion transporters and factors involved in signaling cascades and transcriptional control are essential to counteract stress effects (Wang et al., 2004; Bita and Gerats, 2013). During stress conditions, the initial stress signals arise in the form of osmotic or ionic effects or changes in temperature or membrane fluidity would trigger downstream signaling processes and transcription controls. This further activates various stress-responsive genes and mechanisms to re-establish homeostasis and protect and repair damaged proteins and membranes in plants during stressful conditions (Bohnert et al., 2006). Plants may experience high temperatures even in their natural distribution which would be lethal in the absence of this rapid acclimation response (Wahid et al., 2007). In addition, plants can experience major temperature fluctuations, leading to the acquisition of thermotolerance which may induce more general and variety of mechanisms that contribute to redox control of homeostasis of metabolism on a daily basis (Hong et al., 2003). During high temperature stress, the primary effects are on the plasmalemma, resulting in increased fluidity of lipid bilayer thereby leading to Ca2<sup>+</sup> influx, cytoskeleton reorganization, which results in the up regulation of mitogen activated protein kinases (MAPK) and calcium dependent protein kinases (CDPK), heat shock element (HSE), heat shock proteins (HSPs) and histidine kinase (HSK) (Sung et al., 2003). These different signaling cascades lead to the production of antioxidants and compatible osmolytes for cell water balance and osmotic adjustment, which also maintain redox homeostasis in plant cells (Bohnert et al., 2006). Osmoprotectants accumulation is one of an important adaptive mechanism in plants subjected to extreme temperature conditions (Sakamoto et al., 2000). The accumulation of different osmoprotectants like proline, glycine betaine and soluble sugars is necessary to regulate osmotic activities and protect various cellular structures from temperature stresses by maintaining the cell-water balance, membrane stability and by buffering the cellular redox potential (Farooq et al., 2008). According to studies, higher availability of carbohydrates such as glucose and sucrose during heat stress represents an important physiological trait associated with stress tolerance and acclimation (Liu and Huang, 2000). Also, sugars have been shown to act as antioxidants in plants (Lang-Mladek et al., 2010). However, at lower concentrations, they act as signaling molecules, but at higher concentrations these act as ROS scavengers also (Sugio et al., 2009). For instance, in tomato, the high cell wall and vacuolar invertases activities and increased sucrose import into young fruit contribute to high temperature tolerance through increasing sink strength and sugar signaling activities (Li et al., 2012). Furthermore, secondary metabolites like anthocyanins and carotenoids also help in plant acclimation responses by enhancing their synthesis and by decreasing leaf osmotic potential, resulting in an increased uptake and reduced transpirational loss of water under stress conditions (Wahid et al., 2007). Plants may accumulate phenolics by stimulation of their biosynthesis and inhibition of their catabolism as one of the acclimation mechanisms against temperature stress, as indicated by several studies in tomato and watermelon (Rivero et al., 2001; Wahid et al., 2007). The ability of plants to withstand or to acclimate to extreme temperature conditions results from repair of their heat-sensitive components and also the prevention of further heat injury and redox homeostasis being also maintained during stress (Kaya et al., 2001).

### **LOW TEMPERATURE STRESS**

In cold acclimation, plants acquire stress tolerance on prior exposure to suboptimal, low and non-freezing temperatures however; various plant species differ in their ability to face cold stress, which is governed by appropriate changes in gene expression to alter their metabolism, physiology and growth (Chinnusamy et al., 2010). Plant species acclimate during cold stress, by synthesis of cryoprotective molecules such as soluble sugars (saccharose, raffinose, stachyose, trehalose), sugar alcohols (sorbitol, ribitol, inositol) and low-molecular weight nitrogenous compounds (proline, glycine betaine) (Janska et al., 2009). These molecules stabilize both membrane phospholipids and proteins, and cytoplasmic proteins in conjunction with dehydrin proteins (DHNs), cold-regulated proteins (CORs) and heat-shock proteins (HSPs). Cryoprotective solutes are also involved in maintenance of hydrophobic interactions, homeostasis of ions, protection of the plasma membrane from adhesion of ice, scavenging ROS and consequent damage to cells (Iba, 2002; Wang et al., 2003; Gusta et al., 2004, 2005; Chen and Murata, 2008; Janska et al., 2009).

Also, the increased activity of the antioxidative enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase, ascorbate peroxidase and catalase, as well as the presence of a series of non-enzymatic antioxidants, such as tripeptidthiol, glutathione, ascorbic acid (vitamin C) and alphatocopherol (vitamin E) play important role in cold acclimation and maintanance of cellular redox homeostasis (Chen and Li, 2002). Cold acclimation also affects cell lipid composition by increasing the proportion of unsaturated fatty acids making up the phospholipids, which is necessary for the maintenance of plasma membrane functionality (Rajashekar, 2000; De Palma et al., 2008). Cold-acclimation induced chilling tolerance in chickpea was found to be associated with marked increase in endogenous ABA, cryoprotective solutes, antioxidative enzymes like ascorbate, glutathione, superoxide dismutase and catalase, relative growth rate of roots and significant decrease in electrolyte leakage and oxidative damage (Nayyar et al., 2005a). Some previous observations on this aspect also related higher chilling tolerance imparted by cold acclimation to elevated endogenous ABA (Janowiak et al., 2003), calcium (Knight et al., 1996), carbohydrates (Thomashow, 1999), and proline (Xin and Browse, 1998). During cold acclimation, changes in H2O2 concentrations and GSH/GSSG ratio alter the redox state of cells and activate special defense mechanisms through redox signaling chain (Kocsy et al., 2001). H2O2 generated by NADPH oxidase in the apoplast of plant cells plays a crucial role in cold acclimation induced chilling tolerance in tomato (*Lycopersicon esculentum*; Zhou et al., 2012). Some plants modulate their antifreeze activity by Ca2+, which is either released from pectin or bound to specific proteins and enhance the synthesis of proteins that inhibit the activity of ice nucleators in response to cold stress (Moffatt et al., 2006; Janska et al., 2009). An altered ratio of abscisic acid (ABA) to gibberellin content, in favor of ABA, results in the retardation of growth required for cold acclimation (Juntilla et al., 2002). Gibberellin content is regulated by a family of nuclear growthrepressing proteins called DELLAs, and these are components of the C-repeat (CRT) binding factor 1 (CBF1)-mediated cold stress response. However, the degradation of DELLAs is stimulated by gibberellins (Achard et al., 2008). Various cellular changes induced by temperature stress and metabolic homeostasis are shown in model (**Figure 6**).

### **STRATEGIES FOR THE DEVELOPMENT OF TEMPERATURE STRESS TOLERANCE INVOLVING REDOX HOMEOSTASIS EXOGENOUS MOLECULES IN REDOX HOMEOSTASIS IN PLANTS UNDER TEMPERATURE REGIMES**

Some molecules have the potential to protect the plants from the harmful and adverse effects of temperature stresses (Kaushal et al., 2011; Sharma et al., 2012) and these impart protection by managing the ROS. There are several reports where exogenous application of molecules such as proline (Pro), glycine betaine (GB), trehalose (Tre), brassiosteroids (Brs), polyamines (PAs), salicylic acid (SA), nitric oxide (NO), abscisic acid (ABA) and some trace elements like selenium etc. has shown beneficial effects on plant growth and development under stressful conditions by upregulation of antioxidant capacity (Tausz et al., 2004; Hefny and Abdel-Kader, 2009). Proline (Pro), a non-essential amino acid, is one of the most studied and extensively-reported thermoprotectant. Many studies have indicated a positive relationship between the accumulation of Pro and plant stress tolerance. Chickpea plants grown with exogenous Pro showed less injury to membranes, improved chlorophyll and water contents especially at 45/40◦C due to protection of vital enzymes of antioxidant metabolism under heat stress (Kaushal et al., 2011). Proline, when exogenously applied to tobacco culture cells resulted in decreased lipid peroxidation but increased SOD and catalase activities (Islam et al., 2009). Supplementation with Pro and GB considerably reduced H2O2 production and showed decrease in oxidative injury coupled to elevated levels of antioxidants in sugarcane (Rasheed et al., 2011). According to Gao et al. (2013), under heat stress, pre-treatment with trehalose (Tre) protected proteins in the thylakoid membranes and the photosynthetic capacity, reduced electrolyte leakage, MDA content and hydrogen peroxide levels due to elevated levels of antioxidants. The potential of Tre to induce heat tolerance in other crops needs to be examined as has been reported for inducing cold tolerance. Likewise, induction of cold tolerance by glycine beatine was found to be associated with increase in leaf water content, chlorophyll and sucrose concentrations, reduction in ABA and oxidative damage (Nayyar et al., 2005d). When supplied with exogenous glycine betaine, cold-stressed cucumber plants showed better survival, enhanced photosynthetic efficiency, and reduced MDA content and ROS (Li et al., 2004). Similar cryoprotective effects of exogenously applied GB were also confirmed when applied to *Medicago* seedlings (Zhao et al., 1992), potato (Somersalo et al., 1996), strawberry (Rajashekar et al., 1999), maize (Farooq et al., 2008), and tomato (Park et al., 2006). Foliar application of GB has resulted in induction of tolerance against cold stress in *Medicago sativa* (Zhao et al., 1992), wheat (Allard et al., 1998), strawberry (Rajashekar et al., 1999), and chickpea (Nayyar et al., 2005d).

Abscisic acid (ABA) is a naturally-occurring compound that helps to regulate plant growth and development (Pospisilova et al., 2009). A significant increase in free and conjugated ABA was observed in tomato seedlings at 45/35◦C compared to control plants (25/15◦C), which increased plant tolerance to temperature stress (Daie and Campbell, 1981). Likewise, ABA levels increased in response to heat treatment in tobacco (Teplova et al., 2000), which possibly is linked to redox homeostasis. There are reports where exogenous application of 10μM ABA alleviated heat stress symptoms by increasing SOD, CAT, APX, POX and decreasing H2O2 and MDA contents (Ding et al., 2010). In heat-stressed chickpea, exogenous application of 2.5μM ABA increased growth which was associated with enhanced endogenous ABA levels (Kumar et al., 2012b). Maize seedlings grown for 1–4 days in the presence of ABA were better able to withstand the effects of 3 h sub-lethal (40◦C) and lethal (45◦C) heat shocks to roots and shoots (Bonham-Smith et al., 1988). Pre-treatment of maize with 0.3 mML−<sup>1</sup> ABA at 46◦C improved the thermotolerance under heat stress (Gong et al., 1998). Heat tolerance increased significantly within 24 h of ABA application at 7.6 or 9.5μM in leaves and cell tissue culture in grapes (Abass and Rajshekhar, 1993). An ABA concentration of 10−<sup>5</sup> M inhibited heat-induced effects and enhanced thermostability of thylakoid organization in barley in response to heat stress (Ivanov et al., 1992). In cold-stressed plants too, ABA-treated plants showed significantly less oxidative damage, which was attributed to enhanced activities of various enzymatic and non-enzymatic antioxidants. The studies indicated that these plants showed improved cold tolerance as a result of increase in leaf water content and decrease in oxidative stress (Kumar et al., 2008).

Brassinosteroids (BRs) have a protective function under various abiotic stresses (Vardhini and Rao, 2003), which includes

enhancement of antioxidants. Exogenous application of BR has a promotory effect on the growth of wheat (Shahbaz et al., 2008), French bean (Upreti and Murti, 2004) and is involved in stimulating cell elongation under water stress conditions (Salchert et al., 1998). Supplementation with exogenous 24-BR's in tomato plants showed better responses under heat stress (40/30◦C). Activity of antioxidant enzymes such as SOD, APX, CAT were found to be increased, resulting in increase of shoot weight (Ogweno et al., 2008). A significant increase in net photosynthetic rate was reported by epibrassinosteroid (EBR) application to cucumber (*Cucumis sativum* L.; Yu et al., 2004) and tomato (Singh and Shono, 2005). The treatment of rapeseed and tomato seedlings with 24-epibrassinolide (a type of brassionosteroid) increased their basic thermotolerance (Dhaubhadel et al., 1999). In Indian mustard, application of different concentrations of 24 epibrassinolide (0, 10−6, 10−8, 10−<sup>10</sup> M) on 10-day-old seedlings at 40◦C identified that 10−<sup>8</sup> M was most effective for temperature amelioration due to enhanced activity of antioxidant enzymes (SOD, CAT, APX; Kumar et al., 2012c). Exogenous application of BRs retarded the rate of chlorophyll degradation and proteins associated with these pigments particularly those associated with chloroplast thylakoid membranes (Hola, 2011). In cold-stressed plants too, BRs conferred protection by reducing the oxidative damage. Foliar application of 24-epibrassinolide reduced oxidative damage and accelerated recovery from photoinhibition of PSII by activation of enzymes in Calvin cycle and increased the

antioxidant capacity in cucumber during cold stress (10/7◦C) (Jiang et al., 2013).

Salicylic acid is an important signaling molecule in plant defense responses (Yuan et al., 2008). Exogenous application of SA mitigates the effects of heat stress (Dat et al., 1998; Senaratna et al., 2003). In grape plants, exogenous pre-treatment with 0.1 mM SA maintained relatively higher activities of POX, SOD, APX, GR, and MDHAR indicating that SA can induce intrinsic heat tolerance in grapevines (Wang and Li, 2006). In another study on grapes treated with 100μM SA, exposure to 43◦C resulted in higher RUBISCO activity, increased PSII function and hence photosynthesis (Wang et al., 2010). Likewise, 10−<sup>5</sup> M SA significantly increased all growth parameters, antioxidant activity and Pro levels in Indian mustard growing under heat stress (30◦C and 40◦C) (Hayat et al., 2009). The results were confirmed by Kaur et al. (2009) who reported improved antioxidative abilities of CAT and POX in *Brasscia* species after exogenous application of 10 and 20μM SA at high temperatures (40–55◦C). SA application enhanced SOD activity significantly at 2 and 12 h heat stress and increased CAT activity within 12 h (He et al., 2003). In a study on six chickpea genotypes, seedlings were sprayed with 100μM L−<sup>1</sup> SA at 46◦C significantly reduced membrane injury, and enhanced protein and Pro contents which were accompanied by increased POX and APX activities (Chakraborty and Tongden, 2005). Pre-treatment of heat-stressed mungbean seedlings with SA reduced lipid peroxidation but improved membrane thermostability and antioxidant activity (Saleh, 2007). In cucumber, 1 mM SA foliar spray reduced electrolyte leakage and H2O2 level, and increased catalase activity (Shi et al., 2006). SA application has been found to be effective for improving cold tolerance (Tuteja et al., 2013). For instance, SA can induce cold tolerance in barley (*Hordeum vulgare*) by regulating activities of apoplastic antioxidative enzymes (Mutlu et al., 2013).

Nitric oxide (NO) is considered a signaling molecule involved in the regulation of physiological processes and stress responses in plants (Hasanuzzaman et al., 2013b). NO is a highly reactive, membrane permeant free radical which plays a crucial role in many physiological processes such as seed germination, reduction of seed dormancy, leaf expansion, regulation of plant maturation and senescence (Mishina et al., 2007), suppression of floral transition (He et al., 2004), ethylene emission and stomatal closure (Garcia-Mata and Lamattina, 2002; Neill et al., 2002; Guo et al., 2003), programmed cell death and light-mediated greening (Zhang et al., 2006). Recently, it has attracted wide attention due to its protective role in stress responses in different plant species (Hasanuzzaman et al., 2013b). In wheat, application of 50 and 100μM SNP on two cultivars C306 (heat-tolerant) and PBW550 (heat-sensitive) growing at 33◦C increased the activities of all antioxidant enzymes along with increased membrane thermostability and cellular viability (Bavita et al., 2012). In Mungbean, exogenous NO in the form of SNP during heat shock maintained the stability of chlorophyll a fluorescence, membrane integrity, H2O2 content and antioxidant enzyme activity (Yang et al., 2006). Similarly, exogenous application of 0.5 mM SNP on 8-day-old heat-treated seedlings (38◦C) of wheat for 24 and 48 h significantly reduced the high-temperature-induced lipid peroxidation and H2O2 content but increased the chlorophyll content, ascorbic acid, reduced glutathione (GSH) and the oxidized glutathione (GSSG) ratio (Hasanuzzaman et al., 2012a,b). The protective effect was linked to up**-**regulation of the antioxidant and glyoxalase system (Hasanuzzaman et al., 2012a,b, 2013b). SNP pre-treatment reduced the heat-induced damage in rice seedlings (Uchida et al., 2002) and increased the survival rate of wheat leaves and maize seedlings (Lamattina et al., 2001) thus validating its role in thermotolerance.

Ascorbic acid (AsA) is the most abundant and low molecular weight potential antioxidant having key role in defense against oxidative stress caused by enhanced level of ROS (Horemans et al., 2000; Athar et al., 2008). It can directly quench superoxide (O− <sup>2</sup> ), hydroxyl radicals (OH*.* ), and singlet oxygen (1O2) thereby providing membrane protection and regenerating α-tocopherols from tocopheroxyl radical, thereby, providing protection to membranes (Sharma et al., 2012). According to some reports, overexpression of enzymes involved in AsA biosynthesis confers temperature stress tolerance, as observed in some plants such as *Lycopersicum esculentum*, *Solanum tuberosum* (Chaves et al., 2002; Hemavathi et al., 2010; Radyuk et al., 2010), strawberry (Hemavathi et al., 2009). In Mungbean, plants treated with 50μM ascorbic acid exhibited significant enhancement in germination and growth of seedlings, pertinently under heat stress. AsAtreated plants showed less damage to membranes, cellular respiration, chlorophyll concentration and water status. Moreover, the oxidative stress was significantly reduced as a result of ASA application. Also, the increased activities of SOD, CAT and ascorbate peroxidase were found in AsA treated plants at 40/30 and 45/35◦C (Kumar et al., 2011).

Various signaling molecules providing stress tolerance are shown in **Figure 7**. Some other examples of crops with protective effects of exogenous molecules under stress conditions are shown in **Table 1**.

### **TRANSGENICS**

Plants can sense, transduce and translate the signals associated with ROS into appropriate cellular response depending on cellular redox state (Bhattacharjee, 2005). ROS/redox signaling networks in chloroplast and mitrochondria have important roles in plant adaptations to stresses (Mittler, 2002; Hemantaranjan et al., 2014). These various signals help the plant in cellular homeostasis under stressful conditions by controlling essential processes like transcription, translation, energy metabolism and protein phosphorylation (Mittler et al., 2011; Bita and Gerats, 2013). Various molecular approaches are assisting to understand the concept of temperature stress tolerance in plants (Wang et al., 2003; Hemantaranjan et al., 2014). Plants tolerate stress by modulating multiple genes and by coordinating the expression of genes in different pathways (Vinocur and Altman, 2005; Hasanuzzaman et al., 2013b). The adverse effects of temperature stresses can be mitigated by developing crop plants with improved stress tolerance using various transgenic approaches (Rodriguez et al., 2005). Among different defensive mechanisms, expression of some special types of proteins called heat shock proteins (HSPs) appears to be universal in lower and higher organism (Wahid et al., 2007; Suzuki et al., 2011). Temperature stress-response signal transduction pathways and various defense mechanisms, involving heat shock transcription factors (HSFs) and heat shock proteins (HSPs) are thought to be intimately associated with ROS and help in defense mechanisms in plants by providing stress tolerance (Pnueli et al., 2003; Suzuki and Mittler, 2006; Zhang et al., 2008). According to various studies, an intimate relationship appears to exist between oxidative stress and heat shock response (Pucciariello et al., 2012). HSF's possibly act as direct sensors of ROS, as evidenced by earlier studies on mammals, Drosophila and yeast (Ritossa, 1962). HSP's act as molecular chaperones and stabilize several cellular proteins under temperature stress, which has been reported to be a highly conserved response (Ahn and Thiele, 2003; Suzuki and Mittler, 2006). ROS production leads to the transduction of signals and the expression of heat shock genes in tobacco (Konigshofer et al., 2008). Heat shock proteins (HSP) are present under normal conditions but their expression level increases when the cell is under stress or shock (Robert, 2003). In normal growth conditions, HSPs control cellular signaling, protein folding, translocation and degradation but under high temperature stress they prevent protein misfolding and aggregation, and also protect membranes in plants and maintain redox homeostasis (Bita and Gerats, 2013). These proteins function as molecular chaperones and play crucial role in protecting plants against stress and maintaining homeostasis in cell and helps in its survival during heat stress (Feder and Hofmann, 1999). In addition to the studies concerning expression of sHSPs/chaperones and manipulation of HSF gene expression, transgenic plants

Compatible solutes Pro, GB, and Tre mitigate stress by osmoregulation and production of antioxidants. BRs, PAs, SA, ABA, and other signaling molecules such as NO activate the MAPK cascade leading to regulation

Abbreviations: BRs, Brassinosteroids; Pas, Polyamines; SA, Salicylic acid; ABA, Abscisic acid; NO, Nitric oxide; MAPK, Mitogen-activated protein kinase.

modified with other genes related to heat tolerance have been produced with varied success. Genetic improvement of proteins involved in osmotic adjustments, ROS detoxification, photosynthetic reactions and protein biosynthesis have showed positive results in developing transgenic plants with thermotolerance as shown in **Table 2**.

Diverse crop species tolerate low temperature to a varying degree, which depends on re-programming gene expression to modify their physiology, metabolism and growth (Sanghera et al., 2011). According to various studies, over-expression of combinations of antioxidant enzymes in transgenic plants has synergistic effect on stress tolerance (Kwon et al., 2003). Some of the stress-inducible genes especially encoding proteins which involve detoxification enzymes such as CAT, SOD, APX, GR etc. have been overexpressed in transgenic plants, further producing stress-tolerant phenotypes (Shinozaki et al., 2003). Simultaneous expression of multiple antioxidant enzymes, such as Cu/Zn-SOD, APX, and DHAR in chloroplast has shown to be more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses (Lee et al., 2007). Low temperature limitations have been overcome by the identification of cold- tolerant genes for transfer to genetically transformed crops. Therefore, transgenic plants overexpressing multiple antioxidants have increased emphasis in order to achieve cold tolerance (Sharma et al., 2012). Overexpresson of GR in *Nicotiana tabacum* and *Populus* plants leads to higher foliar AsA contents and improved tolerance to oxidative stress (Aono et al., 1993; Foyer et al., 1995) due to chilling injury. Tobacco plants genetically engineered to over-express chloroplast glycerol-3-phosphate acyltransferase (GPAT) gene (involved in phosphatidyl glycerol fatty acid desaturation), taken from *Arabidopsis* and *Cucurbita maxima*, were found to have enhanced cold tolerance, which was attributed to increase in number of unsaturated fatty acids. Higher lipid de-saturation of membranes



**Table 2 | Transgenic plants having heat tolerance in various plant species and their responses to ROS scavenging.**


is crucial for optimum membrane function in plants (Sanghera et al., 2011). In *Nicotiana tabacum*, chilling tolerance at 1◦C for 7 days was achieved by over-expression of genes encoding chloroplast omega-3-fatty acid desaturase (Kodama et al., 1994). Transgenic rice seedlings overexpressing OsNAC5 (encodes for transcription factor to regulate stress response) or suppression of OsNAC5 expression by RNAi provided low temperature tolerance (Song et al., 2011). Also, the transgenic rice overexpressing Sod1 (encoding Cu/Zn superoxide dismutase) were produced to obtain plants with improved tolerance to oxidative and cold stress (Cruz et al., 2013). Various crops have been genetically engineered to obtain plants with improved low temperature tolerance, which also involves reduction in oxidative stress (**Table 3**).

### **CONCLUSION AND FUTURE PERSPECTIVES**

Extreme temperatures are considered as major abiotic stresses for crop plants and also they are the causes of consequences of present day climate change. Plants growing in temperature range exceeding their limits of adaptation have substantial influence on their metabolism, physiology and yield. A common response in the form of oxidative stress is often showed by plants exposed to extreme temperature conditions (Hasanuzzaman et al., 2013b). During temperature stresses, overproduction of ROS can be a major risk factor to plant cells and also enhance the expression of ROS detoxifying and scavenging enzymes (Hossain and Fujita, 2011). ROS scavenging enzymes or antioxidants form the network, having important roles in redox signaling in chloroplast and mitochondria. This redox signaling maintains a delicate balance of homeostasis between different cellular components and within each organelle (Suzuki and Mittler, 2006; Suzuki et al., 2011). Under stress conditions, various important biological pathways such as regulation of gene expression, energy metabolism and protein phosphorylation are regulated by the cross-talk between different cellular components and redox signaling, further providing essential information on cellular redox state, associated with abiotic stress responses to optimize defense and survival (Foyer and Noctor, 2005, 2009). However, the extent of oxidative damage due to extreme temperature conditions depends largely on the duration of the adverse temperature, exposure of plant and their stage of growth. Therefore, there is need to develop the crop plants with temperature stress tolerance by exploring suitable and necessary strategies to manage oxidative stress. The use of the various exogenous molecules and the development of plants with different transgenes are important strategies to manage oxidative stress and maintain redox cellular state in plants. The ROS networks are interlinked with different networks in plants and control the temperature stress acclimation and tolerance. Various components involved in redox signaling networks may have individual signaling tasks within a given cellular compartment (Foyer and Noctor, 2003). Although in recent studies, the role of ROS and antioxidants in maintaining redox state has been intensively studied, but still there are open questions in this field. Therefore, it needs attention to study in detail the redox changes during cell growth, differentiation and division


**Table 3 | Transgenics for cold tolerance and involvement of ROS scavenging mechanisms.**

and also the specificity of the individual ROS and antioxidants and their interactions with hormone and secondary messengers during temperature-stress conditions. New insights into converging and diverging redox signaling pathways would be provided by the description of the redox-dependent spatial and temporal changes at various organization levels during plant growth and development and evaluation processes. Therefore, it could be useful for the better agriculture to clearly understand the redox control of plant growth, development and flowering. There are numerous research findings which support the notion that induction and regulation of antioxidant defenses are necessary for obtaining substantial tolerance against temperature stresses. Based on the various studies on redox environment, the modification of the cellular redox state may be used to increase the yield and stress tolerance in plants and to improve agriculture.

### **REFERENCES**


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respiratory pathway. *Plant Physiol.* 120, 765–772. doi: 10.1104/pp.120. 3.765


cytosolic ascorbate peroxidase (Apx1)-deficient Arabidopsis plants. *Plant J.* 34, 187–203. doi: 10.1046/j.1365-313X.2003.01715.x


chromosome 4 of rice and their association with anther length. *Theor. Appl. Genet*. 103, 862–868. doi: 10.1007/s001220100661


antioxidant metabolites, and ultrastructure of chloroplasts in two cool-season turf grass species under heat stress. *Environ. Exp. Bot*. 56, 274–285. doi: 10.1016/j.envexpbot.2005.03.002


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 November 2014; accepted: 09 February 2015; published online: 17 March 2015.*

*Citation: Awasthi R, Bhandari K and Nayyar H (2015) Temperature stress and redox homeostasis in agricultural crops. Front. Environ. Sci. 3:11. doi: 10.3389/fenvs. 2015.00011*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2015 Awasthi, Bhandari and Nayyar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants

### *Kaushik Das and Aryadeep Roychoudhury\**

*Post Graduate Department of Biotechnology, St. Xavier's College (Autonomous), Kolkata, India*

#### *Edited by:*

*Naser A. Anjum, University of Aveiro, Portugal*

#### *Reviewed by:*

*Naser A. Anjum, University of Aveiro, Portugal Carmen Arena, University of Naples Federico II, Italy Sarvajeet Singh Gill, Maharshi Dayanand University, India*

#### *\*Correspondence:*

*Aryadeep Roychoudhury, Post Graduate Department of Biotechnology, St. Xavier's College (Autonomous), 30, Mother Teresa Sarani, Kolkata - 700016, West Bengal, India e-mail: aryadeep.rc@gmail.com*

Reactive oxygen species (ROS) were initially recognized as toxic by-products of aerobic metabolism. In recent years, it has become apparent that ROS plays an important signaling role in plants, controlling processes such as growth, development and especially response to biotic and abiotic environmental stimuli. The major members of the ROS family include free radicals like O•− <sup>2</sup> , OH• and non-radicals like H2O2 and 1O2. The ROS production in plants is mainly localized in the chloroplast, mitochondria and peroxisomes. There are secondary sites as well like the endoplasmic reticulum, cell membrane, cell wall and the apoplast. The role of the ROS family is that of a double edged sword; while they act as secondary messengers in various key physiological phenomena, they also induce oxidative damages under several environmental stress conditions like salinity, drought, cold, heavy metals, UV irradiation etc., when the delicate balance between ROS production and elimination, necessary for normal cellular homeostasis, is disturbed. The cellular damages are manifested in the form of degradation of biomolecules like pigments, proteins, lipids, carbohydrates, and DNA, which ultimately amalgamate in plant cellular death. To ensure survival, plants have developed efficient antioxidant machinery having two arms, (i) enzymatic components like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR); (ii) non-enzymatic antioxidants like ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol, carotenoids, flavonoids, and the osmolyte proline. These two components work hand in hand to scavenge ROS. In this review, we emphasize on the different types of ROS, their cellular production sites, their targets, and their scavenging mechanism mediated by both the branches of the antioxidant systems, highlighting the potential role of antioxidants in abiotic stress tolerance and cellular survival. Such a comprehensive knowledge of ROS action and their regulation on antioxidants will enable us to develop strategies to genetically engineer stress-tolerant plants.

**Keywords: antioxidants, oxidative damages, reactive oxygen species, plant redox homeostasis, environmental stress**

### **INTRODUCTION**

Molecular oxygen was introduced to the early reducing atmosphere of the Earth about 2.7 billion years ago by O2- evolving photosynthetic organisms, causing the advent of the reactive oxygen species (ROS) as unwanted byproducts (Halliwell, 2006). Aerobic metabolism constantly generates ROS which are confined to the different plant cellular compartments, like the chloroplast, mitochondria and peroxisomes. Recent findings also shed light on the role of apoplast as a site for ROS generation (Jubany-Marí et al., 2009; Roychoudhury and Basu, 2012). Under favorable conditions, ROS is constantly being generated at basal levels. However, they are unable to cause damage, as they are being scavenged by different antioxidant mechanisms (Foyer and Noctor, 2005). The delicate balance between ROS generation and ROS scavenging is disturbed by the different types of stress factors like salinity, drought, extreme temperatures, heavy metals, pollution, high irradiance, pathogen infection, etc (**Figure 1**). The survival of the plants, therefore depends on many important factors like change in growth conditions, severity and duration of stress conditions and the capacity of the plants to quickly adapt to changing energy equation (Miller et al., 2010). Estimates show that only 1–2% of the O2 consumption by plant tissues, leads to the formation of ROS.

The ROS mainly comprise of 1O2, H2O2, O•− <sup>2</sup> , and OH•. These are very lethal and causes extensive damage to protein, DNA and lipids and thereby affects normal cellular functioning (Apel and Hirt, 2004; Foyer and Noctor, 2005). Redox homeostasis in plants during stressful conditions, is maintained by two arms of the antioxidant machinery—the enzymatic components comprising of the superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione-Stransferase (GST), and catalase (CAT), and the non-enzymatic low molecular compounds like ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol, carotenoids, phenolics, flavonoids, and proline (Gill and Tuteja, 2010; Miller et al., 2010; Gill et al., 2011). The omnipresent nature of both arms of the antioxidant machinery underlies the necessity of detoxification of ROS for cellular survival (Gill et al., 2011). In this review, we will primarily delve deeper into the domains of ROS, the antioxidant machinery and how they synergistically counteract the effects of environmental stress.

### **TYPES OF ROS**

Phototrophs convert light energy from the sun into biochemical energy and therefore are crucial for sustaining life on Earth. The

#### **Table 1 | Different members of the ROS family and their attributes.**

price they have to pay for this is to face the risk of oxidative damages, because of the different types of ROS, namely, 1O2(singlet oxygen), H2O2(hydrogen peroxide), O•− <sup>2</sup> (superoxide radical), and OH• (hydroxyl radical), generated as unwanted byproducts (**Table 1**). These are generated from only 1–2% of total O2 consumed by plants (Bhattacharjee, 2005). The reactions generating the different ROS members are shown (**Figure 2**).

#### **SUPEROXIDE RADICAL (O•− <sup>2</sup> )**

The ROS is being constantly generated in the chloroplasts due to partial reduction of O2 or as a result of transfer of energy to O2. The superoxide radical (O•− <sup>2</sup> ) is formed mainly in the thylakoidlocalized PSI during non-cyclic electron transport chain (ETC), as well as other cellular compartments. Normally, H2O is generated when cytochrome c oxidase interacts with O2. Occasionally, O2 reacts with the different ETC components to give rise to the O•− <sup>2</sup> . It is usually the first ROS to be formed. Superoxide radical (O•− <sup>2</sup> ) can also undergo further reactions to generate other members of the ROS family.

$$\rm O\_2^{\bullet-} + \rm Fe^{3+} \rightarrow \rm {}^{1}O\_2 + \rm Fe^{2+} $$

$$\rm 2O\_2^{\bullet-} + 2H^+ \rightarrow \rm O\_2 + \rm H\_2O\_2Fe^{3+} $$

$$\rm Fe^{2+} + \rm H\_2O\_2 + \rm Fe^{3+} \rightarrow \rm Fe^{3+} + \rm OH^- + \rm OH^- $$

(Fenton Reaction)

O•− <sup>2</sup> being moderately reactive with a short half-life of 2–4 μs, does not cause extensive damage by itself. Instead, it undergoes transformation into more reactive and toxic OH• and 1O2 and cause membrane lipid peroxidation (Halliwell, 2006).

### **SINGLET OXYGEN (1O2)**

Singlet Oxygen is an atypical ROS which is generated not by electron transfer to O2, but rather by the reaction of chlorophyll (Chl)


triplet state in the antenna system with O2.

$$\begin{aligned} \text{Chl} & \rightarrow {}^3\text{Chl} \\ {}^3\text{Chl} + {}^3\text{O}\_2 & \rightarrow \text{Chl} + {}^1\text{O}\_2 \end{aligned}$$

Environmental stresses like salinity, drought and heavy metals cause stomatal closure, leading to insufficient intracellular CO2 concentration. This favors the formation of 1O2. Singlet oxygen can cause severe damages to both the photosystems, PSI and PSII, and puts the entire photosynthetic machinery into jeopardy. Even though 1O2 has a short half-life of about 3 μs (Hatz et al., 2007), it can manage to diffuse some 100 nanometers and causes damage to wide range of targets. These include molecules like proteins, pigments, nucleic acids and lipids (Wagner et al., 2004; Krieger-Liszkay et al., 2008), and is the major ROS responsible for light-induced loss of PSII activity, eliciting cellular death. Plants have managed to efficiently scavenge 1O2 with the help of β-carotene, tocopherol, plastoquinone, and can also react with the DI protein of PSII. Alternatively, singlet oxygen plays a role in up regulating genes which are responsible for providing protection against photo-oxidative stress (Krieger-Liszkay et al., 2008).

### **HYDROGEN PEROXIDE (H2O2)**

Hydrogen peroxide, a moderately reactive ROS is formed when O•− <sup>2</sup> undergoes both univalent reduction as well as protonation. It can occur both non-enzymatically by being dismutated to H2O2 under low pH conditions, or mostly by a reaction catalyzed by SOD.

$$\begin{aligned} \mathrm{2O}\_{2}^{\bullet-} + 2\mathrm{H}^{+} &\to \mathrm{H}\_{2}\mathrm{O}\_{2} + \mathrm{O}\_{2} \\ \mathrm{2O}\_{2}^{\bullet-} + 2\mathrm{H}^{+} &\to \mathrm{H}\_{2}\mathrm{O}\_{2} + \mathrm{O}\_{2} \end{aligned}$$

H2O2 is produced in plant cells not only under normal conditions, but also by oxidative stress, caused by factors like drought, chilling, intense light, UV radiation, wounding, and pathogen infection (Sharma et al., 2012). Due to stomatal closure and low availability of CO2 and its limited fixation, Ribulose 1, 5-bisphosphate (RuBP) oxygenation is favored and thus photorespiration is enhanced. This accounts for more than 70% of the H2O2 produced as a result of drought stress (Noctor et al., 2002). The major sources of H2O2 production in plant cells include the ETC in the chloroplast, mitochondria, ER, cell membrane, β-oxidation of fatty acid and photorespiration. Additional sources comprise of different reactions involving photo-oxidation by NADPH oxidase and xanthine oxidase (XOD).

H2O2 in plants behaves like double-edged sword; it is beneficial at low concentrations, but damaging at higher concentrations in the cell. At low intracellular concentrations, it acts as a regulatory signal for essential physiological processes like senescence (Peng et al., 2005), photorespiration and photosynthesis (Noctor et al., 2002), stomatal movement (Bright et al., 2006), cell cycle and growth and development (Tanou et al., 2009a,b). Due to its significantly longer half-life of 1 ms, compared to other ROS members, it can traverse longer distances and cross plant cell membranes. It can cross membranes via aquaporins and cover considerable lengths within the cell (Bienert et al., 2007) and cause oxidative damage. H2O2 at high intracellular concentration oxidizes both cysteine (-SH) and methionine (-SCH3) residues and inactivates Calvin cycle enzymes, Cu/Zn SOD and Fe-SOD by oxidizing their thiol groups (Halliwell, 2006). It causes 50% loss in activity of different enzymes like fructose 1, 6 bisphosphatase, sedoheptulose 1, 7 bisphosphatase and phosphoribulokinase, at concentrations of 10μM H2O2 and is also responsible for programmed cell death at high cellular concentrations (Dat et al., 2000). However, like O•− <sup>2</sup> , H2O2 is moderately reactive; therefore, its damage is fully realized only when it is converted into more reactive species.

#### **HYDROXYL RADICAL (OH•)**

Among its family members, hydroxyl radical (OH•) is the most reactive and the most toxic ROS known. It is generated at neutral pH by the Fenton reaction between H2O2 and O•− <sup>2</sup> catalyzed by transition metals like Fe (Fe2+, Fe3+).

$$\mathrm{H\_2O\_2 + O\_2^{\bullet-} \to OH^- + O\_2 + OH^\bullet}$$

It has the capability to damage different cellular components by lipid peroxidation (LPO), protein damage and membrane destruction. Since there is no existing enzymatic system to scavenge this toxic radical, excess accumulation of OH• causes the cellular death (Pinto et al., 2003).

### **SITES OF ROS PRODUCTION IN PLANT CELLS**

The ROS is being produced under both normal and stressful conditions at various locations in the chloroplasts, mitochondria, peroxisomes, plasma membranes, ER and the cell wall. In presence of light, chloroplasts and peroxisomes are the major sources of ROS production, while the mitochondrion is the leading producer of ROS under dark conditions (Choudhury et al., 2013).

#### **CHLOROPLAST**

The chloroplast comprises of an extremely ordered system of thylakoid membranes which houses the light capturing photosynthetic machinery as well as anatomical requirements for efficient light harvesting (Pfannschmidt, 2003). The photosystems, PSI and PSII which form the core of the light harvesting system in the thylakoids are the major sources of ROS production. Abiotic stress factors like drought, salinity, temperature extremes, all of which cause water stress and limit CO2 concentrations, coupled with excess light, leads to the formation of O•− <sup>2</sup> at the PS, via the Mehler reaction.

$$\text{2O}\_2 + \text{2Fd}\_{\text{red}} \to \text{2O}\_2^{\bullet-} + \text{2Fd}\_{\text{ox}}$$

Subsequently, a membrane-bound Cu/Zn SOD at the PSI converts O•− <sup>2</sup> into H2O2 (Miller et al., 2010). The other accomplices of leaking electrons from the ETC of PSI are the 2Fe-2S and the 4Fe-4S clusters. In the PSII, seepage of electrons occurs, via the QA and QB electron acceptors and is responsible for the generation of O•− <sup>2</sup> . The superoxide radical then goes onto converting itself into more toxic ROS like OH• via H2O2 intermediate by the Fenton reaction at the Fe-S centers. The PSII is also responsible for the generation of 1O2 and this occurs in two ways. Firstly, when environmental stress upsets the delicate balance between light harvesting and energy utilization, it leads to the formation of triplet Chl (3Chl∗) which on reacting with dioxygen (3O2) liberates singlet oxygen (1O2) (Karuppanapandian et al., 2011). Secondly, when the ETC is over reduced, the light harvesting complex (LHC) at the PSII generates 1O2 (Asada, 2006). The 1O2 accumulating in the chloroplast causes peroxidation of membrane lipids, and especially Polyunsaturated Fatty Acids (PUFA) and damages membrane proteins which put the P680 reaction center of PSII at risk. It could also directly lead to cell death (Møller et al., 2007; Triantaphylidès et al., 2008). The involvement of the chloroplast in oxidative stress-induced programmed cell death was revealed when animal anti-apoptotic Bcl-2 was expressed in transgenic tobacco (Chen and Dickman, 2004). The 1O2 can also initiate a genetic program, via the EXECUTOR1 and EXECUTOR2 pathways and lead to growth inhibition in plants (Lee et al., 2007). Thus, the chloroplast is a major source of ROS production in plants. To ensure the continual survival of plants under stress, controlling and scavenging the ROS in the chloroplast is very essential, as shown in transgenic plants, as well in stress-tolerant cultivars (Tseng et al., 2007).

### **MITOCHONDRIA**

Mitochondria are also the site of generation of harmful ROS, like H2O2 and O•− <sup>2</sup> (Navrot et al., 2007), though in a smaller scale. Plant mitochondria differ from animal counterparts in having O2 and carbohydrate-rich environment (Rhoads et al., 2006) and also being involved in photorespiration. The mitochondrial ETC (mtETC) is the major culprit as it houses sufficiently energized electrons to reduce O2 to form the ROS. The two major components of the mtETC responsible for producing ROS are Complex I and Complex III (Møller et al., 2007; Noctor et al., 2007). The NADH Dehydrogenase or Complex I directly reduces O2 to O•− 2 in its flavoprotein region. The ROS production at Complex I is further enhanced when there is reverse electron flow from Complex III to Complex I due to lack of NAD+-linked substrates. This reverse flow of electrons is controlled by ATP hydrolysis (Turrens, 2003). In Complex III, ubiquinone in its fully reduced form donates an electron to Cytochrome c1 leaving behind an unstable ubisemiquinone semi-radical which favors leakage of electrons to O2, thereby generating O•− <sup>2</sup> (Murphy, 2009). Other sources of ROS production in the mitochondria are the various enzymes present in the mitochondrial matrix. This include enzymes like aconitase which directly produces ROS and others like 1-Galactono-γ-lactone dehydrogenase (GAL) which indirectly produce ROS by feeding electrons to the ETC (Rasmusson et al., 2008). Even though O•− <sup>2</sup> is the leading ROS in the mitochondria, it is converted to H2O2 by the Mn-SOD and the APX (Sharma et al., 2012). Estimates show that 1–5% of the total O2 consumption by the mitochondria is diverted toward production of H2O2. Mitochondrion generally produces ROS during normal conditions, but is greatly boosted at times of abiotic stress conditions (Pastore et al., 2007). Such stressful conditions affect the tight coupling of ETC and ATP synthesis, leading to over reduction of electron carriers like ubiquinone (UQ) pool, thereby generating ROS (Rhoads et al., 2006; Blokhina and Fagerstedt, 2010). Since respiratory rate increases during drought, the mitochondrial ATP synthesis increases to compensate for the lower rate of chloroplast ATP synthesis, enhancing the mitochondrial ROS production (Atkin and Macherel, 2009). To counteract this oxidative stress in the mitochondria, two enzymes, Mitochondrial Alternative Oxidase (AOX) and Mitochondrial SOD (Mn-SOD) are very crucial. The AOX maintains the reduced state of the UQ pool and cuts down the ROS production. Its importance is evident from the fact that *Arabidopsis* lacking a functional AOX is sensitive to drought stress and has altered transcription profiles of different components of the antioxidant machinery (Ho et al., 2008). On the other hand, the higher activity of Mn-SOD clearly made the difference between a salt-tolerant cultivar and a saltsensitive cultivar of tomato under salinity stress (Mittova et al., 2003).

### **PEROXISOMES**

Peroxisomes are single-membrane-bound spherical microbodies and are the major sites of intracellular H2O2 production due to their integral oxidative metabolism (Luis et al., 2006; Palma et al., 2009). They also produce O•− <sup>2</sup> , like chloroplasts and mitochondria during the course of various metabolic process. The O•− <sup>2</sup> is generated at two different locations. The Xanthine oxidase (E.C.1.17.3.2), located in the peroxisomal matrix, metabolizes both xanthine and hypoxanthine into uric acid and generate O•− <sup>2</sup> as a by-product. Second is the NADPH-dependent small ETC, composed of NADH and Cyt b localized in the peroxisomal membrane which utilizes O2 as the electron acceptor and releases O•− <sup>2</sup> into the cytosol. Additionally, Peroxisomal Membrane Polypeptides (PMPs) of molecular masses 18, 29, and 132 kDa are the three integral membrane proteins responsible for O•− <sup>2</sup> production. The NADH acts as the electron donor of the 18 and 32 kDa PMPs, whereas the 29 kDa PMP uses the NADPH as the electron donor to reduce Cytochrome c. During stressful conditions, when the availability of water is low and stomata remains closed, the ratio of CO2 to O2 reduces considerably which causes increased photorespiration leading to glycolate formation. This glycolate is oxidized by the glycolate oxidase in peroxisome to release H2O2, making it the leading producer of H2O2 during photorespiration (Noctor et al., 2002). Besides, there are other supplemental metabolic processes like β-oxidation of fatty acids, flavin oxidase pathway and the disproportionation of O•− <sup>2</sup> radicals for peroxisomal ROS production.

### **APOPLAST**

Apoplast, the diffusible space around the plant cell membrane is responsible for converting the incoming CO2 into a soluble, diffusible form which enters the cytosol to undergo photosynthesis. At times of adverse environmental conditions, stress signals combined with abscisic acid (ABA) make the apoplast a prominent site for H2O2 production (Hu et al., 2006). The NADPH oxidases expressed by the AtRbohD and AtRbohF in the guard cells and the mesophyll cells of *Arabidopsis*, account for generating the apoplastic ROS which is required for ABA-induced stomatal closure (Kwak et al., 2003). Besides these enzymes, there are additional ROS-generating enzymes which comprise of pH dependent peroxidases (POXs), cell wall-linked oxidases, germin-like oxalate oxidases and polyamine oxidases, all of which mainly produce H2O2.

### **PLASMA MEMBRANES**

Plasma membrane which surrounds the entire plant cell plays an important role in interacting with the ever changing environmental conditions and provides information necessary for the continual survival of the cell. The NADPH-dependent-oxidases which are localized in the plasma membrane are in the spotlight due to their gene expression and presence of different homologs during different stress conditions (Apel and Hirt, 2004). The NADPH oxidase produces O•− <sup>2</sup> by transferring electrons from cytosolic NADPH to O2, which either spontaneously dismutates to H2O2 or is catalyzed by SOD. The fact that NADPH oxidase plays an important role in plant defense against pathogenic infection and abiotic stress conditions (Kwak et al., 2003) is well supported.

### **CELL WALLS**

During stress, the cell wall-localized lipoxygenase (LOX) causes hydroperoxidation of polyunsaturated fatty acids (PUFA) making it active source of ROS like OH•, O•− <sup>2</sup> , H2O2, and 1O2. The cell wall-localized diamine oxidases utilize diamines or polyamines to generate ROS in the cell wall. During pathogen attack, lignin precursors undergo extensive cross-linking, via H2O2-mediated pathways to reinforce the cell wall with lignin (Higuchi, 2006).

### **ENDOPLASMIC RETICULUM (ER)**

The NADPH-mediated electron transport involving CytP450, localized in the ER generates O•− <sup>2</sup> (Mittler, 2002). Organic substrate, RH interacts with the CytP450 followed by reduction by a flavoprotein to give rise to a free radical intermediate (Cyt P450 R−). This intermediate promptly reacts with triplet oxygen (3O2) to form an oxygenated complex (Cyt P450-ROO−). The complex may occasionally decompose to Cyt P450-Rh by generating O•− 2 as byproduct.

### **TARGETS OF ROS**

ROS is known to cause damages to biomolecules such as lipids, proteins and DNA (**Figure 3**).

### **LIPIDS**

Lipids form a major portion of the plasma membrane which envelopes the cell and helps it to adapt to the changing environment. However, under stressful conditions, when the level of ROS rise above the threshold value, LPO becomes so damaging that it is often considered as the single parameter to gauge lipid destruction. LPO starts a chain reaction and further exacerbates oxidative stress by creating lipid radicals which damages proteins and DNA. The two main targets of the ROS in membrane phospholipids are the double bond between C-atoms and the ester linkage between glycerol and fatty acids. The PUFA which are important components of the plasma membrane are the hotspots for ROS damage. PUFAs like linoleic and linolenic acid are specifically prone to attack by ROS like 1O2 and OH•. The hydroxyl radical (OH•) is the most damaging member as it has the ability to trigger a cyclic chain reaction and cause further peroxidation of other PUFAs.

The entire process of LPO can be divided into three distinct phases, Initiation, Progression, and Termination. Initiation involves energizing the O2 (a rate limiting step) to give rise to radicals like O•− <sup>2</sup> and OH•. These ROS react with the methylene groups of the PUFA, yielding conjugated dienes, lipid peroxyl radicals and hydroperoxides (Smirnoff, 2000).

PUFA-H + OH• → PUFA•(PUFA alkyl radical) + H2O PUFA• + O2 → PUFA-OO•(Peroxyl radical)

The PUFA peroxyl radical once formed possesses the ability to further propagate the LPO by extracting one H-atom from adjoining PUFA side chains.

PUFA-OO• + PUFA − H → PUFA − OOH + PUFA•

The lipid hydroperoxide (PUFA-OOH) undergoes cleavage by reacting with reduced metals such as Fe2+.

$$\text{PUFA-OOH} + \text{Fe}^{2+} \rightarrow \text{PUFA-O}^{\bullet} + \text{Fe}^{3+} $$

The lipid hydroperoxides can also undergo decomposition to form different reactive species such as lipid alkoxyl radicals, aldehydes, alkanes, lipid epoxides, and alcohols. LPO terminates with the formation of different lipid dimers caused by different lipid derived radicals.

$$\text{PUFA}^{\bullet} + \text{PUFA}^{\bullet} \rightarrow \text{PUFA} + \text{PUFA}$$

$$(\text{Fatty Acid Dimer})$$

$$\text{PUFA}^{\bullet} + \text{PUFA-OO}^{\bullet} \rightarrow \text{PUFA-OO-PUFA}$$

$$(\text{Peroxidebridged Dimer})$$

$$\text{PUFA-OO}^{\bullet} + \text{PUFA-OO}^{\bullet} \rightarrow \text{PUFA-OO-PUFA} + \text{O}\_2$$

$$(\text{Peroxidebridged Dimer})$$

Overall, the LPO increases membrane fluidity causing the membrane to be leaky to substances which otherwise enter the cell through special channels, damage the membrane proteins, deactivate the membrane receptors, membrane-localized enzymes and ion-channels.

### **PROTEINS**

The ROS produced during stress conditions causes the oxidation of proteins. The protein undergoes different types of modifications which may either be direct or indirect. During direct modifications, the activity of the protein becomes varied as a result of different chemical modifications such as nitrosylation, carboxylation, disulfide bond formation, and glutathionylation. Protein carbonylation is often used as a marker for evaluating protein oxidation (Møller et al., 2007). Indirect modification of proteins can occur as a result of interaction with the products of LPO. The ROS concentration, on crossing its threshold value, leads to the site-specific modification of amino acids like Arg, Lys, Pro, Thr, and Trp, and increased susceptibility to proteolytic degradation (Møller et al., 2007). The amino acids differ in their susceptibility to ROS attack. Amino acids containing thiol groups and sulfur are the most vulnerable. The Cys and Met are both prone to damage by the reactive 1O2 and OH•. The enzymes containing iron-sulfur centers are irreversibly inactivated on getting oxidized by O•− <sup>2</sup> . The oxidized proteins thus become better targets for proteolytic digestion by getting primed for ubiquitination-mediated proteosomal degradation.

### **DNA**

Since the plant nuclear DNA is well protected by histones and associated proteins, both mitochondrial and chloroplastic DNA bears the brunt of the ROS attack due to lack of protective histones as well as the close proximity to ROS production machinery. Oxidative damage of DNA as a result of ROS occurs at multiple levels which include oxidation of the deoxyribose sugar residue, modification of the nucleotide base, abstraction of a nucleotide, breaks in either DNA strand, and cross-linking of the DNA and protein. The hydroxyl radical not only damages the deoxyribose sugar backbone by extracting H-atom, but also reacts with double bonds of the purine and pyrimidine bases (Halliwell, 2006). The ROS abstracts the C-4 H-atom of the deoxyribose sugar and forms a deoxyribose radical which reacts further to cause single strand breaks in the DNA (Evans et al., 2004). The damaged products as a result of base oxidation include the most common 8-hydroxyquinine and other less common ones like hydroxyl methyl urea, dehydro-2 -deoxyguanosine, thymine glycol, and thymine and adenine ring opened. The OH• is also notorious for creating DNA-protein cross-links when it reacts with either DNA or associated proteins. These cross-links are not easily reparable and may be lethal to the plant cell, if not repaired in time before commencement of critical cellular processes like replication or transcription.

### **ROS DEFENSE MACHINERY**

The ROS defense mechanism consists of the antioxidant machinery which helps to mitigate the above mentioned oxidative stress-induced damages. The antioxidant machinery has two arms with the enzymatic components and non-enzymatic antioxidants (**Table 2**).

### **ENZYMATIC ANTIOXIDANTS**

The enzymes localized in the different subcellular compartments and comprising the antioxidant machinery include Superoxide Dismutase (SOD), Catalase (CAT), Ascorbate Peroxidase (APX), Monodehydroascorbate reductase (MDHAR), Dehydroascorbate reductase (DHAR), Glutathione Reductase (GR), and Guaiacol Peroxidase (GPX).

### *Superoxide Dismutase (SOD)*

SOD (E.C.1.15.1.1) belongs to the family of metalloenzymes omnipresent in all aerobic organisms. Under environmental stresses, SOD forms the first line of defense against ROS-induced damages. The SOD catalyzes the removal of O•− <sup>2</sup> by dismutating it into O2 and H2O2. This removes the possibility of OH• formation by the Haber-Weiss reaction. SODs are classified into three isozymes based on the metal ion it binds, Mn-SOD (localized in mitochondria), Fe-SOD (localized in chloroplasts), and Cu/Zn-SOD (localized in cytosol, peroxisomes, and chloroplasts) (Mittler, 2002). SOD has been found to be up regulated by abiotic stress conditions (Boguszewska et al., 2010).

$$\text{O}\_2^{\bullet-} + \text{O}\_2^{\bullet-} + 2\text{H}^+ \rightarrow 2\text{H}\_2\text{O}\_2 + \text{O}\_2$$

### *Catalase (CAT)*

CAT (E.C.1.11.1.6) is a tetrameric heme-containing enzyme responsible for catalyzing the dismutation of H2O2 into H2O and O2. It has high affinity for H2O2, but lesser specificity for organic peroxides (R-O-O-R). It has a very high turnover rate (6 <sup>×</sup> <sup>10</sup><sup>6</sup> molecules of H2O2 to H2O and O2 min−1) and is unique amongst antioxidant enzymes in not requiring a reducing equivalent. Peroxisomes are the hotspots of H2O2 production due to β-oxidation of fatty acids, photorespiration, purine catabolism and oxidative stress (Mittler, 2002). However, recent reports suggest that CAT is also found in other subcellular compartments


**Table 2 | List of all the enzymatic and non-enzymatic antioxidants along with their functions and cellular localization.**

such as the cytosol, chloroplast and the mitochondria, though significant CAT activity is yet to be seen (Mhamdi et al., 2010). Angiosperms have been reported to have three *CAT* genes. *CAT1* is expressed in pollens and seeds (localized in peroxisomes and cytosol), *CAT2* predominantly expressed in photosynthetic tissues but also in roots and seeds (localized in peroxisomes and cytosol) and finally *CAT3* is found to be expressed in leaves and vascular tissues (localized in the mitochondria). Stressful conditions demand greater energy generation and expenditure of the cell. This is fulfilled by increased catabolism which generates H2O2. CAT removes the H2O2 in an energy efficient way.

$$\mathrm{H\_2O\_2} \rightarrow \mathrm{H\_2O} + \mathrm{(l/2)O\_2}$$

### *Ascorbate peroxidase (APX)*

APX (E.C.1.1.11.1) is an integral component of the Ascorbate-Glutathione (ASC-GSH) cycle. While CAT predominantly scavenges H2O2 in the peroxisomes, APX performs the same function in the cytosol and the chloroplast. The APX reduces H2O2 to H2O and DHA, using Ascorbic acid (AA) as a reducing agent.

$$\rm H\_2O\_2 + \rm AA \to \rm 2H\_2O + DHA$$

The APX family comprises of five isoforms based on different amino acids and locations, viz., cytosolic, mitochondrial, peroxisomal, and chloroplastid (stromal and thylakoidal) (Sharma and Dubey, 2004). Since APX is widely distributed and has a better affinity for H2O2 than CAT, it is a more efficient scavenger of H2O2 at times of stress.

### *Monodehydroascorbate reductase (MDHAR)*

MDHAR (E.C.1.6.5.4) is responsible for regenerating AA from the short-lived MDHA, using NADPH as a reducing agent, ultimately replenishing the cellular AA pool. Since it regenerates AA, it is co-localized with the APX in the peroxisomes and mitochondria, where APX scavenges H2O2 and oxidizes AA in the process (Mittler, 2002). MDHAR has several isozymes which are confined in chloroplast, mitochondria, peroxisomes, cytosol, and glyoxysomes.

MDHA + NADPH → AA + NADP<sup>+</sup>

### *Dehydroascorbate reductase (DHAR)*

DHAR (M.C.1.8.5.1) reduces dehydroascorbate (DHA) to AA using Reduced Glutathione (GSH) as an electron donor (Eltayeb et al., 2007). This makes it another agent, apart from MDHAR, which regenerates the cellular AA pool. It is critical in regulating the AA pool size in both symplast and apoplast, thus maintaining the redox state of the plant cell (Chen and Gallie, 2006). DHAR is found abundantly in seeds, roots and both green and etiolated shoots.

$$\text{DHA} + \text{2GSH} \rightarrow \text{AA} + \text{GSSG}$$

### *Glutathione Reductase (GR)*

GR (E.C.1.6.4.2) is a flavoprotein oxidoreductase which uses NADPH as a reductant to reduce GSSG to GSH. Reduced glutathione (GSH) is used up to regenerate AA from MDHA and DHA, and as a result is converted to its oxidized form (GSSG). GR, a crucial enzyme of ASC-GSH cycle catalyzes the formation of a disulfide bond in glutathione disulfide to maintain a high cellular GSH/GSSG ratio. It is predominantly found in chloroplasts with small amounts occurring in the mitochondria and cytosol. GSH is a low molecular weight compound which plays the role of a reductant to prevent thiol groups from getting oxidized, and react with detrimental ROS members like 1O2 and OH•.

$$\text{GSSG} + \text{NADPH} \rightarrow \text{2GSH} + \text{NADP}^+$$

### *Guaiacol peroxidase (GPX)*

GPX (E.C.1.11.1.7) is a heme-containing enzyme composed of 40–50 kDa monomers, which eliminates excess H2O2 both during normal metabolism as well as during stress. It plays a vital role in the biosynthesis of lignin as well as defends against biotic stress by degrading indole acetic acid (IAA) and utilizing H2O2 in the process. GPX prefers aromatic compounds like guaiacol and pyragallol (Asada, 1999) as electron donors. Since GPX is active intracellularly (cytosol, vacuole), in the cell wall and extracellularly, it is considered as the key enzyme in the removal of H2O2.

$$\text{H}\_2\text{O}\_2 + \text{GSH} \rightarrow \text{H}\_2\text{O} + \text{GSSG}$$

### **NON-ENZYMATIC ANTIOXIDANTS**

The non-enzymatic antioxidants form the other half of the antioxidant machinery, comprising of AA, GSH, α-tocopherol, carotenoids, phenolics, flavonoids, and amino acid cum osmolyte proline. They not only protect different components of the cell from damage, but also play a vital role in plant growth and development by tweaking cellular process like mitosis, cell elongation, senescence and cell death (de Pinto and De Gara, 2004).

### *Ascorbic Acid (AA)*

AA is the most abundant and the most extensively studied antioxidant compound. It is considered powerful as it can donate electrons to a wide range of enzymatic and non-enzymatic reactions. Majority of AA in plant cells is the result of Smirnoff-Wheeler pathway, catalyzed by L-galactano-γ-lactone dehydrogenase in the plant mitochondria, with the remaining being generated from D-galacturonic acid. 90% of the AA pool is concentrated not only in the cytosol, but also substantially in apoplast, thus making it the first line of defense against ROS attack (Barnes et al., 2002). AA is oxidized in two successive steps, starting with oxidation into MDHA, which if not reduced immediately to ascorbate, disproportionates to AA and DHA. It reacts with H2O2, OH•, O•− <sup>2</sup> , and regenerates α-tocopherol from tocopheroxyl radical, thereby protecting the membranes from oxidative damage (Shao et al., 2005). It also protects and preserves the activities of metalbinding enzymes. AA in its reduced state acts as the cofactor of violaxanthine de-epoxidase and maintains the dissipation of the excess excitation energy (Smirnoff, 2000). AA has also been reported to be involved in preventing photo-oxidation by pHmediated modulation of PSII activity and its down regulation, associated with zeaxanthine formation.

### *Reduced glutathione (GSH)*

Glutathione is a low molecular weight thiol tripeptide (γ-glutamyl-cysteinyl-glycine) abundantly found in almost all cellular compartments like cytosol, ER, mitochondria, chloroplasts, vacuoles, peroxisomes, and even the apoplast. It is involved in a wide range of processes like cell differentiation, cell growth/division, cell death and senescence, regulation of sulfate transport, detoxification of xenobiotics, conjugation of metabolites, regulation of enzymatic activity, synthesis of proteins and nucleotides, synthesis of phytochelatins and finally expression of stress responsive genes (Mullineaux and Rausch, 2005). This versatility of GSH is all due to its high reductive potential. A central cysteine residue with nucleophilic character is the source of its reducing power. GSH scavenges H2O2, 1O2, OH•, and O•− <sup>2</sup> and protects the different biomolecules by forming adducts (glutathiolated) or by reducing them in presence of ROS or organic free radicals and generating GSSG as a by-product. GSH also plays a vital role in regenerating AA to yield GSSG. The GSSG thus generated is converted back to GSH, either by de novo synthesis or enzymatically by GR. This ultimately replenishes the cellular GSH pool. GSH also helps in the formation of phytochelatins via phytochelatin synthase (Roychoudhury et al., 2012a), which helps to chelate heavy metal ions and thus scavenges another potential source of ROS formation in plants (Roy Choudhury et al., 2012b). Therefore, the delicate balance between GSH and GSSG is necessary for maintaining the redox state of the cell.

### **α***-Tocopherol*

The α-tocopherol belongs to a family of lipophilic antioxidants which are efficient scavengers of ROS and lipid radicals, making them indispensable protectors and essential components of biological membranes (Holländer-Czytko et al., 2005; Kiffin et al., 2006). The α-tocopherol has the highest antioxidant capability among the four isomers (α-, β-, γ-, δ-). The tocopherols are synthesized only by photosynthetic organisms and thus only present in green tissues of plants. The α-tocopherol is synthesized from γtocopherol by γ- tocopherol-methyl-transferase (γ-TMT encoded by *VTE4*). Tocopherols are known for their ability to protect lipids and other membrane constituents of the chloroplasts by reacting with O2 and quenching its excess energy, thus protecting the PSII, both structurally and functionally. Tocopherol also serves as an effective free radical trap by halting the chain propagation step of the LPO cycle. It reacts with the lipid radicals RO•, ROO•, and RO∗ at the membrane-water interface, where α-tocopherol reduces them and itself gets converted into TOH•. The TOH• radical undergoes recycling to its reduced form by interacting with GSH and AA (Igamberdiev et al., 2004).

### *Carotenoids*

Carotenoids belong to family of lipophilic antioxidants which are localized in the plastids of both photosynthetic and nonphotosynthetic plant tissues. They are found not only in plants, but also in micro-organisms. They belong to a group of antennae molecules which absorbs light in the 450–570 nm and transfers the energy to the chlorophyll molecule. Carotenoids exhibit their antioxidative activity by protecting the photosynthetic machinery in four ways, (a) reacting with LPO products to end the chain reactions, (b) scavenging 1O2 and generating heat as a byproduct, (c) preventing the formation of 1O2 by reacting with 3Chl<sup>∗</sup> and excited chlorophyll (Chl∗), and (d) dissipating the excess excitation energy, via the xanthophyll cycle.

### *Flavonoids*

Flavonoids are widely found in the plant kingdom occurring commonly in the leaves, floral organs and pollen grains. Flavonoids can be classified into four classes on the basis of their structure, flavonols, flavones, isoflavones, and anthocyanins. They have diverse roles in providing pigmentation in flowers, fruits and seeds involved in plant fertility and germination of pollen and defense against plant pathogens. Flavonoids have been considered as a secondary ROS scavenging system in plants experiencing damage to the photosynthetic apparatus, due to the excess excitation energy (Fini et al., 2011). They also have a role in scavenging 1O2 and alleviate the damages caused to the outer envelope of the chloroplastic membrane (Agati et al., 2012).

### *Proline*

Proline, an osmolyte is also regarded as a powerful antioxidant. It is widely used across the different kingdoms as a nonenzymatic antioxidant to counteract the damaging effects of different ROS members. Proline is synthesized using glutamic acid as a substrate, via a pyrroline 5-carboxylate (P5C) intermediate. This pathway in plants is catalyzed by two enzymes, ð1-pyrroline-5-carboxylate synthetase (P5CS) and Pyrroline-5 carboxylate reductase (P5CR). It is an efficient scavenger of OH• and 1O2 and can inhibit the damages due to LPO. During stress, proline accumulates in plants in large amounts which is either due to enhanced synthesis or reduced degradation (Verbruggen and Hermans, 2008).

### **ANTIOXIDANT REGULATION FOR ENVIRONMENTAL STRESS TOLERANCE**

Increased SOD activity in response to drought stress was detected in three different cultivars of *Phaseolus vulgaris* (Zlatev et al., 2006) and *Oryza sativa* (Sharma and Dubey, 2005a,b). The SOD activity was found to be heightened during drought stress in the leaves of white clover, viz., *Trifolium repens* L. (Chang-Quan and Rui-Chang, 2008). The SOD activity was found to be up regulated during salt stress in many plants like chickpea (Kukreja et al., 2005) and tomato (Gapiñska et al., 2008). All three isoforms of SOD have been found to be expressed in chickpea in response to salinity stress (Eyidogan and Öz, 2007). Transgenic *Arabidopsis* overexpressing Mn-SOD was found to have enhanced salt tolerance (Wang et al., 2004). SOD activity was increased by UV-B radiation in pea, wheat, *Arabidopsis* and rice, but not affected in barley and soybean. In a field study, supplemental UV-B increased SOD activity in wheat and mungbean, and caused differential responses among soybean cultivars (Agrawal et al., 2009). The CAT activity was found to increase especially in drought-sensitive varieties of wheat (Simova-Stoilova et al., 2010). *Cicer arietinum* under salt stress also have increased CAT activity in both leaves (Eyidogan and Öz, 2007) and roots (Kukreja et al., 2005). Increased CAT activity under cadmium stress has been reported in *Phaseolus aureus*, *Pisum sativum*, *Lemna minor*, barley and sunflower (Sreedevi and Krishnan, 2012). When the antioxidant profile of drought-tolerant and drought-susceptible genotypes of wheat were compared, it was found out that the drought-tolerant genotype C306 showed higher APX and CAT activity, and AA content with lower H2O2 and MDA content than the droughtsusceptible genotype, HD2329 (Sairam et al., 1998). When APX was overexpressed in the chloroplasts of *Nicotiana tabacum,* it reduced the toxic effects of H2O2 and generated drought tolerance (Badawi et al., 2004). There was also an enhancement in their tolerance to salt stress. UV-B radiation increased APX activity in *Arabidopsis thaliana* (Rao et al., 1996). The activity of APX positively correlated with Pb treatment in *Eichhornia crassipes* (water hyacinth) seedlings (Malar et al., 2014). Roychoudhury et al. (2012c) reported that the activities of antioxidative enzymes like GPX and APX increased both in IR-29 (salt-sensitive) and Nonabokra (salt-tolerant) rice varieties during CdCl2 stress; however, the activity was more enhanced in Nonabokra. The CAT activity during Cd stress showed a different trend, with a marked decrease in IR-29, while marked increase in Nonabokra at higher Cd concentration. The activity of peroxidase and CAT increased progressively with the increase in CdCl2 concentration in *Vigna radiata* (Roychoudhury and Ghosh, 2013). *Vaccinium myrtillus L*. is regarded as a species which is a successful colonist of acidand heavy metal-contaminated soil. Upon analysis of the antioxidant response of this plant from heavily polluted sites (immediate vicinity of zinc smelter, iron smelter and power plant), it was found that the contents of GSH, non-protein thiols, proline and activity of GPX were elevated. The GPX activity seemed to be universal, sensitive and correlated well with heavy metal stress (Kandziora-Ciupa et al., 2013). Overexpression of MDHAR in tobacco (Eltayeb et al., 2007) and DHAR in *Arabidopsis* (Ushimaru et al., 2006) resulted in improved salt tolerance. Stressed rice seedlings displayed increased activity of the enzymes MDHAR, DHAR and GR, all of which are involved in the regeneration of AA (Sharma and Dubey, 2005a,b). Under salt stress, APX and GR activities were found to be higher in salt-tolerant cultivars of potato, while being markedly diminished in salt-sensitive varieties. This sensitivity was attributed to the reduction of APX and GR activity during saline conditions (Aghaei et al., 2009). Marked drought-induced increase in GPX activity was noted in both the sensitive rice varieties IR-29 and Pusa Basmati (Basu et al., 2010a). Exogenous application of AA to wheat cultivars resulted in higher chlorophyll contents, net photosynthesis and growth, compared to the non-treated plants challenged with drought stress (Malik and Ashraf, 2012). It has also been seen that priming *Carthamus tinctorius* seeds with AA significantly relieved the harsh effects of drought stress on seedling growth (Razaji et al., 2012). When AA was exogenously applied, prior to and during salt stress in tomato seedlings, it helped expedite the recovery process and ensured long-term survival (Shalata and Neumann, 2001). AA also helped to relieve oxidative damage in wheat, by improving photosynthetic capacity and sustaining ion homeostasis (Athar et al., 2008). The greater susceptibility of the sensitive varieties IR-29 and Pusa Basmati to water scarcity was also reflected by considerable decrease in GSH/GSSG ratio, as compared to the tolerant variety Pokkali (Basu et al., 2010b). Both AA and GSH were found to have enhanced levels in salt-tolerant cultivar Pokkali than in the sensitive cultivar Pusa Basmati (Vaidyanathan et al., 2003). Arsenic (III) significantly decreased the GSH content in rice roots, due to its conversion to phytochelatins. The GSH supplementation resulted in partial protection against arsenic stress, reducing the MDA content and restoring the seedling growth of arsenic (V) exposed seedlings (Roychoudhury and Basu, 2012). GSH was also found to lessen the oxidative damage in rice chloroplasts caused due to salinity stress (Wang et al., 2014). Under low UV-B doses, increases in AA and GSH pools, as well as AA regeneration ability functioned to keep the balance of cellular H2O2 (Roychoudhury and Basu, 2012). Studies on heat-acclimated vs. non-acclimated cool season turfgrass species suggested that the former had lower production of ROS, as a result of enhanced synthesis of AA and GSH. In wheat, it was established that heat stress induced the accumulation of GSH levels and increased the activity of the enzymes involved in GSH synthesis and the GSH/GSSG ratio (Hasanuzzaman et al., 2013). When transgenic tobacco overexpressing *Arabidopsis VTE1* (encoding tocopherol biosynthesis enzyme) were subjected to drought conditions, they showed decreased LPO, electrolyte leakage and H2O2 content, but had increased chlorophyll compared with the wild type (Liu et al., 2008). *Arabidopsis vte1* and *vte4* mutants lacking α-tocopherol are particularly sensitive to salt stress, as evident by their reduced growth and increased oxidative stress. This is because α-tocopherol maintains the cellular Na+/K+ homeostasis and hormonal balance (Ellouzi et al., 2013). Acute exposure of UV-B leads to decrease in α-tocopherol levels in plants, possibly reflecting reactions with lipid radicals (Jain et al., 2003). In drought-resistant plants, the number of carotenoid molecules per chlorophyll unit increased under drought stress, thus providing photo-protection from oxidative damages (Munné-Bosch and Alegre, 2000). Water deficit, induced by 20% polyethylene glycol (PEG 6000) treatment to rice seedlings led to increment in antioxidants like flavonoids and phenolics, which were several folds higher in the tolerant cultivar Pokkali, as compared to the sensitive varieties like IR-29 and Pusa Basmati (Basu et al., 2010a). The two isolines of soybean cv. Clark, the normal line with moderate levels of flavonoids and the magenta line with reduced flavonoid levels, were grown in the field with or without natural levels of UV-B. Solar UV-B radiation caused oxidative stress in both the lines and altered ROS metabolism, primarily by decreasing SOD activity and increasing the activities of APX, CAT, and GR. This resulted in decreased AA content and increased DHA content. The magenta line had greater oxidative stress than the normal line, in spite of its enhanced oxidative defense capacity as compared to the normal line, even under UV-B exclusion. These results indicate enhanced sensitivity in the magenta line, especially under UV-B exclusion that was likely due to the absence of flavonoid epidermal screening compounds and subsequent increased penetration of solar ultraviolet radiation into the leaf (Xu et al., 2008). Proline, an osmoprotectant as well as a sink for energy to regulate redox potentials, was found to have increased accumulation in drought-tolerant cultivars of chickpea than sensitive cultivars under both control and drought stress conditions (Mafakheri et al., 2010). In case of rice seedlings, exposed to high salt stress (200 mM NaCl), the antioxidants like anthocyanin and proline showed the highest level in the salt-tolerant cultivar Nonabokra, as compared to the salt-sensitive cultivars like M-1-48 and Gobindobhog (Roychoudhury et al., 2008). The content of flavonoids and proline were also found to be enhanced in salt-tolerant cultivars of indica rice than in the salt-sensitive cultivars, as evident by the reduced membrane damage caused by LPO (Chutipaijit et al., 2009).

### **CONCLUSION**

The ROS plays the double role of being the inevitable by-product of aerobic metabolism on one hand and serving as a marker during stressful conditions on the other hand. They not only serve as agents of damages in plants, but also trigger stresssignaling components to prevent further damages. ROS synthesis is widespread, with production sites being present in both intracellular and extracellular locations. The damage caused by ROS is extensive and the targets include all biomolecules like lipids, proteins and DNA, damaging the integrity of the cell and ultimately leading to its death. However, evolution has equipped plants with a wider range of defense measures which include changes at the morphological, metabolic and genetic level to adapt to the adverse environmental conditions. This review gives an insight into how both arms of the antioxidant machinery; the antioxidant enzymes and the non-antioxidant metabolites, work in conjunction to alleviate the damaging effects of ROS and develop tolerance against various environmental stress conditions. Although significant progress has been achieved in recent years, there are still ambiguities and gaps in our understanding of ROS formation and how they affect plants, primarily due to their short half-life and highly reactive nature. Although the highly compartmentalized nature of antioxidants is well defined, the sensing and response mechanism as well as the control of the delicate balance between production and scavenging need to be better explored. Several issues remain unanswered, like the interaction between ROS and calcium signaling and the regulation of ROS during multiple environmental stresses. In future, advanced imaging techniques like the markers for Ca2<sup>+</sup> imaging can lead to better understanding of ROS metabolism. Advanced functional genomics, coupled with proteomics and metabolomics will offer detailed insights into ROS network and its related responses. There is no doubt that transgenic approach for overexpression of antioxidant gene cassettes can lead to enhanced tolerance to multiple stresses in future (Oztetik, 2012).

### **ACKNOWLEDGMENTS**

Financial support from Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India through the research grant (SR/FT/LS-65/2010) to Dr. Aryadeep Roychoudhury is gratefully acknowledged.

### **REFERENCES**


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 08 October 2014; accepted: 11 November 2014; published online: 02 December 2014.*

*Citation: Das K and Roychoudhury A (2014) Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Front. Environ. Sci. 2:53. doi: 10.3389/fenvs.2014.00053*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2014 Das and Roychoudhury. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Control of cucumber (*Cucumis sativus* L.) tolerance to chilling stress—evaluating the role of ascorbic acid and glutathione

#### *Alexander S. Lukatkin1 \* and Naser A. Anjum2*

*<sup>1</sup> Department of Botany, Physiology and Ecology of Plants, N.P. Ogarev Mordovia State University, Saransk, Russia <sup>2</sup> CESAM-Center for Environmental and Marine Studies and Department of Chemistry, University of Aveiro, Aveiro, Portugal*

#### *Edited by:*

*Adriano Sofo, Università degli Studi della Basilicata, Italy*

#### *Reviewed by:*

*Sarvajeet Singh Gill, Maharshi Dayanand University, India Yogesh Abrol, Bhagalpur University, India*

#### *\*Correspondence:*

*Alexander S. Lukatkin, Department of Botany, Physiology and Ecology of Plants, N.P. Ogarev Mordovia State University, Bolshevistskaja Str., 68. Saransk 430005, Russia e-mail: aslukatkin@yandex.ru*

Chilling temperatures (1–10◦C) are known to disturb cellular physiology, cause oxidative stress *via* creating imbalance between generation and metabolism of reactive oxygen species (ROS) leading finally to cell and/or plant death. Owing to known significance of low molecular antioxidants—ascorbic acid (AsA) and glutathione (GSH) in plant stress-tolerance, this work analyzes the role of exogenously applied AsA and GSH in the alleviation of chilling stress (3◦C)-impact in cucumber (*Cucumis sativus* L. cv. Vjaznikowskij 37) plants. Results revealed AsA and GSH concentration dependent metabolism of ROS such as superoxide (O2•−) and the mitigation of ROS-effects such as lipid peroxidation (LPO) as well as membrane permeability (measured as electrolyte leakage) in *C. sativus* leaf discs. AsA concentration (750µM) and GSH (100µM) exhibited maximum reduction in O2•<sup>−</sup> generation, LPO intensity as well as electrolyte leakage, all of these were increased in cold water (3 and 25◦C)-treated leaf discs. However, AsA, in particular, had a pronounced antioxidative effect, more expressed in case of leaf discs during chilling (3◦C); whereas, at temperature 25◦C, some AsA concentrations (such as 50 and 100 mM AsA) exhibited a prooxidative effect that requires molecular-genetic studies. Overall, it is inferred that AsA and GSH have high potential for sustainably increasing chilling-resistance in plants.

**Keywords: chilling stress, oxidative stress, ascorbic acid, glutathione,** *Cucumis sativus* **L., tolerance**

### **INTRODUCTION**

Sub-optimal (low non-freezing/chilling) temperatures are among the major environmental factors known to impact crop productivity via affecting growth, development and metabolism especially in the tropics and subtropics (Lukatkin et al., 2012; Li et al., 2014). Plant species may exhibit their differential sensitivity to chilling stress, where the exhibition of their incapability to withstand potential impacts of low temperatures has been extensively reported in chilling-sensitive/ non-tolerant plants (reviewed by Lukatkin et al., 2012). At temperatures above the freezing point of tissues but lower than 10◦C (chilling temperature), a range of chilling-sensitive crop plants (such as maize, rice, cotton, tomato, cucumber, and soybeans) may develop external symptoms of injury and/or may succumbed to death (reviewed by Lukatkin et al., 2012). In chilling-sensitive plants, visible symptoms of injury may include wilting leaves and hypocotyls, the appearance of surface pits and large cavities, discoloration of leaves and internal tissues, and leaf necrosis and plants death (Tsuda et al., 2003; reviewed by Lukatkin et al., 2012). At cellular level, chilling-sensitive plant genotypes may exhibit: impaired cell cycle progression (Rymen et al., 2007), multiple disorganizations of the cells' ultrastructure, such as disturbed the formation of chloroplasts, caused destruction of chloroplasts membranes (Gutierrez et al., 1992; Kratsch and Wise, 2000), shift in intracellular pH, and an increase in cell membrane permeability (Kasamo et al., 2000; reviewed by Lukatkin et al., 2012).

Oxidative stress, a physiological condition, where occurs an imbalance between the generation of reactive oxygen species (ROS; such as singlet oxygen, 1O2•<sup>−</sup>; superoxide anion, O2•−; hydroxyl radical, HO•; hydrogen peroxide, H2O2) and their and metabolism via enzymatic and non-enzymatic antioxidants, may also occur in chilling-sensitive plants (Lukatkin, 2002a,b; Xiong et al., 2002; Gill and Tuteja, 2010; Anjum et al., 2012). Non-metabolized ROS may cause considerable damages to membrane lipids (lipid peroxidation, LPO) and other cellular components, and also increase electrolyte leakage (Lukatkin, 2003; Suzuki and Mittler, 2006; Anjum et al., 2010, 2012, 2014a; Gill and Tuteja, 2010; Popov et al., 2010). To efficiently counteract ROS-mediated potential consequences, plants employ enzymatic (SOD, superoxide dismutase; CAT, catalase; GPX, guaiacol peroxidase; GST, glutathione sulfo-transferase; APX, ascorbate peroxidase; MDHAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase) and non-enzymatic (ascorbic acid, AsA; glutathione, GSH; carotenoids; tocopherols; phenolics) antioxidants-based defense system (Anjum et al., 2010, 2012, 2014b; Gill and Tuteja, 2010).

Though the role of AsA and GSH in plant stress-tolerance has been emphasized (Anjum et al., 2010; Noctor et al., 2012), and the literature is full on the significance of AsA (Dolatabadian et al., 2008; Kumar et al., 2011b; Zhang et al., 2011), and also that of GSH (Anjum et al., 2010; Cai et al., 2010), reports on a comparative account of AsA and GSH in chilling exposed plants are rare and/or unsubstantiated. Therefore, this work analyzes the role of exogenously applied AsA and GSH in the alleviation of chilling stress-impact in cucumber (*Cucumis sativus* L. cv. Vjaznikowskij 37) plants.

### **MATERIALS AND METHODS**

### **PLANT CULTURE AND TREATMENTS**

Cucumber (*Cucumis sativus* L. cv. Vjaznikowskij 37) seeds were surface sterilized with 0.5% KMnO4 for 20 min, 6% chloramines for 10 min and 70% ethanol for 1 min then rinsed with sterile water. Sterilized seeds were sown in pots containing 2.0 kg of soil (median-loamy degraded chernozem) at 22–24◦C, 60–80% of full soil water capacity, and with illumination about 200µM photons m−<sup>2</sup> s <sup>−</sup>1) photosynthetic photon flux density (PPFD) and 12 h light day. Leaves were isolated from 18 to 20 day old plants and leaf discs (8 mm in diameter) were punched with a cork-borer. Subsequently, leaf discs were immersed in Petri dishes (about 300 mg leaves discs per 10 ml of water or antioxidant solution (AsA: 0.5, 0.75, 1.5, 10, 50, 100 mM; or GSH: 50, 100, 150µM), all with temperature 25 or 3◦C) and kept from 0.5 to 2 h at these temperatures. The control leaf discs were kept at 25◦C.

### **BIOASSAYS**

The methods adopted and described by Lukatkin (2002a) were employed to estimate O2•<sup>−</sup> generation (based on oxidation of adrenaline), and membrane lipid peroxidation (LPO) (measured as the level of MDA). In brief, homogenate obtained by the homogenization of leaf discs (0.3 g) in distilled was centrifuged for 15 min at 4000 g. To 3.0 ml of supernatant, 100µl of 0.01% adrenaline (epinephrine) solution was added, and the tube was incubated for 45 min at room temperature and 80µM photons m−<sup>2</sup> s <sup>−</sup><sup>1</sup> PPFD. Immediately at the end of incubation, optical density of the adrenochrome formed (as a measure of estimate O2•−) was read against homogenate with water on a UVvisible spectrophotometer (SF-46, LOMO, St. Petersburg, Russia) at 480 nm. For LPO estimation, 10 ml of isolation medium (0.1 M Tris-HCl buffer pH 7.6, containing 0.35 M NaCl) was used to homogenize leaf discs (1.0 g). To 3.0 ml of the obtained

homogenate, 2.0 ml of thiobarbituric acid (TBA) (prepared in 20% trichloracetic acid, TCA) was added, and the solution was heated in a boiling water bath for 30 min, filtered and ice-cooled and centrifuged at 1000 × g for 10 min at 4◦C. Reading in the supernatant was recorded at 532 nm in the UV-Vis spectrophotometer. The rate of LPO was expressed as µmole MDA g−<sup>1</sup> tissue wet weight using a molar extinction coefficient of 1*.*<sup>56</sup> <sup>×</sup> 105 M−<sup>1</sup> cm−1. Additionally, index of chilling injury (ICI) was assessed by measuring electrolyte leakage employing the formula: Injury cofficient = Ld – Lo/100 - Lo× 100 (Lukatkin, 2003). Where, Ld and Lo denote electrolyte losses, respectively from damaged/treated and untreated/control leaf discs. Injury cofficient reflects the electrolyte efflux and was expressed in percent of total loss.

### **STATISTICAL ANALYSES**

All experiments were repeated three-six times in 4–6 biological replicates. The figures and tables represent the means of all experimental data and their standard errors. The significance of differences between treatments was evaluated by Student's *t*-test.

### **RESULTS**

The results presented below describe the significant responses of O2•<sup>−</sup> generation, LPO and electrolyte leakage considering AsA followed by GSH in chilling experiments with leaf discs.

### **SUPEROXIDE ION STATUS IN LEAF DISCS UNDER TEMPERATURE REGIMES (25 AND 3◦C), AND WITH ASCORBIC ACID AND GLUTATHIONE**

The pattern of AsA effect on O2•<sup>−</sup> generation exhibited its dependency on AsA concentration and temperature regimes (**Table 1**). Leaf discs incubated in the distilled water at 25◦C showed a monotonous increase of O2•<sup>−</sup> generation, indicating a graduate intensification of oxidative stress induced by mechanical injuries in leaf disc cells. Though O2•<sup>−</sup> generation in the leaf disc immersed in AsA solutions at 25◦C was enhanced too but almost all concentrations of AsA exhibited their tendency to lowering of O2•<sup>−</sup> generation when compared with leaf discs immersed in water. The best mitigating effect of AsA on O2•<sup>−</sup> was with

**Table 1 | Effect of ascorbic acid (AsA) concentrations on O2•<sup>−</sup> generation (µM g−<sup>1</sup> min−1) in the leaf discs of** *Cucumis sativus* **at 3 and 25◦<sup>C</sup> temperatures.**


750µM AsA solution, where a reduction of 40% in the O2•<sup>−</sup> level was evidenced. However, a comparison among the used AsA concentrations revealed a prooxidative effect of the highest AsA concentrations (50 and 100 mM). On the other hand, incubation of leaf discs in water at 3◦C lead to more intensive increase of O2•<sup>−</sup> generation as compared with temperature 25◦C. The rate of O2•<sup>−</sup> generation was most prominent after 2 h of immersion of leaf discs in cold water, where increase of 3.75 times was noted when compared to initial point. Here also, all the used AsA concentrations lead to diminishing of oxidative stress caused by O2•<sup>−</sup> elevation when compared with distilled water. Among the AsA concentrations used, 0.75 mM AsA exhibited the highest O2•−-decreasing effect. Interestingly, the highest concentration of AsA (100 mM) had the best effect in first 0.5 and 1 h of chilling, with sharp increase of O− <sup>2</sup> generation in next hour.

Experiments considering another non-enzymatic antioxidant—GSH, showed different pattern of O2•<sup>−</sup> generation (**Figure 1**). Here also, leaf discs incubated in the water with 25◦C, as at 3◦C temperatures exhibited an enhanced rate of O2•<sup>−</sup> generation, where all the used GSH concentrations effectively lowered the O2•<sup>−</sup> generation rate almost similarly at both 25 and 3◦C. Notably, GSH at 100µM most effectively lowered O2•<sup>−</sup> generation rate when comparison was made among the used GSH concentrations. In the experiments, where leaf discs were incubated for 4 h in solutions with different GSH concentrations, 100µM GSH was found as the most effective GSH concentration in decreasing the O2•<sup>−</sup> generation rate (**Figure 2**).

### **ELECTROLYTE LEAKAGE AND LIPID PEROXIDATION STATUS IN LEAF DISCS WITH ASCORBIC ACID AND GLUTATHIONE**

Electrolyte leakage from leaf discs was used as criterion of cell membrane damage (**Figure 2B**). At 25◦C, incubation of *C. sativus* leaf discs in AsA solution lead to lowering of electrolyte leakage, but GSH enhanced this parameter. Leaf discs incubated at 3◦C showed a significant increase in the electrolyte leakage when compared to leaf discs incubated in distilled water. The lowest electrolyte leakage was revealed in the case of AsA and GSH-supplemented solutions. Considering LPO, a 3◦C caused a significant increase in leaf disc-LPO level (measured as MDA content) when compared to LPO in leaf discs incubated at 25◦C (**Figure 2A**). However, leaf discs incubated in antioxidant solution at 25◦C exhibited a lower LPO intensity, where decreases of 7.8 and 18.8% were displayed, respectively with GSH and AsA.

### **DISCUSSION**

The current test plant, *C. sativus* is a warm-season vegetable and is known for its susceptibility to low temperatures throughout its growth cycle (Kuk and San Shin, 2007). Therefore, this study was performed to assess the role of major non-enzymatic antioxidants such as AsA and GSH in the control of O2•<sup>−</sup> generation and its consequence (measured herein as LPO and electrolyte leakage). In the present study, temperature regimes (3 and 25◦C) without AsA or GSH differentially impacted leaf discs by significantly enhancing the content of O2•<sup>−</sup> when compared with the control leaf discs. Nevertheless, a gradual time-dependent intensification of oxidative stress (in terms of elevated O2•<sup>−</sup> generation) was perceptible when incubation of leaf discs was done in water at 3◦C (vs. 25◦C). The detailed above observation coincides well with the earlier studies where, chilling stress enhanced the generation of ROS (such as superoxide, O2·) (Lukatkin, 2002a,b, 2003; Popov et al., 2010). Low temperature-mediated differential enhancements in the generation of O− <sup>2</sup> have been evidenced earlier in the leaves of a number of plants including cucumber, maize and millet (Lukatkin, 2002a). If not metabolized, ROS

can initiate damaging cellular membrane by oxidizing membrane biomolecules such as lipids and proteins (Anjum et al., 2010, 2012, 2014a; Gill and Tuteja, 2010; Popov et al., 2010; Lukatkin et al., 2012). To this end, electrolyte leakage and lipid peroxidation are among the major consequences of O2•−-accrued impact on cell membrane (Halliwell and Gutteridge, 2000; Anjum et al., 2014a). This is also true in the present study, where leaf discs incubated at 3◦C exhibited significantly increased electrolyte leakage as well as the content of MDA (a well-known indicator of membrane lipid peroxidation). Nevertheless, enhanced LPO was reported earlier, where the extent of LPO elevation was shown to correlate with species and cultivar sensitivity to chilling, and, therefore, it is believed to be a measure for cold-induced damage to chilling-sensitive plants (Lukatkin, 2002a; reviewed by Lukatkin et al., 2012).

Efficiency of antioxidant defense system to scavenge ROS such as O2•<sup>−</sup> largely decides the plant's sensitivity to various stress factors including chilling (Lukatkin, 2002a,b; Xiong et al., 2002; Gill and Tuteja, 2010; Anjum et al., 2012). AsA and GSH are the most abundant low molecular weight non-enzymatic antioxidants in plant cells participating in ROS scavenging (Anjum et al., 2010; Gill and Tuteja, 2010). In the present study, AsA and GSH concentrations differentially controlled the rate of O2•<sup>−</sup> generation in the leaf discs incubated in solutions with 3 and 25◦C temperatures. In particular, AsA concentration (750µM; 0.75 mM) and GSH (100µM) exhibited maximum reduction in the O2•<sup>−</sup> generation and its consequence measures herein as LPO as well as electrolyte leakage. In fact, apart from the enzymatic antioxidants (such as SOD, CAT, GPX, GST, APX, MDHAR, DHAR, GR), the non-enzymes such as AsA and GSH are among the major nonenzymatic antioxidants significant for efficiently metabolizing major ROS and also for counteracting their consequences (such as electrolyte leakage and LPO) in abiotic stressed plants (Anjum et al., 2010, 2012, 2014b; Gill and Tuteja, 2010). However, AsA, in particular, had a pronounced antioxidative effect, more expressed in case of leaf discs during chilling (3◦C); whereas, at temperature 25◦C, some AsA concentrations (such as 50 and 100 mM AsA) exhibited a prooxidative effect. The observed noticeable result may be a consequence of an interactive effect of exogenous AsA with endogenous Fe or Cu (data not shown) that are known to increase of ROS generation via Fenton reaction.

AsA is widely distributed in plant tissues and is used as a substrate by APX (a major ROS-metabolizing enzyme); therefore, reduced AsA significantly controls of the cellular reducing environment (Davey et al., 2000; Anjum et al., 2010, 2014b). Many environmental stresses including chilling conditions can induce an increase of endogenous AsA (Wang et al., 2004). The AsA contents was more in tolerant to chilling stress chickpea (*Cicer arietinum*) genotypes after chilling at reproductive phase (Kumar et al., 2011a). Overexpression of SIGMEs (*Solnaum lycopersicon* GDP-Mannose 3 ,5 -epimerase) was reported to cause AsA accumulation with enhanced cold tolerance in tomato (Zhang et al., 2011). On the other hand, GHS is a crucial antioxidant associated with regenerating AsA in the AsA-GSH cycle, and thus GSH is also involved in the regulation of ROS (such as H2O2) (Anjum et al., 2010). Based on its redox buffering action and abundance in cells, reduced form of GSH is considered to protect the cell against elevated ROS-mediated oxidative damages (Anjum et al., 2010; Noctor et al., 2012). Extensive reports have revealsed that the GSH pool size in plants and the status of its oxidation and reduction are associated highly with plant resistance to stressed environments (Kumar et al., 2008; Xu et al., 2008; Anjum et al., 2010). A differential elevation in GSH has been reported in a number of chilling exposed plants including cucumber genotypes (Xu et al., 2008) and *C. arietinum* (Kumar et al., 2008). The AsA-GSH cycle is highly related to plant antioxidant defense, and the metabolic intensity of the cycle is directly associated with the capacity of plant resistance to stress. The oxidative and reductive status of AsA and GSH are closely related to the adaptation of plants to stressed environments, and the accomplishment of AsA function depends largely on the available GSH supply and the conditions of oxidation and reduction in cells (Anjum et al., 2010). Lowered extent of oxidative stress and enhanced stress resistance in plants has also been reported with exogenous application (seed treatment, soil influx or foliar spraying) of AsA (Dolatabadian et al., 2008; Dolatabadian and Saleh, 2009; Al-Hakimi and Hamada, 2011; Kumar et al., 2011b) or GSH (Cai et al., 2010; Teh et al., 2014).

### **CONCLUSIONS**

*C. sativus* responded to chilling conditions by exhibiting duration of exposure and temperature level-dependent elevations in O2•<sup>−</sup> and its consequences measured as electrolyte leakage and LPO. Exogenous supply of AsA and GSH to chilling stressed plants can be beneficial in improving plant health and productivity. AsA concentration (750µM) and GSH (100µM) exhibited maximum reduction in the O2•<sup>−</sup> generation, LPO intensity as well as membrane permeability. It can be said that AsA and GSH have high potential in increase of chilling-resistance in plants. However, molecular-genetic research is required to unveil the exact mechanisms underlying the reported herein peroxidative effect of 50 and 100 mM AsA at temperature 25◦C that was not displayed at 3◦C.

### **ACKNOWLEDGMENTS**

Results were obtained in the framework of the state task of the Russian Ministry of Education and Science (project number 6.783.2014K, sanctioned to Alexander S. Lukatkin). Naser A. Anjum gratefully acknowledges financial support received from Portuguese Foundation for Science and Technology (FCT) through post-doctoral research grants (SFRH/BPD/64690/2009; SFRH/BPD/84671/2012).

### **REFERENCES**


two cucumber cultivars under low light. *Physiol. Plant* 132, 467–478. doi: 10.1111/j.1399-3054.2007.01036.x

Zhang, C., Liu, J., Zhang, Y., Cai, X., Gong, P., Zhang, J., et al. (2011). Overexpression of SIGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato. *Plant Cell Rep.* 30, 389–398. doi: 10.1007/s00299-010-0939-0

**Conflict of Interest Statement:** The Associate Editor, Adriano Sofo, declares that, despite co-hosting a Frontiers Research Topic with the author, Naser Anjum, the review process was handled objectively and no conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 24 November 2014; accepted: 04 December 2014; published online: 18 December 2014.*

*Citation: Lukatkin AS and Anjum NA (2014) Control of cucumber (Cucumis sativus L.) tolerance to chilling stress—evaluating the role of ascorbic acid and glutathione. Front. Environ. Sci. 2:62. doi: 10.3389/fenvs.2014.00062*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2014 Lukatkin and Anjum. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Polyamines as redox homeostasis regulators during salt stress in plants

Jayita Saha<sup>1</sup> , Elizabeth K. Brauer 2, 3, Atreyee Sengupta<sup>1</sup> , Sorina C. Popescu2, 3 , Kamala Gupta<sup>1</sup> and Bhaskar Gupta<sup>1</sup> \*

<sup>1</sup> Department of Biological Sciences, Presidency University, Kolkata, India, <sup>2</sup> The Boyce Thompson Institute for Plant Research, Ithaca, NY, USA, <sup>3</sup> Department of Plant Pathology and Plant Microbe Biology, Cornell University, Ithaca, NY, USA

The balance between accumulation of stress-induced polyamines and reactive oxygen species (ROS) is arguably a critical factor in plant tolerance to salt stress. Polyamines are compounds, which accumulate in plants under salt stress and help maintain cellular ROS homeostasis. In this review we first outline the role of polyamines in mediating salt stress responses through their modulation of redox homeostasis. The two proposed roles of polyamines in regulating ROS—as antioxidative molecules and source of ROS synthesis—are discussed and exemplified with recent studies. Second, the proposed function of polyamines as modulators of ion transport is discussed in the context of plant salt stress. Finally, we highlight the apparent connection between polyamine accumulation and programmed cell death induction during stress. Thus, polyamines have a complex functional role in regulating cellular signaling and metabolism during stress. By focusing future efforts on how polyamine accumulation and turnover is regulated, research in this area may provide novel targets for developing stress tolerance.

Keywords: polyamine signaling, ROS, plant abiotic stress, salinity stress, redox homeostasis

### Introduction

Global climate change and agronomic practices have contributed to increased soil salinity, which currently affects an estimated 45 million hectares of irrigated land (Rengasamy, 2010). Salt stress limits crop productivity and is imposed by an accumulation of cations (Na+, K+, Mg2+, Ca2+) and anions (Cl−, SO2<sup>−</sup> 4 , HCO<sup>−</sup> 3 ) originating from water-soluble salts such as Na2SO4, NaHCO3, NaCl, and MgCl<sup>2</sup> as well as less water-soluble salts including CaSO4, MgSO4, and CaCO3.These salts accumulate due to factors such as mineral erosion and crop irrigation with mineralized water or ocean water (Todorova et al., 2013).

High salt concentrations in soil cause both hyperionic and hyperosmotic stress in the intracellular environment. During the initial stages of salt stress, the high external solute concentration decreases the cellular water potential, which eventually imposes turgor loss and pleiotropic physiological responses including stomatal closure, growth inhibition, reduced pollen viability, inhibition of photosynthetic enzyme activity, sucrose accumulation, and inactivation of photosynthetic electron transport (Munns and Tester, 2008; Chaves et al., 2009; Biswal et al., 2011; Silva et al., 2011; Mittal et al., 2012; Shu et al., 2012; Jajoo, 2013). Long-term salt stress results in hyperaccumulation of Na<sup>+</sup> leading to suppression of enzymatic activity, increased H2O<sup>2</sup> and lipid peroxidation that ultimately causes leaf senescence (Sairam et al., 2002; Chinnusamy and Zhu, 2003; Allu et al., 2014).

Under normal conditions, the cytosol contains 100–200 mM of K<sup>+</sup> and 1–10 mM of Na<sup>+</sup> (Taiz and Zeiger, 2002). Excess NaCl is the most common cause of salt stress in plants and induces

#### Edited by:

Naser A. Anjum, University of Aveiro, Portugal

#### Reviewed by:

Naser A. Anjum, University of Aveiro, Portugal Maria Marina, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico Chascomús (UNSAM-CONICET), Argentina Melike Bor, Ege University, Turkey

#### \*Correspondence:

Bhaskar Gupta, Department of Biological Sciences, Presidency University, 86/1 College Street, Kolkata 700073, India bhaskar.dbs@presiuniv.ac.in; bhaskarzoology@gmail.com

#### Specialty section:

This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science

> Received: 11 December 2014 Accepted: 05 March 2015 Published: 01 April 2015

#### Citation:

Saha J, Brauer EK, Sengupta A, Popescu SC, Gupta K and Gupta B (2015) Polyamines as redox homeostasis regulators during salt stress in plants. Front. Environ. Sci. 3:21. doi: 10.3389/fenvs.2015.00021 overaccumulation of Na<sup>+</sup> and Cl<sup>−</sup> and depletion of K<sup>+</sup> ions in the cell. This imbalance in the Na+:K<sup>+</sup> ratio is a result of the competition between the ions for transport into the cell and is thought to produce detrimental effects due to changes in osmotic potential, nutrient limitation and ionic toxicity. Plants counteract these effects using multiple strategies including: (i) producing osmolytes like soluble sugars, organic acids, free amino acids, and accumulating potassium ions (Ahmad and Sharma, 2008; Ahmad et al., 2012), (ii) activating transporters that export sodium from the cell, (iii) limiting Na<sup>+</sup> uptake into roots and leaves, (iv) sequestering Na<sup>+</sup> ions into subcellular compartments, (v) altering photosynthetic rates, (vi) changing membrane structure, (vii) inducing antioxidative enzymes, and (viii) decreasing stomatal conductance (Jithesh et al., 2006; Ozgur et al., 2013). In addition, plant cells rapidly accumulate reactive oxygen species (ROS) in response to salt and other stresses, a response widely known as the "oxidative burst" (Mittler, 2002; Miller et al., 2008). The oxidative burst has an important role in inducing signaling events and is dependent on enzymes located in several subcellular compartments (Foyer and Noctor, 2005; Baxter et al., 2014). However, it is essential that ROS production be regulated, as excess ROS accumulation results in membrane lipid peroxidation, DNA damage, protein denaturation, carbohydrate oxidation, pigment breakdown, and ultimately leads to cell death (Scandalios, 1993; Noctor and Foyer, 1998). To counteract the potentially damaging effects of the oxidative burst, plants produce a diverse set of antioxidants whose regulation is not yet fully understood. While the interplay between ROS turnover and antioxidant accumulation during stress is quite complex, it is essential to understand how this system works for its potential in enhancing plant stress tolerance (Noctor and Foyer, 1998).

### Mechanisms of ROS Production During Salt Stress

ROS are highly reactive forms of molecular oxygen and include the hydroxyl radical (HO. ), superoxide (O2.−), hydrogen peroxide (H2O2), and singlet oxygen (1O2) (Dowling and Simmons, 2009; Shapiguzov et al., 2012). The reactivity and half-life of different ROS species are correlated to their mobility and diffusion distance in the cellular space. Among the ROS species present in plants, hydrogen peroxide is the most stable having a half-life of 1 ms, whereas singlet oxygen (1O2), superoxide (O<sup>2</sup> .−) and hydroxyl radicals (OH• ) are short-lived species with half-lives of 1–4µs to 1 nanosecond (Gechev et al., 2006; Moller et al., 2007). Although numerous subcellular compartments contribute to ROS production, the major sites of ROS generation include the chloroplast, mitochondria, and peroxisome (**Figure 1**) (Foyer et al., 2003; Mittler et al., 2004; Asada, 2006; Rhoads et al., 2006).

The chloroplast produces the highest levels of ROS under both normal conditions and salt stress. ROS generation occurs within both Photosystem I (PSI) and Photosystem II (PSII) reaction centers in the thylakoid membrane. During salt stress, ROS production is enhanced due to changes in membrane fluidity and protein complex formation, blocking the electron transfer from water to PSII (Chaves et al., 2009; Biswal et al., 2011; Silva et al., 2011; Jajoo, 2013). Another important site for ROS production is the mitochondria. During salt stress, mitochondrial respiration is disrupted; over-reduction of the ubiquinone pool facilitate the leakage of electrons from complexes I and III of the mitochondrial electron transport chain to molecular oxygen, resulting in O·− 2 production (Noctor et al., 2007; Miller et al., 2010). Excess O2in the cell also increases the photorespiration rate, which produces O·− 2 and <sup>1</sup>O<sup>2</sup> as by products (Allakhverdiev et al., 2002; Foyer and Noctor, 2003). Peroxisomes, which cater as a site for numerous metabolic processes such as photorespiration, β-oxidation of fatty acid, flavin oxidase pathway, dismutation of superoxide radicals and polyamine catabolism, also contribute significantly to ROS accumulation in plants subjected to salinity stress (Moschou et al., 2008a,b; Mohapatra et al., 2009). The effects of salt stress on peroxisomes and chloroplasts are interconnected. Reduced water availability and stomatal closure during salt stress causes reduction in the CO<sup>2</sup> to O<sup>2</sup> ratio in mesophyll cells. This facilitates the affinity of Rubisco to O2, thus increasing photorespiration and production of glycolate in chloroplasts. The end product of chloroplasts (glycolate) is oxidized by glycolateoxidase in peroxisomes—a major pathway of H2O<sup>2</sup> production (Noctor et al., 2002; Karpinski et al., 2003). In addition to organelles, enzymes localized in other cellular compartments, including the cytosolic polyamine oxidase (PAO) and diamine oxidase (DAO), plasma membrane NADPH oxidases, cell wallassociated peroxidases (POXs) and oxalate oxidases participate in ROS synthesis and may play a minor role in ROS production during salt stress (Kawano, 2003; Parida and Das, 2005; Ahmad and Sharma, 2008).

### Enzymatic and Non-Enzymatic Regulation of ROS in Plants

High levels of ROS can damage the cell by inactivating enzymes, initiating lipid oxidation of membranes, and breaking DNA strands (Van Breusegem et al., 2001; Halliwell, 2006). Plants modulate ROS accumulation during salinity stress via enzymatic and non-enzymatic pathways. The cytosolic enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and enzymes that participate in the ascorbate-glutathione cycle (**Figure 2**). Non-enzymatic antioxidants include the lipid-soluble membrane-associated α-tocopherol, and β-carotene, which are products of lipid peroxidation. Polyamines belong to the category of water-soluble compounds with antioxidative properties alongside glutathione (GSH), ascorbate (ASC), polyphenols (flavonoids, tannins, and anthocyanins), proteinaceous thiols, proline, and glycine-betaine (Mittler, 2002; Ozgur et al., 2013; Todorova et al., 2013). Glycine-betaine is a key regulator in ROS homeostasis, which stabilizes PSII by preventing high salt (Na<sup>+</sup> and Cl−)-induced dissociation of the regulatory extrinsic proteins (Papageorgiou and Murata, 1995). Some plants also use the alternative oxidase enzyme (AOX) to remove electrons from the ubiquinone pool and transfer them to oxygen to form water, thus preventing the over-reduction of ubiquinones and resulting in decrease of salt-induced ROS production in mitochondria (Smith et al., 2009; Miller et al., 2010). Unlike metazoans, plant cells do not have a mechanism to detoxify OH· enzymatically and to regulate the accumulation of OH· , rely on non-enzymatic

antioxidants, and various mechanisms to prevent OH· formation (Bose et al., 2014).

Numerous studies have shown a correlation between antioxidant accumulation and plant salt stress tolerance; however recent evidence hints that this relationship is more complex than previously thought. Several polyols accumulating during salt stress (sorbitol, mannitol, myo-inositol, pinitol, and others) may be involved in scavenging hydroxyl radicals (Williamson et al., 2002). In particular, the osmolyte proline seems to be associated with the activation of ROS-scavenging enzymes during salt stress (Saradhi and Mohanty, 1997; Szabados and Savouré, 2010; Gupta and Huang, 2014). For example, exogenous application of proline improves salt tolerance in melon, and was associated with increased chlorophyll content, photosynthetic rate, reduced O·− 2 , and H2O<sup>2</sup> accumulation, and increased levels of antioxidants (SOD, POD, CAT, APX, DHAR, and GR) (Yan et al., 2011). In addition, heightened levels of proline were observed in salt-tolerant transgenic rice overexpressing the DEAD-box helicase PDH45 which correlated with increased activation of antioxidant enzymes including SOD, APX, GPX, and GR under salt stress (Gill et al., 2013). It has also been shown that exogenous application of compatible solutes like glycine betaine, proline, mannitol, trehalose or myo-inositol, considerably reduced OH·− generated K<sup>+</sup> efflux during salt stress through an unknown mechanism (Cuin and Shabala, 2007).

Thus, ROS production and detoxification during salt stress appears to involve multiple cellular locations and molecular mechanisms. While polyamines are only one of several compounds with antioxidative properties that accumulate in stressed plants, they seem to play a significant role in regulating stress tolerance as outlined below.

### Polyamines

### Overview

Polyamines, small aliphatic amines with proposed antioxidant effect, are ubiquitous across all living organisms (Hussain et al., 2011; Gupta et al., 2013). Endogenous levels of polyamines increase during exposure to abiotic stresses such as drought, salinity, chilling, heat, hypoxia, ozone, UV, and heavy metal exposure and are ubiquitously produced in all cells and tissues

(Alcázar et al., 2010; Gill and Tuteja, 2010). The most abundant plant polyamines include putrescine (Put, 1, 4- diaminobutane), spermidine (Spd, N -3-aminopropyl-1, 4-diaminobutane) and spermine (Spm, bis (N -3-aminopropyl)-1,4-diaminobutane). Beside these, cadaverine (Cad, 1, 5-diaminopentane) has also been detected in several plant species, in particular in Gramineae, Leguminoseae and Solanaceae (Lutts et al., 2013). Another polyamine, thermospermine—a structural isomer of spermine is synthesized by the action of thermospermine synthase (Takano et al., 2012). Putrescine (Put) is primarily synthesized by ornithine decarboxylase using ornithine as a substrate (**Figure 1**). Another alternative pathway for Put synthesis occurs through the action of arginine decarboxylase (ADC) followed by two successive steps catalyzed by agmatine iminohydrolase (AIH) and N-carbamoyl-Put amidohydrolase (CPA) (Fuell et al., 2010). Put can be used as a substrate to generate Spd by spermidine synthase (SPDS) and Spd can then converted to Spm by spermine synthase (SPMS). Other polyamine oxidation products include hydrogen peroxide and γ-aminobutyric acid, which are involved in plant development and stress responses (Tiburcio et al., 2014). The unique polycationic structure of polyamines suggest that they may be free radical scavengers, in line with some observations that their accumulation correlates with plant tolerance to biotic and abiotic stresses (Mehta et al., 2002; Walters, 2003; Groppa and Benavides, 2008; Gill and Tuteja, 2010; Gupta et al., 2013).

The interactions between polyamines, ROS and antioxidants are complex and induce diverse and apparently contradictory physiological effects during stress (Bhattacharjee, 2005; Gill and Tuteja, 2010; Pottosin et al., 2012, 2014; Velarde-Buendia et al., 2012). In particular, increased levels of cellular polyamines during abiotic stress (e.g., salinity) have shown dual effects. On one hand, exogenous polyamine application was correlated with higher plant tolerance to abiotic stress, partly due to the increased ability to inactivate oxidative radicals. On the other hand, polyamines were reported to decrease plant's capacity to withstand stress, possibly due to the increased levels of H2O<sup>2</sup> resulted from polyamines' catabolism (Minocha et al., 2014). Indeed, both the anabolism and catabolism of the polyamine species were reported to increase during abiotic stress, with the net effect of raised cellular levels of ROS as well as antioxidant enzymes and metabolites (Pottosin et al., 2012, 2014; Minocha et al., 2014). In this review we have attempted to clarify the complex relationship between polyamines and ROS, focusing on the potential role of polyamine as a redox homeostasis manager during plant abiotic stress response.

### Polyamines: One of the Prominent Regulators in ROS Homeostasis during Salt Stress

Plant polyamines are thought to contribute to cellular responses during salt stress through modulation of ROS homeostasis via two distinct mechanisms (Takahashi and Kakehi, 2010). First, polyamines promote ROS degradation by scavenging free radicals and activating antioxidant enzymes during stress conditions (Gupta et al., 2013). Free polyamines are responsible for the detoxification of superoxide anions and hydrogen peroxide, while the conjugated polyamines are involved in scavenging other ROS (Langebartels et al., 1991; Kubis, 2005). Kuznetsov and Shevyakova (2007) have reported that conjugated polyamines show more antioxidant ability than free polyamines. Second, polyamines promote ROS production through polyamine catabolism in the apoplast (Yoda et al., 2006; Marina et al., 2008; Mohapatra et al., 2009; Campestre et al., 2011). While it is difficult to determine which of these mechanisms is most important during salt stress, manipulation of the polyamine biosynthetic pathways is correlated to abiotic stress resistance in several studies. For example, impaired expression of ADC1 or ADC2 significantly decreased Put levels and increased susceptibility to salt stress (Urano et al., 2004). When mouse ornithine decarboxylase (ODC) was introduced in Nicotiana tabacum, free polyamine content increased by 2-4 fold and germination increased by 33–45% on high salt medium (Kumria and Rajam, 2002). Transgenic Nicotiana tabacum plants overexpressing a S-adenosylmethionine decarboxylase (SAMDC) gene also demonstrated enhanced of soluble polyamines as well as increased seed weight, photosynthetic rate and expression of antioxidant enzymes (APX, MnSOD, and glutathione S-transferase) relative to untransformed lines (Wi et al., 2006). Increased polyamine accumulation (4–7%) was also observed in tobacco plants expressing the S-adenosylmethionine synthetase (SsSAMS2) gene, which supported up to 20% higher photosynthetic rates and biomass accumulation compared to the control (Qi et al., 2010). Similarly, introduction of SAMDC cDNA from Tritordeum into Oryza sativa produced higher free polyamine content (Put, Spd, Spm), and a reduction in salt-induced shoot growth repression compared to non-transgenic rice plants (Roy and Wu, 2002). Ectopic expression of SPDS orthologs from different source plants also improved growth and survival of young plants in Arabidopsis, European pear (Pyrus communis L.) and tomato suggesting the importance of this enzyme to cope up with saline environmental condition across diverse plant species (Kasukabe et al., 2004; Wen et al., 2008; Neily et al., 2011). Exogenous application of polyamines has also been shown to have a significant effect on the plant, and has been suggested to be a potential strategy to increase plant survival during salt stress. For example, Spm application promoted osmotic and salt stress tolerance in Arabidopsis and rice, which was thought to be due to enhanced polyphenol accumulation, CAT, and SOD enzyme activities (Sreenivasulu et al., 2000; Cheruiyot et al., 2007; Roychoudhury et al., 2011; Zrig et al., 2011; Radhakrishnan and Lee, 2013). In cucumber, Spm treatment enhanced salt tolerance (growth, photosynthetic rates) in a salt-sensitive cultivar, which was correlated to higher antioxidative enzyme activity and proline accumulation (Duan et al., 2008). Put application also increased the activity of antioxidant enzymes and carotenoids in leaf tissues of salt stressed Brassica juncea seedlings and enhanced seedling growth relative to the untreated controls (Verma and Mishra, 2005). Together, these studies indicate that altering polyamine accumulation through manipulation of biosynthetic pathways or direct application could have an effect on physiological responses to salt stress. **Table 1** summarizes the effect of endogenously formed and exogenously applied polyamines in alleviating salt resistance via the modulation of cellular antioxidative components (enzymatic or non-enzymatic).

Engineering consistent polyamine accumulation may not be so simple however, as plants also exhibit increased polyamine degradation during salt stress and thus polyamine turnover appears to be highly regulated. During salt stress, intracellular polyamines are exported from the cytosol to the apoplast, against the electrochemical gradient, and oxidized by DAO and/or PAO to generate hydrogen peroxide that is further converted to OH. via the Fenton reaction (Pottosin et al., 2014). For example, polyamine degradation occurs through oxidative deamination catalyzed by aminooxidases such as the copper-containing DAO and flavoprotein-containing PAO. DAO exhibits high affinity for diamines, while PAO oxidizes secondary amine groups from Spd and Spm (Alcazar et al., 2006). While dicotyledonous plants predominantly accumulate DAO, monocotyledonous plants usually accumulate more PAO than DAO (Šebela et al., 2001; Cona et al., 2006). The oxidative deamination of Put produces 1 1 pyrroline, H2O2, and NH<sup>3</sup> by DAO whereas activity of PAO resulted in the formation of 11–pyrroline (from Spd oxidation) or 1-(3-aminopropyl)-pyrroline (from Spm oxidation), along with 1, 3-diaminopropane and H2O<sup>2</sup> (Federico and Angelini, 1991). Both DAO and PAO are localized to the cytoplasm and cell wall and are involved in production of the hydrogen peroxide required for cell wall stiffening (Cona et al., 2003; Kuznetsov and Shevyakova, 2007) (**Figure 1**). These enzymes seem to contribute to changes in growth during salt stress since increased PAO accumulation in the expansion zone of maize leaves enhanced both ROS accumulation and elongation (Rodríguez et al., 2009; Shoresh et al., 2011). Moreover, high salt (400 mM NaCl) or ROS application induces DAO activity in the leaves and roots of the halophyte Mesembryanthemum crystallinum further implicating that these enzymes play a role in salt stress (Shevyakova et al., 2006).

### Polyamines as Modulators of Ion Homeostasis

Polyamines are also hypothesized to promote salt stress tolerance through their direct or indirect effects on ion transport (**Figure 3**) (Demidchik and Maathuis, 2007; Pandolfi et al., 2010; Bose et al., 2011). For instance, polyamines including Spd, Spm, and Put affect ion transport indirectly by interacting with plasma membrane phospholipids and enhancing membrane stability. Polyamine-enhanced membrane stability has been shown



to have a significant effect on both H+/ATPase and Ca <sup>2</sup>+/ATPase transporters during salinity stress (Roy et al., 2005; Pottosin and Shabala, 2014). Ca2<sup>+</sup> channel regulation mediated by polyamines and H2O<sup>2</sup> in response to salt stress leads to the rapid rise in the intracellular concentration of Ca2<sup>+</sup> that, subsequently, enforces a positive feedback on the ROS production via the membranelocalized NADPH-oxidase (Takeda et al., 2008; Bose et al., 2014). Sudden exposure to salt stress is reflected in the alterations of turgor that is sensed by rapid increase in cellular cGMP, produced by the action of receptor kinase cyclase. This in turn activates the root-localized cyclic nucleotide-gated channels allowing the inward flow of Ca2+, thus cGMP signal is converted to Ca2<sup>+</sup> signal during salinity (Demidchik and Maathuis, 2007). On the other hand, a rise in cGMP can directly inactivate root voltageindependent non-selective cation channels (VI-NSCC) by reducing the influx of toxic Na<sup>+</sup> (Rubio et al., 2003). Salt-stress elicited Ca2<sup>+</sup> signals activate signaling molecules including the SOS3 calcium-binding protein and the serine/threonine protein kinase SOS2 which in turn activate the membrane Na+/H<sup>+</sup> antiporter SOS1 leading to Na<sup>+</sup> efflux (Zhu, 2003). If we consider the above mentioned reports, one can easily observe an indirect cumulative effect of polyamines and ROS in regulating the cellular Ca2<sup>+</sup> that is important for salt response. In contrast, Spm may directly affect ion transport during salt stress by blocking inwardrectifying K<sup>+</sup> channels (KIRC) and non-selective cation channels (NSCCs), limiting Na<sup>+</sup> influx, and K<sup>+</sup> efflux (Liu et al., 2000; Shabala et al., 2007; Zhao et al., 2007; Zepeda-Jazo et al., 2008). Put and Spm have shown strong potential in reducing the hydroxyl radical-induced K(+) efflux and the respective non-selective current. This synergistic effect between ROS and polyamines was much more pronounced in a salt-sensitive barley variety than salt-tolerant one (Velarde-Buendia et al., 2012). Subsequently, an increased external [Ca2+] activated depolarization-activated NSCCs (DA-NSCCs), inhibited Na+ -induced K+ efflux, thus ameliorating Na<sup>+</sup> toxicity in plants (Shabala et al., 2006). During salinity, exogenous application of spermidine has been found to block VI-NSCC reducing the inward flow of Ca2<sup>+</sup> and Na<sup>+</sup> and the outward flow of K<sup>+</sup> in barley seedlings (Zhao et al., 2007). It has been reported that polyamine accumulation under salt stress has a tendency to make the overall tonoplast cation conductance more K<sup>+</sup> selective, thus considered to lead to higher vacuolar Na<sup>+</sup> sequestration and an improved cytosolic K+/Na<sup>+</sup> homeostasis (Zepeda-Jazo et al., 2008). Absence of Spm causes an imbalance in Ca2<sup>+</sup> homeostasis in the Arabidopsis mutant plant and showed hypersensitivity to salinity, suggesting its involvement in modulating the activity of certain Ca2+- permeable channels and changes in Ca2<sup>+</sup> allocation compared to unstressed state, which may prevent Na<sup>+</sup> and K<sup>+</sup> entry into the cytosol, enhance Na<sup>+</sup> and K<sup>+</sup> influx into the vacuole, or suppress Na<sup>+</sup> and K<sup>+</sup> release from the vacuole (Yamaguchi et al., 2006). Moreover, vacuolar Cation/H<sup>+</sup> Exchangers (CAX) are found to be overexpressed and both FV and SV channels (FV, fact-activating vacuolar channel; SV, slow-activating vacuolar channel) suppressed during salinity, resulting into an overall increase in vacuolar Ca2<sup>+</sup> (Cheng et al., 2004; Pottosin et al., 2004). Dobrovinskaya et al. (1999) reported that cellular polyamines strongly inhibited FV and SV channels whose reduced activity is essential for conferring salinity tolerance in the facultative halophyte Chenopodium quinoa (Bonales-Alatorre et al., 2013). However, more research is required to understand this interaction as well as the putative interactions between polyamines and vacuolar transport systems (Pottosin and Shabala, 2014).

### Cross Talk between Polyamines, ROS, NO, and ABA

Plants employ multi-level signal transduction to induce stress responses. The coordinated actions of hormones such as abscisic acid (ABA), ethylene, jasmonate, and auxin along with other signaling molecules like Ca2+, cyclic nucleotides, ROS and reactive nitrogen species such as NO form a complex signaling network (Neill et al., 2003; Tuteja and Sopory, 2008). Interestingly, ABA was found to be involved in regulating both biosynthetic and catabolic pathways for polyamines in Arabidopsis (Urano et al., 2004; Hussain et al., 2011). For example, exogenous application of ABA has been found to modulate the transcription and biosynthesis of polyamine metabolic enzymes such as ADC2, SPDS, and SPMS during stress (Alcazar et al., 2006; Hussain et al., 2011). On the other hand Put has been found to serve as a modulator of indispensable ABA increase under cold stress thus representing and reciprocal relationship between Put and ABA biosynthesis during the period of stress in order to increase plant adaptive potential (Cuevas et al., 2008, 2009; Urano et al., 2009). The transgenic tobacco plants overexpressing the ABA-biosynthetic enzyme 9-cis-epoxycarotenoid dioxygenase is associated with the ABAinduced production of H2O2, NO, and the subsequent induction of antioxidant enzymes conferring salt tolerance (Zhang et al., 2009). Recently, it has been shown that polyamines can induce the production of NO that serves as a signal-inducing salt resistance by increasing the K<sup>+</sup> to Na<sup>+</sup> ratio by stimulating the expression of the plasma membrane H+-ATPase and Na+/H+ antiport in the tonoplast (Zhao et al., 2004; Tun et al., 2006; Yamasaki and Cohen, 2006; Zhang et al., 2006). It was suggested that NO production induced by polyamines could be mediated either by H2O2, one reaction product of oxidation of polyamines by DAO and PAO, or by unknown mechanisms involving polyamines, DAO and PAO (Wimalasekera et al., 2011). Pre-treatment with H2O<sup>2</sup> or sodium nitroprusside (NO donor) induced major antioxidant defense (SOD, catalase, APX, and GR), reduced protein carbonylation and accumulated leaf S-nitrosylated proteins, suggesting an overlap relation between NO and H2O<sup>2</sup> signaling pathways in salinity acclimation (Tanou et al., 2009a,b).

channel; VI-NSCCs, Voltage-independent nonselective cation channels.

In the light of these observations we have made an attempt to explore the interconnection(s) between polyamines, NO, ABA, and ROS as potential mediator(s) of stress responses. More research is needed to determine the exact nature of these intricate connections in the context of salt stress.

### Polyamines and Programmed Cell Death

Plant cells employ dynamic activation of ROS production to regulate defense responses during stress. When ROS accumulation crosses a threshold value, cells enter into a genetically programmed necrotic process that leads to cellular suicide, which restricts the oxidative damage to a controlled number of cells and triggers pathways for nutrient recycling (de Pinto et al., 2006; Stowe and Camara, 2009). The key regulator of the switch between the cellular endurance and programmed cell death (PCD) under salt stress could be controlled by the interplay between polyamine and ROS homeostasis; specifically, the precise modulation of polyamine levels by the shift between polyamine anabolism and catabolism may result a lower polyamine concentration which, in turn may facilitate PCD (Moschou et al., 2008a; Toumi et al., 2010).

We have already discussed in our previous section that polyamines act as important regulators of ion homeostasis during salt stress. Modulation of the cellular K<sup>+</sup> and Ca2<sup>+</sup> concentrations regulate stress-related PCD pathways in plants (Moschou and Roubelakis-Angelakis, 2014). Plant polyamines are found to affect intracellular dynamics of both ions, thus suggesting their direct involvement in PCD (Wu et al., 2010; Zepeda-Jazo et al., 2011). Low cellular concentrations of K<sup>+</sup> were shown to increase the activity of metacaspases and nucleases, thus promoting ROS- and salt-induced PCD (Demidchik et al., 2010). Salt stress led to high cytosolic [Ca2+] which promoted the opening of mitochondrial permeability transition pore (MPTP) and PCD induction in tobacco protoplasts (Lin et al., 2005). Mitochondrial depolarization and cytochrome-c release is a hallmark event during the PCD (Logan, 2008; Andronis and Roubelakis-Angelakis, 2010). Takahashi's group showed that 0.5 mM Spm pretreatment of tobacco leaf discs induced expression of the Salicylic acid (SA)-induced Protein Kinase

(SIPK) and Wound-Induced Protein Kinase (WIPK) and caused mitochondrial dysfunction similar to the one observed during PCD in tobacco leaves (Takahashi et al., 2003).

Accumulation of metabolic derivatives of polyamines may also indirectly control PCD pathways (Moschou and Roubelakis-Angelakis, 2014). For example, tobacco plants with reduced or increased PAO expression demonstrated increased salt tolerance or PCD depending on the availability of intracellular polyamines (Moschou et al., 2008c). Expression of the Spm Oxidase (SMO) can also be linked to hydrogen peroxide production and PCD, providing additional support to the above presented view of PAO-induced PCD (Moschou and Roubelakis-Angelakis, 2014). It has also been reported that overexpression of PAO activates mitogen-activated protein kinases (MAPK)-mediated pathways during biotic stress (Moschou et al., 2009).

In sum, a connection between polyamine metabolism and PCD can be inferred, but more work is needed to determine the molecular mechanisms underlying this relationship.

### Conclusions and Future Prospects

This review outlines our current understanding of polyamines and their contributions to ROS homeostasis during salt stress, summarized in **Figure 4**. The figure depicts the possible cellular pathways by which polyamines modulate ROS homeostasis during salinity and the probable mode of action of endogenous and exogenous PAs into a single frame, so that one can easily view the current state of the field.

### References


Our literature review suggests that the regulation of polyamine metabolism is a complex process where the exact roles of polyamines in regulating ROS, ion transport and PCD are still to be discovered. For the field to progress there is a need to address several important aspects: (i) The identity of the cellular components that mediate the link between ROS synthesis, ROS signaling and polyamines; (ii) The mechanisms that these mediator components employ; and (iii) The potential organ- or tissue-specific differences in the composition and regulation of polyamine-ROS networks.

To solve these questions one should focus on several relevant processes including polyamine biosynthesis, transport and catabolism in parallel with the tissue-, species-, and salt stress dependent expression of various ion channels and transporters. Additionally, one should consider the nature of various ROS and polyamine species that accumulate in plants under stress and the sites of their subcellular synthesis, alongside changes in the polyamine and ROS scavenging systems.

Salt stress constitutes a serious challenge to overcome in the quest of global increase in crop productivity. Understanding the underlying molecular mechanism of salt stress adaptation is the key to successful crop biotechnology.

### Acknowledgments

The authors acknowledge the support of technical facilities available at Presidency University, Kolkata. Financial assistance from DBT (RGYI) (Govt. of India) and W.B. State DST (Govt. of West Bengal) to BG and KG and DST-SERB (Govt. of India) to KG are also gratefully acknowledged.


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Saha, Brauer, Sengupta, Popescu, Gupta and Gupta. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Brassinosteroids make plant life easier under abiotic stresses mainly by modulating major components of antioxidant defense system

#### *Bojjam V. Vardhini <sup>1</sup> \* and Naser A. Anjum2*

*<sup>1</sup> Department of Botany, Telangana University, Nizamabad, India*

*<sup>2</sup> CESAM-Centre for Environmental and Marine Studies and Department of Chemistry, University of Aveiro, Aveiro, Portugal*

#### *Edited by:*

*Rene Kizek, Central European Institute of Technology, Czech Republic*

#### *Reviewed by:*

*Harminder Pal Singh, Panjab University, India Yogesh Abrol, Bhagalpur University, India*

#### *\*Correspondence:*

*Bojjam V. Vardhini, Department of Botany, Telangana University, Dichpally, Nizamabad 503322, India e-mail: drvidyavardhini@ rediffmail.com*

Various abiotic stress factors significantly contribute to major worldwide-losses in crop productivity by mainly impacting plant's stress tolerance/adaptive capacity. The latter is largely governed by the efficiency of antioxidant defense system for the metabolism of elevated reactive oxygen species (ROS), caused by different abiotic stresses. Plant antioxidant defense system includes both enzymatic (such as superoxide dismutase, SOD, E.C. 1.15.1.1; catalase, CAT, E.C. 1.11.1.6; glutathione reductase, GR, E.C. 1.6.4.2; peroxidase, POD, E.C. 1.11.1.7; ascorbate peroxidase, APX, E.C. 1.11.1.11; guaiacol peroxidase, GPX, E.C. 1.11.1.7) and non-enzymatic (such as ascorbic acid, AsA; glutathione, GSH; tocopherols; phenolics, proline etc.) components. Research reports on the status of various abiotic stresses and their impact on plant growth, development and productivity are extensive. However, least information is available on sustainable strategies for the mitigation of abiotic stress-mediated major consequences in plants. Brassinosteroids (BRs) are a novel group of phytohormones with significant growth promoting nature. BRs are considered as growth regulators with pleiotropic effects, as they influence diverse physiological processes like growth, germination of seeds, rhizogenesis, senescence etc. and also confer abiotic stress resistance in plants. In the light of recent reports this paper: (a) overviews major abiotic stresses and plant antioxidant defense system, (b) introduces BRs and highlights their significance in general plant growth and development, and (c) appraises recent literature available on BRs mediated modulation of various components of antioxidant defense system in plants under major abiotic stresses including metals/metalloids, drought, salinity, and temperature regimes. The outcome can be significant in devising future research in the current direction.

**Keywords: abiotic stress, reactive oxygen species, antioxidant defense system, tolerance, brassinosteroids**

### **INTRODUCTION**

### **ABIOTIC STRESSES AND PLANT ANTIOXIDANT DEFENSE SYSTEM**

Plants are inevitably exposed to various environmental stress factors of like abiotic and biotic types. In particular, abiotic stresses such as temperature (heat, chilling, and freezing), water (drought, water logging), salt, heavy metals, light (intense and weak), radiation (UV-A/B) etc. are serious threats to agriculture worldwide (Bray et al., 2000). Elevation in the generation of various reactive oxygen species (ROS; such as superoxide radicals, O2−; hydroxyl radicals, OH−; perhydroxyl radicals, HO2−; alkoxy radicals, RO−; hydrogen peroxide, H2O2; singlet oxygen, 1O2.) is a common consequence in plants growing under abiotic stresses (Anjum et al., 2010, 2012, 2014; Gill and Tuteja, 2010). Important signal transduction functions and triggering and/or orchestration of plant responses to varied (abiotic) stresses can be possible with minimal levels of ROS. However, major ROS and their reaction products that escape antioxidant-mediated scavenging can disturb the ROS/antioxidant homeostasis in plant cells, cause oxidative stress, bring critical damages to the primary metabolites of plants viz., proteins, lipids, carbohydrates and DNA and halt cellular metabolism (Anjum et al., 2010, 2012, 2014; Gill and Tuteja, 2010). To survive such stresses, plants have evolved many intricate defense mechanisms to increase their tolerance and survive under such extreme environmental conditions. Plant stress tolerance requires the activation of complex metabolic activities including antioxidative pathways, especially ROS-scavenging systems within the cells that in turn can contribute to continued plant growth under stress conditions (El-Mashad and Mohamed, 2012). Plant antioxidant defense system consists of the enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POD: EC 1.11.1.11), ascorbate peroxidase (APX: E.C. 1.11.1.11), glutathione reductase (GR: EC 1.6.4.2), glutathione sulfo-transferase (GST: EC), polyphenol oxidase (PPO: E.C. 1.14.18.1), guaiacol peroxidase (GPX: EC 1.11.1.7), monodehydroascorbate reductase (MDHAR: EC 1.1.5.4), dehydroascorbate reductase (DHAR: EC 1.8.5.1) etc. Whereas, non-enzymatic components may include osmolytes like proline, glycine betaine, sorbital, mannitol etc., and reduced glutathione (GSH), ascorbic acid (AsA) that are needed for osmotic adjustment, stabilization of membranes, and ROS-scavenging (Anjum et al., 2010, 2012, 2014; Gill and Tuteja, 2010) (**Figure 1**).

### **BRASSINOSTEROIDS**

### **OVERVIEW**

Brassinosteroids (BRs) are a new type of polyhydroxy steroidal phytohormones with significant growth-promoting influence (Vardhini, 2012a,b; Bajguz and Piotrowska-Niczyporuk, 2014). Mitchell et al. (1970) discovered BRs which were later extracted from the pollen of *Brassica napus* by Grove et al. (1979). BRs can be classified as C27, C28 or C29 BRs according to the number of carbons in their structure (Vardhini, 2013a,b). Sixty BRs related

compounds have also been identified (Haubrick and Assmann, 2006). However, brassinolide (BL), 28-homobrassinolide (28- HomoBL) and 24-epibrassinolide (24-EpiBL) are the three bioactive BRs those are widely used in most physiological and experimental studies (Vardhini et al., 2006) (**Figure 2**). BRs are considered ubiquitous in plant kingdom as they are found in almost all the phyla of the plant kingdom like alga, pteridophyte, gymnosperms, dicots and monocots (Bajguz, 2009). BRs are considered also as a new group of plant growth hormones that perform a variety of physiological roles like growth, seed germination, rhizogenesis, senescence, and resistance to plants against various abiotic stresses (Rao et al., 2002).

### **SIGNIFICANCE IN GENERAL PLANT GROWTH AND DEVELOPMENT**

BRs have to their credit a host of roles in general plant growth and development. BRs can activate the cell cycle during seed germination (Zadvornova et al., 2005), control progression of cell cycle (González-Garcia et al., 2011), induce exaggerated growth in hydroponically grown plants (Arteca and Arteca, 2001), and also control proliferation of leaf cells (Nakaya et al., 2002). In addition, reports are also available on the role of BRs in growth promotion of apical meristems in potato tubers (Meudt et al., 1983), acceleration of rate of cell division in isolated protoplasts of *Petunia hybrida* (Ho, 2003) and cell division and leaf expansion (Zhiponova et al., 2013). Initially BRs were identified based on their growth promoting activities; however, subsequent physiological and genetic studies revealed additional functions of BRs in regulating a wide range of processes, including source/sink

relationships, seed germination, photosynthesis, senescence, photomorphogenesis, flowering and responses to different abiotic and biotic stresses (Deng et al., 2007). The work with BR biosynthetic mutants in *Arabidopsis thaliana* (Tao et al., 2004) and *Pisum sativum* (Nomura et al., 1997) have provided strong evidences that BRs are essential for plant growth and development and BR- signaling plays a positive in plant growth and development (Fábregas and Caño-Delgado, 2014). A simple BR- analog 2α, 3α-dihydroxy-17β-(3-methyl butynyloxyl) 7-oxa-B-homo-5α androstan-6-one induces bean second node splitting which is considered as the prominent physiological feature of BRs (Strnad and Kohout, 2003). Dwarf and de-etiolated phenotypes and BR deficient species of some *Arabidopsis* mutants were rescued by application of BRs (Bishop and Yakota, 2001). Even *Pharbitis nil*, *Uzukobito* was a defective BR- biosynthetic dwarf mutant strain (Suzuki et al., 2003) which emphasized that BR-deficient and defective BR-biosynthetic species exhibited abnormal growth. Friedrichsen et al. (2002) also reported that three redundant BR genes encode transcription factors which are required for normal growth, indicating the necessity of BRs for normal growth. Similarly, the inhibition of growth (Asami et al., 2000) and secondary xylem development (Nagata et al., 2001) of cress (*Lepidius sativus*) by brassinozole, a specific inhibitor of BL synthesis was reversed by the exogenous application of BL, further emphasizing the necessity of BRs for normal plant growth.

BRs also exhibit synergistic effect with other phyohormones in regulating the plant growth and metabolism. BRs interact with auxins, cytokinins, gibberellins (Domagalska et al., 2010), abscisic acid (ABA) (Domagalska et al., 2010), ethylene (ET) (Manzano et al., 2011) salicylic acid (SA) (Divi et al., 2010) and jasmonic acid (JA) (Creelman and Mullet, 1997; Peng et al., 2011) to promote plant growth and metabolism. Ability of 24-EpiBL to control the basic thermotolerance and salt tolerance of the mutants has been evidenced (Divi et al., 2010). In addition, these authors also reported synergistic role of 24-EpiBL, where treatment with 24-EpiBL increased expression of various hormone marker genes in both wild type and mutant *Arabidopsis* seedlings those were either deficient in or insensitive to ABA, ET, JA, and SA. Notably, BRs may be applied/supplied to plants at different stages of their life cycle viz., vegetative stage (Vardhini and Rao, 1998), flowering stage (Vardhini, 2012a, 2013a), meiosis stage (Saka et al., 2003), grain filling stage (Vardhini, 2012a), anthesis stage (Liu et al., 2006) etc. as foliar spray (Vardhini et al., 2008), seed treatment (Zhang et al., 2007; Kartal et al., 2009), root application (Shang et al., 2006; Song et al., 2006) and even as shot gun approach (Hayat et al., 2010a). Examples of a range of other major functions of BRs and related compounds reported in different plants can be found in **Figure 3**.

### **BRASSINOSTEROIDS-MEDIATED MODULATION OF PLANT ANTIOXIDANT DEFENSE SYSTEM UNDER MAJOR ABIOTIC STRESS**

Extensive research over the years' has established stress-impactmitigating role of BRs and associated compounds in different plants exposed to various abiotic stresses such as high temperature (Zhou et al., 2004; Kurepin et al., 2008; Janeczko et al., 2011), low temperature in terms of chilling (Divi and Krishna, 2010; Liu et al., 2011; Wang et al., 2014) as well as freezing (Janeczko et al., 2009). Reports are available on the significance of BRs and associated compounds in different plants exposed to salinity (Avalbaev et al., 2010; Abbas et al., 2013), light (Wang et al., 2010, 2012; Kurepin et al., 2012; Li et al., 2012a), drought (Anjum et al., 2011; Li et al., 2012b; Mahesh et al., 2013), flooding (Lu et al., 2006; Liang and Liang, 2009), metals/metalloids (Arora et al., 2010a,b; Ashraf et al., 2010; Bajguz, 2010), herbicides (Sharma et al., 2013a), pesticides (Xia et al., 2006), insecticides (Xia et al., 2009b, 2011), organic pollutants (Ahammed et al., 2012a, 2013a), newly reclaimed sandy soil (Ahmed and Shalaby, 2013) and preservatives (Hu et al., 2014).

Hereunder, recent reports on the role of BRs (and associated compounds) in the modulation of both enzymatic and non-enzymatic components of antioxidant defense system in abiotic stressed plants are critically appraised. The discussion will consider primarily metals/metalloids followed by temperature regimes (high and low), drought, salinity and other major abiotic stresses.

### **METAL/METALLOID STRESS**

Foliar application of homoBL was reported to improve Cdtolerance in *Brassica juncea* through increasing activity of antioxidative enzymes (such as CAT, POD, SOD) and the content of osmolyte (such as proline) (Hayat et al., 2007). Improved Cdtolerance in *Phaseolus vulgaris* was possible as a result of 24-epiBL (5μM)-mediated increased activity of antioxidative enzymes, and proline content and subsequent improvements in the membrane stability index (MSI), relative leaf water content (RLWC) (Rady, 2011). Nullification of the damaging effect of Cd was reported in totamato cultivars (K-25 and Sarvodya) as a result of 28 homoBL/24-epiBL (10(−8) M)-mediated improvement in photosynthetic machinery and antioxidant defense system (Hasan et al., 2011). Application of BRs (10−<sup>8</sup> M) to Cd (3.0, 6.0, 9.0, and 12 mg kg−1) stressed *Solanum lycopersicum* plants enhanced antioxidant system activity and improved fruit yield and quality (Hayat et al., 2012). Cd-impact-ameliorative role of 24-epiBL and 28-homoBL (3.0μM) was evidenced in *Raphanus sativus*, where these BRs enhanced levels of free proline, antioxidant enzymes CAT, SOD, APX, GPX, and also reduced the activity of POD and AAO (Anuradha and Rao, 2007b). In Cd (0.5, 1.0, and 1.5 mM)-exposed *Raphanus sativus*, a diminished oxidative stress via 24-epiBL (10−7, 10−9, and 10−<sup>11</sup> M)-supplementationmediated elevation in the activity of GST and PPO enzymes was reported (Sharma et al., 2012). Earlier, these authors evidenced 28-homoBL (10−11, 10−9, and 10−<sup>7</sup> M)-assisted amelioration of Cd (0.5, 1.0, and 1.5 mM) impacts in *Raphanus sativus*, where improved biomass and seedling growth was argued as a result of 28-homoBL-mediated regulation of the activity of APX, CAT, GR, POD, and SOD (Sharma et al., 2010). Hasan et al. (2008) also reported 28- homoBL-mediated elevated activity of CAT, POD, and SOD and the protection of *Cicer arietinum* against Cd (50, 100, or 150μM).

Application of 24-epiBL ameliorated Ni-stress in *Brassica junce*a by enhancing mainly the activity of antioxidant enzymes (Kanwar et al., 2013). Earlier, these authors reported BRs (24- EpiBL, CS, dolicholide and typhasterole)-mediated significant

**compounds reported in plants.** 1Zadvornova et al., 2005; 2González-Garcia et al., 2011; 3Hartwig et al., 2011; 4Manzano et al., 2011; 5Yamamoto et al., 2001; 6Jin et al., 2014; 7Carange et al., 2011; 8Pokotylo et al., 2014; 9Vardhini and Rao, 1999; 10Upreti and Murti,

mitigation of Ni (0.2, 0.4, and 0.6 mM)-stress in *Brassica juncea* plants by elevating the activity of ROS-metabolizing enzymes (and also via lowering the metal uptake) (Kanwar et al., 2012). Significantly elevated activity of antioxidant enzymes (such as GPX, CAT, GR, APX, and SOD) in *Brassica juncea* seedlings emerged from the homoBL (0.01, 1.0,and 100 nM)-treated seeds was argued to provide tolerance of this plant to Ni concentrations (25, 50, and 100 mg dm−3) (Sharma et al., 2008). In *Brassica juncea*, the spraying of homoBL was evidenced to partially neutralize the toxic effect of 50 or 100 μM Ni, where elevated activity of POD and CAT, and content of proline was observed in leaves and roots (Alam et al., 2007). Spraying of 24-epiBL (1.0μM) to Ni-exposed *Brassica juncea* was reported to detoxify Ni-impacts (Ali et al., 2008a). Elevated CAT, POD, and SOD activity via the spray of 0.01μM of 28-homoBL was argued to protect five wheat (*Triticum aestivum*) cultivars (PBW-373, UP-2338, DL-LOK-01, DL-373, and HD-2338) against Ni concentrations (50 and 100μM) (Yusuf et al., 2011b). *Raphanus sativus* seedlings emerged from seeds pre-soaked in 24-epiBL, Bonato-Negrelle, 2012; 14Vardhini and Rao, 2002; 15Weng et al., 2007; 16Vogler et al., 2014; 17Nakaya et al., 2002; 18Zhiponova et al., 2013; 19Malabadi and Nataraja, 2007; 20Aydin et al., 2006; 21Cheng et al., 2014; 22Haubrick et al., 2006; 23Xia et al., 2014.

exhibited elevated activity of APX, SOD, CAT, GPX, MDHAR, DHAR, and GR; that eventually resulted in reducing lipid peroxidation, enhanced proline and protein contents, and improved enhancing the root/shoot length, fresh biomass under Ni exposure (Sharma et al., 2011a). Application of 10−<sup>6</sup> M 24-epiBL as shotgun approach (pre-sowing seed soaking) to the Ni-stressed T-44 (Ni-tolerant) and PDM-139 (Ni-sensitive) varieties of *Vigna radiata* plants improved biological yield, number of nodules, leghemoglobin content, and the activity of CAT, POD, and SOD enzymes. The 24-epiBL-mediated up-regulation of antioxidant enzyme activity and the elevated level of proline (osmolyte) were argued to confer Ni-tolerance and improve growth, nodulation and yield attributes (Yusuf et al., 2012). Recently, these authors reported BRs-mediated improved antioxidant defense (and also nitrogen metabolism) in two contrasting cultivars of *Vigna radiata* under different levels of Ni (Yusuf et al., 2014).

The role of BRs and associated compounds in the mitigation of elevated levels of Cu has also been reported in plants. To this end, treatment of *Brassica juncea* seedlings with 10−10, 10−8, and 10−<sup>6</sup> M homoBL improved growth and photosynthetic traits via decreased H2O2 and elevated activity of CAT, POD, and SOD (Fariduddin et al., 2009b). Recently, these authors reported an improved growth of NaCl+Cu (100 mg kg−1) stressed *Cucumis sativus* via epiBL-mediated enhancements in the activity of CAT, POD, and SOD (Fariduddin et al., 2013a). Supplemantation of 10−7, 10−9, and 10−<sup>11</sup> M 24-epiBL to *Raphanus sativus* ameliorated the oxidative stress caused by Hg (0.5, 1.0, and 1.5 mM) by enhancing the activity of ROS-metabolizing enzymes such as GST and PPO (Sharma et al., 2012). Recently, 24-epiBL (10−7, 10−9, 10−<sup>11</sup> M)-mediated increased activity of antioxidative enzymes such as SOD, CAT, APOX, GPX, GR, MDHAR and DHAR, and also the contents of GSH were argued to help radish plants to counteract the consequences of Hg (Kapoor et al., 2014).

Supplementation of 24-epiBL reduced Pb toxicity and enhanced the growth in radish (*Raphanus sativus* L.) seedlings by increasing the activities of antioxidant enzymes like CAT, APX, GPX, SOD and reducing POD activity (Anuradha and Rao, 2007a). Mitigation of the consequences of Pb (100 or 200μM) was reported in tomato plants as a result of 24-epiBL-mediated increases in the activities of SOD, CAT, APX and GR, and the contents of AsA and GSH (Rady and Osman, 2012). 24-epiBL ameliorated Cr (VI) stress in 7-d old *Raphanus sativus* L. cv. "Pusa chetki" seedlings by enhancing the pools of reduced GSH and AsA, the activity of GR, SOD, and APX enzymes, and also the contents of phytochelatins, proline, and glycinebetaine (Choudhary et al., 2011). Co-application of epiBL and spermidine (polyamine) was also evidenced to remarkably enhance the titers of antioxidants (GSH, AsA, proline, glycine betaine and total phenols) and the activity of GPX, SOD, and GR) in Cr-stressed *Raphanus sativus* (Choudhary et al., 2011). Seed pre-soaking treatment of 28-homoBL at 10(−7) M was effective in ameliorating Cr(VI) stress in *Raphanus sativus* L. (Pusa Chetaki), where an increased activity of antioxidative enzymes (except GPX) and the contents of proline and proteins but reduced lipid peroxidation were evidenced (Sharma et al., 2011b). 24-EpiBL-mediated diminution of Cr-toxicity in *Brassica juncea* was reported, where increased activity of GPX, CAT, GR, APX, SOD, MDHAR, and DHAR was argued to improve plant health (Arora et al., 2010b). Amelioration of Al-impacts was evidenced through epiBL or homoBL spraying to mung bean (*Vigna radiata*), where these BRs increased RLWC, water use efficiency, photosynthetic rate via enhancing the activity of antioxidative enzymes such as CAT, POD, and SOD in leaves (Ali et al., 2008b). Plant tolerance to Pb (and also to Cu and Cr) is possible via BL-mediated significant activation of enzymes (such as SOD, CAT, APX, and GR) and non-enzymes (such as reduced GSH, total AsA) (Bajguz, 2010) (**Table 1**).

Least reports are available on the role of BRs in plants under Zn, Bo, Co, Mn, and As stress. Supplementation of 28-homoBL to *Raphanus sativus* seedlings was reported to help this plant to tolerate Zn toxicity by enhancing antioxidative enzyme activities, strengthening GSH metabolism and redox status, and improving the contents of non-enzymatic antioxidants and proteins (Ramakrishna and Rao, 2013). The role of 28-homoBL (Arora et al., 2008b) and that of 24-epiBL (Arora et al., 2010a) was reported respectively in *Zea mays* and *Brassica juncea* under Zn stress. In the previous studies, increased activity of SOD, CAT, APOX, GPX, GR, MDHAR and DHAR and the contents of GSH were reported to control Zn-accrued lipid peroxidation. Application of 28-homoBL (10(−8) M) to Bo (0.50, 1.0, and 2.0 mM)-exposed *Vigna radiata* improved the growth, water relations, photosynthesis by enhancing antioxidant enzymes (such as CAT, POD and SOD) (Yusuf et al., 2011a). Foliar spray treatment with 24-epiBL (0, 10−10, 10−8, and 10−<sup>6</sup> M) alleviated the stress generated by Co (0, 5 <sup>×</sup> <sup>10</sup>−4, 10−3, 1.<sup>5</sup> <sup>×</sup> <sup>10</sup>−3, and 2 <sup>×</sup> <sup>10</sup>−<sup>3</sup> M) ion in *Brassica juncea* through significantly improving the activities of SOD, CAT, POD, GR, APOX, MDHAR, and DHAR enzymes (Arora et al., 2012). Under elevated lelevs of Mn, epiBL application was reported to enhance the activities of SOD, POD, CAT, APX, DHAR, and GR, and the contents of AsA, and GSH that eventually controlled lipid peroxidation and metabolized superoxide radical and H2O2 in *Zea mays* (Wang et al., 2009). Recently, Raghu et al. (2014) reported BRs-mediated improved As-tolerance in *Raphanus sativus* as a result of increased activity of SOD and CAT.

### **TEMPERATURE REGIMES**

BRs and associated compounds have been extensively reported to modulate different components of antioxidant defense system and to play a positive role in the mitigation of the consequences in different plants exposed to both high (Mazorra et al., 2002, 2011; Zhou et al., 2004; Cao and Zhao, 2007; Ogweno et al., 2008; Hayat et al., 2010b) and low (Janeczko et al., 2007; Liu et al., 2009; Kumar et al., 2010; Aghdam et al., 2012; Wang et al., 2012; Hu et al., 2013; Xi et al., 2013; Aghdam and Mohammadkhani, 2014) temperatures (**Table 1**).

Young seedlings of two Indica rice (*Oryza sativa*) cultivars namely *Xieqingzao* B (heat-sensitive) and 082 (heat-tolerant), subjected to high temperature; sprayed with 0.005 mg L−<sup>1</sup> of BR exhibited significant enhancement in activities of POD and SOD isozyme expression levels, reduction in MDA level and leakage of leaf electrolytes (Cao and Zhao, 2007). Supplementation with 28-homoBL to *Vigna radiata* c.v. T-44 plants detoxified the stress generated by high temperature by improving the membrane stability index (MSI), leaf water potential (ψ) via increased the activities of antioxidative enzymes and the level of proline (Hayat et al., 2010b). Pre-treatment of 24-epiBL to *Lycopersicon esculentum* Mill. cv. 9021 plants exposed to high temperature (40/30◦C; for 8 days) significantly alleviated high-temperaturecaused inhibition of photosynthesis by increasing the activities of SOD, APX, GPX, and CAT, and reducing total H2O2 and MDA contents (Ogweno et al., 2008). Pre-incubation of tomato leaf discs with 24-epiBL or MH5 (polyhydroxylated spirostanic analog of BR) (for 24 h) stimulated the activities of CAT, POD and SOD, controlled cell damage under heat stress (40◦C) (Mazorra et al., 2002). EpiBL-induced tolerance to heat shock (HS) in tomato seedlings (BR-deficient mutant, *extreme dwarf d(x)*), a partially BR-insensitive mutant *curl*3(-abs) allele (*curl*<sup>3</sup> altered brassinolide sensitivity; and a line overexpressing the dwarf, BR-biosynthesis gene, 35SD) was argued as a result of reduced ion leakage, lipid peroxidation through enhanced activities of antioxidative enzymes (Mazorra et al., 2011).


#### **Table 1 | Summary of representative studies on brassinosteroids (BRs) and related compounds in the mitigation of major abiotic stress-impacts in different plant species.**

*(Continued)*

#### **Table 1 | Continued**


Literature is full also on the role of BRs in plants under low temperature stress. BR infiltration prior to cold treatment can reduce the ion leakage in chilling-exposed rape plants (Janeczko et al., 2007); whereas, 24-epiBL can increase the antioxidant defense (and also osmoregulation) in chilling stressed young grapevines (Xi et al., 2013). Application of 24- EpiBL to suspension cultured cells of low temperature (4 and 0◦C)-exposed *Chorispora bungeana* alleviated oxidative damage through enhancing the activity of ROS-metabolizing enzymes such as APX, CAT, POD and SOD and the content of AsA (Liu et al., 2009). In chilling (4◦C) exposed *Brassica juncea* seedlings, exogeniously applied 24-epiBL alleviated the toxic effect of H2O2 through increasing the activities of various enzymes involved in antioxidant defense system like CAT, APX, and SOD (Kumar et al., 2010). Hu et al. (2010) reported that exogenous application of 24-epiBL alleviated the 12/8◦C chilling-induced inhibition of photosynthesis in cucumber (*Cucumis sativus*) by reducing ROS generation and accumulation through increasing the activities of SOD, APX. In another study on cucumber pretreated with 24-epiBL (0.3 and 1.0 mmol·L−<sup>1</sup> chlorpyrifos) and exposed to chilling stress, these authors reported elevations in the activities of APX, GR, CAT, and GPX that eventually alleviated the chillingaccrued phytotoxicity (Hu et al., 2013). 28-homoBL (10−8, or 10−<sup>6</sup> M)-mediated significantly increased activities of antioxidant enzymes like CAT, POD, and SOD (and also the elevated content of proline) in cucumber (*Cucumis sativus*) were reported to improve tolerance of this plant to chilling temperatures (10/8◦C, 5/3◦C) (Fariduddin et al., 2011; BRs 5, 10, and 15μM) effectively reduced chilling injury of pepper fruit during 18-day storage at 3◦C by reducing the electrolyte leakage, MDA content; increasing the activities of antioxidant enzymes including CAT, POD, APX, and GR (Wang et al., 2012). Aghdam et al. (2012) reported that treatments with 3.0 and 6.0μM BRs to tomato fruits stored at 1◦C for 21 days reduced the chilling injury, electrolyte leakage, MDA content; enhanced proline, total phenol contents, phenylalanine ammonia-lyase (PAL) activity and maintained membrane integrity. In a recent work, these authors reported that application of 0, 3 and 6μM BL to tomato fruits subjected to 1◦C chilling stress can inhibit the activities of phospholipase D (PLD) and lipoxygenase (LOX), major causes of chilling injury induction in tomato fruits (Aghdam and Mohammadkhani, 2014). BRs protected the photosynthetic apparatus from cold-induced damage in *Cucumis sativus* plants by activating the enzymes of Calvin cycle and increasing the antioxidant capacity, which in turn mitigated the photo-oxidative stress and plant growth inhibition during the recovery of chilling injury (Jiang et al., 2013).

### **DROUGHT STRESS**

Reports are extensive on the role of BRs and related compounds in plant drought tolerance (Li and Van Staden, 1998a,b; Li et al., 1998, 2008, 2012b; El-Khallal, 2002; Vardhini and Rao, 2003a,b, 2005; Zhang et al., 2008; Behnamnia et al., 2009; Fariduddin et al., 2009a; Farooq et al., 2010; Yuan et al., 2010; Anjum et al., 2011; Mahesh et al., 2013). Field and pot experiments of 0.2 mg L−<sup>1</sup> BL application to 1-year-old *Robinia pseudoacacia* seedlings grown under drought stress increased the activity of SOD, POD and CAT, and the contents of soluble sugars and free proline (Li et al., 2008). Application of 0.1μM 24-epiBL increased the resistance in drought-stressed *Chorispora bungeana* by reducing the lipid peroxidation (measured in terms of MDA content), membrane permeability as a result of increased activities of antioxidative enzymes and the pools of non-enzymatic antioxidants such as AsA and GSH (Li et al., 2012b). BL ameliortaed the negative effect water stress (Poly Ethylene Glycol:PEG for 24 h) on callus tissues of drought-resistant (PAN 6043) and drought-sensitive (SC 701) cultivars of *Zea mays* by enhancing the activities of SOD, CAT, APX, POD, and GR (Li and Van Staden, 1998a,b). Earlier also, BL was reported to increase the activities of SOD, CAT, and APX eznymes, and the contents of AsA and total carotenoids in seedlings of drought-resistant (PAN 6043) and drought-sensitive (SC 701) cultivars of *Zea mays* under water stress (−1.0 MPa PEG 6000) (Li et al., 1998). Exogenous application of BL alleviated the detrimental effects of drought in *Zea mays* by enhancing enzymatic antioxidant enzyme activities and the contents of proteins, relative leaf water and proline (Anjum et al., 2011). 28-HomoBL and 24-epiBL ameliorated the negative impact of PEG-imposed osmotic/water stress in CSH-14, ICSV (Vardhini and Rao, 2003a) and CSH-15 (Vardhini and Rao, 2005) varieties of *Sorghum vulgare* seedlings by increasing the activity of CAT and the contents of free proline and nucleic acids. Seedlings of *Triticum* cultivars Sakha 69 (drought-resistant) and Giza 164 (drought-sensitive) subjected to water stress (by soaking the roots for 48 h in 30% PEG 6000; −0.9 MPa) and BR treatment, exhibited higher RWLC, MSI, proline, regulation of expression of water stress-inducible proteins as well as induced *de-novo* synthesis of specific polypeptides (El-Khallal, 2002). Exogenously applied 24 epiBL (0.01μM) improved the drought tolerance in rice (*Oryza sativa*) cultivar *Super-Basmati* which was sturdily associated with the greater tissue water potential, increased synthesis of metabolites and enhanced capacity of antioxidant system (Farooq et al., 2010). Spraying with HBL (0.01μM) to 30-day stage seedlings of *Brassica juncea* subjected to drought stress (for 7 days at the 8–14 (DS1)/15–21 (DS2) days stage of growth) improved the activities of antioxidant enzymes such as CAT, POD and SOD, and the content of proline (Fariduddin et al., 2009a). Foliar application of BRs elevated the activities of POD and SOD, increased the concentrations of soluble sugars and proline that eventualy resulted into decreased MDA concentration and electrical conductivity in the leaves of drought exposed *Glycine max* (Zhang et al., 2008). *Lycopersicon esculentum*, subjected to drought stress and pretreated with BR showed increased activities of POD, SOD, CAT and APX, and the contents of non-enzymatic antioxidants such as AsA and proline (Behnamnia et al., 2009). Yuan et al. (2010) also reported that 1.0μM 24-epiBL treatment significantly alleviated water stress and increased the activities of antioxidant enzymes such as CAT, APX, and SOD that decresaed the levels of H2O2 and MDA in two *Lycopersicon esculentum* genotypes viz., Mill. cv. Ailsa Craig (AC) and its ABA-deficient mutant notabilis (not). 24-epiBL and 28-homoBL-mediated reduction in the inhibitory effect of water stress on seed germination and seedling growth of radish (*Raphanus sativus*) subjected to water stress (imposed by 15% (w/v) PEG) was a result of elevated levels of SOD, CAT and APX and the free proline content (Mahesh et al., 2013) (**Table 1**).

### **SALINITY STRESS**

Modulation of various components of antioxidant defense system via BRs and associated compounds in salinity exposed plants has been extensively reported (Nunez et al., 2003; Özdemir et al., 2004; Song et al., 2006; Shahbaz and Ashraf, 2007; Zhang et al., 2007; Ali et al., 2008b; Arora et al., 2008a; El-Khallal et al., 2009; Hayat et al., 2010b; Rady, 2011; Vardhini, 2011; Ding et al., 2012; El-Mashad and Mohamed, 2012; Abbas et al., 2013; Fariduddin et al., 2013b; Lu and Yang, 2013; Sharma et al., 2013b) (**Table 1**). BL mitigated the negative impact of salt stress in *Zea mays* by inducing the activities of different antioxidant enzymes (El-Khallal et al., 2009). Application of 28-homoBL (10−7, 10−9, and 10−<sup>11</sup> M) for 7 days improved seedling growth, lipid peroxidation via elevating antioxidative enzyme activities (SOD, CAT, GR, APX, and GPX) in the seedlings of *Zea mays* (var. Partap-1) subjected to salt (25, 50, 75, and 100 mM NaCl) stress (Arora et al., 2008a). 24-EpiBL applied as a foliar spray could alleviate the adverse effects of salt on two hexaploid wheat (*Triticum aestivum*) cultivars, S-24 (salt tolerant) and MH-97 (moderately salt sensitive), grown in saline conditions (150 mM of NaCl) by enhancing the activity of POD and CAT (Shahbaz and Ashraf, 2007). BL treatment increased the activities of CAT, SOD and GR; reduced the activities of POD and PPO of two varieties of sorghum plants ("CSH-5" and "CSH-6") grown in two saline experimental sites of Karaikal (Varchikudy and Mallavur), thus indicating its ability to counteract the negative impact of saline stress (Vardhini, 2011). Exogenous BL (0. 01 mg × L (−1)) markedly decreased the salt stress index, mortality rate, MDA, electrolyte leakage via enhancing the activities of SOD, POD, and CAT in *Cucumis sativus* seedlings (Song et al., 2006). Exogenous BR (0.005, 0.01, 0.05, 0.1, and 0.2 mg/L−1) protected *Cucumis sativus* seedlings against salt stress by elevating the activity of SOD, POD and CAT, and that in turn distinctly lowered the salt injured index (40.2%) and increased the contents of free-proline, soluble sugars (Shang et al., 2006). Application of epiBL to salinity-exposed *Cucumis sativus* seedlings decreased leaf superoxide anion production rate, H2O2, MDA, cell membrane permeability, improved seedlings growth as a result of increased the activities of SOD, POD, CAT (Lu and Yang, 2013). Application of epiBL to the Cu+NaCl (150 mM) stressed seeds of two cultivars (Rocket and Jumbo) of *Cucumis sativus* plant enhanced the activities of various antioxidant enzymes viz., CAT, POD, SOD, that eventually improved growth, carbonic anhydrase activity, photosynthetic efficiency (Fariduddin et al., 2013b). Seed priming with 5.0μM L−<sup>1</sup> BL was reported to improve the seed germination and seedling growth of 3 lucerne (*Medicago sativa* L.) varieties, viz., *Victoria*, *Golden Empress*, and *Victor* by significantly increasing the activities of POD, SOD, and CAT under salt stress (13.6 dS/m NaCl solution) (Zhang et al., 2007).

In salinity (120 mM NaCl) exposed IR-28 *Oryza sativa* seedlings, 24-EpiBL considerably alleviated oxidative damage and improved seedling growth by increasing APX activity and reducing lipid peroxidation (Özdemir et al., 2004). A polyhydroxylated spirostanic brassinosteroid analog (BB-16; 0.001 or 0.01 mg dm−3) application to salinity (75 m NaCl)-exposed *O. sativa* seedlings showed significant increases in the activities of CAT, SOD, and GR (Nunez et al., 2003). Exogenous application of 24-epiBL to *Oryza sativa* var *Pusa Basmati-1*, grown under salt stress conditions exhibited improvement in growth, levels of protein, proline contents and antioxidant enzymes activities through expression of various BRs (OsBRI1, OsDWF4) and salt (SalT) responsive genes (Sharma et al., 2013b). Eggplant seedlings, when exposed to 90 mM NaCl with 0, 0.025, 0.05, 0.10, and 0.20 mg dm−<sup>3</sup> of epiBL for 10 days exhibited decreased electrolyte leakage, superoxide production, MDA, H2O2 probably as a result of increased activities of SOD, GPX, CAT and APX enzymes and the contents of non-enzymatic antioxidants such as AsA and GSH (Ding et al., 2012). 24-epiBL decreased the adverse effects of salinity stress on two varieties of pepper (*Capsicum annuum*) arguably by increasing the activities of antioxidative enzymes and the contents of proline, total anthocyanins and minerals (Abbas et al., 2013). Spraying of 1.0μM of 24-epiBL to NaCl-exposed *Brassica juncea*detoxified the stress generated by NaCl by enhancing antioxidative enzymes and the level of proline (Ali et al., 2008b). Supplementation of *Vigna radiata* c.v. T-44 plants with 28-homoBL detoxified the stress generated by NaCl by elevating the activities of antioxidative enzymes and the proline content that in turn improved the MSI, leaf water potential (ψ) (Hayat et al., 2010b). In a similar study, Rady (2011) reported that spraying 5μM of 24-epiBL to NaCl-exposed *Phaseolus vulgaris* improved the MSI, RLWC as a result of significant elevations in the activities of antioxidative enzymes and proline content. Imbibition with 24-epiBL to pea (*Pisum sativum* L.) cv. climax seeds, subjected to sodium chloride stress significantly elevated the activity of SOD, POD, and CAT enzymes the helped plants to improve fresh and dry biomass, seedling height, photosynthetic rate, stomatal conductance, and the total chlorophyll content (Shahid et al., 2011). Treatment with 0.05 ppm BL as foliar spray mitigated salt stress-impacts in cowpea (*Vigna sinensis*) by inducing the activities of antioxidant enzymes such as SOD, POD, PPO and GR and the contents of AsA (El-Mashad and Mohamed, 2012).

### **OTHER ABIOTIC STRESSES**

Apart from the discussed above major abiotic factors, BRs and associated compounds can also play significant roles in plants under a range of other abiotic stress factors such as photoinhibition/light stress, waterlogging/flooding stress, pesticides, neonicotinoid insecticide, imidacloprid (IMI) etc. (Kang et al., 2006, 2009; Lu et al., 2006; Xia et al., 2006, 2009a,b; Liang and Liang, 2009; Hayat et al., 2010c; Ogweno et al., 2010; Ahammed et al., 2012c; Lu and Guo, 2013; Sharma et al., 2013a,b). 24-BL (0.01 mg l−1) has been benefitted tomato (*Lycopersicon esculentum* Mill.) to maintain net photosynthetic rate (Pn), quantum efficiency of PSII (-PSII) and photochemical quenching (qP) under photoinhibition/light stress by decreasing lipid peroxidation as a result of efficient ROS-metabolism via enhanced activity of SOD, GPX, CAT, and APX enzymes (Ogweno et al., 2010). In another study, exogenous application of 24-epiBL was reported to enhance the tolerance of elite Indica *O. sativa* variety (*Pusa Basmati-1* seedlings) to stress generated by neonicotinoid insecticide, imidacloprid (IMI) by elevating the activity of antioxidative enzyme such as SOD, APX, CAT, GR and MDHAR, upregulating the expression of most of the genes like Cu/Zn-SOD, Fe-SOD, Mn-SOD, APX, CAT and GR, and decreasing lipid peroxidation (Sharma et al., 2013a). In 80 mM Ca(NO3)2-exposed *Cucumis sativus* cv. Jinyou No. 4, EpiBL (0.1μM) protected the photosynthetic membrane system by up-regulating the ROSscavenging capacity of the antioxidant system (Yuan et al., 2012). Folair spray of epiBL or homoBL to *Lycopersicon esculentum* Mill. cv. K-21 showed lowered sodium nitroprusside (SNP) concentration (10−<sup>5</sup> M) and improved growth and the content of pigment contents via strengthning antioxidant system (Hayat et al., 2010c). Application of 24-epiBL-mediated increased H2O2-metabolism and decreased lipid peroxidation via enhanced activity of GST and the content of GSH were argued to help *Solanum lycopersicum* seedlings to counteract three-ringed PAH (phenanthrene-PHE)-accrued consequences (Ahammed et al., 2012a,c). Alleviation of impacts caused by phenanthrene and pyrene phytotoxicity in tomato plants has been evidenced as a result of 24-epiBL-mediated increased activities of GPX, CAT, APX and GR and decreased content of MDA) (Ahammed et al., 2012b). Recently, these authors reported that 24-epiBL (100μM) can alleviate PCB (polychlorinated biphenyls)-induced oxidative stress in tomato plants by enhancing the activities of antioxidant enzymes, and maintaining photochemical efficiency of PSII Fv/Fm), the quantum efficiency of PSII photochemistry [-(PSII)] and photochemical quenching coefficient (Pq) (Ahammed et al., 2013b). The 24-epiBL-mediated strengthning of antioxidant defense system and eventually decreased membrane lipid peroxidation was reported in plants exposed to phenanthrene + Cd co-contamination (Ahammed et al., 2013a). Pretreatment of *Cucumis sativus* with 24-epiBL alleviated the phytotoxicities of nine pesticides (paraquat, fluazifop-p-butyl, haloxyfop, flusilazole, cuproxat, cyazofamid, imidacloprid, chlorpyrifos, and abamectin) by increasing the activities of antioxidant enzymes, and CO2 assimilation capacity (Xia et al., 2006). Significant role of 24-epiBL was also reported in plants exposed to Chlorpyrifos (a widely used insecticide), where elevated activity of GST, POD, and GR was argued to regulate net photosynthetic rate and quantum yield of PSII [Phi(PSII)] (Xia et al., 2009a). BRs and associated compounds were reported to provide tolerance to waterlogging/flooding stress in different crops including soybean (Lu et al., 2006), cucumber (Kang et al., 2006, 2009; Lu and Guo, 2013) and oilseed rape (Liang and Liang, 2009) mainly as a result of decreased oxidative damage via enhanced activities of SOD and POD.

### **CONCLUSION AND FUTURE PROSPECTS**

It is a well-established fact that environmental stresses are the primary cause of crop loss worldwide, reducing average yields for most major crop plants adversely affecting the global crop production and the adverse impacts are getting more serious in the past few decades. Environmental stresses induce the production of ROS, alter the activity of antioxidant system and adversely affect the process of photosynthesis. The crop physiologists and scientists have employed strategies to mitigate the elevated ROS (and their reaction products)-accrued oxidative stresses/damages via strengthening antioxidant defense system in plants exposed to various abiotic/stress factors. In this regard, the use of different plant growth regulators (PGRs) has been considered as a better sustainable alternative, and also as a technically simpler approach (Khan et al., 2012; Iqbal et al., 2013; Asgher et al., 2014). To this end, in addition to playing significant roles under general plant growth, development and metabolism, BRs and associated compounds have been extensively reported to counteract consequences of various abiotic stresses including temperature (heat, chilling, and freezing), water (drought, water logging), salt, heavy metals, light (intense and weak) and radiation (UV-A/B). Though much has been achieved in the current context, integrated approach is required to investigate more insights into molecular-genetic mechanisms of BRs and associated compounds-mediated modulation of various components of antioxidant defense system and subsequently the control of abiotic stress-consequences in plants.

### **ACKNOWLEDGMENTS**

Financial support to B. V. Vardhini from University Grants Commission (UGC), New Delhi [MRP-655/05 (UGC-SERO); Link No 1655.0] India is gratefully acknowledged. Naser A. Anjum gratefully acknowledges the partial financial support received from Portuguese Foundation for Science and Technology (FCT) through post-doctoral research grants (SFRH/BPD/64690/2009; SFRH/BPD/84671/2012), and the Aveiro University Research Institute/Centre for Environmental and Marine Studies (CESAM).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 November 2014; accepted: 19 December 2014; published online: 12 January 2015.*

*Citation: Vardhini BV and Anjum NA (2015) Brassinosteroids make plant life easier under abiotic stresses mainly by modulating major components of antioxidant defense system. Front. Environ. Sci. 2:67. doi: 10.3389/fenvs.2014.00067*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2015 Vardhini and Anjum. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Managing the pools of cellular redox buffers and the control of oxidative stress during the ontogeny of drought-exposed mungbean (*Vigna radiata* L.)—role of sulfur nutrition

#### *Naser A. Anjum1,2, Shahid Umar 1, Ibrahim M. Aref <sup>3</sup> and Muhammad Iqbal <sup>1</sup> \**

*<sup>1</sup> Department of Botany, Faculty of Science, Hamdard University, New Delhi, India*

*<sup>2</sup> Department of Chemistry, CESAM-Centre for Environmental and Marine Studies, University of Aveiro, Aveiro, Portugal*

*<sup>3</sup> Plant Production Department, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia*

#### *Edited by:*

*Adriano Sofo, Università degli Studi della Basilicata, Italy*

#### *Reviewed by:*

*Yogesh Abrol, Bhagalpur University, India Kumar Ajit, University of South Australia, Australia*

#### *\*Correspondence:*

*Muhammad Iqbal, Department of Botany, Faculty of Science, Hamdard University, New Delhi, 110062, India e-mail: iqbalg5@yahoo.co.in*

Impacts of increasing environmental stresses (such as drought) on crop productivity can be sustainably minimized by using plant-beneficial mineral nutrients (such as sulfur, S). This study, based on a pot-culture experiment conducted in greenhouse condition, investigates S-mediated influence of drought stress (imposed at pre-flowering, flowering, and pod-filling stages) on growth, photosynthesis and tolerance of mungbean (*Vigna radiata* L.) plants. Drought stress alone hampered photosynthesis functions, enhanced oxidative stress [measured in terms of H2O2; lipid peroxidation (LPO); electrolyte leakage (EL)] and decreased the pools of cellular redox buffers (namely ascorbate (AsA); glutathione (GSH)], and the overall plant growth (measured as leaf area and plant dry mass), maximally at flowering stage, followed by pre-flowering and pod-filling stages. Contrarily, S-supplementation to drought-affected plants (particularly at flowering stage) improved the growth- and photosynthesis-related parameters considerably. This may be ascribed to S-induced enhancements in the pools of reduced AsA and GSH, which jointly manage the balance between the production and scavenging of H2O2 and stabilize cell membrane by decreasing LPO and EL. It is inferred that alleviation of drought-caused oxidative stress depends largely on the status of AsA and GSH via S-supplementation to drought-stressed *V. radiata* at an appropriate stage of plant growth, when this nutrient is maximally or efficiently utilized.

**Keywords: cellular buffers, drought stress, mungbean ontogeny, oxidative stress, sulfur,** *Vigna radiata*

### **INTRODUCTION**

Recognized as one of the major environmental stress factors, and as a main constraint for crop production worldwide, drought affects virtually every aspect of plant growth, physiology and metabolism (Harb et al., 2010). In particular, at the wholeplant level, drought stress affects mainly the plant photosynthetic functions, causing imbalance in "CO2 fixation and electron transport." This facilitates transfer of electrons to reactive oxygen species (ROS), including H2O2, as a result of over-reduction of the electron-transport-chain components (Anjum et al., 2008a; Lawlor and Tezara, 2009). Additionally, high concentration of ROS causes oxidative damage to photosynthetic pigments, biomolecules such as lipids, proteins and nucleic acids, and leakage of electrolytes via lipid peroxidation (LPO), leading to cessation of normal plant cellular metabolism (Anjum et al., 2012a). The ascorbate-glutathione (AsA-GSH) pathway constitutes the major part of antioxidant defense system in plants where a number of ROS are effectively metabolized and/or detoxified by a network of reactions involving enzymes and metabolites with redox properties. Both AsA and GSH (tripeptide GSH; γ-glutamate-cysteine-glycine) are cellular redox buffers closely linked in major physiological functions. Nevertheless, in conjunction with other components of AsA-GSH pathway, both AsA and GSH together determine the lifetime of varied ROS and their reaction products within the cellular environment and provide crucial protection against oxidative damage (Anjum et al., 2010, 2012a, 2013). In recent studies, exogenous application of AsA or GSH was reported to help plants to withstand consequences caused by a range of abiotic stresses including Cd (Cai et al., 2011; Son et al., 2014), salinity (Wang et al., 2014) and high temperature (Nahar et al., 2015).

Maintenance of the status of mineral nutrients in plants is important for increasing the crop productivity and plant resistance to environmental stress (Cakmak, 2005; Anjum et al., 2008b, 2012b). The cumulative role of mineral nutrients in modulating cellular levels of AsA and GSH, and in strengthening the plant antioxidant defense system has been discussed extensively (Anjum et al., 2010, 2012a, 2013; Gill et al., 2011). Sulfur (S) is the fourth essential macronutrient for plants, after N, P and K, and plays a vital role in the regulation of plant growth, development and productivity (Hawkesford, 2000), via affecting leaf chlorophyll, N content and photosynthetic enzymes. Sulfur is required for protein synthesis, incorporated into organic molecules in plants, and is located in thiol (−SH) groups in proteins (cysteine-residues) or non-protein thiols (glutathione, GSH), the potential modulators of stress response (Anjum et al., 2008b; Lunde et al., 2008). Significance of plant ontogeny in the modulation of plant responses to abiotic stress factors such as drought (Anjum et al., 2008a) and heavy metals (Anjum et al., 2008c) has been reported. Also, considering a single plant growth-stage, the role of S nutrition in the improvement of plant growth, development and yield (Ahmad et al., 2005), and tolerance to stresses (such as Cd; Anjum et al., 2008b) has been evidenced. However, information is meager on the Smediated control of plant responses to drought stress during plant ontogeny.

Given the paucity of information on drought sensitivity of legume crops, and on the physiological basis of mineralnutrient-(like S)-assisted management of crop growth and productivity, the current study was undertaken (i) to screen the drought-sensitive stage(s) during plant ontogeny, (ii) to identify the plant-growth stage when S helps plants maximally to improve the pools of both cellular redox buffers (AsA, GSH) and mineral-nutrients (K, S, and Mg) in order to counteract the drought-accrued oxidative stress (measured as electrolyte leakage (EL), membrane lipid peroxidation and H2O2 levels). Mungbean (*Vigna radiata* L. Wilczek) was chosen as a model plant system for the current study, because it is a potential pulse crop in the Indian sub-continent due to its ready market, N2-fixation capability, early maturity and the ability to fit well in croprotation program (Anjum, 2006). Additionally, S-requirement of the pulse crops, for maintaining their normal growth and development, stands just second to that of the oil-yielding crops.

### **MATERIALS AND METHODS EXPERIMENTAL MATERIALS, PROCEDURE AND SOIL CHARACTERISTICS**

Seeds of mungbean (*Vigna radiata* L. Wilczek) cultivar Pusa 9531 were sown in 30-cm-diameter earthen pots filled with 8 kg soil. The soil was sandy loam in texture, with 7.8 pH 7.8, 0.38 dsm electrical conductivity, 0.43% organic carbon, 70 mg kg−<sup>1</sup> soil available K and 5 mg kg−<sup>1</sup> soil available S. Nitrogen (N; 120 mg kg−<sup>1</sup> soil) and phosphorus (P; 30 mg kg−<sup>1</sup> soil) were applied at the time of sowing. S was applied to *V. radiata* plants at the rate of 40 mg kg−<sup>1</sup> soil, in the form of solution, 5 days before droughtstress imposition at various growth stages. The sources of N, P, K, and S were urea, single super phosphate, gypsum, and muriate of potash, respectively. After germination, three plants per pot were maintained until harvest. The pots were kept in green house under semi-controlled condition. A polythene plastic film was used to thwart the effects of rainfall, which allowed transmittance of 90% of visible wavelength (400–700 nm) under natural day and night conditions with a day/night temperature 25/20 ± 4◦C and relative humidity of 70 ± 5%. All experiments were performed using completely expanded leaves from the second youngest nodes from the top of the plants.

### **DROUGHT STRESS IMPOSITION AND SULFUR (S) APPLICATION SCHEDULE**

Drought was imposed at pre-flowering (15 d after sowing) (group 1), flowering (30 d after sowing) (group 2) and at pod filling (50 d after sowing) (group 3) by withholding water for 5 days; this was followed by normal watering (without S). Other three groups (4– 6) as well as the controls were supplied with an equal amount of S solution (40 mg S kg−1). All these (1–6) plant groups, and the control group, were maintained until harvest, and watered on alternate days. Soil moisture content was measured gravimetrically on dry weight basis at the time of pre-flowering, flowering, and post-flowering (pod-filling) stages (**Table 1**). Samplings were done after re-watering the drought-exposed plants for 5 days at the given growth stage i.e., at 25, 40, and 60 days after sowing. The treatments were arranged in a randomized complete block design, and each treatment was replicated five times.

### **PLANT GROWTH, PHOTOSYNTHESIS AND BIOCHEMICAL ESTIMATIONS**

Leaf area was measured with a leaf area meter (LI-3000A, LI-COR, Lincoln, NE). Plant dry mass was determined after drying the plant at 80◦C to a constant weight with the help of an electronic balance (SD-300). Net photosynthetic rate (P*n*), stomatal conductance (G*s*) and intercellular CO2 concentration (C*i*) were recorded in fully expanded leaves of second youngest nodes, using infra-red gas analyzer (IRGA, LI-COR, 6400, Lincoln, NE) on a sunny day between 10:00 and 11:00 h. Chlorophyll content was estimated in fully expanded young leaves at each stage using the method given by Hiscox and Israelstam (1979). Estimation of soluble protein content was done according to Bradford (1976) using bovine serum albumin as standard.

### **OXIDATIVE STRESS TRAITS**

We considered electrolyte leakage (EL), membrane lipid peroxidation and H2O2 levels as the biomarkers of oxidative stress. Cellular membrane integrity in leaves was assayed by measuring the EL according to Anjum et al. (2013). In brief, fresh leaves (1.0 g) were kept in glass vials containing 10 ml deionized water.

**Table 1 | Soil moisture content (%) measured in the control and drought-stressed conditions [with and without sulfur (S) supply], at pre-flowering, flowering and pod-filling stages of mungbean (***Vigna radiata***) plants. Values are the means of five replicates ± standard deviation.**


*Significant differences are: avs. Control; bvs. Drought (pre-flowering); <sup>c</sup> vs. Drought (flowering).*

The vials, covered with plastic caps, were placed in a shaking incubator at a constant temperature of 25◦C for 6 h and the electrical conductivity (EC) of the solution was measured (EC1) using an electrical conductivity meter (WTW Cond 330i/SET, Weilheim, Germany). Subsequently, the same vials were kept in water bath shaker at 90◦C for 2 h, cooled and EC2 was measured. EL was expressed following the formula EL = EC1/EC2 × 100.

Membrane lipid peroxidation was estimated in terms of thiobarbituric acid reactive substances (TBARS) contents adopting the method of Dhindsa et al. (1981) as described by Anjum et al. (2013). Briefly, fresh leaves (1.0 g) were ground in liquid nitrogen, mixed with 0.73% 2-thiobarbituric acid in 12% trichloroacetic acid, incubated for 30 min in boiling water, ice-cooled, centrifuged at 1000×g for 10 min at 4◦C and the absorbance measured in the supernatant at 532 nm. The rate of lipid peroxidation was expressed as nmoles of TBARS formed per gram of fresh weight, using a molar extinction coefficient of 1.55 <sup>×</sup> 105 M−<sup>1</sup> cm−1. Leaf-H2O2 content was determined following the method of Loreto and Velikova (2001) as adopted and described by Dipierro et al. (2005). In brief, leaf tissues (1.0 g) were homogenized in 2 ml of 0.1% (w/v) TCA. The homogenate was centrifuged at 12,000×g for 15 min and 0.5 ml of the supernatant were mixed with 0.5 ml of 10 mM K-phosphate buffer pH 7.0 and 1 ml of 1 M KI. The H2O2 content of the supernatant was evaluated by comparing its absorbance at 390 nm with a standard calibration curve.

### **DETERMINATION OF CELLULAR BUFFERS**

Both reduced GSH and AsA were considered as representative cellular redox buffers. The content of reduced glutathione (GSH) was estimated following the method of Anderson (1985). Fresh leaf tissues (1.0 g) were homogenized in 2 ml of 5% (w/v) sulphosalicylic acid at 4◦C. The homogenate was centrifuged at 10,000×g for 10 min. To a 0.5 ml of supernatant, 0.6 ml of Kphosphate buffer (100 mM, pH 7.0) and 40μl of 5 5 -dithiobis-2-nitrobenzoic acid (DTNB) were added, and absorbance was recorded after 2 min at 412 nm on a UV-VIS spectrophotometer (Lambda Bio 20, Perkin Elmer, MA, USA). The method of Law et al. (1983) was followed for estimation of reduced ascorbate (AsA). In brief, fresh leaf (0.5 g) was homogenized in 2.0 ml of K-phosphate buffer (100 mM, pH 7.0) containing 1 mM EDTA and centrifuged at 10,000×g for 10 min. To a 1.0 ml of supernatant, 0.5 ml of 10% (w/v) trichloroactetic acid (TCA) was added, thoroughly mixed and incubated for 5 min at 4◦C. Then, 0.5 ml of NaOH (0.1 M) was mixed with 1.5 ml of the above solution and centrifuged at 5000×g for 10 min at 20◦C. The aliquot thus obtained was equally distributed into two separate microfuge tubes (750μl each). For estimation of AsA, 200μl of K-phosphate buffer (150 mM, pH 7.4) was added to 750μl of aliquot. For DHA estimation, 750μl of aliquot was added to 100μl of 1,4-dithiothreitol (DTT), followed by vortex-mixing, incubation for 15 min at 20◦C, and addition of 100μl of 0.5% (w/v) NEM. Both the microfuge tubes were then incubated for 30 s at room temperature. To each sample tube, 400μl of 10% (w/v) TCA, 400μl of H3PO4, 400μl of 4% (w/v) bipyridyl dye (N'N-dimethyl bipyridyl) and 200μl of 3% (w/v) FeCl3 were added and thoroughly mixed. Absorbance was recorded at 525 nm after incubation for 1 h at 37◦C.

### **K, S, AND Mg CONTENT DETERMINATIONS**

The method of Lindner (1944) was followed to estimate K content in digested samples using flame photometry; whereas, for S determination, 100 mg of dried fine powder of leaf was digested in a mixture of concentrated HNO3 and 60% HClO4 (85:1, v/v) and the content of sulfate was estimated using the turbidimetric method of Chesnin and Yien (1950). Leaf Mg content was determined by digesting samples in 5 ml of 96% H2SO4 and 3 ml of 30% H2O2 at 270◦C; thereafter, Mg content was assayed by atomic absorption spectrometry at 285.2 nm wavelengths.

### **DATA ANALYSIS**

SPSS (PASW statistics 18, Chicago, IL, USA) for Windows was used for statistical analysis. One-Way analysis of variance (ANOVA) was performed, followed by all pairwise multiple comparison procedures (Tukey test). Mann-Whitney *U*-test and Levene's test were performed in order to check the normal distribution and the homogeneity of variances, respectively. The data are expressed as mean values ± SD of five independent experiments with at least five replicates for each. The significance level was set at *P* ≤ 0*.*05.

### **RESULTS**

Significant results related to plant growth, photosynthetic functions, soluble-protein content, oxidative stress, cellular reducing buffers, plant mineral nutrients, and yield attributes are presented here, highlighting the significant changes observed at different (pre-flowering, flowering, and pod-filling) stages of plant growth.

### **PLANT GROWTH AND PHOTOSYNTHETIC FUNCTIONS**

Under drought stress, plant growth, in terms of leaf area and plant dry mass, decreased significantly at pre-flowering stage (vs. control, C). On application of S, significant change was noted in the drought-induced reduction in leaf area only. Drought stress imposed during pre-flowering stage also caused significant decrease in photosynthetic functions (viz., photosynthetic rate, *Pn*; stomatal conductance, *Gs*; intercellular CO2, C*i*; chlorophyll content), as compared with the control. Supplementation of S significantly increased the drought-induced reductions in *Pn*, G*s,* and C*i* (**Tables 2, 3**).

During the flowering/reproductive stage, significant decrease in leaf area and plant dry mass was perceptible under drought stress alone (vs. C), whereas S application significantly increased the drought-induced reductions in these parameters. *Pn*, G*s,* C*i* and the chlorophyll content displayed significant decreases due to drought stress (vs. C); whereas, supplementation of S improved these traits (vs. drought at flowering). During the pod-filling stage, leaf area and plant dry mass decreased significantly due to imposition of drought stress (vs. C) and S application deepened the decline in leaf area and plant dry mass. Likewise, *Pn*, *Gs* and C*i* and chlorophyll content displayed significant decreases due to drought stress imposed at the pod-filling stage (vs. C). The decrease in *Pn*, C*i* and chlorophyll was significantly ameliorated with S supplementation (**Tables 2, 3**).

### **OXIDATIVE STRESS AND MODULATION OF THE POOLS OF CELLULAR REDOX BUFFERS**

With-holding water for 5-days during pre-flowering stage led to significant increases in electrolyte leakage (EL) and in the contents of thiobarbituric-acid-reactive substances (TBARS) and H2O2 (vs. C). However, S-application significantly decreased the impact of drought stress-impact at pre-flowering by reducing EL and the contents of TBARS and H2O2 (**Figure 1**). The pools of cellular redox buffers namely reduced ascorbate (AsA) and glutathione (GSH) declined significantly due to pre-flowering drought stress (vs. C); S-supplementation was insignificant to mitigate these declines. Oxidative stress traits such as EL, and the contents of TBARS and H2O2 significantly increased due to drought stress created at flowering stage (vs. C); however, S-application significantly decreased this drought-caused oxidative stress. In contrast,

**Table 2 | Leaf area (cm<sup>2</sup> plant−1) and plant dry mass (g plant−1) in mungbean (***Vigna radiata***) as influenced by drought stress and by drought <sup>+</sup> sulfur (S) application (mg kg−<sup>1</sup> soil) at pre-flowering, flowering, and pod-filling stages.**


*Values are the means of five replicates* ± *standard deviation. Significant differences within the same growth stage are: avs. Control; bvs. Drought (pre-flowering); <sup>c</sup> vs. Drought (flowering); <sup>d</sup> vs. Drought (pod-filling).*

the contents of reduced AsA and GSH contents declined significantly (vs. C) due to drought stress at this stage, and supplementation of S significantly ameliorated these declines. Drought stress imposed at pod-filling stage significantly increased EL and the contents of TBARS and H2O2 (vs. C); whereas S-application significantly reduced the levels of H2O2 and EL elevated by drought at this stage. The reductions in AsA and GSH contents due to the drought stress imposed at pod-filling stage were insignificant (vs. C), and the effect of S supplementation in mitigating the impact of drought stress was also insignificant (**Figure 2**).

### **MINERAL NUTRIENTS**

Plant nutrients, such as K, S, and Mg, displayed significant reductions due to drought stress imposed at pre-flowering stage (vs. C); however, no significant difference was observed when droughtstressed plants were supplemented with S. Drought imposition during flowering stage caused significant reduction in K, S, and Mg levels in the leaf tissue (vs. C); whereas their contents significantly increased when plants facing drought at flowering stage were supplemented with S. Among the plant nutrients studied, only K content displayed a significant reduction due to drought at pod-filling stage (vs. C); whereas S and Mg contents did not differ significantly under the stress of drought alone or drought + S imposed at pod-filling stage (vs. C). Moreover, the K content significantly increased when drought-stressed plants were supplemented with S (**Table 4**).

### **DISCUSSION**

### **PLANT GROWTH AND PHOTOSYNTHETIC FUNCTIONS**

Plant growth is the outcome of coordination of major physiological/biochemical processes in plants. In the present study, plant dry mass and leaf area showed a significant relationship with the severity of water deficit stress, irrespective of the phase of plant ontogeny. Earlier, plant growth in terms of dry mass accumulation and leaf area has been used as a tool for the assessment of crop productivity (Sundaravalli et al., 2005; Anjum et al., 2008a). Cell division, enlargement and differentiation and also the plant

**Table 3 | Net photosynthetic rate (P***n***;**μ**mol CO2 m−<sup>1</sup> s−1), stomatal conductance (G***s***; mol m−<sup>2</sup> s−1), intercellular CO2 concentration (C***i***;**μ**mol mol−1), chlorophyll (Chl) content (mg g−<sup>1</sup> fresh weight, f.w.) and soluble protein content (mg g−<sup>1</sup> f.w.) in mungbean (***Vigna radiata***), as influenced by drought stress and by drought <sup>+</sup> sulfur (S) application (mg kg−<sup>1</sup> soil) at pre-flowering, flowering, and pod-filling stages.**


*Values are the means of five replicates* <sup>±</sup> *standard deviation. Significant differences within the same growth stage are: avs. Control; bvs. Drought (pre-flowering); <sup>c</sup> vs. Drought (flowering); <sup>d</sup> vs. Drought (pod-filling).*

**peroxidation (LPO; nmol thiobarbituric acid reactive substances, TBARS g−<sup>1</sup> f.w.) (B) and electrolyte leakage (%) (C) in the mungbean (***Vigna radiata***) leaf as influenced by drought stress and by drought + sulfur (S) application (mg kg−<sup>1</sup> soil) at pre-flowering, flowering, and pod-filling stages of plant growth.** Values are the means of five replicates ± standard deviation. Significant differences within the same growth stage are: avs. Control; bvs. Drought (pre-flowering); cvs. Drought (flowering); and dvs. Drought (pod-filling).

genetic make-up are significantly influenced by water-deficit stress, which in turn affects plant growth (Aref et al., 2013). In the present study, previously mentioned processes might be impacted by drought tress severely during vegetative/flowering stage which coincides with drought-mediated considerable decreases in leaf area and photosynthesis, as observed earlier also (Sundaravalli et al., 2005; Anjum et al., 2008a; Husen et al., 2014). However, the

**FIGURE 2 | The reduced ascorbate (AsA) (A) and reduced glutathione (GSH) (B) contents (nmol g−<sup>1</sup> fresh weight) in the mungbean (***Vigna radiata***) leaf as influenced by drought stress and sulfur (S) application (mg kg−<sup>1</sup> soil) at pre-flowering, flowering, and pod-filling stages of plant growth.** Values are the means of five replicates ± standard deviation. Significant differences within same growth stage are: avs. Control; bvs. Drought (pre-flowering); cvs. Drought (flowering); and dvs. Drought (pod-filling).

drought-induced huge reduction in leaf area (a major component of plant growth) may be a strategy that plants adopt to adjust with water-deficit stress. Earlier, the reduced leaf-expansion/area was evidenced to conserve the internal water/moisture through the reduced rate of transpiration (reviewed by Mahajan and Tuteja, 2005).

Photosynthesis (P*n*) and its related variables (*Gs*, C*i*, chlorophyll content) are highly regulated multi-step processes and exhibit great sensitivity to drought stress (Zlatev et al., 2006; Lawlor and Tezara, 2009; Husen et al., 2014). In the current study, drought stress alone significantly decreased P*n*, G*s*, C*i* and chlorophyll content irrespective of the plant ontogenetic stages. In fact, photosynthesis and its related variables are tightly interwoven and hence changes in one component significantly affect the performance of others (Lawlor and Tezara, 2009). Our findings on the drought stress-accrued reductions in G*s* and leaf C*i* coincide with those of Zlatev et al. (2006) and Meyer and Genty (1998), who considered *Gs* as the major factor for controlling C*i* and hence the P*n*. Additionally, unavailability of chlorophyll **Table 4 | Potassium (K), sulfur (S), and magnesium (Mg) contents (µmol g−<sup>1</sup> dry weight) in mungbean (***Vigna radiata***) leaves as influenced by drought stress and by drought +S application (mg kg−<sup>1</sup> soil) at pre-flowering, flowering, and pod-filling stages.**


*Values are the means of five replicates* ± *standard deviation. Significant differences within same growth stage are: avs. Control; bvs. Drought (pre-flowering); <sup>c</sup> vs. Drought (flowering); <sup>d</sup> vs. Drought (pod-filling).*

also contributes to drought-induced decrease in P*n* (Lawlor and Tezara, 2009). The drought-induced decrease in chlorophyll content has been reported earlier also due to reduction in the lamellar content of the light-harvesting chlorophyll a/b protein, inhibition in biosynthesis of chlorophyll-precursors and/or degradation of chlorophyll (Khanna-Chopra et al., 1980). Our findings on drought-mediated decrease in P*n*, G*s*, C*i* and the content of chlorophyll confirm some earlier reports (Khanna-Chopra et al., 1980; Anjum et al., 2008a; Husen et al., 2014).

Regardless of irrigation treatments, our results also revealed that S-application significantly increased the growth and chlorophyll content and *Pn*. It was more effective when applied at flowering stage of the plant. The adequate and balanced supply of mineral nutrients has been shown to play a vital role in sustaining food security (Cakmak, 2005). S is involved in the light reaction of photosynthesis as an integral part of ferredoxin, a non-haem iron-sulfur protein (Marschner, 1995). Additionally, it plays essential roles in mechanisms like vitamin co-factors, GSH in redox homeostasis, and detoxification of xenobiotics (Anjum et al., 2012b). The S requirement by plants varies with growth stage and with species, varying normally between 0.1 and 1.5% of dry weight. Anjum et al. (2008b) suggested that adequate S supply may improve the pools of these compounds in plants to a great extent that may lead to increased photosynthetic efficiency, dry mass and crop yield. Sufficient S supplies improved photosynthesis and growth of *Brassica juncea* through regulating N assimilation (Khan et al., 2005). The maximum utilization of S in *Brassica campestris* crop takes place when applied at flowering stage (Ahmad et al., 2005; Anjum, 2006). Application of S increased the seed yield and attributing characters in other crops also (Anjum et al., 2012b).

### **OXIDATIVE STRESS AND MODULATION OF THE POOLS OF CELLULAR REDOX BUFFERS AND MINERAL NUTRIENTS**

Production of ROS, such as H2O2*,* is mediated by O2 reduction and subsequent oxidative damages in drought-exposed plants (Khanna-Chopra and Selote, 2007; Anjum et al., 2012a). Plant membrane is regarded as the first target of many plant stresses due to increase in its permeability and loss of integrity under environmental stresses including the drought stress (Candan and Tarhan, 2003). In the present study, the drought-stress sensitivity of the reproductive phase of drought-exposed *V. radiata* was evidenced by significantly high levels of H2O2, the content of TBARS (the cytotoxic products of lipid peroxidation and indicator of extent of stress-led ROS-mediated high oxidative stress) and the EL (the measure of stress-mediated changes in membrane leakage and injury) at flowering stage, followed by pre-flowering and postflowering stages. These results are in close agreement with the findings of Qureshi et al. (2007). Earlier, the least peroxidation of membrane lipids and the ability of cell membranes to tightly control the rate of ion movement in and out of cells have been used as tests of damage to a great range of tissues (Candan and Tarhan, 2003). However, the drought-stressed plants exhibited least contents of H2O2, TBARS and percent EL, when supplemented with S at their flowering and pod-filling stages. These results suggested that the S-mediated decrease in contents of H2O2, TBARS and percent EL depends on application of S to drought-stressed plants at appropriate growth stage when S is efficiently and differentially utilized to strengthen plants to withstand the enhanced lipid peroxidation and subsequent leakage of electrolytes due to elevated levels of H2O2. Thus, S-application protected differentially the drought-stressed plants against H2O2-mediated localized oxidative damage, disruption of metabolic functions, LPO and leakage of electrolytes (Zlatev et al., 2006). Our observations on drought alone-mediated significant increases in H2O2 content, lipid peroxidation (in terms of TBARS content) and percent EL in *V. radiata* plants coincide with the findings of Sreenivasulu et al. (2000) and Selote and Khanna-Chopra (2006) on different crop plants.

Plant resistance to stresses is closely associated with the efficiency of the antioxidant defense system (comprising both enzymatic and non-enzymatic components of AsA-GSH pathway) in the maintenance of the balance between the basal production of ROS and their elimination (Anjum et al., 2010, 2012c). In this perspective, AsA and GSH are important water-soluble non-enzymatic antioxidants and major cellular redox buffers in plants (Anjum et al., 2010, 2012c, 2014). Both are interlinked in terms of their physiological role in AsA-GSH pathway for effective elimination of ROS (such as H2O2) in plant cells (Anjum et al., 2010, 2012a,c). Contrary to an earlier report (Shehab et al., 2010) on drought-induced increase in AsA and GSH levels in different plant species, our study revealed a significant decrease in the contents of both AsA and GSH in *V. radiata,* irrespective of the growth stage at which the drought stress was imposed. However, our findings are in conformity with those of Khanna-Chopra and Selote (2007) on drought-exposed *Triticum aestivum*. The application of S improved the AsA and GSH contents and was thus beneficial when applied to drought-stressed plants at their flowering or post-flowering stages. It was, therefore, significant for protection of *V. radiata* against ROS-mediated oxidative stress. This substantiates our earlier report suggesting improved AsA and GSH contents in Cd-stressed *Brassica campestris* plants by S supplementation (Anjum et al., 2008b). However, it is imperative to mention here that exhibition of higher levels of AsA and GSH in plants receiving drought stress + S supply at pod-filling stage may be due to S-mediated maintenance of elevated activities of AsA-GSH-regenerating enzymes such as dehydroascorbate reductase, monodehydroascorbate reductase, and GSH reductase (Eltayeb et al., 2007; Anjum et al., 2008b). Moreover, as AsA and GSH are key players in cellular redox homeostasis; the S-mediated improvement in their reduced pools must help plants to run normally the ascorbate peroxide-dependent H2O2 metabolism under drought-stress conditions. Therefore, S application mitigated, although partially, the drought-induced decrease in AsA content by maintaining elevated activities of dehydroascorbate reductase and monodehydroascorbate reductase (data not shown)—the key components in maintaining the reduced pool of AsA and hence the plant tolerance to oxidative stress (Eltayeb et al., 2007). Considering K, S, and Mg responses to drought and S, the uptake of the available nutrient ions dissolved in the soil solution by

plants depends upon water flow through the soil-root-shoot pathway. It also depends on root growth and nutrient mobility in the soil (Fageria et al., 2002). In this study, drought stress significantly impacted the contents of K, S, and Mg in leaves contingent upon the plant-growth stage exposed. However, as reported also in earlier studies (Abdin et al., 2003; Malvi, 2011), a synergistic interaction of S with K and Mg was revealed herein, where Sapplication ameliorated drought-induced reductions in the leaf K, S, and Mg contents, maximally when applied at reproductive stage.

### **CONCLUSIONS**

Drought stress in isolation enhanced ROS generation and decreased the cellular redox buffers (AsA and GSH) and eventually hampered photosynthetic functions. These results were significant at flowering stage, followed by the pre-flowering and post-flowering (pod-filling) stages (**Figure 3**). However, improvements in these parameters due to S application was apparent (at the flowering/reproductive stage), which enhanced the pools of cellular redox buffers (AsA and GSH), which in turn managed a balance between the production and scavenging of H2O2 and stabilized the cell membrane by decreasing LPO (**Figure 3**). Overall, the study inferred that supplementation of S to drought-exposed plants at their flowering stage can improve their growth, photosynthesis and related variables via efficiently being utilized, and in turn managing the pools of AsA and GSH, and subsequently controlling the drought-accrued oxidative stress.

### **ACKNOWLEDGMENTS**

Partial financial support received from the Hamdard National Foundation (HNF), New Delhi, India (DSW/HNF-18/2006) and the Portuguese Foundation for Science and Technology (FCT) (SFRH/BPD/64690/2009; SFRH/BPD/84671/2012) is gratefully acknowledged.

### **REFERENCES**


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 11 December 2014; accepted: 17 December 2014; published online: 08 January 2015.*

*Citation: Anjum NA, Umar S, Aref IM and Iqbal M (2015) Managing the pools of cellular redox buffers and the control of oxidative stress during the ontogeny of droughtexposed mungbean (Vigna radiata L.)—role of sulfur nutrition. Front. Environ. Sci. 2:66. doi: 10.3389/fenvs.2014.00066*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2015 Anjum, Umar, Aref and Iqbal. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Are plant endogenous factors like ethylene modulators of the early oxidative stress induced by mercury?

*M. Belén Montero-Palmero1,2, Cristina Ortega-Villasante1, Carolina Escobar <sup>2</sup> and Luis E. Hernández <sup>1</sup> \**

*<sup>1</sup> Laboratory of Plant Physiology, Department of Biology, Universidad Autonoma Madrid, Madrid, Spain*

*<sup>2</sup> Plant Physiology, Department of Environmental Sciences, Universidad de Castilla-La Mancha, Toledo, Spain*

#### *Edited by:*

*Naser A. Anjum, University of Aveiro, Portugal*

#### *Reviewed by:*

*Naser A. Anjum, University of Aveiro, Portugal Nafees A. Khan, Aligarh Muslim University, India*

#### *\*Correspondence:*

*Luis E. Hernández, Laboratory of Plant Physiology BS13, Department of Biology, Universidad Autonoma Madrid, Edif. Biológicas, C/Darwin 2, Campus Universitario Cantoblanco, 28049 Madrid, Spain e-mail: luise.hernandez@uam.es*

The induction of oxidative stress is one of the quickest symptoms appearing in plants subjected to metal stress. A transcriptional analysis of the early responses of alfalfa (*Medicago sativa*) seedlings to mercury (Hg; 3µM for 3, 6 and 24 h) showed that up-regulation of genes responding to ethylene were up-regulated, a phytohormone known to mediate in the cellular redox homeostasis. In this mini-review we have compared these quick responses with two other concurrent transcriptomic analysis in Barrel medic (*Medicago truncatula*) and barley (*Hordeum vulgare*) under Hg stress. Besides ethylene, ABA, and jasmonate related genes were up-regulated, all of them are endogenous factors known to intervene in oxidative stress responses. The information obtained may target future work to understand the cellular mechanisms triggered by Hg, enabling biotechnological approaches to diminish Hg-induced phytotoxicity.

**Keywords: ethylene, homeostasis, hormones, mercury, oxidative stress, transcription**

### **BACKGROUND**

Mercury (Hg) is a natural component of the Earth crust that is released by mainly geothermal activity, but accumulates in land and water ecosystems, mainly as a consequence of different human activities, such as mining and industry (Nriagu, 1996; Järup, 2003). This represents a serious problem to the environment and risks for human health (Tchounwou et al., 2012) as occurs in the mining district of Almadén (Spain), which contains the largest deposits of Hg in the World, with soils heavily polluted which require *in situ* and economically feasible cleaning procedures (Millán et al., 2006). Different strategies to clean-up metal polluted soils have been developed; among them, phytoremediation is considered one of the most economic and environmental friendly procedures to restore soil fertility (Alkorta et al., 2004). This biotechnical approach relies on the innate capability of plants to uptake and to accumulate metals from the soil, but it requires plants able to tolerate Hg accumulation in their organs and to prevent the general oxidative damage induced by this metal (Cho and Park, 2000; Patra et al., 2004; Ortega-Villasante et al., 2005). The maintenance of the cellular redox homeostasis in cells, where antioxidant enzymes and metabolites ameliorate the accumulation of oxidant Reactive Oxygen Species (ROS), would modulate the final tolerance response to Hg (Rellán-Álvarez et al., 2006; Zhou et al., 2008; Sobrino-Plata et al., 2009). Cross-talk of oxidative stress signaling cascades and endogenous factors, like ethylene, jasmonate, auxin, or abscisic acid, is pivotal for plant acclimation to stress and development (Potters et al., 2007), where antioxidants modulate ROS production (Considine and Foyer, 2014). In particular, ethylene through the family of APETALA 2/Ethylene Response Element Binding Protein (AP2/EREBP) transcription factors is known to mediate in hormone and redox signaling processes in context of abiotic stresses (Dietz et al., 2010). Understanding the mechanisms controlling acclimation to hazardous environmental will help to optimize tolerance to Hg in plant cells, knowledge that has been elusive (Chen and Yang, 2012). However, recent research using improved transcriptomics is now paving the way to identify mechanisms involved in the early responses to Hg, putatively involved in the tolerance to this toxic metal, in particular with regards to redox homeostasis.

### **EARLY OXIDATIVE STRESS INDUCED BY METAL TOXCITY**

In spite of high concentration of Hg in polluted soils, only a modest amount is taken up by plants, a function of the predominant edaphic conditions (Xuexum and Linhai, 1991). Moreover, Hg translocation from roots to shoots occurs normally at low rates, and most toxic effects are found in roots (Boening, 2000). Mercury reduces dramatically the root growth, diminishes the nutrients uptake rates and enhances cell death, and induces an early oxidative burst (Cho and Park, 2000; Patra and Sharma, 2000; Patra et al., 2004; Ortega-Villasante et al., 2005). A strong lipid peroxidation and protein oxidation occurred after shortterm exposure to Hg in maize (Rellán-Álvarez et al., 2006) and pea plants (Cho and Park, 2000), which represent chronic toxic effects with several alterations in cellular functions, such as cross-linking at the cell wall that may led to its stiffening and cell growth inhibition (Cargnelutti et al., 2006). Localization of Hg in plant tissues using X-ray synchrotron imaging showed that this metal enter the plant at the root tip, and accumulates in the vascular bundle, where vascular parenchyma cells showed corrugated morphology probably due to water balance alteration (Carrasco-Gil et al., 2013).

The knowledge about physiological responses of plants exposed to Hg has increased in the last few years as Hg, which has been compared frequently with the phytotoxicity caused by other toxic elements like Cd (Gallego et al., 2012). With regard to the induction of the oxidative burst, several authors observed alterations in antioxidant enzymes activities such as catalase (CAT), ascorbate peroxidase (APX), or superoxide dismutase (SOD) mainly in roots (Rellán-Álvarez et al., 2006; Zhou et al., 2008). Interestingly, Hg-specific responses were found in the activity of glutathione reductase (GR), key enzyme to maintain the redox balance of glutathione (GSH) that is strongly inhibited by Hg, while under Cd or As it is induced (Sobrino-Plata et al., 2009, 2013); enzyme that has been suggested recently as a biomarker of Hg accumulation (Sobrino-Plata et al., 2013). A significant and early induction of ROS, such as superoxide anion (O− <sup>2</sup> ) and hydrogen peroxide (H2O2), has been observed in *Brassica juncea* plants exposed to Hg (Meng et al., 2011). Microscale experiments with alfalfa seedlings showed that the generation of ROS by Hg occurs within minutes (Ortega-Villasante et al., 2007), possibly associated with the induction of plasma membrane NADPHoxidases responsible of the accumulation of H2O2 in the root apoplast (Montero-Palmero et al., 2014). This mechanism of ROS production may differ from that of triggered by Cd, possibly more related with mitochondrial electron transfer chain malfunction (Heyno et al., 2008). The *Respiratory Burst Oxidase Homolog* (Rboh)/NADPH-oxidases in plants has been reported as regulatory mechanisms of biotic and abiotic stress mediating in ROS production. Recently, the characterization of *Arabidopsis* RbohD, RbohF, and RbohC family members has been useful to understand better their role under stress conditions using *atrbohD*/*atrbohF* and *atrbohC* mutant plants, which demonstrated their relevance in the signaling network involved in stress cellular homeostasis (Torres and Dangl, 2005). In this sense, *Arabidopsis atrbohD/atrbohF* mutants and a *35S::AtrbohD* overexpressor suggest the involvement of Rboh/NADPH-oxidases in the generation of H2O2 under Hg stress (Montero-Palmero et al., 2014).

ROS are considered as components of a signal cascade capable of triggering the induction of defense genes to cope with abiotic and biotic stresses. For example, Mittler et al. (2004) reviewed a list of more than 150 genes in *Arabidopsis* that participate in a complex network to regulate ROS levels after an oxidative burst. The identification of common components in the stress responses as key factors of cell homeostasis has been a major research effort recently (Kreps et al., 2002). Among others, the zinc-finger superfamily of transcription factors are one of the best functionally characterized group, which mediates in both biotic and abiotic stresses (Kodaira et al., 2011; Figueiredo et al., 2012). In this sense, the transcription factor Zat12 may canalize the oxidative burst signaling in *Arabidopsis*, as was observed when the tolerance was altered by interfering the expression of genes regulated by Zat12 (Davletova et al., 2005b). This regulatory role was shared with WRKY transcription factors, which are thought to regulate the expression of several stress-related genes, such as those encoding several APXs (Davletova et al., 2005a; Vanderauwera et al., 2005; Miao and Zentgraf, 2007; Chen et al., 2012).

Consistent with some physiological symptoms of Hg stress, changes in the transcription of genes needed for the regeneration of the photosynthetic apparatus and antioxidant enzymes were detected in *Arabidopsis thaliana* and tomato seedlings exposed to Hg (Cho and Park, 2000; Heidenreich et al., 2001). Similarly, there was an up-regulation of genes encoding peroxidases, NADHdehydrogeneases and enzymes of the sulfur assimilatory pathway, as well as genes involved in secondary metabolism in Hgtreated pea plants (i.e., biosynthesis of salicylic acid (SA) and isoflavonoids; Sävenstrand and Strid, 2004). Additionally, heme oxygenases (HOs) may mediate in the Hg-related responses in *Brassica napus* (Shen et al., 2011), which are related with pathogenesis related proteins or small heat shock proteins (SHSPs) (Didierjean et al., 1996; Wollgiehn and Neumann, 1999).

Recent evidences suggest that metal homeostasis depend on a complex crosstalk between different signaling processes, where ROS signals are integrated with phytohormones signaling. Therefore, ROS are considered as important clues for development and ontogeny of plant cells (Mittler et al., 2011). It is possible that hormone and ROS signaling are playing their role at the same level in the stress response (Fujita et al., 2006), but they could also be involved in different steps of signaling cascade. Thus, phytohormones could alter ROS production or, in the contrary, ROS could be promoting the hormone cascade activation (Bartoli et al., 2013). Therefore, more complete studies of massive transcriptional analysis are required to understand the complex levels of responses normally studied using a heuristic incomplete approach, which has been recently undertaken as discussed below.

### **CHARACTERIZATION OF THE MASSIVE TRANSCRIPTIONAL PATTERN UNDER Hg STRESS**

Recent bioinformatics and technological advances based on "omics" research have revitalized the integration at the transcriptional level of many physiological processes in plants (Mochida and Shinozaki, 2011). In this sense, DNA microarrays technology is a powerful tool used widely in the last decades after genome sequencing projects, that are enabling a more complete understanding of the global transcriptional changes under different environmental conditions and effectors, endogenous signals, interaction with pathogens, and so on (Amaratunga et al., 2014). With regard to metal homeostasis, a substantial effort has been done to characterize the primary cellular mechanisms involved in the heavy metal stress perception and defense mechanisms using different RNA-DNA microarray technologies. *Arabidopsis* DNA chips have been used to identify global transcriptional pattern in response to metals such as Zn (Becher et al., 2004), As (Abercrombie et al., 2008) or Cd (Herbette et al., 2006; Weber et al., 2006); where in most cases the transcriptional response of *Arabidopsis thaliana* has been compared with that of the metalliferous *Arabidopsis halleri.* Transcriptional activity of the metal Zn/Cd accumulator *Noccaea* (*Thlaspi) caerulescens* has also been compared with *Arabidopsis* in response to Cd (Van De Mortel et al., 2008). Apart from *Arabidopsis*, sensitive and tolerant cultivars of rice (*Oryza sativa*) have been used to assess their transcriptional response to As (Norton et al., 2008; Chakrabarty et al., 2009; Huang et al., 2012; Yu et al., 2012), Cu (Sudo et al., 2008) using different DNA microarray platforms. In addition, the transcriptional responses of Cd were compared with those of essential trace micronutrients like Cu in *Arabidopsis* (Zhao et al., 2009) or in rice roots (Lin et al., 2013).

With respect to Hg, three very recent concurrent studies were completed to characterize the massive transcriptional profile in seedlings of *Medicago sativa* (Montero-Palmero et al., 2014), *Medicago truncatula* (Zhou et al., 2013), and *Hordeum vulgare* (Lopes et al., 2013). The main purpose of these transcriptional studies was to obtain a comprehensive understanding of the metabolic pathways involved in the Hg-stress response, which would shed light in the tolerance mechanisms involved. Genes encoding enzymes of the plant secondary metabolism, and other known to participate in biotic and abiotic stresses genes were differently expressed in the three transcriptomic studies performed with different plant species. For example, there was a clear up-regulation of genes encoding enzymes of the lignin biosynthesis pathway, such as those producing lignin precursors like coumarins, caffeoyl, and other monolignols (**Table 1**). It is known that lignin polymerization promotes cell wall stiffening (Passardi et al., 2004); lignin cross-linking reactions that may be responsible of the observed rapid root growth inhibition under Hg stress (Ortega-Villasante et al., 2007; Montero-Palmero et al., 2014). In addition, these phenolic metabolites are known antioxidants under metal stress conditions (Van De Mortel et al., 2008), which would counteract the rapid ROS induction by Hg Moreover observed in our experiments (Ortega-Villasante et al., 2007; Montero-Palmero et al., 2014). Other strongly regulated genes, common to all three plant species, fall in several stressrelated categories such as glutathione-S-transferases, heat shock proteins or pathogenesis related proteins (**Table 1**). It is thought that toxic metabolites, protein instability and other alterations in the cellular components may compromise cell survival in plants subjected to different types of environmental stresses (Mittler et al., 2011), indicating that a general unspecific response is also triggered by Hg. Interestingly, several phytohormone signaling pathways seemed to operate under Hg stress: ethylene, ABA and auxin related genes are among those significantly up-regulated after a short-term treatment (**Table 1**), which may be key players in metal perception and homeostasis.



*Differential expressed genes (DEGs) from the microarray of Medicago sativa root-seedlings exposed to 3µM Hg during 3, 6, or 24 h (FDR < 0.01), compared to the transcriptomics analyses made in Medicago truncatula seedlings treated with 10µM HgCl2 during 6, 12, 24, and 48 h (FDR < 0.001; Fold Change over 1) and in Hordeum vulgare root-seedlings exposed to approximately 300µM Hgin sand semi-hydroponics for 15 days (P < 0.05), and classified into the main functional MapMan categories.*

### **INVOLVEMENT OF ETHYLENE IN THE OXIDATIVE BURST INDUCED BY Hg**

Plant cells exposed to toxic metals experience drastic metabolic changes, ranging from primary signaling events, biochemical and metabolic responses to transcriptional activation, as outlined in **Figure 1A**. An important feature of the early responses to toxic metals is the induction of ROS accumulation and oxidative stress (Baier et al., 2005), which occur minutes after the exposure of root epidermal cells to Cd and Hg (Hernández et al., 2012). Metal perception by plant cells is normally accomplished by a stress signaling network that would involve the activation of a Ca-signaling cascade (DalCorso et al., 2010), accumulation of ROS and reactive nitrogen species (RNS; i.e., nitric oxide or NO), or with the accumulation of certain stress-related phytohormones like salicylic, jasmonate and oxylipins (Rodríguez-Serrano et al., 2009; Tamás et al., 2010). Downstream signaling events include changes in the activity of several antioxidant enzymes, such as APX, GR, or SOD, along with modified concentration of antioxidant metabolites, such as ascorbate and GSH (Jozefczak et al., 2012), activation of Ca-dependent calmodulins, and mitogen-activated protein kinases (MAPKs; Jonak et al., 2004; Ye et al., 2013). Ethylene accumulated in *Brassica juncea* leaves when exposed to Ni and Zn, phytohormone that promoted the activation of APX and GR enzymatic activities and augmented the pool of reduced GSH, conceivably required to enhance the antioxidant defensive barriers against metal stress (Khan and Khan, 2014). On the other hand, ethylene mediates in the assimilation process and the nutrition balance of sulfur, a fundamental macronutrient for plant acclimation to stress *via* GSH metabolism (Iqbal et al., 2013). The defenses to metal stress also comprise transcriptional changes, necessarily orchestrated by certain families of transcription factors, in particular some responding to ethylene, ABA, jasmonate, or auxin (i.e., ERF/AP2, WRKY, MYB, and ARF, respectively), that would recognize different *cis*-DNA regulatory motifs to control the transcription of genes involved in metal detoxification and tolerance (Thapa et al., 2012; **Figure 1B**). Expression of different metal transporters (for example the HMA1-4 and CDF families), enzymes of sulfur metabolism and GSH biosynthesis, and SHSPs are among the cellular defenses activated upon the commented transcriptional activation (Gallego et al., 2012; **Figure 1**).

Stress related phytohormones like SA or jamonate (derived from oxylipins) are known effectors that modulate responses to toxic metals (Xiang and Oliver, 1998; Zhou et al., 2009). In fact, several hormone responsive genes and genes involved in hormones synthesis are up-regulated, indicating that phytohormones play an important role in the Hg-induced response (**Table 1**). SA, brassinosteroids, cyotokinins, gibberellins, or IAA have been described stimulating the antioxidant response in terrestrial and aquatic plants exposed to Cd, Cu, or Pb (Hayat et al., 2007; Noriega et al., 2012; Piotrowska-Niczyporuk et al., 2012). On the contrary, jasmonate is known to trigger ROS production under metal stress (Maksymiec and Krupa, 2006), and accumulated in leaves of *Arabidopsis* and *Phaseolous coccineus* under Cu and Cd stress (Maksymiec et al., 2005). Interestingly, jasmonateinduced ROS is mediated by the oxidative status of GSH, as has been shown in GR defective mutants (Mhamdi et al., 2010). Subsequently, the transcriptional activity due to jasmonate has been recently associated with the oxidative burst led by changes in the redox potential of GSH in plant cells (Han et al., 2013). In addition, jasmonate entwines with ethylene in a complex signaling cascade that results in ROS production (Mittler, 2006). The stress response induced by ethylene may be associated with the jasmonate pathway, mediated by the COI1-jasmonate receptor as was shown in the root meristematic activity (Adams and Turner, 2010). This draws a rather complex picture where redox unbalance in the cell is required in turn for cysteine and GSH synthesis, possibly as part of a positive feedback mechanism where jasmonate or ethylene may intervene (Queval et al., 2009).

Ethylene plays also a complex role along with ROS in the defense responses to biotic and abiotic stresses (Mittler et al., 2011). Insensitive plants to ethylene, such as *Arabidopsis ein2- 5*, were unable to promote the oxidative burst after a pathogen elicited response (De Jong et al., 2002), highlighting the contribution of this phytohormone in the oxidative burst that precedes plant immune responses (Mersmann et al., 2010). This hormone could also interfere in the ROS signaling in *Arabidopsis* exposed to Cu and Cd (Arteca and Arteca, 2007) and Al (Sun et al., 2010). Moreover, the Hg-induced release of H2O2 by roots was attenuated when the ethylene perception was blocked in alfalfa and *Arabidopsis* seedlings, implying that ethylene is required by the activation of NADPH-oxidases to generate ROS under metal stress (Montero-Palmero et al., 2014).

Ethylene is also a phytohormone that determines root architecture and controls defense responses of plants to stress (Swarup et al., 2007). The rapid root growth inhibition observed under Hg (Ortega-Villasante et al., 2007) was counteracted when ethylene perception was blocked in alfalfa and *Arabidopsis*, implying a direct relationship between this phytohormone and the root architecture (Montero-Palmero et al., 2014). It is feasible that this role occurs under other metal stress conditions, as it was shown that ethylene synthesis antagonists alleviated the Alinduced arrest of root elongation (Tian et al., 2014). This control is exerted in combination of other hormones like auxins, salicylic acid, jasmonate, ABA, or strigolactones, contributing to a general mechanism of tolerance and resistance to a wide range of biotic and abiotic stresses (Bari and Jones, 2009). For instance, a downstream regulation of auxin-related genes (such as YUCCA, PIN, or ARF), and cell cycle related-genes (CDKs and cyclins), are also interconnected with a H2O2 signaling cascade under Cd stress in rice plants (Zhao et al., 2012) and barley roots (Liptakova et al., 2012). Interestingly, there are clear evidences that auxins accumulation in roots depends partially on ethylene metabolism, which may affect ultimately the architecture of roots by promoting the appearance of secondary roots emergence (Ruzicka et al., 2007; Swarup et al., 2007). Uptake of mineral nutrients and exudation of malate could be also modulated by ethylene and auxin, possibly via a transcriptional regulation, which could be related with root architecture under metal stress (Tian et al., 2014). Similarly, jasmonate mediates in ASA1 expression, a protein involved in auxin synthesis and distribution-related, which also modulates root development (Wasternack and Hause, 2013). All these endogenous factors would interact with oxidative stress promoted signaling, composing a complex transduction network that regulates cell division, expansion, and ultimately root

**FIGURE 1 | (A)** Sequence of events occurring upon metal exposure. Known events that appeared in plants treated with metals, with emphasis in those triggered by Hg (shaded boxes). For more information, refer to the following literature: (1) Lin et al., 2013; (2) Zhou et al., 2007, 2008; (3) Sobrino-Plata et al., 2009; Sobrino-Plata et al., 2014a,b; (4) Lopes et al., 2013; (5) Zhou et al., 2013; (6) Montero-Palmero et al., 2014. **(B)** Cellular responses in plants treated with metals (divalent toxic metal cation; HM2+). Metal cations can interact with cell wall components and then enter the cytoplasm *via* ion Ca2<sup>+</sup> channels or active transporters (HMA). Once inside the cell, ROS production (H2O2 or O<sup>−</sup> <sup>2</sup> ) is induced possibly by NADPH-oxidases, in addition with electron transfer

reactions in the chloroplast and mitochondria. Metals may be chelated with phytochelatins (PC) before are transferred to the vacuole. Antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidases (APX), are activated to maintain the cellular redox homeostasis. At the transcriptional level, the expression of certain stress genes (i.e., heat shock proteins, pathogen related proteins, or cell wall stiffening) is up-regulated, including stress-related transcription factors, antioxidantant proteins, microRNAs, and sulfur metabolism related genes. ROS/phytohormones crosstalk in response to metal stress would modulate the overall transcriptional profile, promoting the expression of the corresponding transcription factors.

architecture (Considine and Foyer, 2014), which would be altered in plants exposed to toxic metals.

### **FUTURE PERSPECTIVES AND CHALLENGES**

Current evidences support the concept that phytohormones are playing a significant role in the perception and response to toxic metals, possibly by interacting with a ROS-dependent signaling pathway. Being said that, the current understanding of the crosstalk between phytohormones and ROS networks is still obscure and very limited. Thus, future work should be directed to describe in detail the genetic network that regulates the perception of the stress induced by metals, and how different phytohormones and signaling components interact using the available collection of mutants with inhibited or blocked receptors, together with current massive transcriptomic profile analyses, and bioinformatic tools to obtain an integrated picture. This will allow the development of biotechnical strategies to enhance tolerance of plants to metal toxicity.

### **ACKNOWLEDGMENTS**

This work was funded by the Ministry of Economy and Competitiveness (Project PROBIOMET AGL2010-15151), Junta Comunidades Castilla-La Mancha (FITOALMA2, POII10-007- 6458), and Comunidad de Madrid (EIADES consortium S2009/ AMB-1478).

### **REFERENCES**


versus plasma membrane NADPH-oxidase. *New Phytol.* 179, 687–699. doi: 10.1111/j.1469-8137.2008.02512.x


appear in the metallophyte *Silene vulgaris* when grown hydroponically. *RSC Adv.* 3, 4736–4744. doi: 10.1039/c3ra40357b


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 May 2014; accepted: 16 July 2014; published online: 08 August 2014. Citation: Montero-Palmero MB, Ortega-Villasante C, Escobar C and Hernández LE (2014) Are plant endogenous factors like ethylene modulators of the early oxidative stress induced by mercury? Front. Environ. Sci. 2:34. doi: 10.3389/fenvs.2014.00034 This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2014 Montero-Palmero, Ortega-Villasante, Escobar and Hernández. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Monoterpenoid indole alkaloids and phenols are required antioxidants in glutathione depleted *Uncaria tomentosa* root cultures

Ileana Vera-Reyes <sup>1</sup> , Ariana A. Huerta-Heredia<sup>1</sup> , Teresa Ponce-Noyola<sup>1</sup> , Carlos M. Cerda-García-Rojas <sup>2</sup> , Gabriela Trejo-Tapia<sup>3</sup> and Ana C. Ramos-Valdivia<sup>1</sup> \*

<sup>1</sup> Departamento de Biotecnología y Biongeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, D. F., Mexico, <sup>2</sup> Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, D. F., Mexico, <sup>3</sup> Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos,

#### *Edited by:*

Instituto Politécnico Nacional, Yautepec, Mexico

Rene Kizek, Central European Institute of Technology in Brno, Czech Republic

#### *Reviewed by:*

Naser A. Anjum, University of Aveiro, Portugal Zbynek Heger, Central European Institute of Technology in Brno, Czech Republic Ondrej Zitka, Mendel University in Brno, Czech Republic

#### *\*Correspondence:*

Ana C. Ramos-Valdivia, Departamento de Biotecnología y Biongeniería, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional, A. P. 14-740, México, D. F. 07000, Mexico aramos@cinvestav.mx

#### *Specialty section:*

This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science

> *Received:* 06 January 2015 *Accepted:* 20 March 2015 *Published:* 09 April 2015

#### *Citation:*

Vera-Reyes I, Huerta-Heredia AA, Ponce-Noyola T, Cerda-García-Rojas CM, Trejo-Tapia G and Ramos-Valdivia AC (2015) Monoterpenoid indole alkaloids and phenols are required antioxidants in glutathione depleted Uncaria tomentosa root cultures. Front. Environ. Sci. 3:27. doi: 10.3389/fenvs.2015.00027 Plants cells sense their environment through oxidative signaling responses and make appropriate adjustments to gene expression, physiology and metabolic defense. Root cultures of Uncaria tomentosa, a native plant of the Amazon rainforest, were exposed to stressful conditions by combined addition of the glutathione inhibitor, buthionine sulfoximine (0.8 mM) and 0.2 mM jasmonic acid. This procedure induced a synchronized two-fold increase of hydrogen peroxide and guaiacol peroxidases, while the glutathione content and glutathione reductase activity were reduced. Likewise, in elicited cultures, production of the antioxidant secondary metabolites, monoterpenoid oxindole, and glucoindole alkaloids, were 2.1 and 5.5-fold stimulated (704.0 ± 14.9 and 845.5 ± 13.0µg/g DW, respectively) after 12 h, while phenols were three times increased. Upon elicitation, the activities and mRNA transcript levels of two enzymes involved in the alkaloid biosynthesis, strictosidine synthase and strictosidine β-glucosidase, were also enhanced. Differential proteome analysis performed by two-dimensional polyacrylamide gel electrophoresis of elicited and control root cultures showed that after elicitation several new protein spots appeared. Two of them were identified as thiol-related enzymes, namely cysteine synthase and methionine synthase. Proteins associated with antioxidant and stress responses, including two strictosidine synthase isoforms, were identified as well, together with others as caffeic acid O-methyltransferase. Our results propose that in U. tomentosa roots a signaling network involving hydrogen peroxide and jasmonate derivatives coordinately regulates the antioxidant response and secondary metabolic defense via transcriptional and protein activation.

Keywords: oxidative stress, *Uncaria tomentosa*, proteome, antioxidant responses, glutathione

### Introduction

Oxidative stress arises from disruption in redox balance due that the amount of reactive oxygen species (ROS) exceeds the ability of the cell to accomplish an effective antioxidant response. Unlike other ROS, hydrogen peroxide (H2O2) is a non-radical species, containing no net charge, with a relatively long half-life. Because of these properties, H2O<sup>2</sup> acts as a long-distance signaling molecule and is a physiological indicator of the intensity of biotic and/or abiotic stress (Apel and Hirt, 2004). In turn, to prevent the harmful effects of ROS, plants have evolved coordinate antioxidant mechanisms that include superoxide dismutase, peroxidases, the ascorbate-glutathione cycle, and other antioxidant responses (Noctor and Foyer, 1998).

Glutathione is a low molecular weight tripeptide useful in protecting plant cells from oxidative injury due to its redox buffering capacity and relative abundance. In response to environmental stress through the ascorbate–glutathione pathway, the redox potential of the reduced glutathione (GSH) pool is altered and converted to the disulfide form (GSSG) without net consumption (Meyer and Fricker, 2002). It has been reported that H2O2, produced in response against various stimuli, would be acting as a signaling molecule, regulating the expression of selected genes, including those involved in the defense pathways and participating in the crosstalk between other metabolic signals (Quan et al., 2008). Several studies suggest that, as the result of adaptation responses of plants to oxidative stress, changes occur not only in the primary defense mechanisms but also in the profile of secondary metabolism (Apel and Hirt, 2004).

Alkaloids represent one of the most active natural product groups against a wide range of organisms. The main role of these substances is generally linked to plant defense mechanisms from predators, besides the important ecological factors associated to them. However, the close relationship between alkaloids and the oxido-reduction processes in plants containing them strongly suggests that these compounds play a fundamental role in protecting plants when they are subjected to oxidative stress (Ramos-Valdivia et al., 2012). Furthermore, polyphenols are the most abundant and widely distributed group of naturally occurring compounds. Their functions are critical to the maintenance of the plant, being relevant in the defense against herbivores, for protection to different types of biotic or abiotic stress, as well as signals in interactions either with other plants or with microbes (Buer et al., 2010).

GSH deficit may occur in plants as a consequence of increased cellular consumption and/or due to biosynthetic disorders. However, GSH depletion of GSH can occur by addition of L-buthionine-(S,R)-sulphoximine (BSO). This nontoxic substance is a specific inhibitor of γ-glutamylcysteine synthetase (Ruegsegger et al., 1990; May and Leaver, 1993). Treatment of plant tissue with BSO has been used as an elicitor of secondary metabolites since this substance weakens the antioxidant defense mechanisms, provoking endogenous accumulation of H2O<sup>2</sup> and oxidative stress (Berglund and Ohlsson, 1993; Guo et al., 1993; Vera-Reyes et al., 2013).

Uncaria tomentosa, which belongs to the Rubiaceae family, is an Amazon rainforest species known as cat's claw. This plant produces the highly oxidized monoterpenoid oxindole alkaloids (MOA) isopteropodine, mitraphylline, isomitraphylline and rhynchophylline, which exhibits immunomodulatory, anti-AIDS, cytotoxic, and antileukemic properties (Laus, 2004). In previous work, it was found that root suspension cultures of this species produced MOA and accumulated 3α-dihydrocadambine (Huerta-Heredia et al., 2009), a glucoindole alkaloid with hypotensive and antioxidant activities (Endo et al., 1983) and dolichantoside (Luna-Palencia et al., 2013), a N-β-methylated strictosidine with potent anti-malarial effect (Frédérich et al., 2000). Moreover, the antioxidant response and alkaloid production stimulation have been correlated with oxidative stress (Trejo-Tapia et al., 2007) triggered by H2O<sup>2</sup> treatment (Huerta-Heredia et al., 2009; Vera-Reyes et al., 2013) and by combined addition of the glutathione inhibitor, buthionine sulfoximine and jasmonic acid (Vera-Reyes et al., 2013). It has been suggested that monoterpenoid indole alkaloids (MIA) are precursors of MOA whose transformation may take place through oxidation of the indole ring system. The central precursor of the MIA pathway is the glycosylated indole alkaloid strictosidine, which is formed through the condensation of the indole precursor tryptamine with secologanin catalyzed by the enzyme strictosidine synthase (STR; EC 4.3.3.2). Then, strictosidine β-D-glucosidase (SGD; EC 3.2.1.105) hydrolyzes the glucose moiety present in strictosidine forming an aglycone, which is rapidly converted to a dialdehyde intermediate. In some plants such as Catharanthus roseus, this substance is reduced by NADPH to ajmalicine or their isomers through cathenamine (Kutchan, 1995). Strictosidine also participates in the biosynthesis of other glucoindole alkaloids characteristic of the Rubiaceae family such as isodihydrocadambine (Szabó, 2008).

Both STR and SGD are encoded by single genes (McKnight et al., 1990), even though the STR from C. roseus has shown several isoforms due to post-translational modifications (De Waal et al., 1995; Jacobs et al., 2005). Vera-Reyes et al. (2013) reported that in U. tomentosa root cultures, the increase of oxindole and glucoindole alkaloids observed under oxidative stress, is provoked by the regulatory mechanisms at the level of enzyme activities and gene expression of STR and SGD. Thus, proteomics provides a promising approach for the study of the protein response to oxidative stress in general and its relation with the secondary metabolism production (Ramos-Valdivia et al., 2012). Particularly, comparative proteomic studies based on contrasting plant cultures on stressed and non-stressed conditions are essential for understanding the stress-related defense mechanisms.

In order the study the regulatory mechanisms functioning in the monoterpenoid indole alkaloid production in U. tomentosa root cultures, activities and mRNA transcript levels of two enzymes involved its alkaloid biosynthesis, antioxidant defense and comparative proteome analysis in response to oxidative stress were examined.

### Materials and Methods

### Root Cultures and Elicitation

Root cultures of U. tomentosa (line Utr-3) arising from micropropagated plantlets (Luna-Palencia et al., 2013) were grown in 250-mL Erlenmeyer flasks (covered with aluminum foil) with 100-mL of MS medium (Murashige and Skoog, 1962), 2% sucrose without plant growth regulators and pH 6.4 adjusted prior to sterilization. The cultures were incubated at 25 ± 2 ◦C, using orbital agitation at 110 rpm, and under continuous light intensity 13µmol m−<sup>2</sup> s −1 . The cultures were sub-cultivated every 20 days and uniform inocula for the experiments were developed in 1000 mL Erlenmeyer flasks containing 400-mL of culture medium. A selection of 20-days-old roots were cut in pieces of ∼5 cm length and kept in deionized water until they were inoculated (2 g FW) into 250-mL shaken flasks containing 100-mL culture medium. Roots were elicited at day 13 with simultaneous addition of 0.8 mM BSO and 0.2 mM jasmonic acid (BSO-JA) and were incubated as indicated above. Three control cultures and three elicited flask cultures were harvested after 12 h.

### Extraction of Total Proteins for 2D SDS-PAGE

Ten grams of frozen roots were ground using a mortar and a pestle and were cooled with liquid N2. A solution (20 mL) of cold (−20◦C) 10% TCA in acetone with 0.07% β-mercaptoethanol was poured over the sample (Jacobs et al., 2005). The mixture was kept at 20◦C overnight to enable a complete precipitation. After centrifugation for 15 min at 3000 g, samples were washed twice with a cold solution (−20◦C) of acetone and 0.07% βmercaptoethanol for removing TCA. The precipitate was solubilized in ReadyPrep rehydration/sample buffer BioRad [8 M urea, 2% CHAPS, 50 mM dithiothreitol (DTT), 0.2% (w/v) Bio-Lyte <sup>R</sup> 3/10 ampholytes, and bromophenol blue (trace)] completed with 2 M thiourea. The mixture was vortexed and centrifuged (5 min, 16,000 g) several times during 1 h. The supernatant was recovered and cleaned up using a Micro Bio-Spin <sup>R</sup> column (BioRad, USA) and stored at −80◦C. The concentration of protein was measured with a 2D Quant kit (Amersham Biosciences, USA).

### 2D-PAGE

About 250µg of protein was loaded into 11-cm strips with a pH gradient between 4 and 7 (IPG, immobilized pH gradient, Bio-Rad) by in-gel rehydration during 12 h. Isoelectric focusing (IEF) was carried out on a Protean IEF apparatus (Bio-Rad, USA) at 20◦C by application of a voltage gradient from 0 to 250 V for 1 h, 250 to 500 V for 1 h, 1000 to 8000 V for 1 h, from 8000 to 20,000 V for 2 h, and 500 V for 2 h. The protein IPG strips were equilibrated before applying a sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) procedure using an equilibration buffer I (50 mM Tris HCl [pH 8], 8 M urea, 30% glycerol, 2% SDS, and 0.3% DTT) for 10 min. The strips were then soaked for 10 min in the equilibration buffer II containing 50 mM Tris HCl (pH 8), 8 M urea, 30% glycerol, 2% SDS, and 4.5% iodoacetamide. SDS-PAGE was done using polyacrylamide 12% acrylamide gels. Electrophoresis was carried out at 25 mA for 45 min and 35 mA for 2.5 h (SE 600 Ruby™; GE Healthcare Life Science, USA). Protein samples were visualized by staining with Sypro Ruby (BioRad, USA).

### Gel Analysis

At least three independent 2-D experiments were repeated at minimum four times to confirm reproducibility. Image analysis was achieved by visual inspection and the observed changes were qualitative using Melanie 7.0 gel analysis platform (GE Healthcare). The volume of each spot was normalized as a relative volume to compensate for the variability in gel staining. Manual editing was carried out after the automated detection and matching for each spot, achieving this procedure with a minimum of four gels for each sample. Only those spots that showed significant and reproducible changes (at least 1.3-fold) were taken in to account as differentially expressed proteins, ANOVA (p < 0.05). The Scaffold program (version 4.0.6.1 from Proteome Software Inc., Portland, OR) was employed for protein identification. The validation was done if the probability was greater than 99.0% and contained at least 2 identified peptides. Protein probabilities were allocated by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that could not be differentiated based on MS/MS analysis were grouped to satisfy the principles of parsimony. The estimated experimental Mr/pI was useful to rise the identification confidence.

### In-Gel Digestion, MALDI-TOF MS and Database Search

Excised SYPRO <sup>R</sup> Ruby (BioRad)-stained protein gel spots following 2D SDS-PAGE were digested with trypsin (10µg/mL) at 37◦C for 12 h. Tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS) analysis of in-gel trypsin digestedproteins (Shevchenko et al., 1996) was performed in a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) furnished with an Advion nanomate ESI source (Advion, Ithaca, NY). ZipTip (Millipore, Billerica, MA) C18 sample cleanup was achieved as indicated in the manufacturer's instructions. The peptide fraction was eluted from a C18 precolumn of 100 µm id × 2 cm (Thermo Fisher Scientific) and loaded onto an analytical C18 column of 75-µm ID × 10 cm C18 (Thermo Fisher Scientific) eluting with solvent A (water and 0.1% formic acid) and a 5–10% gradient of solvent B (acetonitrile, 0.1% formic acid) for 5 min, followed by a 10–35% gradient of solvent B for 35 min, 35–50% gradient of solvent B for 20 min, 50–95% gradient of solvent B for 5 min, and 95% solvent B for 5 min, all elutions were run at a flow rate of 400 nl/min. Data dependent scanning (m/z 400–1600) was carried out in the Orbitrap analyzer, followed by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) of the 14 most intense ions in the linear ion trap analyzer using the Xcalibur v 2.1.0 software (Andon et al., 2002) and a mass scan of 60,000 resolution. The precursor ions were chosen by the monoisotopic precursor selection (MIPS) setting the acceptance or rejection of ions thought a ±10 ppm window. Dynamic exclusion was established to place any selected m/z peak on an exclusion list for 45 s after a single MS/MS. All MS/MS spectra were explored against asterids proteins downloaded from Uniprot or from NCBI on October 09, 2012 or June 20, 2013, respectively, using Thermo Proteome Discoverer 1.3 (Thermo Fisher Scientific). The UniprotKB protein database of all species was also used in searching the data independently. Variable modifications considered during the search included methionine oxidation, adding 15.995 Da, and/or cysteine carbamidomethylation, adding 57.021 Da. At the time of the search, asterids database from Uniprot or NCBI contained 65,406 and 102,843 entries, respectively (UniprotKB database contained 452,768 entries as of October 10, 2012). Proteins were identified with a confidence level of 99% with XCorr score cut-offs (Qian et al., 2005) as determined by a reversed database search. The results were displayed with the Scaffold program v 3.6.1 (Proteome Software Inc., Portland OR) that depends on various search engines (Sequest, X!Tandem, MASCOT) using Bayesian statistics (Keller et al., 2002; Nesvizhskii et al., 2003).

### Quantification of Phenolic Compounds

Powdered roots (0.20 g) were frozen in liquid N2, pulverized and sonically extracted with 5 mL of methanol-water (8:2 v/v) and centrifuged. A supernatant aliquot of 0.2 mL was mixed with 0.2-mL Folin-Ciocalteu reagent diluted 1:1 (v/v) with water, 0.6 mL of sodium carbonate (Na2CO3) saturated solution and 4 mL of deionized water. The mixture was intensively shaken, left at room temperature for 25 min, and centrifuged at 5000 rpm for 10 min. The absorbance of supernatant was registered at 725 nm in a Genesys 10V spectrophotometer (Thermo Scientific). Total phenols were expressed in terms of D-catechin equivalents. Quantification of individual phenols was done by HPLC analysis according (Pavei et al., 2010) using a 3-caffeoylquinic acid (chlorogenic acid) calibration curve.

### Extraction and Quantification of Alkaloids

Alkaloid extraction and quantification were performed as described previously (Vera-Reyes et al., 2013). Briefly, frozen roots (liquid N2) were pulverized and sonically extracted with 5% hydrochloric acid. Alkaloids from the acid-solutions or culture media were extracted twice with chloroform adjusting the pH to 8-9 using a NH4OH solution. The organic layer was vacuum evaporated and the solid residue was dissolved in a 9:11 mixture of acetonitrile and 10 mM phosphate buffer at pH 7. The solutions were filtered and injected into a Varian ProStar 333 HPLC system equipped with a photodiode array detector (Varian, Walnut Creek, CA) using a reverse-phase C18 column (Waters Spherisorb 5 mm ODS2 of 250 mm length 4.6 mm i.d.). Elution was carried out with the same 9:11 mixture of acetonitrile and phosphate buffer at 0.7 mL/min flow rate and detecting at 244 nm. For quantification of MOA and glucoindole alkaloids, mitraphylline and 3α-dihydrocadambine respectively, were used as the standard compound to determine the calibration curve.

### Statistical Analysis

All measurements were done in triplicate and the statistical evaluation was achieved with Anova, taking p ≤ 0.05 as significant.

### Protein Extracts and Enzyme Assays

Roots (1 g) were homogenized in a pre-chilled mortar under liquid N<sup>2</sup> with 1–2% (w/w) polyvinylpyrrolidone. Extraction buffer (0.1 M potassium phosphate pH 6.3, containing 3 mM EDTA and 6 mM DTT) was added in a 1:1 ratio (v/w) shaking to obtain a homogeneous mixture. For GR assay, the extraction buffer was 0.1 M potassium phosphate pH 7.5, with 1 mM EDTA. Centrifugation at 18,000 g was done for 10 min at 4◦C and the supernatant was collected and desalted on Bio-Rad Micro Bio-Spin <sup>R</sup> P-30 columns. The eluted samples were employed for the enzymatic assays.

The protein fractions were kept frozen at −20◦C until use. The total protein content was determined following the procedure described by Peterson (1977) with bovine serum albumin as the standard.

### Antioxidant Enzyme Assays

Guaiacol peroxidases were measured as oxidation of guaiacol (8.26 mM, ∈ = 26.6 mM−<sup>1</sup> cm−<sup>1</sup> ) according to Pütter (1974). Enzyme extract was incubated in 100 mM phosphate buffer pH 6.0 containing 3 mM H2O2. The reaction was started by addition of 15 mM guaiacol and the absorption was measured for 2 min at 470 nm using a Beckmann spectrophotometer (DU 7500, Munich). Rates were corrected by chemical control experiments. Peroxide activity was determined as the amount of protein that produces 1µmol of oxidized guaiacol. The activity of glutathione reductase was measured using the Glutathione Reductase Assay Kit (Sigma-Aldrich, St. Louis, USA), which was determined by the absorbance decrease caused by NADPH oxidation at 340 nm. One enzyme unit (U) catalyzes the oxidation of 1 µmol of NADPH per min at 25◦C.

### Strictosidine-Related Enzyme Assays

The assay of strictosidine synthase (STR) activity depends on the enzymatic condensation of secologanin and tryptamine to produce strictosidine. Strictosidine formation was quantified by HPLC using a strictosidine standard (Phytoconsult, The Netherlands) for constructing the calibration curve. Strictosidine glucosidase (SGD) activity was determined by measuring the glucose release using Amplex Red <sup>R</sup> (Invitrogen) assay kit. Both enzyme assays were previously described (Vera-Reyes et al., 2013).

### mRNA Extraction, cDNA Synthesis and Semiquantitative RT-PCR Analysis

RNA isolation, DNA treatment, reverse transcription, and semiquantitative-PCR amplification were achieves as reported previously (Vera-Reyes et al., 2013), as well as the primers used for the genes: STR (strictosidine synthase), SGD (strictosidine glucosidase and the control 18S rRNA. The relative gene expression was analyzed using a Kodak Image Station 2200R, DU <sup>R</sup> 730 equipped with Molecular Imaging Software version 1.4 (Kodak) on a 1.2% agarose gel. The gene expression analysis is represented in arbitrary units employing average values of semi-quantitative RT-PCR assays in triplicate with respect to the corresponding non-treated cultures.

### Determination of H2O<sup>2</sup>

Roots (500 mg) were frozen and pulverized under liquid N2. The powder was extracted with 5 mL of 0.1% TCA (w/v), mixed with ice for 5 min, and pelleted by centrifugation at 10,000 g at 4◦C for 10 min. The supernatant was neutralized with 0.2 M NH4OH to pH 8.0 and was centrifuged at 3000 g for 2 min to sediment the insoluble material. The quantification of H2O<sup>2</sup> in the extracts was done with the Amplex Red Hydrogen Peroxide Assay kit (Molecular Probes, Invitrogen), according to the manufacturer instructions. A total of 50µL of extract was combined with an equal volume of 50 mM sodium phosphate buffer pH 7.4 containing 0.1 U/mL of horseradish peroxidase and incubated for 1 h at room temperature, measuring the absorbance at 560 nm. The H2O<sup>2</sup> concentration for each sample was determined with a standard curve obtained with known concentrations of H2O2.

### Glutathione Assay

The levels of total glutathione (GSH + GSSG) were determined with a glutathione assay kit (Sigma) following the manufacturer's protocol. Roots were frozen in liquid N<sup>2</sup> and pulverized until obtaining fine particles. A solution of 5% 5-sulfosalicylic acid (500µL) was added to 0.1 g of the powder to deproteinize the sample. Glutathione was measured in a kinetic assay based on the reduction of 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) to yellow TNB, which was spectrophotometrically measured at 412 nm. The amount of total glutathione was determined with a standard curve of reduced glutathione.

### Results

### Hydrogen Peroxide and Antioxidant Response to BSO-JA Elicitation

U. tomentosa roots induce their antioxidant defense to scavenge excess of ROS in response to combined addition of BSO-JA. After 12 h of elicitation, a two-fold increase of H2O<sup>2</sup> concentration (from 0.48 ± 0.05 to 0.96 ± 0.03µmol/g FW) and POD activity (from 243.9 ± 15.4 to 370.8 ± 8.9µM/mg.min protein) were found (**Figures 1A,C**). In these elicited cultures, glutathione concentration was significantly reduced in a 55%, while the GR activity was slightly lower (17%) than non-treated roots (**Figures 1B,D**). Noteworthy, biomass concentration (6.33 ± 0.20 g DW/L) and viability of roots after the elicitation remained essentially the same as in controls.

### Activities of Strictosidine-Related Enzymes, mRNA Expression Levels, and Production of Phenols and Alkaloids in Response to BSO-JA Elicitation

After 12 h of elicitor treatment, MOA, 3α-dihydrocadambine and dolichantoside production (**Figure 2A**) were rapidly increased by 2.1-, 5.5-, and 2.6-fold, respectively, compared with control cultures (329.7 ± 39.8µg/g DW; 152.4 ± 27.9µg/g DW; 14.0 ± 1.8µg/g DW). Concurrently, BSO-JA treatment increased STR activity by three times in relation to untreated roots (38.7 ± 4.0 pKat/mg protein), while SGD activity had 4.2 times more activity than the control (65.8 ± 2.9 pKat/mg protein) (**Figure 2B**). Upon elicitation, STR and SGD transcripts increased during the first 12 h after treatment reaching 5.8- and 9.7-fold higher, respectively, compared to the control levels (**Figures 2C,D**).

TABLE 1 | Polyphenols accumulation in *Uncaria tomentosa* root cultures growing in Erlenmeyer flasks 12 h after BSO-JA elicitor addition.


\*Values are the mean of three replicates ± standard error of means.

In correlation with the alkaloid induction after BSO-JA addition, total polyphenol content in U. tomentosa root cultures increased from 3.40 ± 0.12 mg/g to 11.45 ± 0.02 mg/g DW. In these elicited roots, the content of 3-caffeoylquinic acid, caffeic acid, catechin, and epicatechin were increased by 210.5, 50.8, 21.7, and 58.8%, respectively (**Table 1**).

### Detection of Differentially Expressed Proteins after BSO-JA Elicitation

One of the key approaches of proteomic analysis is to identify differential protein expression between control and experimental samples. Hence, four replicate gels of U. tomentosa protein extracts from 12 h after BSO-JA addition were compared with the

same number of replicates from non-treated root cultures. Although the gels showed the same profile, the control gels exhibited more proteins than the elicited ones. The control gel with higher protein spots (480) was used for the analysis as standard reference gel. An 87% of the protein spots on the other three gels from untreated roots coincided with those found in the reference gel, whereas those from elicited extracts were 85% coincident. The new proteins that appeared after elicitation and those proteins from the region pI 5–6 and 30–35 kDa (**Figure 3**) that, as previously reported correspond to alkaloid biosynthesis enzymes (Jacobs, 2005), were selected for sequenciation. The 14 identified proteins (**Table 2** and Supplementary Table 1) can be classified into several functional categories, including energy metabolism and photosynthesis: two triosephosphate isomerases (chloroplastic and cytoplasmic; spots 1, 2, and 8), as the same protein in multiple spots differing in pI and M<sup>r</sup> , and ribulose 1,5-bisphosphate carboxylase (Rubisco) large chain (spot 4). Protein synthesis: some proteins involved in the sulpur amino acid biosynthesis such as cysteine synthase (spot 11) and methionine synthase (spot 10) were up-regulated in BSO-JA conditions. Secondary metabolism: oxidative stress increased the expression of protein spots 7 and 9, identified as strictosidine synthase (STR) isoforms, and spot 12 identified as caffeic acid O-methyltransferase. ROS

scavenging, defense and stress: abundance of defense-related proteins as ascorbate peroxidase (spot 3), proteasome alpha subunit (spot 6), universal stress protein (spot 13), and pathogenesisrelated protein (spot 14) were altered during oxidative stress condition.

### Discussion

### Induction of Hydrogen Peroxide and Antioxidant Responses by BSO-JA Elicitation

The high increase in H2O<sup>2</sup> combined with reduction of glutathione concentration in U. tomentosa roots 12 h after addition of BSO-JA may reflect that oxidative stress conditions were present. The plant cell protection of reduced glutathione (GSH) against the oxidative injury is established by its redox buffering activity and abundance. Therefore, treatment of plant cell or tissue with the glutathione biosynthesis inhibitor BSO can lead to weakened antioxidative defenses; an increase in the concentration of endogenous H2O2; and secondary metabolites stimulation (Berglund and Ohlsson, 1993; Guo and Ohta, 1993; Guo et al., 1993). Furthermore, H2O<sup>2</sup> is a secondary messenger that mediates hormonal responses, biotic/abiotic environmental stresses, and developmental signals (Neill et al., 2002). Thus, the jasmonate signaling is mediated by H2O<sup>2</sup> (Orozco-Cárdenas et al., 2001), and is controlled via a suitable antioxidant response to neutralize its adverse effects. The increase of peroxidase activity in elicited cell and plant cultures has been found as a primary response to oxidative stress (Quan et al., 2008), whereas glutathione reductase plays a key role in the antioxidant defense processes by reducing oxidized glutathione (GSSG) to glutathione (GSH), thus allowing the maintenance of a high GSH/GSSG ratio (Foyer and Noctor, 2005). Roots grown in the presence of BSO would be unprotected by the glutathione diminution. Nevertheless, the non-induction of GR activity at 12 h after elicitation could be caused by the increase in polyphenol content as previously reported (Zhang et al., 1997).

Under the assayed conditions, BSO-JA addition did not affect the biomass concentration and root viability, probably because jasmonic acid would be inducing the biosynthesis and activity of other defense responses (Sasaki-Sekimoto et al., 2005) offsetting the antioxidant diminishing caused by BSO.

### Relations among Activities of Strictosidine-Related Enzymes, mRNA Expression Levels, and Production of Phenols and Alkaloids in BSO-JA Elicited Roots

In cell or plant cultures, a synergistic effect of elicitors on secondary metabolites production may occur (Zhao et al., 2005). It has been reported that BSO induces oxidative stress by depletion of glutathione (Noctor and Foyer, 1998), JA can induce ROS production, and JA signaling is important for oxidative stress tolerance (Sasaki-Sekimoto et al., 2005; Pauwels et al., 2008). Separate application of JA or BSO in U. tomentosa roots also elicited the production of alkaloids but in smaller quantities (Vera-Reyes et al., 2013). An increase in secondary metabolite production was also obtained in carrot cells when BSO was used alone or in combination with a yeast glucan elicitor, stimulating an increase in the H2O<sup>2</sup> at cellular level (Guo and Ohta, 1993). In U. tomentosa cell cultures growing in bioreactors, a positive correlation among the increment of endogenous H2O<sup>2</sup> level, activities of NAD(P)H oxidase and peroxidases, and MOA production was reported (Trejo-Tapia et al., 2007). Moreover, H2O<sup>2</sup> treatment induced oxidative stress and alkaloid production in U. tomentosa roots (Huerta-Heredia et al., 2009; Vera-Reyes et al., 2013).

In C. roseus, STR and strictosidine are confined inside the vacuole (McKnight et al., 1990) separated from the activity of the nuclear localized SGD (Guirimand et al., 2010). In U. tomentosa root cultures, a probable cell compartmentalization for alkaloids has been suggested (Vera-Reyes et al., 2013) as MOA were mainly found in the culture medium, while the glucoindole alkaloids 3αdihydrocadambine and dolichantoside were always found inside the roots. Furthermore, alkaloid biosynthesis includes multiple oxidations catalyzed in a stereo- and regiospecific fashion, indicating that specific oxidases are involved in the in vivo biosynthesis. It has been found that peroxidases, microsomal cytochrome P-450-dependent enzymes, 2-oxoglutarate dependent dioxygenases and flavoproteins catalyze some of these oxidations with high substrate specificity enzymes (Kutchan, 1995). However, in vitro studies have revealed the ability of plant peroxidases to accept alkaloids as substrates as well as a number of vacuolar metabolites, such as phenols and flavonoids (Sottomayor et al., 2004; Takahama, 2004). In response to the BSO-JA elicitation, polyphenols production in U. tomentosa root cultures, mainly 3-O-caffeoylquinic acid and catechins, was highly stimulated due to the prevailing oxidative stress. Therefore, polyphenols, as flavonols and phenylpropanoids present in vacuoles and the apoplast, can metabolize H2O<sup>2</sup> as an electron donor for phenol peroxidases. This change results in the formation of the respective phenoxyl radicals, which can be regenerated by a nonenzymatic reaction with ascorbate (**Figure 4**). Thus, in C. roseus it


TABLE 2 | Protein identification through MALDI-TOF from *Uncaria tomentosa* root cultures under BSO-JA treatment.

has been suggested that vacuolar alkaloids, peroxidases, and phenolic derivatives can function as a hydrogen peroxide scavenging system (Ferreres et al., 2011).

### Differentially Expressed Proteins after BSO-JA Elicitation

Identification of proteins that differ in stressed and control plants has revealed groups of proteins that respond to oxidative stress conditions with different roles. Nevertheless, the crucial limitation for protein identification using mass spectrometry analysis is the lack of the sequence data of genes and proteins of U. tomentosa. The SWISS-PROT database (November 2014) only contains five protein entries for this species. Consequently, identification of proteins from 2D-gels requires the knowledge of the sequence data and not relying solely on peptide masses. Several studies reported that oxidative stress provoked different responses such as induction or more often repression of the enzymes involved in carbon metabolism. Therefore, plants must be required to make an economical use of their metabolites and energy to deal with adverse environments (Zhang et al., 2012).

It has also been reported that under conditions of oxidative stress, Rubisco was differentially regulated even though its activity decreased having transcriptional and translational repression thereof caused by jasmonates (Weidhase et al., 1987). Moreover, JA stimulates the glutathione, ascorbate and cysteine accumulation while increases dehydroascorbate reductase activity. This last is a relevant enzyme involved in the ascorbate recycling system (Sasaki-Sekimoto et al., 2005). Cysteine synthase is a key enzyme in cysteine biosynthesis, which constitutes one of the significant factors limiting GSH biosynthesis in plants (Vierling, 1991).

Proteolysis-related proteins like proteasome alpha subunit were also more abundant in stressed conditions because they are necessary for degradation of damaged proteins and for maintaining cellular protein homeostasis (Kurepa et al., 2009). Evidence obtained in U. tomentosa BSO-JA elicited cultures indicates posttranslational modifications of STR proteins in correlation with the three times increase in the STR enzyme activity. Six isoforms of the glycosylated enzyme STR have been detected in C. roseus (De Waal et al., 1995), while in these cell cultures five STR isoforms were induced after elicitation with P. aphanidermun (Jacobs et al., 2005). It is known that jasmonic acid acts as a signal for the biosynthesis of MIA, and is involved in the activation of transcription factors such as ORCA, which have shown to activate transcription of the STR (Peebles et al., 2009). Another interesting protein identified as up-accumulated in the present study was caffeic acid-O-methyl transferase, one of the key enzymes that catalyzes O-methylation of the hydroxyl group at C5 in phenolic rings (Tu et al., 2010). In general, most methyltransferases possess a broad substrate permissiveness, which also includes several alkaloid N-methyltransferases (Zubieta et al., 2003; Nomura and Kutchan, 2010).

The ascorbate peroxidase, which constitutes one of the most important antioxidant systems for removal of H2O<sup>2</sup> generated in the cell, was also up-expressed by BSO-JA addition. Deficiency of cytosolic ascorbate peroxidase occasioned accumulation of H2O<sup>2</sup> and consequently damage in specific proteins of leaf cells (Davletova et al., 2005).

In U. tomentosa root cultures, BSO-JA elicitation induced intracellular JA and H2O<sup>2</sup> accumulation by glutathione depletion (**Figure 4**). They act as a signal transducers and secondary messengers, triggering signaling cascades and activating certain late genes that regulate the activity of detoxifying enzymes associated with antioxidant compounds. Therefore, production of alkaloids and specific phenylpropanoids is also activated,

protecting roots from oxidative stress damage. Identification of proteins with diverse roles that are present in oxidative stress conditions evidences the complexity of the responses. This approach contributes to the understanding of the metabolic mechanisms operating in U. tomentosa subjected to oxidative stress and the manner how this plant produces the appropriate adjustments for tolerating them.

### Acknowledgments

This research was financed by CINVESTAV-IPN and CONACYT-Mexico (222097). IV thank CONACYT-Mexico for a doctoral (173034) fellowship. Authors wish to thank C. Fontaine for technical support. We appreciate the proteomics analytical support of Dr. George Tsaprailis (Arizona Proteomics Consortium). Mass spectrometry and proteomics data were acquired by the Arizona Proteomics Consortium supported by NIEHS grant ES06694 to the SWEHSC, NIH/NCI grant CA023074 to the UA Cancer Center and by the BIO5 Institute of the University of Arizona.

### Supplementary Material

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fenvs. 2015.00027/abstract

### References


micropropagated plantlets and root cultures. Biotechnol. Lett. 35, 791–797. doi: 10.1007/s10529-012-1128-8


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Vera-Reyes, Huerta-Heredia, Ponce-Noyola, Cerda-García-Rojas, Trejo-Tapia and Ramos-Valdivia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## NADPH-generating dehydrogenases: their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditions

#### *Francisco J. Corpas <sup>1</sup> \* and Juan B. Barroso2*

*<sup>1</sup> Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain*

*<sup>2</sup> Group of Biochemistry and Cell Signaling in Nitric Oxide, Department of Biochemistry and Molecular Biology, University of Jaén, Jaén, Spain*

### *Edited by:*

*Naser A. Anjum, University of Aveiro, Portugal*

#### *Reviewed by:*

*Naser A. Anjum, University of Aveiro, Portugal Margarete Baier, Freie Universität Berlin, Germany Yogesh Abrol, Bhagalpur University, India*

#### *\*Correspondence:*

*Francisco J. Corpas, Profesor Albareda 1, 18008-Granada, Spain e-mail: javier.corpas@eez.csic.es*

NADPH is an essential reductive coenzyme in biosynthetic processes such as cell growth, proliferation, and detoxification in eukaryotic cells. It is required by antioxidative systems such as the ascorbate-glutathione cycle and is also necessary for the generation of superoxide radicals by plant NADPH oxidases and for the generation of nitric oxide (NO) by L-arginine-dependent nitric oxide synthase. This coenzyme is principally re-generated by a group of NADP-dehydrogenases enzymes including glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), both belonging to the pentose phosphate pathway, the NADP-malic enzyme (NADP-ME), and NADP-isocitrate dehydrogenase (NADP-ICDH). In this study, current perspectives on these enzymes in higher plants under different stress situations are reviewed and it is also pointed out that this group of NADPH-generating dehydrogenases is a key element in supporting the mechanism of response to nitro-oxidative stress situations.

**Keywords: G6PDH, 6PGDH, ICDH, NAPDH, NADP-ME, nitric oxide, nitrosative stress, oxidative stress**

### **INTRODUCTION**

The supply of reducing equivalents in the form of NADPH (the reduced form of the nicotinamide adenine dinucleotide phosphate) is essential in all living organisms (Pandolfi et al., 1995; Barroso et al., 1998; Ying, 2008). Thus, NADPH is required for cell growth and proliferation which are necessary in several metabolic pathways including fatty acid biosynthesis, biosynthesis of sugars in the Calvin cycle, biosynthesis of carotenoids, conversion of ribonucleotide (RNA) to deoxy-ribonucleotide (DNA) and regulation of chloroplast protein import via the metabolic redox status of the chloroplast, specifically in the Tic62, (a component of the translocon at the inner envelope of chloroplasts, Tic complex) (Stengel et al., 2008; Kovács-Bogdán et al., 2010). NADPH is also required by NADPH-cytochrome P450 reductases (Ro et al., 2002), the generation of superoxide radicals by the NADPH oxidase (NOX) (Sagi and Fluhr, 2006) and is a necessary cofactor for the generation of nitric oxide (NO) by Larginine-dependent nitric oxide synthase (NOS) activity (Corpas et al., 2009). NADPH is also essential by different antioxidative systems including the activity of glutathione reductase (GR), a key enzyme in the ascorbate-glutathione cycle to protect against oxidative damage (Noctor et al., 2006; Gill et al., 2013), and by

**Abbreviations:** FNR, ferrodoxin-NADP reductase (FNR ferrodoxin-NADP reductase); G6PDH, glucose-6-phosphate dehydrogenase; GR, glutathione reductase; NADKs, NAD kinases; NADP-ICDH, NADP-isocitrate dehydrogenase; NADP-ME, NADP-malic enzyme; NO, nitric oxide; NOS, nitric oxide synthase; NOX, NADPH oxidase; NTRs, NADPH-dependent thioredoxin reductases; ONOO−, peroxynitrite; 6PGDH, 6-phosphogluconate dehydrogenase; ROS, reactive oxygen species; RNS, reactive nitrogen species; Tic, The Inner envelope of Chloroplasts.

NADPH-dependent thioredoxin reductases (NTRs) in the regulation of metabolic pathways through thiol group reduction (Spinola et al., 2008; Cha et al., 2014). Curiously, in this last case it has been reported that the chloroplastic G6PDH activity can undergo a redox regulation by thioredoxin (Née et al., 2014) which suggests a complex interaction between the source of NADPH and the NTR system. In consequence, the ultimate antioxidant capacity of the cell must be determined by the availability of reducing equivalents. **Figure 1** summarizes the main pathways in plant cells where NADPH is required.

There are several enzymatic components involved in the maintenance of the pool of NADP and NADPH. NAD kinases (NADKs) catalyze the direct phosphorylation of NAD to NADP and therefore contribute to the generation of the cellular NADP pool (Pollak et al., 2007; Agledal et al., 2010). On the other hand, ferrodoxin-NADP reductase (FNR) in photosynthetic cells during the light phase is recognized as a principal source of NADPH. However, in non-photosynthetic cells during the dark phase of photosynthesis, the main enzymes capable of generating power reduction in the form of NADPH are the following: glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) (both belonging to the pentose phosphate pathway), NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42) and NADP-malic enzyme (NADP-ME, EC 1.1.1.40), also known as NADP-malate dehydrogenase. This mini-review will focus on these groups of NADPH recycling dehydrogenases, principally in relation to their role as second lines of defense against nitro-oxidative stress.

### **SUBCELLULAR NADP-DEHYDROGENASE COMPARTMENTALIZATION AS A NADPH SUPPLY REGULATION MECHANISM**

The NADPH pool is required in many processes while the contribution of each NADP-dehydrogenase in specific situations is difficult to determine. However, cell compartmentalization is required as an additional control mechanism in order to keep the NADPH supply close to the system when required, particularly given that NADPH is part of a network containing other energy-rich molecules such as NADH and ATP (Scheibe and Dietz, 2012). In addition, given that NADPH is not easily transported across membranes but, rather, operates through indirect shuttle systems, all these NADP-dehydrogenases usually have different isozymes which are localized in the different subcellular compartments. Although the localization of some of these NADPdehydrogenases in the different organelles has been described in different plant species (Gálvez and Gadal, 1995; Corpas et al., 1998, 1999; Debnam and Emes, 1999; Hodges et al., 2003; Kruger and von Schaewen, 2003; Leterrier et al., 2007), the availability of genomes in higher plants such as *Arabidopsis thaliana* and *Oryza sativa* has facilitated a more systematic analysis of different NADP-dehydrogenases (Chi et al., 2004; Wakao and Benning, 2005; Wheeler et al., 2005).

### **FUNCTION OF NADP-DEHYDROGENASES UNDER ENVIRONMENTAL STRESS CONDITIONS**

Under diverse biotic and abiotic stress conditions, plants have developed a whole battery of response mechanisms in order to overcome any potential cellular damage. In many cases, these processes could be accompanied by an uncontrolled increase in reactive oxygen and nitrogen species (ROS and RNS) which might generate nitro-oxidative stress (Corpas et al., 2007; Corpas and Barroso, 2013). As all these processes usually involve a redox response, an additional NADPH supply may be required for all the pathways using it.

To support this hypothesis, there is a body of evidence to show that, under specific stress conditions, one or more NADPdehydrogenases are regulated at the level of activity and protein/gene expression (Valderrama et al., 2006; Liu et al., 2007, 2013; Marino et al., 2007; Mhamdi et al., 2010; Airaki et al., 2012). Moreover, the importance of some of these NADPdehydrogenases has been confirmed by reverse genetic studies (Scharte et al., 2009; Dal Santo et al., 2012; Voll et al., 2012; Siddappaji et al., 2013).

In olive plants (*Olea europaea*) under salinity-induced nitrooxidative stress, a general increase in the activity of the main antioxidative systems (catalase, superoxide dismutase and enzymes of the ascorbate-glutathione cycle) was accompanied by a significant increase in the activity and protein expression of G6PDH, NADP-ME, and NADP-ICDH (Valderrama et al., 2006, 2007). Similar behavior has been reported in leaves from pepper plants (*Capsicum annum*) exposed to cadmium stress which generates oxidative stress and a concomitant increase in the activity of all NADP-dehydrogenases (G6PDH, 6PGDH, NADP-ME, and NADP-ICDH) (León et al., 2002). In pepper plant leaves exposed to low temperatures (8◦C) for different periods of time (1–3 d) after 24 h treatments, we observed alterations in the metabolism of ROS and RNS (an increase in lipid oxidation and protein nitration) and a general rise in the activity of the main NADPH-generating enzymes (G6PDH, 6PGDH, NADP-ME, and NADP-ICDH) which appeared to contribute to cold acclimation (Airaki et al., 2012). Arabidopsis seedlings grown under salinity conditions (100 mM NaCl) also displayed nitro-oxidative stress. Among the NADPH-generating dehydrogenases (G6PDH, 6PGDH, NADP-ME, and NADP-ICDH) analyzed under these conditions, NADP-ICDH showed maximum activity levels, mainly attributable to the root NADP-ICDH (Leterrier et al., 2012c). Another study of NADP-ICDH activity in Arabidopsis has demonstrated that this enzyme's kinetic parameters vary depending on the organ involved, being the specific activity much higher in roots than in leaves. *In vitro* analysis of NADP-ICDH activity in the presence of different ROS and RNS showed that H2O2 does not affect this activity in either organ; however, reduced glutathione (GSH) inhibited activity in leaves but not in roots. On the other hand, *S*-nitrosoglutathione, a cellular *S*-nitrosothiol used as a NO donor, and peroxynitrite (ONOO−) depressed NADP-ICDH activity in leaves and roots (Leterrier et al., 2012b). Modulation of NADP-ICDH activity by RNS was also observed in pea roots (*Pisum sativum*) during natural senescence which is associated with nitro-oxidative stress since there are increases in the ONOO− levels and in the number of nitrated proteins. Thus, cytosolic NADP-ICDH activity was shown to be inhibited by nitration at Tyr392 during senescence in a process mediated by peroxynitrite (Begara-Morales et al., 2013).

Depending on the plant species involved, the organs analyzed and the intensity of stress, the response of the NAPDdehydrogenases could also be vary. Thus, in tomato roots (*Solanum lycopersicum*) under salinity conditions (120 mM NaCl) accompanied by oxidative stress, an overall decrease in NADPH content and the enzymatic activities of the main NADPH-generating dehydrogenases has been reported, especially NADP-ICDH activity which recorded a drastic reduction of 94% (Manai et al., 2014). This could be explained by the sensitivity of this enzyme to post-translational modification mediated by ONOO− as observed in pea roots during senescence (Begara-Morales et al., 2013). However, in *Arabidopsis thaliana* seedlings exposed to arsenic (1 mM KH2AsO4) which also generates nitro-oxidative stress based in the concomitant increase of tyrosine-nitration and lipid peroxidation, the activity of NADPdehydrogenases (G6PDH, 6PGDH, and NADP-ICDH) did not vary significantly, suggesting that the supply of NAPDH was sufficient to withstand this stress (Leterrier et al., 2012a). Alternatively, the involvement of Arabidopsis cytosolic NADP-ICDH in leaves has been demonstrated to contribute to the maintenance of redox homeostasis under biotic stress caused by *Pseudomonas syringe* (Mhamdi et al., 2010). In the leaves of tobacco plants (*Nicotiana tabacum*), NADP-ME activity increased significantly in response to drought (Doubnerová-Hısková et al., 2014). On the other hand, in *Lotus japonicus* exposed to water stress, differential and spatially distributed nitro-oxidative stress was reported in roots and leaves. Analysis of NADP-dehydrogenase activities in roots revealed that, whereas G6PDH and NADP-ICDH activity decreased 6.5- and 1.5-fold, respectively, 6PGDH and NADP-ME increased 1.5- and 1.3-fold, respectively. However, no leaf NADP-dehydrogenase appeared to be affected, except for G6PDH which decreased by around 50% under water stress conditions (Signorelli et al., 2013). **Table 1** summarizes some examples of the response of NADP-dehydrogenases to nitro-oxidative stresses generated by different abiotic stresses.

As mentioned above, certain post-translation modifications could negatively affect activity under stress conditions although up-regulation has also been reported. For example, in *Arabidopsis* *thaliana* under salinity (150 mM NaCl) stress conditions, the cytosolic G6PDH isozyme (G6PD6) is targeted by phosphorylation at Thr-467 whose activity increased. The important role played by this dehydrogenase was corroborated using *Arabidopsis thaliana* knockout mutants of cytosolic G6PDH (G6PD6) where the cellular redox state was altered and plants were more sensitive to salt stress (Dal Santo et al., 2012). The importance of cytosolic G6PDH in the leaves of tobacco plants (*Nicotiana tabacum*) at an early stage of defense against the *Phytophthora nicotianae* pathogen which is accompanied by oxidative burst has also been reported. This was demonstrated using a genetic approach involving over-expression of this G6PDH isozyme which improved NADPH provision for pathogen-activated NOXs at the plasma membrane during early oxidative burst (Scharte et al., 2009). In addition, these tobacco plants showed heightened resistance to drought stress. In the same way, transgenic tobacco plants over-expressing the cytosolic G6PDH from *Populus suaveolens* have enhanced cold (4◦C) tolerance. Beside of the increased G6PDH activity, these transgenic plants showed lower level of lipid oxidation and higher activity of antioxidant enzymes such as superoxide dismutase and peroxidase. Moreover, these plants have activated the expression of stress-related genes. Therefore, these data clearly show the regulatory function of G6PDH during low temperature stress (Lin et al., 2013).

There are other examples of certain specific NADPdehydrogenases being regulated at the level of activity and gene expression under diverse stress conditions. For instance, *G6PDH* mRNA expression in wheat seedlings under salt stress conditions of 150 mM NaCl reached a maximum level at 12 h of the treatment (Nemoto and Sasakuma, 2000). A similar response was observed in the expression of the *6PGDH* gene which was up-regulated in rice shoots under salt stress (150 mM NaCl) (Huang et al., 2003). By using the Arabidopsis cytosolic NADP-ICDH knockout mutant, it has been reported that the


**Table 1 | Examples of the response of NADP-dehydrogenases to nitro-oxidative stresses generated by different abiotic stresses.**

loss of this isozyme function does not markedly affect the response of Arabidopsis to ozone. However, other cytosolic NADPH-producing enzymes (G6PDH and NADP-ME) showed a significant increase which contributed to maintaining the status of NADPH redox (Dghim et al., 2013a). A similar increase in G6PDH and NADP-ME has also been reported in hybrid poplar leaves in response to ozone (Dghim et al., 2013b).

### **CONCLUSIONS**

Together with NADH, NADPH participates in the equilibrium of cellular redox homeostasis and also maintains certain antioxidant systems such as the ascorbate-glutathione cycle and NTRs. Thus, NADP-dehydrogenase systems should be regarded as a second line of defense in order to maintain the effective functioning of the main antioxidative systems. Biochemical and genetic approaches provide a strong data basis to confirm the essential involvement of NADP-dehydrogenases in the mechanism of response to nitro-oxidative stress situations. Organ distribution and subcellular compartmentalization are regarded as additional regulatory mechanisms of these systems to ensure that the NADPH supply is at the required location. Future research will be essential to identify the specific involvement of each NADP-dehydrogenase in the different organs and cellular compartments supporting a particular pathway as all these enzymes are also involved in nitrogen and carbohydrate metabolisms.

### **ACKNOWLEDGMENTS**

Work in our laboratories is supported by ERDF-cofinanced grants from the Ministry of Science and Innovation (BIO2012-33904 and Recupera 2020-20134R056). We apologize for all the authors we could not cite in this mini review article due to space limitation.

### **REFERENCES**


analysis under abiotic stress conditions. *Free Radic. Res.* 41, 191–199. doi: 10.1080/10715760601034055


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 02 October 2014; accepted: 14 November 2014; published online: 02 December 2014.*

*Citation: Corpas FJ and Barroso JB (2014) NADPH-generating dehydrogenases: their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditions. Front. Environ. Sci. 2:55. doi: 10.3389/fenvs.2014.00055*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2014 Corpas and Barroso. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Mitochondrial isocitrate dehydrogenase is inactivated upon oxidation and reactivated by thioredoxin-dependent reduction in *Arabidopsis*

### *Keisuke Yoshida1,2 and Toru Hisabori 1,2\**

*<sup>1</sup> Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan*

*<sup>2</sup> Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan*

#### *Edited by:*

*Adriano Sofo, Università degli Studi della Basilicata, Italy*

#### *Reviewed by:*

*Shailendra Pratap Singh, Michigan State University, USA Aran Incharoensakdi, Chulalongkorn University, Thailand*

#### *\*Correspondence:*

*Toru Hisabori, Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan e-mail: thisabor@res.titech.ac.jp*

Regulation of mitochondrial metabolism is essential for ensuring cellular growth and maintenance in plants. Based on redox-proteomics analysis, several proteins involved in diverse mitochondrial reactions have been identified as potential redox-regulated proteins. NAD+-dependent isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid (TCA) cycle, is one such candidate. In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. In contrast to mammalian and yeast counterparts reported to date, recombinant IDH in *Arabidopsis* mitochondria did not show adenylate-dependent changes in enzymatic activity. Instead, IDH was inactivated by oxidation treatment and partially reactivated by subsequent reduction. Functional IDH forms a heterodimer comprising regulatory (IDH-r) and catalytic (IDH-c) subunits. IDH-r was determined to be the target of oxidative modifications forming an oligomer via intermolecular disulfide bonds. Mass spectrometric analysis combined with tryptic digestion of IDH-r indicated that Cys128 and Cys<sup>216</sup> are involved in intermolecular disulfide bond formation. Furthermore, we showed that mitochondria-localized *o*-type thioredoxin (Trx-*o*) promotes the reduction of oxidized IDH-r. These results suggest that IDH-r is susceptible to oxidative stress, and Trx-*o* serves to convert oxidized IDH-r to the reduced form that is necessary for active IDH complex.

**Keywords:** *Arabidopsis***, isocitrate dehydrogenase, mitochondria, redox regulation, thioredoxin**

### **INTRODUCTION**

Mitochondria play a pivotal role in providing ATP required for various cellular events in all eukaryotes. In mitochondrial respiration, the tricarboxylic acid (TCA) cycle generates NADH and FADH2 by metabolizing organic acids. These products are then used to drive electron transport in the respiratory chain and coupled ATP production. In addition to this fundamental energy conversion process, mitochondria host a large number of metabolic pathways. Flexible regulation of these mitochondrial reactions is important for ensuring proper cellular function, particularly in plants, which cannot escape exposure to adverse environmental conditions. Although our knowledge of the regulatory mechanisms of plant mitochondria has advanced, much still remains to be solved, particularly the question of how each mitochondrial enzyme is controlled at the post-translational level (Millar et al., 2011; Tcherkez et al., 2012; Lázaro et al., 2013; Nunes-Nesi et al., 2013).

In the last decade, the progress of redox-proteomics analysis has provided hints that a wide variety of biological processes are governed by the redox state of their responsible enzymes (Hisabori et al., 2007; Montrichard et al., 2009; Lindahl et al., 2011). Thioredoxin (Trx), a small ubiquitous protein, plays a crucial role in redox regulation. Trx has a conserved WCGPC motif at an active site, enabling a dithiol–disulfide exchange reaction with the target enzyme. Based on the subcellular localization and sequence similarity, plant Trxs are classified into seven subtypes (*f*-, *m*-, *h*-, *o*-, *x*-, *y*-, and *z*-type). Although it has been recognized that the Trx-*o* resides in plant mitochondria (Laloi et al., 2001), information about the target proteins of Trx-*o* is limited to date. Using Trx affinity chromatography (Motohashi et al., 2001), we recently performed the systematic screening of Trx-targeted proteins in plant mitochondria and identified a list of target candidates (Yoshida et al., 2013). Redoxproteomics studies by other groups have also suggested that diverse proteins involved in manifold mitochondrial processes are redox-regulated via the interaction with Trx (Balmer et al., 2004; Winger et al., 2007). However, careful biochemical study is needed to determine whether these candidate proteins are actually redox-regulated.

NAD+-dependent isocitrate dehydrogenase (IDH) is one of the proteins that were captured by Trx affinity chromatography (Yoshida et al., 2013). IDH catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate coupled to NADH generation, and thus supports the TCA cycle flux. In yeast, it is well established that the minimal functional unit of IDH is a heterodimer comprising regulatory (IDH-r) and catalytic (IDH-c) subunits (Panisko and McAlister-Henn, 2001). Furthermore, detailed biochemical and structural analyses have provided evidence that yeast IDH is allosterically activated by AMP and inactivated by intermolecular disulfide bond formation between IDH-c subunits (Lin and McAlister-Henn, 2003; Taylor et al., 2008; Garcia et al., 2009). In contrast, to our knowledge, there are few reports on the biochemical analysis of plant IDH, although plant IDH has been also suggested to be active in a heterodimeric form based on sequence comparison with its yeast counterpart and the complementation test of yeast IDH mutants with plant *IDH* genes (Lancien et al., 1998; Lemaitre and Hodges, 2006). The regulatory mechanism of plant mitochondrial IDH thus remains poorly characterized.

In this study, we focused on the molecular basis for controlling IDH activity in *Arabidopsis* mitochondria with special attention to redox regulation. The results indicate that IDH-r forms intermolecular disulfide bonds upon oxidation, leading to a drastic decrease in IDH activity. We also showed that Trx-*o* assists in the reduction of oxidized IDH-r. Based on these findings, a novel regulatory mode of plant mitochondrial IDH is discussed.

### **MATERIALS AND METHODS**

### **PREPARATION OF EXPRESSION PLASMIDS FOR IDH-r AND IDH-c**

Total RNA was isolated from *Arabidopsis thaliana* as described in Yoshida and Noguchi (2009) and used as a template for RT-PCR. The *IDH1* (*At4g35260*, encoding IDH-r) and *IDH5* (*At5g03290*, encoding IDH-c) gene fragments encoding the mature protein region (Val26-Asp367 and Ile44-Leu374, respectively) were amplified with the following oligonucleotide primer combination; 5- -AACTGCAGCATATGGTGACTTACAT GCCCAGACC-3- (NdeI) and 5- - GCGAATTCAGTCTAGTTTTG CAATGA-3- (EcoRI) for *IDH1*, 5- - AACTGCAGCATATGATCA CCGCAACTCTCTTCCCT-3- (NdeI) and 5- -AAGGATCCTCAG AGATGATCACAGATTG-3- (BamHI) for *IDH5*. The restriction sites for the enzyme shown in parentheses are underlined. Each of the amplified DNA was ligated into the pET23c expression vector (Novagen). The sequences were confirmed by DNA sequencing (3730xl DNA Analyzer; Applied Biosystems).

### **PROTEIN EXPRESSION AND PURIFICATION**

The *IDH1* and *IDH5* expression plasmids described above were transformed into *E. coli* strain BL21 (DE3) to express IDH-r and IDH-c proteins, respectively. The transformed cells were grown at 37◦C until A600 = 0*.*4–0.8. Expression was induced by adding 0.5 mM IPTG, followed by further culture at 21◦C overnight. The *E. coli* cells were disrupted by sonication. After centrifugation (125,000 ×g for 40 min), the resulting supernatant was used to purify the protein of interest. Each protein was purified by a combination of anion exchange chromatography, using a DEAE-Toyopearl 650M column (Tosoh) and Q-Toyopearl 600C column (Tosoh), and hydrophobic interaction chromatography, using a Butyl-Toyopearl 650M column (Tosoh), as described in Yoshida et al. (2013). Purification was performed in a medium containing 25 mM Tris-HCl (pH 7.5–8.1), 1 mM EDTA, and 0.5 mM DTT, but EDTA and DTT were removed by dialysis after purification. All of the procedures during purification were carried out at 4◦C. The protein concentration was determined with a BCA protein assay (Pierce).

### **IDH ACTIVITY MEASUREMENT**

Prior to the assay, 2μM IDH-r and 2μM IDH-c were mixed in a medium containing 25 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 20 mM isocitrate. After incubation at 25◦C for 30 min, the mixed solution was used for activity measurement. IDH activity was monitored as an increase in absorbance at 340 nm due to NAD+ reduction. The molar extinction coefficient for NADH of 6.22 mM−<sup>1</sup> was used for calculation of the amounts of generated NADH. Assays were performed at 25◦C in a medium containing 25 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM NAD+, and indicated concentrations of isocitrate. IDH-r and IDH-c were added at 40 nM each.

To test the adenylate effects on IDH activity, each adenylate (AMP, ADP, and ATP) was added at 1 mM to the media described above.

### **PEPTIDE MAPPING ANALYSIS**

After separation by non-reducing SDS-PAGE, stained protein bands of interest were excised from the gel and fully destained

**FIGURE 1 | Activity measurements of** *Arabidopsis* **IDH recombinant protein. (A)** SDS-PAGE profiles of purified recombinant IDH-r and IDH-c. **(B)** Monitoring of NAD+-reducing activity of IDH. Time course of absorbance change at 340 nm (Abs340) is shown. IDH-r and/or IDH-c (40 nM each) were added to a reaction medium. Isocitrate concentration was 5.4 mM. The reaction was initiated by the addition of NAD+ (2 mM) at the time point indicated by arrow. See Materials and Methods for details. **(C)** The effect of adenylate on IDH activity under several concentrations of isocitrate. Each adenylate (AMP, ADP, ATP) was added at 1 mM. Each value represents the mean ± *SD* (*n* = 3).

with 50 mM NH4HCO3 and 50% (v/v) acetonitrile. The gel slice was then incubated in 55 mM iodoacetamide and 100 mM NH4HCO3 for protecting free Cys residues. The gel slice was dried completely and then incubated with 50 mM NH4HCO3 containing 20 ng μl <sup>−</sup><sup>1</sup> trypsin at 37◦C overnight. Tryptic peptides were extracted from the gel with 0.1% (v/v) trifluoroacetic acid with 50 and 75% (v/v) acetonitrile, continuously. Whole extracts were concentrated using a centrifugal concentrator and desalted using Solid Phase Extraction C-TIP (Nikkyo Technos). The peptide sample was spotted onto the matrix (α-cyano-4 hydroxycinnamic acid) and air-dried on a MALDI plate (MTP 384 target plate ground steel BC, Bruker Daltonics). MALDI mass spectra were obtained using an UltrafleXtreme-TK2 spectrometer (Bruker Daltonics). Results were queried with the Mascot search engine (http://www*.*matrixscience*.*com/) to identify matched peptides.

#### **Trx-DEPENDENT REDUCTION OF OXIDIZED IDH-r**

For oxidation treatment, IDH-r was incubated in 50μM CuCl2 for 15 min at 25◦C. After dialysis for the removal of CuCl2, the oxidized IDH-r was incubated in a medium containing 25 mM Tris-HCl (pH 7.5), indicated concentrations of DTT, and indicated concentrations of Trx-*o*1 for 30 min at 25◦C. The IDHr redox state was assayed by non-reducing SDS-PAGE. IDH-r equivalent to 1μg was loaded into each lane. The *Arabidopsis* recombinant Trx-*o*1 protein was prepared and confirmed to be efficient in dithiol–disulfide exchange reaction in our previous study (Yoshida et al., 2013).

### **RESULTS**

#### *ARABIDOPSIS* **IDH IS INSENSITIVE TO ADENYLATES**

In *Arabidopsis*, there are five genes encoding mitochondrial IDH subunits. Based on the similarity with yeast IDH, three genes (*IDH1*, *IDH2*, and *IDH3*) are regarded as the genes encoding the IDH-r subunit, whereas two genes (*IDH5* and *IDH6*) encode the IDH-c subunit (Supplementary Fig. S1). Primary amino acid sequences of these gene products show high identity among each isoform (*IDH1-3*; *>*84%, *IDH5-6*; 90%, mature protein region except for targeting peptide). Given that *IDH1* and *IDH5* gene products appear to be more abundantly expressed in *Arabidopsis* (Yoshida et al., 2013), we prepared recombinant proteins from these genes as representative IDH-r and IDH-c, respectively



*aThe Km and Vmax values for isocitrate were calculated using a Hanes–Woolf plot.*

*bEach adenylate was added at 1 mM.*

*Each value represents the mean* ± *SD (n* = *3).*

(**Figure 1A**). NAD+-reducing activity was observed in the presence of both subunits, whereas neither subunit alone showed any catalytic activity (**Figure 1B**). This finding clearly shows that both IDH-r and IDH-c are essential for functional IDH in *Arabidopsis*.

It has been well documented that ADP and AMP act as allosteric activators of IDH in mammal and yeast (Nunes-Nesi et al., 2013). For example, the *Km* value for isocitrate in yeast IDH is drastically lowered in the presence of AMP (Lin and McAlister-Henn, 2003). However, it remains unclear whether this regulatory mechanism is common for plant IDH. We accordingly investigated the effects of adenylates on IDH activity. No adenylates (AMP, ADP, and ATP) affected the saturation velocity curve (**Figure 1C**) or correspondingly the *Km* and *V*max values (**Table 1**) for isocitrate. These results imply that changes in the mitochondrial adenylate energy charge have no direct impact on IDH activity in *Arabidopsis*.

### **IDH-r FORMS INTERMOLECULAR DISULFIDES, LEADING TO A LOSS OF IDH CATALYTIC ACTIVITY**

We next investigated whether IDH activity is controlled by the redox state of the enzyme molecule itself. Prior to the

**FIGURE 2 | Redox regulation of** *Arabidopsis* **IDH recombinant protein. (A)** Redox-dependent change in IDH activity. For oxidation (Ox) treatment, IDH-r or IDH-c was incubated in 50μM CuCl2 for 15 min. For re-reduction (Ox → Red) treatment, IDH-r after Ox treatment was incubated in 50 mM DTT for 30 min. Each value represents the mean ± *SD* (*n* = 4). **(B)** Non-reducing SDS-PAGE profiles of IDH-r after reduction (Red) treatment (5 mM DTT for 15 min) or Ox treatment. **(C)** Non-reducing SDS-PAGE profiles of IDH-r after Ox → Red treatment.

assay, IDH-r or IDH-c was incubated in the presence of 50μM CuCl2 to oxidize the possible thiols on the protein molecule. While the oxidation treatment of IDH-c exerted no significant effect, the IDH-r oxidation nearly completely suppressed enzymatic activity when the oxidized IDH-r was mixed with untreated IDH-c (**Figure 2A**). As revealed by nonreducing SDS-PAGE, IDH-r was shifted to dimeric, trimeric, and higher-order oligomeric forms mediated by intermolecular disulfide bonds under oxidative conditions (**Figure 2B**). The reduction of oxidized IDH-r restored IDH activity to approximately half of the control level (**Figure 2A**), accompanied by the cleavage of the intermolecular disulfide bonds (**Figure 2C**). It thus appeared that *Arabidopsis* IDH is reversibly inactivated in response to oxidative stress via oligomer formation of IDH-r.

### **Cys<sup>128</sup> AND Cys<sup>216</sup> CONSERVED IN IDH-r PLAY A CRITICAL ROLE IN THE REDOX REGULATION OF IDH**

In plant IDH-r, six Cys residues are commonly conserved (Supplementary Fig. S1). We attempted to identify the Cys residues involved in intermolecular disulfide bond formation of IDH-r. For this purpose, proteins in the reduced monomeric and the oxidized trimeric forms (the prevalent form of IDH-r oligomer) were in-gel digested using trypsin after non-reducing SDS-PAGE (**Figure 2B**). Mass spectra of the resulting peptides were then acquired (**Figure 3**). Three major peptides (*m/z*: 1280.6, 2276.2, and 2404.3) were specifically detected in the reduced monomeric form but not in the oxidized trimeric form. By searching for matching peptides using Mascot, the peptides were determined to correspond to Leu207-Arg217, Glu118-Arg137, and Lys117-Arg137 (calculated masses: 1279.6, 2275.2, and 2403.3, respectively) in IDH-r, respectively. The peptide Leu207-Arg217 contained Cys216, whereas Glu118-Arg137 and Lys117-Arg137 contained Cys128. These Cys residues are thus likely to be primarily involved in redox changes in the *Arabidopsis* IDH-r molecule.

### **Trx-***o* **PROMOTES THE REDUCTION OF OXIDIZED IDH-r**

Finally, we addressed the involvement of Trx-*o* in redox regulation of IDH-r (**Figure 4**). The reduction patterns of oxidized IDH-r under several concentrations of DTT (0–500μM) were compared in the presence and absence of 5μM Trx-*o*1 (an isoform of Trx-*o*). When Trx-*o*1 was added to a reaction medium, IDHr was reduced back to monomer even at lower concentrations of DTT (**Figure 4A**). We further analyzed the reduction of oxidized IDH-r with varying Trx-*o*1 concentration (0–5μM) under low concentration of DTT (50μM). The efficiency of IDH-r reduction was highly dependent on Trx-*o*1 concentration (**Figure 4B**). These findings suggest that Trx-*o*1 can efficiently reduce oxidized IDH-r.

### **DISCUSSION**

Mitochondrial respiration is controlled at multiple levels from transcriptional to post-translational to enzyme function levels (Millar et al., 2011). It has been reported that several TCA cycle enzymes are regulated by mitochondrial NAD(P)H/NAD(P)+ ratio, adenylate energy charge, and TCA cycle intermediates (Noctor et al., 2007; Nunes-Nesi et al., 2013). Using partially purified IDH from pea leaves, Igamberdiev and Gardeström (2003) demonstrated that IDH activity is negatively regulated by NAD(P)H. However, further biochemical studies of plant IDH

have not been performed to date, and accordingly the regulatory mechanisms of this enzyme at the molecular level remain to be fully characterized. Previous studies using Trx affinity chromatography have raised the possibility that IDH activity is redox-regulated via the Trx system in plant mitochondria (Balmer et al., 2004; Yoshida et al., 2013). Based on this research background, we addressed in this study the biochemical characteristics of *Arabidopsis* IDH, focusing particularly on redox regulation.

The biochemical properties of IDH, including action mechanisms and regulatory factors, have been best characterized in yeast (Panisko and McAlister-Henn, 2001; Lin and McAlister-Henn, 2003; Taylor et al., 2008; Garcia et al., 2009). The minimal functional unit of yeast IDH is reported to be a heterodimer comprising IDH-r and IDH-c. This form is considered to be common in plant IDH, but there has been only indirect supporting evidence to date in the form of growth restoration of yeast IDH mutants by complementation with plant *IDH* genes (Lancien et al., 1998; Lemaitre and Hodges, 2006). By using the recombinant subunits, we clearly showed that both IDHr and IDH-c are essential for ensuring the catalytic activity of *Arabidopsis* IDH (**Figure 1B**). An intriguing finding is that, in contrast to the animal and yeast counterparts, *Arabidopsis* IDH activity is unaffected by any adenylates (**Figure 1C**, **Table 1**). In yeast IDH, critical residues for allosteric activation by AMP have been determined by a survey using site-directed mutagenesis (Lin and McAlister-Henn, 2003). Alignment between plant and yeast IDH indicated that plant IDH does not conserve some of these residues (Supplementary Fig. S1), and the lack of these critical residues may account for the insensitivity of *Arabidopsis* IDH to adenylates.

Redox-proteomics studies using Trx affinity chromatography have provided a comprehensive list of potentially redox-regulated proteins in plant mitochondria (Balmer et al., 2004; Yoshida et al., 2013). It has been shown that, among these candidate proteins, two stress-related proteins, namely alternative oxidase and peroxiredoxin IIF, are reduced and activated in a Trx-*o*-dependent manner (Barranco-Medina et al., 2008; Martí et al., 2009; Yoshida et al., 2013). In addition, the TCA cycle enzyme citrate synthase has recently been reported to be redox-regulated by Trx-*o* (Schmidtmann et al., 2014). However, most plant mitochondrial proteins suggested as Trx targets remain to be further analyzed by detailed biochemical assays. In the present study, IDH was newly confirmed to be a Trx-*o*-targeted redox-regulated protein in *Arabidopsis* mitochondria. Upon oxidation of IDH-r, IDH activity was largely diminished via intermolecular disulfide-mediated oligomer formation of IDH-r (**Figures 2A,B**). Trx-*o* was effective in the reduction of oxidized IDH-r, likely leading to a recovery of IDH activity (**Figures 2**, **4**). It should be noted that IDH activity was restored to only half of the control level, even after the reduction and monomerization of oxidized IDH-r (**Figure 2**). This result implies that IDH-r also undergoes irreversible oxidative modifications, which could not be revealed in the present study. Further investigation is required for concluding this possibility.

Yeast IDH activity is also controlled in a redox-dependent manner (Garcia et al., 2009). However, the molecular basis of IDH redox regulation is different between *Arabidopsis* and yeast. In the case of yeast IDH, IDH-c forms intermolecular disulfide bonds under oxidative conditions, leading to the inactivation of IDH (Garcia et al., 2009). The critical Cys residue for the intermolecular disulfide bond formation of yeast IDH-c is not conserved in plant IDH-c (Supplementary Fig. S1). In contrast, we identified Cys<sup>128</sup> and Cys<sup>216</sup> as the Cys residues responsible for oxidative modification of *Arabidopsis* IDH-r (**Figure 3**). These Cys residues are highly conserved in the plant kingdom but not in yeast (Supplementary Fig. S1). This fact implies that the mode of IDH redox regulation disclosed in this study is unique to plants.

As summarized in **Figure 5**, our biochemical analysis suggested that IDH-r is a redox-sensitive protein and that the redox change affects IDH activity. This finding implies the significance of the mitochondrial Trx system for the regulation of TCA cycle performance. However, for several redox-regulated protein candidates identified by redox-proteomics studies, it remains to be determined whether they are "pseudo" or "true" redoxregulated proteins. In order to understand the mitochondrial redox network in more detail, biochemical studies of the individual enzymes should be performed one by one. Another key step in dissecting the mitochondrial redox regulation system is the elucidation of the working dynamics or biological significance of the mitochondrial Trx system in living plants. Future studies addressing the visualization of *in vivo* redox states of redox-regulated proteins, as shown in our recent study (Yoshida et al., 2014), and effects of Trx-*o* deficiency on mitochondrial performance will provide physiological insights into the mitochondrial redox regulation system in plants.

### **ACKNOWLEDGMENTS**

This work was supported in part by the Core Research of Evolutional Science and Technology program (CREST) from the Japan Science and Technology Agency (JST) and a Grant-in-Aid for Scientific Research (grant number 24870010 to Keisuke Yoshida) from the Japan Society for the Promotion of Science.

### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www*.*frontiersin*.*org/journal/10*.*3389/fenvs*.* 2014*.*00038/abstract

### **REFERENCES**


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 July 2014; accepted: 08 September 2014; published online: 23 September 2014.*

*Citation: Yoshida K and Hisabori T (2014) Mitochondrial isocitrate dehydrogenase is inactivated upon oxidation and reactivated by thioredoxin-dependent reduction in Arabidopsis. Front. Environ. Sci. 2:38. doi: 10.3389/fenvs.2014.00038*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2014 Yoshida and Hisabori. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Redox homeostasis in plants under abiotic stress: role of electron carriers, energy metabolism mediators and proteinaceous thiols

*Dhriti Kapoor\*, Resham Sharma , Neha Handa , Harpreet Kaur , Amandeep Rattan , Poonam Yadav , Vandana Gautam , Ravdeep Kaur and Renu Bhardwaj*

*Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India*

#### *Edited by:*

*Nafees Ahmad Khan, Aligarh Muslim University, India*

#### *Reviewed by:*

*Mohammad Mobin, University of Tabuk, Saudi Arabia Noushina Iqbal, Aligarh Muslim University, India*

#### *\*Correspondence:*

*Dhriti Kapoor, Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar-Punjab, 143005 Amritsar, India*

*e-mail: renubhardwaj82@gmail.com*

Contemporaneous presence of both oxidized and reduced forms of electron carriers is mandatory in efficient flux by plant electron transport cascades. This requirement is considered as redox poising that involves the movement of electron from multiple sites in respiratory and photosynthetic electron transport chains to molecular oxygen. This flux triggers the formation of superoxide, consequently give rise to other reactive oxygen species (ROS) under adverse environmental conditions like drought, high, or low temperature, heavy metal stress etc. . . that plants owing during their life span. Plant cells synthesize ascorbate, an additional hydrophilic redox buffer, which protect the plants against oxidative challenge. Large pools of antioxidants also preside over the redox homeostasis. Besides, tocopherol is a liposoluble redox buffer, which efficiently scavenges the ROS like singlet oxygen. In addition, proteinaceous thiol members such as thioredoxin, peroxiredoxin, and glutaredoxin, electron carriers and energy metabolism mediators phosphorylated (NADP) and non-phosphorylated (NAD+) coenzyme forms interact with ROS, metabolize and maintain redox homeostasis.

**Keywords: abiotic stress, reactive oxygen species, ascorbate, tocopherol, glutathione**

### **INTRODUCTION**

During cellular respiration, the accretion of molecular oxygen (O2) in earth's environment allows aerobic organisms to utilize O2 as the terminal electron acceptor that gives a higher amount of energy than fermentation (Dismukes et al., 2001). O2 gives rise to prolific reactive excited states, like reactive oxygen species (ROS) and their derivatives during normal metabolic activity as a consequence of various environmental perturbations (Scandalios, 2005). ROS is a product of cellular metabolism; however, the balance between the generation and removal of ROS is disturbed in cellular components of plants under stress conditions (Karuppanapandian et al., 2011). ROS like hydroxyl radical (OH· ), superoxide radical (O·− <sup>2</sup> ), hydrogen peroxide (H2O2), hydroperoxyl radical (HO· 2), alkoxy radical (RO· ), peroxy radical (ROO· ), singlet oxygen (1O2) and excited carbonyl (RO∗), are cytotoxic to plants (Vellosillo et al., 2010). Attack of ROS may results in serious damage to cellular components, DNA lesions and mutations and this frequently leads to irretrievable metabolic dysfunction and cell death (Karuppanapandian et al., 2011). ROS are scavenged by various antioxidative defense systems under steady state conditions (Navrot et al., 2007).

Overproduction of ROS in plants is induced by various environmental perturbations like drought, heat, high light intensity (HL), salinity, chilling, herbicides, heavy metals, pathogens, wounding, ozone (O3), atmospheric pollutants, and photosensitizing toxins which causes oxidative cellular damage (Karuppanapandian and Manoharan, 2008; Mafakheri et al., 2010). Reduction of a single electron from O2 results in the production of the superoxide radicals (O·− <sup>2</sup> ), which is quite reactive. Consequently, it cannot cross biomembranes and may readily dismutated to H2O2. O·− <sup>2</sup> also react with NO· , another very dominant signaling free radical species that gives rise to peroxynitrite (OONO−). O·− <sup>2</sup> leads to the formation of HO· <sup>2</sup> by protonation in aqueous solutions that can cross biomembranes and subtract hydrogen atoms from polyunsaturated fatty acids (PUFAs) and lipid hydroperoxides, therefore initiating lipid autooxidation (Halliwell and Gutteridge, 2000). H2O2 is a relatively long-lived molecule and moderately reactive, which can disseminate short distances away from its production site. H2O2 causes inactivation of enzymes by oxidizing their thiol groups. H2O2 enables it to diffuse the damage and also act as a messenger in the stress signaling response and thus can travel freely across membranes (Moller et al., 2007). It may also trigger the production of OH· , the most reactive oxidant in the ROS family and also considered as one of initiation radicals for lipid peroxidation, via Haber-Weiss/Fenton reactions that utilize the suitable transition metals, especially, iron (Fe) (Lee et al., 2007). The products of OH· reactions may extract signaling responses and cells sequester the catalytic metals to metallochaperones efficiently avoiding OH· , though it does not have signaling function (Moller et al., 2007). It can potentially react with all biomolecules like, proteins, lipids, pigments and DNA and almost all constituent of cells. Excess production of ROS leads to programmed cell death (PCD), as plant cells are not capable to scavenge these ROS (Manoharan et al., 2005).

### **CONCEPT OF REDOX HOMEOSTASIS**

Contemporaneous presence of both oxidized and reduced forms of electron carriers are required by competent flux through electron transport cascades of plant. In photosynthetic and respiratory electron transport chains, the requirement of regular flux of electrons to molecular oxygen from multiple sites is known as redox poising. Despite specific oxidases catalyze the specialized water producing reactions. Primary product of this flux is superoxide and other ROS are produced consequently. Superoxide or H2O2 are generated by various enzyme systems. Due to the reactive nature of these intermediates they are able to act as signaling molecules; therefore their accumulation must be regulated. Accumulation of ROS is resoluted by the antioxidative system, which further balance the metabolism of organism by maintaining the proteins and other cellular components in an active state (Foyer et al., 2005). Large pools of these antioxidants govern the redox homeostasis, which absorb the reductants and oxidants. ROS signaling pathways are generated by homeostatic regulation that is achieved by antioxidant redox buffering. Antioxidants determine the lifetime and the specificity of the ROS signal. Generation of superoxide, H2O2 and even singlet oxygen are coped by plant cells (Wagner et al., 2004). Moreover, due to signaled induction of other defense systems, plants adapt very well for the depletion of antioxidants. Cytoplasmic thiols in the reduced state are balanced in the plants because of the low thioldisulfide redox potential, which is imposed by the thiol buffer, glutathione. Though, plant cells make an additional hydrophilic redox buffer namely ascorbate (vitamin C), which provides strong protection against oxidative damage. However, plants also synthesize tocopherols (vitamin E) that perform as key liposoluble redox buffers. Tocopherol is considered as an effective scavenger of other ROS including singlet oxygen species and in this case the reduced scavenging form is regenerated by ascorbate (Foyer et al., 2005). Moreover, it raises the array of efficient superoxide scavenging as the tocopherol redox couple acts as affirmative midpoint potential than that of the ascorbate pool. In plant cells, the capability of glutathione, tocopherol and ascorbate pools to act as redox buffers is one of significant attributes. Low activities of ascorbate peroxidase and catalase in plants show less harsh symptoms of stress than those plants which lack either one of these enzymes (Rizhsky et al., 2002). For example, tocopherol-deficient *Arabidopsis* vte mutant seedlings possess large amounts of lipid peroxides, whereas mature plants show slightly abnormal phenotype (Kanwischer et al., 2005). It is estimated that DNA repair and rapid protein turnover is enhanced to recompense for improved oxidation or loss of antioxidants.

### **ROLE OF REDOX-HOMEOSTASIS MANAGERS AGAINST VARIOUS ABIOTIC STRESSES ASCORBATE**

Ascorbate (L- Ascorbic Acid/Vitamin C/AsA), a water soluble antioxidant of universal distribution in higher plants, has been studied for its biosynthesis, localization and presence within plant cells, metabolic involvement and biochemistry with respect to other antioxidants (Khan et al., 2011; Szarka et al., 2012; Gallie, 2013; Lisko et al., 2014; Venkatesh and Park, 2014; Foyer, 2015). AsA is concentrated in photosynthetic tissues, meristematic tissues, flowers, young fruits, root tips etc. . . (Gest et al., 2013). The AsA biosynthetic pathway considers D-mannose and L- galactose as primary substrates through various enzymatic reactions (Müller-Moulé, 2008). In addition to this being the main scheme of ascorbate generation (Smirnoff-Wheeler pathway), three other pathways namely the L-gulose (Gul) shunt, the D-galacturonate (GalU) pathway, and the myo-inositol (MI) route have also been identified in plants (Venkatesh and Park, 2014). AsA is generated on the inner mitochondrial membrane; and further transported to different cellular components including the apoplast for consumption, degeneration, and recycling (Green and Fry, 2005) (**Figure 1**). Its transport within the plant system is mediated by facilitated diffusion or active transport systems (Ishikawa et al., 2006). Since this is such a ubiquitous antioxidant present in plant system, it plays a wide array of roles such as scavenging of deleterious ROS produced during all sorts of abiotic and biotic stress (Teixeira et al., 2004), central role in photosynthesis (Smirnoff, 1996), as a major participant in detoxification mechanisms focused in chloroplasts such as the water-water cycle—WWC or the Mehler peroxidase reaction (Neubauer and Yamamoto, 1992) and the xanthophyll cycle (Yabuta et al., 2007).

The hydrogen peroxide and superoxide radicals generated in this reaction are reduced to water by AsA in the presence of ascorbate peroxidase (APX). MDA is a by-product released in this reaction and is further converted to ascorbate either by reduced ferredoxin of PSI (photosystem I) or by MDHA reductase using NADH or NADPH as electron donor (Sano et al., 2005). AsA has also been identified as an alternative electron donor of PSII; thereby retarding the photo inactivation and ROS activity in the thylakoid and providing protection to the entire photosynthetic machinery (Tóth et al., 2011; Gururani et al., 2012). In addition to all this, AsA participates in the regeneration of vitamin E and acts as a substrate for synthesis of important organic acids such as; L-glyceric, L-oxalic acids, L-tartaric, and L-threonic acid (Debolt et al., 2007). AsA however plays the most important

role of guarding cells and organelles against oxidative damage by eliminating ROS which are produced by aerobic metabolic processes such as photosynthesis and respiration or by environmental stresses like salt, drought, cold, and excess light; hence becoming an imperative molecule for overall plant health and well-being. Although there are many experimental and factual evidences from various mutant and transgenic studies for the same, here we discuss the relevant ones with special reference to abiotic stresses. Elevated AsA levels and induction of cytosolic APX have been reported in plants subjected to high light oxidative stress (Yabuta et al., 2007). Many cytosolic and chloroplast centered APX genes have been identified in *Arabidopsis* spp, *Oryza* spp ,*Lycopersican* spp. etc. . . (Koussevitzky et al., 2008; Najami et al., 2008; Lazzarotto et al., 2011; Pang et al., 2011). Similarly, plants under water deficit stress also over produce ascorbate to counter fall of physiological parameters and plant survival (Dolatabadian et al., 2009). Sesame seeds coated with AsA–PEG were subjected to drought stress and surprisingly, recorded for a good germination percentage and index, dry weight and seedling length (Tabatabaei and Naghibalghora, 2013). Another stress countered by AsA is the ozone stress resulting from industrial activities causing extensive leaf damage, fallen stomatal conductance and photosynthesis rates in plants (Sanmartin et al., 2003). The ROS accumulation caused due to ozone entering the apoplastic and symplastic components through stomata leads to severe damage to the photosynthetic machinery (Cho et al., 2011). Apoplastic AsA forms the primal barrier against this stress by direct reaction with free radicals formed by ozone (Chen and Gallie, 2005). Ozone resistant plant species showed enhanced apoplastic ascorbate levels (Feng et al., 2010). Exogenously applied AsA prevented chances of foliar injury and checked loss of photosynthetic activity caused by ozone stress (Zheng et al., 2000; Maddison et al., 2002). Similarly, plants with over expressing DHAR genes also showed an increased ozone tolerance and higher level of photosynthetic activity (Chen and Gallie, 2005). ROS generated due to high/low temperature is also well balanced by AsA metabolic responses in many transgenic plants such as potato, tomato, rice, etc. . . (Tang et al., 2006; Sato et al., 2011). Increased tolerance to temperature stress was also observed in transgenic tobacco plants over expressing the thylakoid-bound APX gene from tomato (Sun et al., 2010). During chilling and heat stresses, the photochemical/oxidative efficiency of PSII in the transgenic lines was observed to be higher than that of non-transformed/wild-type plants (Wang et al., 2011), *Arabidopsis* spp. despite showing a small increase in Asc content, over expressed rice DHAR gene and showed more salt tolerance (Ushimaru et al., 2006). Tomato seedlings over expressing a chloroplast-targeted tomato MDHAR gene reduced membrane damage and resulted in a higher net photosynthetic rate, higher maximal PSII photochemical efficiency and increased fresh weight when subjected to low or high temperatures (Li et al., 2010). The role of AsA in countering metal stress is well confirmed by transgenic and mutant studies. AsA and related enzymes such as DHAR have been expressed by genes from *Arabidopsis* in tobacco for inducing greater tolerance to aluminum and thus resulting in elevated AsA concentration in roots after exposure to Al toxicity (Yin et al., 2010). Similarly, coexpression of a Cu-Zn SOD and APX gene lead to an enhanced tolerance to metal and salt stress localized within the chloroplast of tobacco transgene constructs (Lee et al., 2007; Le Martret et al., 2011), indicating that the beneficial effect of increasing DHAR expression can be used in a combinatorial approach with other enzymes involved in oxidative stress. The adverse effects of Cu toxicity treatments on root and shoot growth was partially alleviated by the treatment of test plants with AsA, thiamine (vitamin B1) and salicylic acid (Al-Hakimi and Hamada, 2011).

### **GLUTATHIONE**

Glutathione (GSH), a low molecular weight thiol (γglutamylcysteinylglycine) is one of the most important metabolites of the living systems (Gill and Tuteja, 2010; Noctor et al., 2012). It has a vital role in sulfur metabolism and translocation (Hell, 1997). It is also reported to play a significant role in cellular metabolism and as a reductant in scavenging of radicals in intracellular environment (Gill and Tuteja, 2010). GSH also functions as reactive nucleophiles which help in detoxification of toxins of electrophilic nature. It is also reported to have signaling function which responds to changes in extracellular environment and is known for its role in regulation of gene expression (Sanchez-Fernandez et al., 1997). It is also involved in synthesis of phytochelatins in which it acts a precursor and aids in binding heavy metals (Grill et al., 1989).

Biosynthesis of GSH occurs in two step process. In first reaction, L-glutamate and L-cysteine are converted to γ-glutamylcysteine (γ-EC) with the help of enzyme γglutamylcysteine synthase (γ-ECS). γ-EC is further converted to GSH by addition of glycine by enzyme glutathione synthase (**Figure 2**). Both these reactions are carried in the presence of ATP (Meister, 1988). Inside the cell, GSH is localized usually

in cytoplasm, endoplasmic reticulum, vacuoles, mitochondria, chloroplasts and peroxisomes (Mittler and Zilinskas, 1992; Jimenez et al., 1998). In cellular environment, glutathione is mainly present in reduced state. Hence, oxidized form of glutathione (GSSG) is present in very low proportions. Therefore, under optimum conditions, a high GSH:GSSG ratio is maintained (Mhamdi et al., 2010; Noctor et al., 2011). In stressed conditions, GSH along with ascorbate (AsA) plays a central role in scavenging of ROS.

Many studies have indicated a correlation between levels of H2O2 and glutathione (Noctor et al., 2012). H2O2 metabolism *via* GSH involves three types of peroxidases which are ascorbate peroxidase (APOX), peroxiredoxin (PRX) and glutathione-S-transferases (GSTs). APOX is a class I heme based peroxidase and is specific to H2O2. It is involved in AsA-GSH radical scavenging pathway that uses NADPH to reduce H2O2 *via* AsA-GSH pools (Noctor et al., 2012). In this process, APOX reduces H2O2 to H2O and AsA acts as a reductant which changes to monodehydroascobate (MDHA). MDHA is unstable and is converted to AsA in the presence of enzyme monodehydroascobate reductase (MDHAR) and NADPH. MDHA can also lead to the formation of dehydroascobate (DHA) which further gets reduced to AsA with the help of the enzyme dehydroascobate reductase (DHAR) (Winkler, 1992). The reductant is this case is GSH which gets oxidized to GSSG thereby indicating that GSH has an imperative role in maintaining AsA pool in the cellular environment (Noctor et al., 1998). GSSG is reduced back to GSH by glutathione reductase (GR) in presence of NADPH.

The second type of peroxidases i.e., peroxiredoxins (PRX) are thiol peroxidases which can reduce H2O2 as well as other peroxides thereby indicating their low specificity to H2O2 (Dietz, 2003; Tripathi et al., 2009). PRXs are of four types and out of these, PRX II uses GSH as a reductant while other three either use thioredoxin (TRX) or NADPH-thioredoxin reductase (NTR) (Pulido et al., 2010). Glutathione peroxidases (GPX) are also included in PRXs as they TRX-dependent peroxiredoxins (Iqbal et al., 2006; Navrot et al., 2006). In plants, GPXs are less likely to be involved in peroxide mediated oxidation of GSH and it is suggested that peroxidation could be carried out by GST (Wagner et al., 2002; Dixon et al., 2009). Many GSTs found in plants have been shown to possess GPX like activity. Type I and type III class of GSTs have been identified in many plants and have been reported to actively respond to oxidative stress (Dixon et al., 1998). Some other enzymes such as methionine sulphoxide reductase (MSR) are also reported to carry out ROS-stimulated oxidation of GSH (Tarrago et al., 2009). Hence, reduction of H2O2 and other peroxides by GSH occurs by both AsA-GSH radical scavenging pathway and AsA independent pathway (**Figure 3**).

One of the important roles of GSH is synthesis of phytochelatins. These are organic ligands that have the ability to bind heavy metals and then these metal complexes are transported to vacuole (Cobbett and Goldsbrough, 2002). Enzyme phytochelatin synthase (PCS) catalyzes the formation of phytochelatins either from GSH or homologous bithiols (Ha et al., 1999; Vatamaniuk et al., 1999). During increased concentrations of heavy metals, γ-glutamylcysteine moiety from one GSH molecule and glutamic acid from another GSH molecule are condensed by PCS thereby releasing glycine as residue and forming phytochelatin molecule which are then able to form metal complexes (Clemens, 2006). Hence, GSH and related enzymes play a crucial role in maintaining the homeostasis of cellular environment and protect the plant system from adverse effects of various stresses.

### **TOCOPHEROL**

Tocopherol is found ubiquitously in the plant kingdom and occurs in all the plant parts. It plays a key role in signal transduction pathways and in the gene expression regulation in different processes such as plant defense and export of photoassimilates (Falk and Munne-Bosch, 2010). It acts as a key lipid soluble redox buffer. It is an important scavenger of singlet oxygen species and also scavenges other ROS (Foyer et al., 2005). Tocopherol role is important under the conditions of severe stress (Havaux et al., 2005) and when stress conditions are not severe, other antioxidants play their protective roles. Tocopherol antioxidant activity depends on its ability of donation of its phenolic hydrogen to free radicals. The α-tocopherol has the highest antioxidant activity of all the tocopherol types, the δ-tocopherol has the lowest and the β- and γ-tocopherols has the intermediate activity (Kamal-Eldin and Appelqvist, 1996; Evans et al., 2002). Tocopherol amount is tightly controlled in the photosynthetic membranes to properly regulate the membrane stability. Role of tocopherol in preventing lipid peroxidation has been noticed in many reports. Lipid peroxyl radicals, which are involved in the propagation of lipid peroxidation, are scavenged by tocopherol (Liebler, 1993). It regulates the expression of genes involved in lipid peroxidation (Sattler et al., 2006). Tocopherol deficiency led to an enhancement in the lipid peroxidation in the transgenic tobacco leaves (Abbasi et al., 2009). It was estimated that the one molecule of tocopherol by using resonance energy transfer could degrade 120 singlet oxygen molecules (Fahrenholzt et al., 1974). Hydroperoxydienone intermediates this reaction, which further gives rise to tocopherol quinone and tocopherol quinol epoxides (Murkovic et al., 1997; Kobayashi and Dellapenna, 2008). Oxidized tocopherol can be recycled back to its reduced form. α-tocopherol quinone has been shown to convert back to α-tocopherol in *Arabidopsis thaliana* chloroplasts (Kobayashi and Dellapenna, 2008). Interaction between carotenoids and α-tocopherol also plays significant role during photooxidative stress in plants. Their interaction was shown to protect the photosystem—II of *Chlamydomonas reinhardtii* from the damage of singlet oxygen species under herbicides stress (Trebst et al., 2002). In the membranes, tocopherol can form complexes with polyunsaturated fatty acid (PUFA). OH• oxidizes PUFA and form lipid peroxyl radical from superoxide. Tocopherol gives rise to the formation of lipid hydroperoxide from lipid peroxyl radicals. It is efficient in breaking the chain reactions of free radicals of PUFA produced by lipid peroxidation (Havaux et al., 2005).

Tocopherol works cooperatively with the other antioxidants such as glutathione, ascorbate, carotenoids etc. . . and helps in the maintenance of appropriate redox state inside the chloroplast under various adverse environmental conditions (Munne-Bosch, 2005). Low molecular weight antioxidants such as tocopherol, glutathione, and ascorbate form a triad and provide protection

against abiotic stresses (Szarka et al., 2012). The increase in the amount of these antioxidants can be achieved in two ways i.e., by increasing their biosynthesis or by increasing their biological efficacy by increasing their redox recycling. Their interdependency plays an important role during the electron transfer step inside a cell (Ahmad et al., 2010; Gill and Tuteja, 2010). The increase in the α-tocopherol occurred with the increase in the ascorbate content under the conditions of Cu stress (Collin et al., 2008). Simultaneous loss of glutathione and α-tocopherol more severely affected the photosynthetic apparatus stability and efficiency in *A. thaliana* plants (Kanwischer et al., 2005). Coordinated action of these three antioxidants helps in the maintenance of the redox homeostasis in a more efficient way.

### **THIOREDOXIN**

Thioredoxins are small (12–14 kDa) and low molecular mass proteins, which are involved in cell redox regulation and are ubiquitously present in all organisms from prokaryotes to eukaryotes (Schurmann and Jacquot, 2000). These were firstly discovered in *Escherichia coli* as an electron donor for ribonucleotide reductase, an enzyme required for DNA synthesis (Moore, 1967). There are two distinct families (family I and II) of thioredoxin which are distinguished on the basis of their amino acid sequences. Family I includes proteins that consist of one distinct thioredoxin domain, whereas family II is composed of proteins with one or more thioredoxin domains coupled to additional domains (Gelhaye et al., 2004). Plants contain a comprehensive thioredoxin system and it is divided into six major groups: thioredoxin f, h, m, o, x, and y on the basis of their sequence and are localized in chloroplast, mitochondria, cytosol and even in the nucleus. Among these thioredoxin m, x, and y are related to prokaryotic thioredoxin and f, h, and o are specific to eukaryotic organisms (Gelhaye et al., 2005; Collet and Messens, 2010).

Thioredoxin plays important role in plants as they are involved in multiple processes, such as photorespiration, lipid metabolism, membrane transport, hormone metabolism, and ATP synthesis (Balmer et al., 2004). They also play important role against various environmental stresses and also protects proteins from oxidative aggregation and inactivation (Holmgren, 1995). A Thioredoxin "h" is required during nodule development to reduce the ROS level in soybean roots (Lee et al., 2005).

Role of mitochondrial thioredoxin PsTrxo1 was reported in providing tolerance against the salt stress (Marti et al., 2011). Plants respond against salinity stress by increasing the mitochondrial thioredoxin activity and protect the mitochondria from oxidative stress by stimulating the activities of antioxidative enzymes. Thioredoxin "h" promote the mobilization of carbon and nitrogen of the endosperm early in grain germination (Wong et al., 2002; Shahpiri et al., 2009). Abiotic stresses elevate thioredoxins either on the gene level or on protein level. Data on proteomics study showed that thioredoxins genes were upregulated in rice under Cu stress (Song et al., 2013). Genomic study of rice, revealed the significant differences in the gene expression of thioredoxin under biotic and abiotic stress conditions (Nuruzzaman et al., 2012).

Thioredoxins play fundamental role in plant tolerance of oxidative stress. They are involved in combating the oxidative damage by transferring reducing power to reductases for the detoxification of lipid hydroperoxides and thus repairing the oxidized proteins (Santos and Rey, 2006). They scavenge the ROS by modulating the antioxidative enzymes and also involved in oxidative stress associated signaling pathway through the control of glutathione peroxidase (Vivancos et al., 2005). Further Serrato et al. (2004) reported a role of NADPH thioredoxin reductase (NTR) in plant protection against oxidative stress. Deficiency of NTR caused growth inhibition and hypersensitivity in reponse to salinity stress, whereas NTRC knock-out mutant in *Arabidopsis* expressed the role of thioredoxins against salt stress. Thus, thioredoxins protect the plants from oxidative damage and indicate that thioredoxins involves in antioxidative defense system.

### **PEROXIREDOXINS**

Peroxiredoxins (Prxs) are a group of antioxidative enzymes including catalase, superoxide dismutase, ascorbate peroxidase, and glutathione peroxidase. These were found firstly in barley plants when genes Hv-1-CysPrx and Hv-2-CysPrx were cloned from the plant (Stacy et al., 1996). Later on it was cloned in various other plants such as *Arabidopsis*, *Oryza sativa*, *Riccia fluitans*, *Spinacia oleracea*, *Populus* spp., *Nicotiana tabacum* and *Secale cereal.* Prxs show alike structure with basic protein and a thioredoxin fold, and have molecular mass ranging from 17 to 22 kDa. On the basis of sequence similarity and catalytic mechanisms Prx proteins are classified into four categories- (a) 1-Cys Prx, (b) Prx II, (c) 2-Cys Prx and (d) PrxQ (Rouhier and Jacquot, 2002, 2005; Dietz, 2003).

Abiotic stresses (drought stress, salinity stress, heavy metals etc. . . ) are the prime threat found these days to the plants due to changing climate and industrial revolution. During abiotic stress, ROS production increases and represents a fundamental problem for the regular metabolism of plants. PrxQ, a type of peroxiredoxins have been identified in photosynthetic cells, and was noted to be participating in protection of plants against ROS (Foyer and Noctor, 2005). Decrease in chlorophyll in PrxQ knockout of *A. thaliana* was observed, suggesting its role in protection of photosynthetic enzymes (Lamkemeyer et al., 2006). The expression profile of four *Prx* genes were observed under various stresses such as NaCl, NaHCO3, PEG, CdCl2, and abscisic acid in roots, stems and leaves of *Tamarix hispida.* Enhanced expression of all the *ThPrx* was reported under both NaCl and NaHCO3. Temporal and spatial specificity expression patterns were observed under PEG and CdCl2 stress. ABA treatment has showed different expression of *ThPrxs,* and it point that these *Prxs* are involved in the ABA signaling pathway (Gao et al., 2012). Genes have been identified and characterized by Vidigal et al. (2013) encoding for Prxs in *Vitis vinifera* using quantitative real time PCR under irradiance, heat and water stress. Seven *vvprx* genes were identified, out of which two were more responsive toward water stress, followed by heat stress and without major change under high irradiance. The *vvprxII-2*, a recognized *PrxII* was most responsive toward the heat stress. It was targeted in the chloroplasts and thought to be correlated with abscisic aciddependent thermotolerance. Similarly, *vvprxIIF* was identified and targeted to mitochondria and was responsive to water stress and supposed to involve in drought tolerance through H2O2 signaling. Guan et al. (2014) tried to investigate the protective role of PrxQ during abiotic stress in *Eustoma grandiflorum* Shinn. The capacity of biosynthesis of PrxQ was increased in plant by using the overexpression of the PrxQ gene (SsPrxQ) from *Suaeda salsa*. This SsPrxQ gene was expressed in *E. grandiflorum.* Enhanced antioxidant activity and thioredoxin dependent peroxidase activity was shown by rPrx proteins. Improved tolerance to salt and high light intensity was also noticed due to overexpression of SsPrxQ. It has been reported that in Chinese cabbage under heat shock and oxidative stress, 2-Cys Prx change its protein structure from a low molecular weight to high molecular weight (Kim et al., 2009). Enhanced tolerance to methyl viologen-mediated oxidative stress and high temperature was observed by the overexpression of At2-cys Prx in potato (*Solanum tuberosum*) (Kim et al., 2011). It has been observed by Jing et al. (2006) that tolerance to the salt and cold stress improves with the overexpression of PrxQ from *S. salsa* in *A. thaliana.* Overexpression of PrxQ in transgenic maize indicated the increased stress tolerance against oxidative stress and fungal diseases (Kiba et al., 2005). Similarly, overexpression of an *Arabidopsis* 2-Cys Prx in transgenic tall fescue (*Festuca arundinacea)* showed more resistance against heat and methyl viologen stress in comparison to control plants. In these plants, less electrolyte leakage and thiobarbituric acid-reactive substances (TBARS) were also observed (Kim et al., 2010). A gene VrPrx which encodes for the 2-Cys Prx has been isolated from the mungbean and studied for the antioxidant activity *in vitro.* Overexpression of VrPrx in transgenic *Arabidopsis* showed enhanced antioxidant activities and photosynthetic efficiency under abiotic stress (Cho et al., 2012).

### **GLUTAREDOXIN**

Glutaredoxins (GRX) are omnipresent proteins of approximately 100 amino-acid residues (Fernandes et al., 2005). Glutaredoxins act as redox enzymes to catalyze the reduction of disulfides by using reduced glutathione (GSH) as a cofactor (Holmgren, 1988, 1989; Holmgren and Gleason, 1988). Glutaredoxins get oxidized by substrates, and are reduced non-enzymatically by glutathione. There is no explicit oxidoreductase to reduce glutaredoxins therefore; oxidation of glutathione is required to reduce the glutaredoxins. The oxidized glutathione is then renewed by GR and these mechanisms collectively make up the glutathione system (Holmgren and Fernandes, 2004). The line of function of glutaredoxin is analogous to thioredoxin. GRX holds an active center disulfide bond. According to their redox-active center, they are subgrouped in six classes of the CSY[C/S]-, CGFS-, CC-type, and three groups with additional domain of unknown function. The CC-type GRXs are only found in higher plants (Nilsson and Foloppe, 2004). In *A. thaliana*, about 30 GRX isoforms are discovered whereas 48 are discovered in *O. sativa* L. (Rouhier et al., 2008). GRX operates in antioxidant defense by reducing dehydroascorbate, peroxiredoxins, and methionine sulfoxide reductase. The glutathione/glutaredoxin system is one of the important cellular factors that have been implicated in the regulation of redox homeostasis (Grant, 2001).

GRX can be engineered to attain enhanced oxidative stress tolerance in plants and using the transgenic plants to investigate redox-controlled processes in temperature stress tolerance. Transgenic expression of fern *Pteris vittata* glutaredoxin PvGrx5 in *A. thaliana* increases plant tolerance to high temperature stress and reduces oxidative damage to proteins (Sundaram and Rathinasabapathi, 2010). It is observed that homozygous lines expressing PvGRX5 possess considerably better tolerance to high temperature stress than the vector control and wild-type, and this is related to leaf glutaredoxin specific activities. Cheng et al. (2011) reported that *Arabidopsis* monothiol glutaredoxin, AtGRXS17, is critical for temperature-dependent postembryonic growth and development via modulating auxin response. Further, AtGRXS17 has played role in anti-oxidative stress and thermotolerance in both yeast and plants (Wu et al., 2012). Ectopic expression of Arabidopsis glutaredoxin AtGRXS17 increases thermotolerance in tomato. Ectopic expression of AtGRXS17 in tomato plants reduces photo-oxidation of chlorophyll and decrease oxidative injury of cell membrane systems under heat stress (Wu et al., 2012).

A glutaredoxin gene SlGRX1 regulates plant responses to oxidative, drought and salt stresses in tomato (Guo et al., 2010). A novel cDNA fragment (SlGRX1) from tomato was isolated and characterized. This fragment encoded a protein containing the consensus GRX family domain with a CGFS active site. SlGRX1 was articulated in all places in tomato including root, stem, leaf, and flower. The expression of SlGRX1 could be induced by oxidative, drought, and salt stresses. Enhancement in sensitivity to oxidative and salt stresses with reduced relative chlorophyll content, and decreased tolerance to drought stress with decreased relative water content were observed after applying virus-induced gene silencing of SlGRX1 in tomato. Quite the opposite, resistance of plants to oxidative, drought, and salt stresses increased considerably by over-expression of SlGRX1 in Arabidopsis plants. The study clearly suggested that the glutaredoxin gene SlGRX1 plays an important role in regulating abiotic tolerance against oxidative, drought, and salt stresses. GRXs also attribute to the high tolerance of in *Caulobacter crescentus* to heavy metals specifically cadmium and chromate (Hu et al., 2005). A GRX of the fern *P. vittata* PvGRX5 is involved in arsenic tolerance (Sundaram and Rathinasabapathi, 2010). It acts as a sensor of oxidative stress mediated by H2O2 (Song et al., 2002).Glutaredoxin GRXS13 plays a key role in protection against photo-oxidative stress in *Arabidopsis* as its expression reduces the photo-oxidative stress generated free radicals (Laporte et al., 2012).

### **NAD/NAD(P)**

Nicotinamide adenine dinucleotide (NAD) and its derivative nicotinamide adenine dinucleotide phosphate (NADP) are pyridine nucleotide coenzymes that act as cardinal metabolites involved in plant cellular redox homeostasis (Hashida et al., 2009). They occur ubiquitously in all living cells (Noctor et al., 2006). These coenzymes occur as redox couples, NAD+/NADP+ are oxidized forms and there counter reduced forms are NADH/NADPH. The ratio of oxidized to reduced form i.e., NAD(P)+/NAD(P)H is known as redox state of a cell and is important signal connecting metabolic state of cell and its gene expression (Schafer and Buettner, 2001; Jambunathan et al., 2010). NADH plays central role as electron shuttle between TCA cycle and mitochondrial electron transport chain. NADP+/NADPH acts as important energy storage and transferring molecule in light and dark photosynthetic reactions. NADH and NADPH also act as reducing equivalents in various catabolic and anabolic processes like nucleic acid and lipid synthesis (Potters et al., 2010). Besides their role as cofactors in energy producing and other metabolic reaction they play key role in redox signaling associated with stress and development by modulating both ROS generation and ROS scavanging (Noctor et al., 2006; Hashida et al., 2009). ROS scavenging is also partly maintained by ascorbate-glutathione cycle and NADP(H) maintains redox flux in this cycle (Noctor et al., 2006). NAD also regulates cellular processes like calcium signaling via NAD derived cyclic ADP-ribose and transcription and microtubule metabolism via deacetylation and/or mono/poly(ADP-ribosy)lation (Hashida et al., 2009).

As, NAD(H) and NADP(H) play discrete physiological roles, maintenance of balance between NAD(H)/NADP(H) is essential for cell survival under normal and stress conditions (Takahara et al., 2010). NADP is generated by adenosine triphosphate (ATP) dependent phosphorylation of NAD catalyzed by NAD kinases (NADK). Decline in levels of pyridine nucleotide as caused under stress induces NAPK which in turn increases NADP(H) levels at expense of NAD(H) (Grose et al., 2006). In *Arabidopsis,* oxidative stress caused by stressed environmental conditions induces expression of NADK1 and NADK3 gene (Berrin et al., 2005; Chai et al., 2005).

Biotic and abiotic stresses cause oxidative stress in plants due to over accumulation of ROS. Oxidative stress may cause damage to organelles, lower antioxidant levels, oxidize proteins, DNA nicking, and ultimately leading to cell death. Among various defense responses against ROS is plants is poly(ADPribosyl)ation (PAR) reaction (Ishikawa et al., 2009). PAR is a post-translational protein modification catalyzed by poly(ADP-Rib)polymerase (PARP) utilizing NAD+ and ATP. PARP catalyzes addition of branched polymers of ADP-Rib on a target protein synthesizing a protein-bound poly(ADP-ribose). These PARP proteins confers resistance to oxidative stress by regulating important cellular processes such as DNA synthesis and repair, chromatin synthesis, cell death, and stress responses to genotoxic stress (Noctor et al., 2006; Ogawa et al., 2009). One of the early responses toward DNA damage caused due to oxidative stress is activation of PARP (Ame et al., 1999). Plant possesses two PARP genes; *parp1* and *parp2* are induced under oxidative stress (Block et al., 2005). Level of PARP induced in plants under stress is directly proportional to severity of stress (Ha and Snyder, 1999). Since these defense responses like PAR over consumes NAD(P), they lead to depletion of NAD concentration. Inhibition of PARP alleviates NAD depletion and ATP consumption diminishing cell death and enhanced tolerance to abiotic stresses (Noctor et al., 2006). Silencing of PARP in *Arabidopsis* and oil seed produced lines that were resistant to broad spectrum of abiotic stresses due to reduce NAD+ consumption and alteration in abscisic levels (Block et al., 2005; Vanderauwera et al., 2007).

Nudix (nucleoside diphosphates linked to some moiety X) hydrolases, hydrolyse nucleoside diphosphate derivates. They act house-cleaning enzymes and play role in maintains PAR and NAD(H) homeostasis (Ge and Xia, 2008). Twenty nine hydrolases have been identified in *A. thaliana* (Kraszewska, 2008). In *A. thaliana* overexpression of AtNUDX2 enhanced tolerance toward oxidative stress by hydrolyzation of ADP-ribose thereby maintaining NAD+ and ATP levels (Ogawa et al., 2009). Similarly AtNUDT7 is found to play an important role in maintaining redox homeostasis by regulating balance between NADH and NAD+ via modulating PAR reaction. Thus, it regulates defense/stress signaling and cell death pathways under oxidative stress (Ishikawa et al., 2009; Jambunathan et al., 2010). In conclusion, NAD/NADP are involved in several signaling pathways that are colligative with stress tolerance and defense reactions.

### **CONCLUSION**

Aerobic life possesses a worldwide characteristics of redox signal transduction honed through evolution to poise information from metabolism and the environment. Information regarding plant health, principally in terms of strength for defense is fulfilled by both oxidants and antioxidants signaling. Between plant cell stress perception and physiological responses, antioxidants play significant role as a signaling compounds as they also possess a vibrant metabolic interface. Redox homeostasis managers set thresholds for apoplastic and cytoplasmic signaling also act as intermediary of the intracellular redox potential.

### **REFERENCES**


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varieties differing in Cu tolerance. *Plant Soil* 366, 647–658. doi: 10.1007/s11104- 012-1458-2


the photosynthetic electron transport chain but independent of sugars in Arabidopsis. *J. Exp. Bot*. 58, 2661–2671. doi: 10.1093/jxb/erm124


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 21 December 2014; accepted: 12 February 2015; published online: 04 March 2015.*

*Citation: Kapoor D, Sharma R, Handa N, Kaur H, Rattan A, Yadav P, Gautam V, Kaur R and Bhardwaj R (2015) Redox homeostasis in plants under abiotic stress: role of electron carriers, energy metabolism mediators and proteinaceous thiols. Front. Environ. Sci. 3:13. doi: 10.3389/fenvs.2015.00013*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2015 Kapoor, Sharma, Handa, Kaur, Rattan, Yadav, Gautam, Kaur and Bhardwaj. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Redox homeostasis via gene families of ascorbate-glutathione pathway

Prachi Pandey 1, 2 \*, Jitender Singh2, 3, V. Mohan Murali Achary <sup>2</sup> and Mallireddy K. Reddy <sup>2</sup>

*<sup>1</sup> Plant Microbe Interaction Lab, National Institute of Plant Genome Research, New Delhi, India, <sup>2</sup> Plant Molecular Biology, International Center for Genetic Engineering and Biotechnology, New Delhi, India, <sup>3</sup> School of Life Sciences, Jawaharlal Nehru University, New Delhi, India*

The imposition of environmental stresses on plants brings about disturbance in their metabolism thereby negatively affecting their growth and development and leading to reduction in the productivity. One of the manifestations of different abiotic and biotic stress conditions in plants is the enhanced production of reactive oxygen species (ROS) which can be hazardous to cells. Therefore, in order to protect themselves against toxic ROS, plant cells employ the anti-oxidant defense system. The ascorbate-glutathione pathway (Halliwell-Asada cycle) is an indispensible component of the ROS homeostasis mechanism of plants. This pathway entails the antioxidant metabolites: ascorbate, glutathione and NADPH along with the enzymes linking them. The ascorbate-glutathione pathway is functional in different subcellular compartments and all the enzymes of this pathway exist as multiple isoforms. The expression of different isoforms of the enzymes of ascorbate-glutathione pathway is developmentally as well as spatially regulated. Moreover, various abiotic and biotic stress conditions modulate the expression of the enzyme- isoforms differently. It is the intricate regulation of expression of different isoforms of the ascorbate-glutathione pathway enzymes that helps in the maintenance of redox balance in plants under various abiotic and biotic stress conditions. The present review provides an insight into the gene families of the ascorbate-glutathione pathway, shedding light on their role in different abiotic and biotic stress conditions as well as in the growth and development of plants.

Keywords: reactive oxygen species, abiotic stress, redox homeostasis, ascorbate-glutathione pathway, isoforms, gene families

### Introduction

When plants are exposed to various biotic and abiotic stresses, they exhibit characteristic increase in the production of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide (O•− 2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH• ) (Mittler et al., 2004). These ROS are capable of causing uncontrolled oxidation of various cellular components that can lead to oxidative damage of the cell (Asada, 1999; Dat et al., 2000). Thus, enhanced production of ROS during stress can be hazardous to cells. ROS have also been acknowledged as central players in complex signaling cascades as they act as signals for the activation of various stressresponsive and defense pathways (Knight and Knight, 2001; Mittler et al., 2011). Apart from playing important roles in stress signaling, ROS like H2O<sup>2</sup> are also involved in plants' growth and developmental processes like differentiation of cellulose rich cell wall, mediation of aleuronic

#### *Edited by:*

*Naser A. Anjum, University of Aveiro, Portugal*

#### *Reviewed by:*

*Brahma B. Panda, Berhampur University, India Ashwani Pareek, Jawaharlal Nehru University, India Dibyendu Talukdar, Raja Peary Mohan College (Affiliated to University of Calcutta), India*

#### *\*Correspondence:*

*Prachi Pandey, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, Delhi 110067, India prachipndy@gmail.com*

#### *Specialty section:*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science*

> *Received: 18 January 2015 Accepted: 13 March 2015 Published: 31 March 2015*

#### *Citation:*

*Pandey P, Singh J, Achary VMM and Reddy MK (2015) Redox homeostasis via gene families of ascorbate-glutathione pathway. Front. Environ. Sci. 3:25. doi: 10.3389/fenvs.2015.00025* cell death and stimulation of somatic embryogenesis (Neill et al., 2002; Slesak et al., 2007). Additionally, the transient accumulation of H2O<sup>2</sup> following pathogen infection leads to localized programmed cell death or hypersensitive (HR) response and stimulates crosslinking of cell wall proteins, thereby preventing pathogen spread in the plant (Grant and Loake, 2000; Kuzniak and Skłodowska, 2005). Considering the ambivalent role of ROS, a delicate balance between their production and scavenging is of utmost importance for proper growth and development of plants.

Plants have an efficient anti-oxidant defense system which scavenges the excess ROS produced in the cell under different oxidative stress conditions. The anti-oxidant safe guard system in plants comprises of non-enzymatic and enzymatic components (Noctor and Foyer, 1998; Scandalios, 2005). The nonenzymatic components include the major cellular redox buffers: ascorbate (AsA) and glutathione (γ-glutamyl-cysteinyl-glycine, GSH) as well as tocopherol, flavonoids, alkaloids, and carotenoids (Arora et al., 2000; Grace and Logan, 2000; Foyer and Noctor, 2003; Gomathi and Rakkiyapan, 2011). The enzymatic components of the anti-oxidative defense system consist of a number of anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX) and the enzymes of ascorbate-glutathione (AsA-GSH) cycle namely, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) (Mittler et al., 2004; Scandalios, 2005). AsA-GSH cycle forms the main H2O2-detoxification system operating in cytosol, chloroplasts and mitochondria of plant cells (Noctor and Foyer, 1998; Shigeoka et al., 2002; Mittler et al., 2004). Since the discovery of the AsA-GSH cycle in the mid-1970s, the enzyme-catalyzed reactions of this pathway have been studied intensively (Noctor and Foyer, 1998; Asada, 1999; Polle, 2001). Studies with mutants and transgenic plants over- or under-expressing enzymes or metabolites of the AsA-GSH pathway have proved very well the co-relation between this pathway and stress tolerance (Gill and Tuteja, 2010). The AsA-GSH cycle not only combats oxidative stress, but also plays an important role in other plant developmental processes (Chen and Gallie, 2006; Eastmond, 2007).

Each enzyme of the AsA-GSH pathway has various subcellular isoforms, which differ from each other with respect to their spatial and temporal expression. Moreover, these isoforms are differentially regulated by different types of stresses. For example, it has been found that under salt stress, various Oryza sativa APX (OsAPX) isoforms are differentially regulated. While some of them are characteristically up-regulated, the others are down-regulated at the same time (Texeira et al., 2006; Yamane et al., 2010). This suggests that the expression of different isoforms of the AsA-GSH pathway enzymes is under intricate regulation. However, the mechanisms underlying the regulation of these isoforms are not completely understood. The present review provides an overview of gene families encoding AsA-GSH pathway in plants and imparts an insight into their role in conferring tolerance to various abiotic and biotic stresses. A brief discussion on the functional importance of this pathway in growth and development of plants is also provided.

### The Ascorbate-Glutathione (AsA-GSH) Pathway

The AsA-GSH pathway is composed of four enzymes namely, APX, MDHAR, DHAR and GR (**Figure 1**) and two anti-oxidants, AsA and GSH. APX, which is the first enzyme of the cycle, detoxifies H2O<sup>2</sup> by bringing about the peroxidation of AsA and yielding monodehydroascorbate radical (MDHA). MDHA is either directly reduced back to AsA by MDHAR or undergoes non-enzymatic disproportionation to AsA and dehydroascorbate (DHA). The next step of the cycle involves DHAR mediated

reduction of DHA into AsA using GSH as the reductant (**Figure 1**). DHA can undergo irreversible hydrolysis to 2, 3 diketogulonic acid, if not reduced by DHAR. Thus, DHAR helps in regeneration of AsA and plays an important role in maintaining the cellular AsA pool (Gallie, 2012). Like AsA, the regeneration of GSH is also important. GSH is regenerated from the oxidized glutathione dimers (GSSG) by NADPH-dependent GR (Gill and Tuteja, 2010). The concentration of AsA and GSH varies in different subcellular compartments of the cell (**Table 1**). Both the redox buffers are known to accumulate in certain cellular compartments under different abiotic stress conditions. The compartment specific role of both the buffers under abiotic stress conditions has been discussed exhaustively in recent reviews (Foyer and Noctor, 2011; Gest et al., 2013; Zechmann, 2014), and we do not focus on this aspect in the present review. The AsA-GSH cycle not only detoxifies toxic H2O<sup>2</sup> but also contributes toward the maintenance of cellular AsA and GSH pools in different compartments of the cell. Existing in all the organelles, the AsA-GSH pathway protects the cell from the toxic effects of ROS generated under a variety of abiotic and biotic stresses (Anjum et al., 2010, 2014; Gill and Tuteja, 2010) (**Figure 2**).

### Ascorbate Peroxidase

APX (EC 1.11.1.11) is the first enzyme of the AsA-GSH pathway. It catalyzes the conversion of H2O<sup>2</sup> to H2O and O<sup>2</sup> using AsA as specific electron donor (Asada, 1999). APX, thus, prevents the accumulation of toxic levels of H2O<sup>2</sup> in the cell. APX belongs to class I peroxidase family of proteins which are characterized by the presence of heme prosthetic groups. APXs are extremely sensitive to the concentration of AsA, which is reflected by the rapid decline in their activity at very low concentration (less than 20µM) of AsA (Shigeoka et al., 2002). The enzyme has been identified in a number of higher plants, algae and cyanobacteria (reviewed in Caverzan et al., 2012). APX gene family comprises of different isoenzymes with different characteristics. Till date, five APX isoforms namely, cytosolic, mitochondrial, peroxisomal/glyoxysomal and chloroplastic have been identified in plants (D ˛abrowska et al., 2007). In Arabidopsis thaliana, the reported eight isoenzymes of APX can be categorized into three groups: soluble cytosolic (APX1, APX2,



*The intracellular AsA and GSH levels in young rosette leaves of Arabidopsis thaliana plants determined using AsA and GSH specific immunogold labeling (Gest et al., 2013; Zechmann, 2014).*

and APX6), microsome–membrane bound (APX3, APX4, and APX5) and chloroplastic (sAPX and tAPX) (D ˛abrowska et al., 2007; Panchuk et al., 2002) (**Table 2**). Similarly, the identification of APX gene family in Lycopersicum esculentum revealed the presence of seven APX genes: three cytosolic, two peroxisomal, and two chloroplastic (Najami et al., 2008). In O. sativa, eight members of the APX gene family have been reported; encoding two cytosolic, two peroxisomal, three chloroplastic, and one mitochondrial isoforms (Texeira et al., 2004, 2006). Mitochondrial APX isoforms have also been reported in Solanum tuberosum and Pisum sativum (Jimenez et al., 1997; Leonardis et al., 2000).

The APX isoforms are stress sensitive and are regulated by nearly all kinds of abiotic and biotic stress conditions (Shigeoka et al., 2002). The expression of APX isoforms can be activated by specific factors such as pathogen attack, mechanical pressure, injury, ultraviolet (UVB) radiation, water deficiency, salt stress, low or high temperature, atmospheric pollution, and excess metal ions (reviewed in Shigeoka et al., 2002; D ˛abrowska et al., 2007). The stress conditions also modulate the kinetic properties of the enzyme. For example, the exposure of A. thaliana wild type and flavonoid mutant (tt5) plants to UVB radiation led to a significant decrease in KAsA <sup>m</sup> as well as synthesis of new isoforms of cytosolic APX (Rao et al., 1996). The over-expression of APX has been shown to confer tolerance to various abiotic stresses (Xu et al., 2008; Sun et al., 2010; Sato et al., 2011). For example, Jatropha curcas plants over-expressing a chloroplastic APX were found to be salt tolerant (Liu et al., 2014). Similarly, over-expression of the peroxisomal APX from the halophyte Salicornia brachiata conferred salt and drought stress tolerance to transgenic Arachis hypogea plants (Singh et al., 2014). Transgenic L. esculentum plants over-expressing cytosolic APX exhibited improved tolerance to chilling, salinity, heat and UVB stress (Wang et al., 2005, 2006). A. thaliana vtc mutants deficient in AsA are reported to be hypersensitive to drought stress (Pastori et al., 2003; Faize et al., 2011).

### Monodehydroascorbate Reductase

MDHAR (EC 1.6.5.4) recycles MDHA molecules into AsA. The exposure of plants to environmental stress conditions like high light leads to very quick oxidation of AsA to MDHA in chloroplast (Polle, 2001). It is, therefore, necessary for the survival of plants that MDHA is reduced back to regenerate AsA. In chloroplast, MDHA is reduced to AsA by photoreduced ferredoxin at a high rate and this is likely to constitute the main pathway of AsA regeneration in the vicinity of the thylakoid membrane. Away from the thylakoid membrane, reduction of MDHA can occur via two enzymes in the AsA-GSH pathway; DHAR and MDHAR (Asada, 1999). MDHAR reduces MDHA directly by using NAD(P)H as an electron donor. Alternatively, two molecules of MDHA can react non-enzymatically to generate AsA and DHA. The majority of MDHA is, however, found to be reduced by MDHAR (Polle, 2001). MDHAR enzyme activity is found across the entire plant and animal kingdom. Plant MDHARs exhibit high level of sequence similarity with

prokaryotic flavoenzymes. MDHAR activities are reported to be present in algae (Haghjou et al., 2009), bryophytes (Lunde et al., 2006) and in all higher plants (Yoon et al., 2004; Leterrier et al., 2005). Higher plants' MDHARs belong to a multigene family constituting several sub-cellular isoforms. MDHAR activity has been detected in several cell compartments, such as chloroplasts, mitochondria, peroxisomes and cytosol (Jimenez et al., 1997; López-Huertas et al., 1999; Mittova et al., 2003; Kavitha et al., 2010). In A. thaliana, six isoforms of MDHAR are present among which two are peroxisomal, two are cytosolic and one is dually targeted chloroplastic/mitochondrial isoform (Lisenbee et al., 2005) (**Table 2**). The L. esculentum MDHAR family consists of three isoforms (Stevens et al., 2007). A total of three cytosolic isoforms of MDHARs have been reported in the moss Physcomitrella patens (Lunde et al., 2006). Physcomitrella apparently lacks any chloroplastic isoform indicating that

AsA reduction in the plant exclusively occurs in cytosol (Drew et al., 2007).

In order to protect against the deleterious effects of ROS, the AsA pools are required to be maintained in a reduced state. Thus, ascorbate reductases like MDHARs, which are responsible for the reduction of AsA have considerable roles in stress tolerance. The activity of MDHAR proteins as well as MDHAR gene expression has been found to be differentially affected by various stress conditions. The increase in MDHAR activity has been reported in stress conditions like salinity, high light, UV radiation, boron toxicity and low temperature (Mittova et al., 2003; Cervilla et al., 2007). Transgenic studies have also confirmed the vital role of this enzyme in conferring tolerance to various abiotic stresses. For example, over-expression of A. thaliana MDHAR in Nicotiana tabacum enhanced tolerance of transgenic plants to ozone, salt and dehydration stress (Eltayeb et al.,



*The table enlists the representative members of the gene families encoding AsA-GSH pathway with their corresponding locus names, localization details and role in abiotic and biotic stress tolerance.*

2007). The over-expression of Acanthus ebracteatus cytoplasmic and Malpighia glabra chloroplastic MDHAR genes improved salt stress tolerance in O. sativa and N. tabacum, respectively (Eltelib et al., 2012; Sultana et al., 2012). Similarly, over-expression of chloroplastic MDHAR from L. esculentum and Avicennia marina, respectively, was shown to confer resistance to high temperature and methyl viologen-mediated oxidative stress in transgenic L. esculentum (Li et al., 2010) and to salt stress in transgenic N. tabacum plants (Kavitha et al., 2010).

### Dehydroascorbate Reductase

AsA, which is a major anti-oxidant in plants, is oxidized to DHA via successive reversible electron transfers with MDHA as a free radical intermediate. DHA, so produced, is reduced to AsA by DHAR with GSH as an electron donor (EC 1.8.5.1). DHAR is the key enzyme to regenerate AsA. DHARs have been isolated and characterized from higher plants like A. thaliana, N. tabacum and agricultural crops such as oleracea, O. sativa and Pennisetum glaucum (Urano et al., 2000; Shimaoka et al., 2000; Ushimaru et al., 2006; Pandey et al., 2014). In A. thaliana five different DHARs (At1g19550, At1g19570, At1g75270, At5g36270, At5g16710) have been identified, with their presence either in an organelle (chloroplast or mitochondrion) or in the cytosol (Chew et al., 2003) (**Table 2**). Recently the At1g19570 isoform has been found to be associated with peroxisomes (Kataya and Reumann, 2010). Two different DHAR isoforms have been discovered in Spinacia oleracea leaves with one isoform located in chloroplasts whereas the other being present in the sub-cellular compartment other than chloroplasts (Shimaoka et al., 2000). DHAR activity has also been found in mitochondria, chloroplasts and peroxisomes of both leaf and root cells of the cultivated L. esculentum (M82) and its wild salt-tolerant relative, L. pennellii (Lpa) (Mittova et al., 2000). Two DHAR genes encoding for cytosolic and chloroplastic DHARs have also been identified in Eucalyptus spp. (Teixeira et al., 2005).

DHAR also plays an important role in abiotic stress tolerance and its expression is activated by a number of abiotic stress factors (Ali et al., 2005; Lu et al., 2008; Fan et al., 2014). Moreover, enhanced tolerance to various abiotic stresses was observed in Pandey et al. Ascorbate-glutathione pathway and stress tolerance

plants over-expressing DHAR (Kwon et al., 2003; Ushimaru et al., 2006; Wang et al., 2010). For example, the over-expression of A. thaliana cytosolic DHAR has been shown to impart tolerance to aluminum stress in transgenic N. tabacum plants (Yin et al., 2010). In yet another report, it was shown that the over expression of DHAR which led to enhanced AsA accumulation conferred oxidative and salt stress tolerance to L. esculentum plants (Li et al., 2012). The simultaneous expression of chloroplastic O. sativa DHAR and E. coli GR in N. tabacum plants resulted in enhanced tolerance to salt and cold stress (Le Martret et al., 2011).

Additionally, DHAR plays an important role in plant growth and development (Chen and Gallie, 2006). The lack of DHAR resulted in the quick loss of AsA from O. sativa plants and led to slower rate of leaf expansion consequently affecting plant growth and development (Ye et al., 2000).

### Glutathione Reductase

GR (NADPH: oxidized glutathione oxidoreductase; EC 1.6.4.2) maintains the cellular redox state by regenerating the reduced form of GSH, thereby, maintaining the balance between reduced GSH and AsA pools (Noctor and Foyer, 1998; Reddy and Raghavendra, 2006). GR is a flavo-protein oxidoreductase ubiquitously present in both prokaryotes and eukaryotes (Romeo-Puertas et al., 2006). The protein has been purified and characterized from a variety of organisms (Rao and Reddy, 2008; Achary et al., 2014). Although localized mainly in the chloroplasts, the enzyme is also found in cytosol (Edwards et al., 1990), mitochondria and peroxisomes (Jimenez et al., 1997; Romeo-Puertas et al., 2006).

Multiple isoforms of GR have been reported in a number of plants (Edwards et al., 1990; Lascano et al., 2001; Contour-Ansel et al., 2006; Rao and Reddy, 2008; Trivedi et al., 2013). Modulation in the expression profile of various GR isoforms have been known to occur under various stress conditions (reviewed in Yousuf et al., 2012; Gill et al., 2013). Transgenic N. tabacum plants over-expressing E. coli GR in the cytoplasm and chloroplast exhibited enhanced GR activity and tolerance to methyl viologen-mediated oxidative stress (Aono et al., 1991, 1993). Similarly, the over-expression of GR in chloroplasts of N. tabacum plants led to enhanced accumulation of GSH and AsA and the transgenic plants were found to be more tolerant to high light and chilling stress (Foyer et al., 1995). Overproduction of chloroplastic GR led to reduced photoinhibition under chilling stress in transgenic Gossypium hirsutum plants (Kornyeyev et al., 2003). Transgenic N. tabacum plants with reduced expression of GR were shown to display enhanced sensitivity to oxidative stress (Ding et al., 2009).

### AsA-GSH Pathway in Chloroplasts

The AsA-GSH cycle plays a critical role in maintaining ROS homeostasis in chloroplasts. These organelles are devoid of catalases and the AsA-GSH cycle acts as the major H2O<sup>2</sup> metabolizing pathway in these photosynthetic organelles. The photoreduction of O<sup>2</sup> in chloroplast via photosystem–I (PSI) leads to the formation of superoxide ions, which rapidly dismutate to H2O<sup>2</sup> spontaneously or by the action of superoxide dismutases (Asada, 1999). Chloroplasts contain relatively higher levels of AsA and GSH as compared to the other sub cellular organelles (Noctor and Foyer, 1998; Gest et al., 2013; Zechmann, 2014). Thus, the AsA-GSH pathway in chloroplast is imperative in protecting it from the deleterious effects of excess ROS production. Among the four enzymes of the AsA-GSH pathway in chloroplasts, the chloroplastic APX (chAPX) which consists of thylakoid (tAPX) and stromal (sAPX) isoforms scavenges the H2O<sup>2</sup> generated during photosynthesis. The stromal and thylakoid-bound APXs have been identified and purified from several plant species (Ishikawa et al., 1996, 1998). tAPX is characterized by the presence of an extended C-terminal sequence that makes it 5 KDa larger than the sAPX (Asada, 1999). This sequence is responsible for binding of the protein to the membrane. sAPX has been shown to be predominantly important for photo-protection in young leaves. tAPX and sAPX isoforms are apparently functionally redundant and contribute to oxidative stress tolerance in chloroplasts. A sudden exposure to high light stress in tapx and sapx double mutant of A. thaliana led to a characteristic decline in the photochemical efficiency of PSII (Kangasjärvi et al., 2008). Likewise, the over-expression of tAPX in N. tabacum plants helped in maintaining photosynthetic efficiency of plants under high light and low temperature stress, thereby, substantiating the role of chloroplastic APX in stress resistance (Yabuta et al., 2002). The MDHA formed in the lumen by the oxidation of AsA disproportionates to DHA and moves into the stroma through the thylakoid membrane. MDHA produced by both stromal and thylakoid bound APX isoforms is reduced by stromal MDHAR. MDHAR has not been reported in the lumen of chloroplast (Obara et al., 2002). Along with the regeneration of AsA from MDHA, chloroplastic MDHAR also brings about the photo-reduction of dioxygen to O •− 2 in absence of MDHA (Miyake et al., 1998; López-Huertas et al., 1999). DHAR and GR activities convert the DHA translocated from the lumen and the DHA generated in the stroma to AsA (Asada, 1999).

### AsA-GSH Pathway in Mitochondria

The presence and activity of AsA-GSH cycle enzymes in mitochondria of plant cells have been established, and this cycle plays an important role in protecting mitochondrion against the toxic ROS regularly produced in respiratory chain reactions (Leonardis et al., 2000; Chew et al., 2003; Mittova et al., 2004; Lázaro et al., 2013). The mitochondrial AsA-GSH cycle deals with both photosynthetic as well as stress-induced oxidative stress (Jimenez et al., 1997). The mitochondrial AsA-GSH cycle also plays an important role in eliminating the mitochondrial-derived radicals, thereby protecting the heme of leghemoglobin in N2-fixing legume root nodules (Iturbe-Ormaetxe et al., 2001; Loscos et al., 2008). The mitochondrial APX is known to be membrane-localized in plants (Leonardis et al., 2000; Iturbe-Ormaetxe et al., 2001). The best collective evidence for the presence of MDHAR, DHAR, and GR in mitochondria is from P. sativum leaves (Jimenez et al., 1997) and Phaseolus valgaris nodules (Iturbe-Ormaetxe et al., 2001).

### AsA-GSH Pathway in Cytoplasm

In A. thaliana, the cytosolic AsA-GSH pathway is characterized by the presence of one cytosolic APX (APX1), with an additional stress inducible APX (APX2) (Panchuk et al., 2002), along with the other enzymes (Mittler et al., 2004). It has been shown that the cytosolic APX imparts cross compartment protection of the other sub-cellular organellar APXs like mitochondrial APX, thylakoidal and stromal APXs hinting toward the fact that cytosolic AsA-GSH pathway plays an important role in protecting the other organelles during periods of stress (Davletova et al., 2005). Notably, cytosolic APX accounts for up to 0.9% of the total soluble protein of nodules and is particularly abundant in infected cells and nodule parenchyma of Medicago sativa (Dalton et al., 1998).

### AsA-GSH Pathway in Peroxisome

Peroxisomes are single membrane-bound subcellular organelles being involved in production as well as the degradation of H2O<sup>2</sup> and are sites for photorespiration, fatty acid β-oxidation, glyoxylate cycle and ureide metabolism (Corpas et al., 2001; Mano and Nishimura, 2005). The four enzymes of the AsA-GSH cycle, APX, MDHAR, DHAR and GR have been reported to be expressed in peroxisomes of roots and leaves of P. sativum and L. esculentum (Jimenez et al., 1997; Mittova et al., 2000; Leterrier et al., 2005). The presence of reduced AsA and GSH, and their oxidized forms, DHA and GSSG, respectively, was demonstrated by high performance liquid chromatography (HPLC) analysis in intact peroxisomes of P. sativum leaves (Jimenez et al., 1997). cDNAs encoding peroxisomal APX have been isolated from Gossypium spp. (Bunkelmann and Trelease, 1996), A. thaliana (Zhang et al., 1997) and S. oleracea (Ishikawa et al., 1998). The deduced amino acid sequence of peroxisomal APX has a high degree of identity with cytosolic APX, but it has a C-terminal amino acid extension containing a single, putative membrane−spanning region (Mullen et al., 1999). DHAR and GR were also found in the soluble fraction of peroxisomes, whereas membrane bound APX proteins have been shown to be present in P. sativum, Cucurbita maxima, and L. esculentum (Yamaguchi et al., 1995; Bunkelmann and Trelease, 1996; López-Huertas et al., 1999).

### Role of Gene Families of AsA-GSH Pathway in Abiotic Stresses

### Drought Stress

Drought stress leads to the production of ROS (mainly H2O2) in chloroplasts and mitochondria of plant cells (Dat et al., 2000). Drought stress causes varied effects on the enzymes of the AsA-GSH cycle, the response being dependent on the plant species, the developmental and metabolic state of plant, and the duration and intensity of the stress (Sofo et al., 2010). In majority of cases, drought stress led to an increase in the activity of enzymes of AsA-GSH cycle (Reddy et al., 2004; Sofo et al., 2005; Pukacka and Ratajczak, 2006; Bian and Jiang, 2009). For example, desiccation of recalcitrant seeds of Acer saccharinum was characterized by increased O<sup>−</sup> 2 and H2O<sup>2</sup> production, elevation in AsA and GSH contents as well as increased activity of the AsA-GSH enzymes (Pukacka and Ratajczak, 2006). Similarly, subjecting five Morus alba cultivars to drought stress led to an increase in the activity of AsA-GSH cycle enzymes (Reddy et al., 2004). During prolonged drought treatment in Prunus spp, the activities of the AsA-GSH enzymes were up-regulated, AsA/DHA ratio was decreased and the ratio of GSH/GSSG was increased suggesting an important role of the AsA-GSH pathway in combating drought stress (Sofo et al., 2005). Polyethylene glycol (PEG) induced drought stress to Cucumis sativus seedling roots led to increased activity of APX. However, the activities of DHAR and MDHAR first decreased (24 h) and then increased. The activity of GR was found to decrease at all time points (Fan et al., 2014). Drought stress differentially affected the antioxidant levels in the genotypes of plants which were contrasting with respect to drought tolerance. For example, the drought tolerant cultivars exhibited enhanced antioxidant enzyme activity under drought stress in comparison with sensitive cultivars of Dendranthema grandiflorum (Sun et al., 2013). The effect of drought stress on different isoforms of AsA-GSH cycle genes is extremely variable among different plant species. For example, drought stress was shown to decrease the activity of cytosolic isoform of APX whereas it led to increased activity of the chloroplastic isoform in Helianthus annuus. In the same study, it was shown that drought stress did not affect the activity of both the cytosolic and chloroplastic isoforms of APX in Sorghum bicolor (Zhang and Kirkham, 1996).

### Salt Stress

In plants, salinity stress leads to cellular dehydration, which enhances the production of ROS causing oxidative stress and thereby leading to enhanced expression of ROS scavenging enzymes. The expression levels of all enzymes of AsA-GSH pathway have been shown to be affected by salt stress (Mittova et al., 2004; Jebara et al., 2005). However, activities of AsA-GSH pathway enzymes were found to be differentially altered by salinity stress in the salt tolerant and sensitive varieties. For example, O. sativa L. cv. Pokkali which is a salt-tolerant genotype, showed enhanced activity of AsA-GSH cycle enzymes, whereas, the saltsensitive, O. sativa L. cv. BRRI dhan 29 exhibited decreased APX activity, increased DHAR activity and unchanged MDHAR and GR activity (Hossain et al., 2013). However, salinity stress in Triticum aestivum and O. sativa resulted in increased activities of MDHAR (Sairam et al., 2002; Vaidyanathan et al., 2003). All the isoforms of MDHAR, viz. mitochondrial, peroxisomal, chloroplastic, and cytosolic have been found to be sensitive to salt stress. For instance, salinity stress leads to increased activities of mitochondrial and peroxisomal MDHARs in Lycopersicon pennellii, which is a salt tolerant wild variety (Mittova et al., 2003). An increased GR activity has been reported in the roots and leaf of Cicer arientinum under salt stress (Eyidogan and Oz, 2005).

### Temperature Stress

High temperature in plants enhances the generation of ROS, consequently inducing oxidative stress (Yin et al., 2008). Under high temperature, RuBisCO can lead to the enhanced production of H2O<sup>2</sup> as a result of its oxygenase reaction (Kim and Portis, 2004). Tolerance to heat stress has been ascribed to elevated antioxidant enzymes' activity in many crop plants (Rainwater et al., 1996; Sairam et al., 2000; Sato et al., 2001; Rizhsky et al., 2002; Vacca et al., 2004; Almeselmani et al., 2006). The AsA-GSH pathway was found to be upregulated in response to heat stress in Malus domestica as reflected by increased gene expression and activities of APX, DHAR and GR enzymes (Ma et al., 2008). Under heat stress, the response of antioxidant enzymes activity varied amongst different genotypes of plants. For example, the analysis of gene expression of APX in a thermo-tolerant and thermo-susceptible variety of Brassica spp, T. aestivum, and Vigna radiata revealed increased activity of the enzyme under heat stress in all the genotypes. However, the elevation in transcript level was found to be higher in case of thermo-tolerant genotypes (Almeselmani et al., 2006; Rani et al., 2013). Heat stress induced elevation in transcript level of APX has also been reported in Poa pratensis by He and Huang (2007). Similar to APX, GR activity was also found to be enhanced by 50% in thermo-tolerant and 33% in thermo-susceptible genotypes of Brassica spp under heat stress (Rani et al., 2013). Exposure of N. tabacum cell suspension to elevated temperature (55◦C) also resulted in increased GR activity (Locatto et al., 2009). However, Ma et al. (2008) reported the initial increase and then decrease in GR activity in M. domestica leaves during prolonged exposure to heat stress. The activities of DHAR and GR were also found to be increased under heat stress in temperature sensitive orchid Phalaenopsis (Ali et al., 2005). The activity of MDHAR was found to be repressed under heat stress in the same study. This study also indicated a differential effect of heat on the activity of the antioxidant enzymes in roots and shoots. For example, the activity of GR was doubled at 40◦C in leaf but was drastically reduced in roots at the same temperature. The authors attributed the decrease in GR activity in roots to reduced availability of NADPH (Ali et al., 2005).

Similar to heat stress, low temperature stress also induces H2O<sup>2</sup> production in cells (Suzuki and Mittler, 2006) and is known to up-regulate transcripts, protein level and activities of different ROS-scavenging enzymes (Prasad et al., 1994; Saruyama and Tanida, 1995; Sato et al., 2001). In Pinus spp, enhanced freezing tolerance during cold acclimation was characterized by elevated levels of APX, GR, MDHAR, and DHAR (Tao et al., 1998). In leaves of Eupatorium adenophorum, the activity of APX and GR increased with decreasing temperatures. However, upon cold stress treatment to leaves of thermo-tolerant E. odoratum, the activity of APX reached a peak value at 15◦C and then declined, whereas GR activity was not affected. MDHAR activity in leaves of the cold-treated E. adenophorum was not significantly different from the controls, whereas the activity was found to be decreased in leaves of E. odoratum. DHAR activity in leaves of the two species was found to increase with both heat and cold stresses (Lu et al., 2008).

### Role of Gene Families of AsA-GSH Pathway in Biotic Stress

The production of ROS constitutes one of the first responses of plant cells to infection (Torres et al., 2006). The apoplastic generation of ROS occurs mainly by enzymes like membrane NADPH-dependent oxidase, cell wall peroxidase or polyamine oxidases (Bolwell et al., 2002). ROS generated upon pathogen attack can either enhance the harmful effect of infection or may contribute to plant defense by causing hypersensitive response (Levine et al., 1994). ROS can also serve as signal molecules for the activation of local and systemic resistance (Grant and Loake, 2000; Kuzniak and Skłodowska, 2005). The ROS-mediated plant defense response is further more complex and is dependent on factors like the life style of pathogen (biotrophy/necrotrophy), the type of plant–pathogen interaction (compatible/incompatible interactions) and the stage of plant development (Govrin and Levine, 2000; Huckelhoven and Kogel, 2003). For maintaining ROS homeostasis, it becomes important to have an intricate and tightly regulated balance between ROS production and removal. Pathogen induced changes in antioxidant enzyme levels have been shown in a number of plants (**Table 3**). For example, in Hordeum vulgare leaves challenged with the powdery mildew fungus, Blumeria graminis, the fungal infection led to a significant decrease in APX and GR activity in whole-leaf extracts


of resistant variety but caused no significant change in the susceptible one. However, there was no change in the activities of MDHAR and DHAR (Vanacker et al., 1998). Kuzniak and Skłodowska (2005) showed that Botrytis cinerea infection differentially affected the AsA-GSH gene families in L. esculentum. Upon infection, APX activity was found to increase in chloroplasts and decrease in mitochondria and peroxisomes 2 days after infection (dpi). The activity of peroxisomal MDHAR increased considerably at 1 dpi followed by subsequent decrease in activities of all MDHAR isoforms. A significant reduction in the activity of DHAR was observed in whole leaf extract at all time points. The chloroplastic DHAR activity was not affected, whereas the mitochondrial and peroxisomal DHAR activities were distinctly decreased starting from the third day after pathogen challenge. The GR activity on the other hand was found to increase in the chloroplasts. The peroxisomal and mitochondrial GR activities were repressed in response to infection by the pathogen. The decline in the activity of mitochondrial and peroxisomal isoforms points toward the "fungus-promoted precocious senescence" that led to the disease development (Kuzniak and Skłodowska, 2005). Similarly, Sesamum orientale plants, upon infection with the fungus Alternaria sesami displayed an initial increase in the activity of APX, MDHAR, and GR followed by a gradual decrease in the corresponding activities (Shereefa and Kumaraswamy, 2014). The expression of cytosolic MDHAR and DHAR was shown to be upregulated in A. thaliana seedlings co-cultivated with the root-colonizing endophytic fungus Piriformospora indica suggesting an important role of the enzyme in the maintenance of mutualistic plant- fungal interaction (Vadassery et al., 2009). However, knockdown of T. aestivum MDHAR resulted in improved resistance to Puccinia striiformis in wheat (Feng et al., 2014) suggesting that plants with compromised activity of the antioxidant enzymes have improved resistance against pathogens.

### Role of Gene Families of AsA-GSH Pathway in Physiological and Developmental Processes of Plants

Apart from the important role in protecting the plants from the stress induced ROS, the enzymes of AsA-GSH pathway also play a part in growth and development of plants. AsA and GSH have been known to play important roles in organ developmental processes of plants (Arrigoni and De Tullio, 2002). The peroxisomal MDHAR in A. thaliana has been shown to be important in mobilization of lipid reserves during early growth following germination by removing H2O<sup>2</sup> generated by β-oxidation (Eastmond, 2007). The transcript profiles of certain enzymes of the pathway are known to be spatially and developmentally regulated. Expression of A. thaliana cytosolic APX (APX1) in leaves and roots is relatively high as compared to the cytosolic APX2 isoforms (Panchuk et al., 2005; Hruz et al., 2008). A. thaliana apx1 mutant plants exhibit delayed development, late flowering and altered stomatal responses (Pnueli et al., 2003). The study of Correa-Aragunde et al. (2013) suggests the participation of APX1 in the redistribution of H2O<sup>2</sup> accumulation during root growth and lateral root development in A. thaliana. The transcripts of APX1 in Ipomoea batata were detected clearly in leaves, weakly in stems, and not in non-storage and storage roots. The expression level appeared to be higher in mature leaves than in immature leaves, suggesting its growth-stage specific expression (Park et al., 2004). Expression of APX2, another cytosolic isoform was found to be limited to bundle sheath cells in leaves exposed to excess light (Fryer et al., 2003). Like APX, DHAR also plays an important role in developmental processes. It has been reported that suppression of DHAR expression results in a preferential loss of chlorophyll a and less CO<sup>2</sup> assimilation, resulting in decreased rate of leaf expansion, reduced foliar dry weight and premature leaf aging. Furthermore, the over-expression of DHAR which led to reduced lipid peroxidation in the transgenic plants led to delayed leaf aging in O. sativa (Chen and Gallie, 2006).

### Summary and Perspectives

Despite their deleterious effects, ROS at low concentrations play crucial roles in stress perception, regulation of photosynthesis, pathogen recognition, programmed cell death, and plant development. The antioxidant enzymes of AsA-GSH pathway help in maintaining ROS homeostasis in cells by avoiding the potential cytotoxicity of ROS and allowing them to function as signal molecules. Considering the different levels and intensities of AsA and GSH production in the different organelles of cell under normal and stress conditions, the regulation of antioxidant enzymes also differs. There are different subcellular isoforms of each of the antioxidant enzymes and each isoform differentially responds to different stress and developmental cues. The mechanism of regulation of each isoforms by different stresses and developmental stages is yet to be completely understood. Further studies are required to decipher the complex regulation of expression of different isoforms of the AsA-GSH pathway enzymes in order to bolster our understanding of ROS homeostasis in plants. Understanding the intricate regulation of the various isoforms under various stress conditions can facilitate deeper insights into the stress tolerance mechanism of plants. This will also help in designing better strategies for the development of plants with improved abiotic and biotic stress tolerance.

### Acknowledgments

The authors acknowledge financial support from Department of Science and Technology to PP and Department of Biotechnology to AVM. The authors also deeply acknowledge the support from National Institute of Plant Genome Research and International Centre for Genetic Engineering and Biotechnology.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Pandey, Singh, Achary and Reddy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Oxidative stress homeostasis in grapevine (*Vitis vinifera* L.)

### Luísa C. Carvalho, Patrícia Vidigal and Sara Amâncio\*

LEAF Research Centre, Departamento de Recursos Naturais, Ambiente e Território (DRAT), Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal

Plants can maintain growth and reproductive success by sensing changes in the environment and reacting through mechanisms at molecular, cellular, physiological, and developmental levels. Each stress condition prompts a unique response although some overlap between the reactions to abiotic stress (drought, heat, cold, salt or high light) and to biotic stress (pathogens) does occur. A common feature in the response to all stresses is the onset of oxidative stress, through the production of reactive oxygen species (ROS). As hydrogen peroxide and superoxide are involved in stress signaling, a tight control in ROS homeostasis requires a delicate balance of systems involved in their generation and degradation. If the plant lacks the capacity to generate scavenging potential, this can ultimately lead to death. In grapevine, antioxidant homeostasis can be considered at whole plant levels and during the development cycle. The most striking example lies in berries and their derivatives, such as wine, with nutraceutical properties associated with their antioxidant capacity. Antioxidant homeostasis is tightly regulated in leaves, assuring a positive balance between photosynthesis and respiration, explaining the tolerance of many grapevine varieties to extreme environments. In this review we will focus on antioxidant metabolites, antioxidant enzymes, transcriptional regulation and cross-talk with hormones prompted by abiotic stress conditions. We will also discuss three situations that require specific homeostasis balance: biotic stress, the oxidative burst in berries at veraison and in vitro systems. The genetic plasticity of the antioxidant homeostasis response put in evidence by the different levels of tolerance to stress presented by grapevine varieties will be addressed. The gathered information is relevant to foster varietal adaptation to impending climate changes, to assist breeders in choosing the more adapted varieties and suitable viticulture practices.

Keywords: ROS, ascorbate-glutathione cycle, oxidative burst, peroxiredoxins, hormone stress signals, *in vitro* stress, biotic stress

### Introduction

Plants are able to maintain growth and reproductive success by sensing changes in the surrounding environment and reacting through mechanisms at the molecular, cellular, physiological, and developmental levels. These response mechanisms enable plants to react rapidly, within hours or days, to extreme environmental conditions that could otherwise be injuring or lethal. Understanding stress responses is one of the most important issues in plant research nowadays. Both biotic and abiotic stresses can promote the onset of oxidative stress through the accumulation of reactive oxygen species (ROS). Worldwide, extensive agricultural

#### *Edited by:*

Margarete Baier, Freie Universität Berlin, Germany

#### *Reviewed by:*

Iris Finkemeier, Max Planck Institute for Plant Breeding Research, Germany Roy Navarre, United States Department of Agriculture, USA Carmen Arena, University of Naples Federico II, Italy

*\*Correspondence:*

Sara Amâncio, LEAF Research Centre, Departamento de Recursos Naturais, Ambiente e Território (DRAT), Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, Calçada da Tapada, 1349-017 Lisboa, Portugal samport@isa.ulisboa.pt

#### *Specialty section:*

This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science

> *Received:* 28 November 2014 *Accepted:* 02 March 2015 *Published:* 19 March 2015

#### *Citation:*

Carvalho LC, Vidigal P and Amâncio S (2015) Oxidative stress homeostasis in grapevine (Vitis vinifera L.). Front. Environ. Sci. 3:20. doi: 10.3389/fenvs.2015.00020 losses are attributed to drought, often in combination with heat (Mittler, 2006). The available scenarios for climate change suggest increases in aridity in Mediterranean climate regions (Jones et al., 2005), where grapevine traditionally grows. This species is an extremely important crop worldwide, at the economic and cultural levels. In Southern Europe, post flowering phases of the growth cycle usually occur under high temperatures, excessive light and drought conditions at soil and/or atmospheric level. In such situations plants are affected by a combination of abiotic and biotic stresses, triggering synergistic or antagonistic physiological, metabolic or transcriptomic responses unique to each stress combination. Oxidative stress also arises in in vitro propagation commonly applied to ornamental species, also used to rapidly propagate grapevine scions for grafting (Carvalho and Amâncio, 2002).

The ultimate "price" to pay for photosynthetic O<sup>2</sup> release and plant aerobic metabolism is the production of ROS. ROS production can also be increased by stress conditions (Apel and Hirt, 2004). When photosynthesis is inhibited, absorption of light energy can be in excess to what can be used by the photosynthetic processes, resulting in ROS production and accumulation. The same is true when stress induced slowdown of other ROS processing metabolic mechanisms results in ROS accumulation. Climate change forecasts indicate a high probability of extreme temperature episodes, both high and low, a decrease in water availability as well as increases in carbon oxide and ozone in the atmosphere. All these factors impact plant growth and development by negatively affecting antioxidant homeostasis, hampering the adaptation to environmental stressors (Munné-Bosch et al., 2013).

A molecule is classified as "antioxidant" when it is able to quench ROS without itself undergoing conversion into a destructive radical, thus interrupting the cascades of uncontrolled oxidation. In that category are included, among others, the metabolites ascorbic acid (AsA, also termed vitamin C), glutathione (GSH), and carotenoid pigments. The ROS signaling or degradation pathways depend on antioxidant redox buffering enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxiredoxins (Prx), ascorbate peroxidase (APX), and glutathione reductase (GOR) (Carvalho et al., 2006; Vidigal et al., 2013).

Upon abiotic stress gene expression profiles are altered and usually genes assigned to the functional categories "protein metabolism and modification," "signaling" and "antioxidative response" undergo significant changes (Carvalho et al., 2011; Rocheta et al., 2014), thus enhancing the common attributes of abiotic stress defense pathways.

The nutraceutical properties of the grape berry and its derivatives, namely wine, are commonly associated with the antioxidant properties of the phenolic compounds they contain (Tenore et al., 2011; De Nisco et al., 2013), from simple flavonoids like anthocyanins to condensed proanthocyanidins (PAs, tannins), which can be solubilized into the vacuole or linked to cell wall polysaccharides, so, it is of great interest to understand their antioxidant mechanisms in planta.

### Metabolites Involved in Antioxidant Homeostasis: ROS, AsA, GSH, Pigments, and Proline

The different levels of tolerance to stress presented by grapevine varieties relates directly to the genetic plasticity of the antioxidant homeostasis. Some varieties keep low basal levels of antioxidant metabolites thus having to synthesize them at the onset of stress. Such varieties have a slower response than those with higher basal levels of antioxidant metabolites (Carvalho et al., 2014). This is put in evidence by the different pattern of antioxidant metabolites in normal growth conditions, as shown in **Table 1**.

### ROS

The first players in antioxidant homeostasis, which in normal conditions induce the need for detoxification/scavenging, are ROS themselves. In chloroplasts O<sup>−</sup> 2 produced in the Mehler reaction occurs in normal conditions, commonly increasing upon stress. It is now believed that O<sup>−</sup> 2 formation is the first step in a chain reaction leading to the control and regulation of several cellular processes (Apel and Hirt, 2004), with ROS integrated into signaling pathways (Mullineaux et al., 2006), often in crosstalk with hormonal regulation (Fujita et al., 2006). A mechanism of acclimation of Nicotiana benthamiana to high light, driven by the hormone abscisic acid (ABA) and by the accumulation of H2O2, also involving Prxs was recently described in Vidigal et al. (2014).

When the energy from triplet excited chlorophylls is transferred to molecular oxygen, <sup>1</sup>O<sup>2</sup> is formed. This ROS is a strong electrophile agent that can react with lipids, proteins, and DNA (Triantaphylidès and Havaux, 2009). However, <sup>1</sup>O<sup>2</sup> also reacts with target mediator molecules which trigger signaling cascades that lead either to programmed cell death or to acclimation (Ramel et al., 2012). In grapevine leaves, <sup>1</sup>O<sup>2</sup> and also H2O<sup>2</sup> are generated in trace-element stress, such as that caused by boron in excess (Gunes et al., 2006).

Peroxisomes are probably the major sites of intracellular H2O<sup>2</sup> production, although O<sup>−</sup> 2 and nitric oxide radicals (NO· ) are also produced in peroxisomes. The photoinhibition that is verified in grapevine leaves upon drought and salinity stress is accompanied of an increase in transcription of genes coding for peroxisome glycolate oxidase, catalase and several photorespiratory enzymes (Cramer et al., 2007).

### AsA and GSH

L-ascorbic acid (AsA) is an abundant metabolite playing important roles in plant stress physiology as well as in growth and development. AsA is a key antioxidant (Conklin and Loewus, 2001), able to directly eliminate several different ROS (Potters et al., 2002). Both the chloroplastic lipophilic antioxidant α-tocopherol (vitamin E) and carotenoid pigments (carotenes and xanthophylls) depend on AsA for regeneration from oxidized radicals (Potters et al., 2002). AsA is also the most important H2O<sup>2</sup> reducing substrate, acting together with glutathione (GSH, γ-L-Glu-L-Cys-Gly) in the ascorbate-glutathione cycle (see Section The Ascorbate-Glutathione Cycle) (Noctor and Foyer, 1998). GSH is a multifunctional metabolite in plants, being


1|Concentrationofantioxidativemetabolitesandactivitiesofantioxidativeenzymesquantifiedinseveralgrapevinevarietiesandin

\*

or

◦ in each column were obtained from the reference labeled accordingly in the Reference line.

Values followed by

a major reservoir of non-protein reduced sulfur, and a crucial element in cellular defense and protection, preventing the denaturation of proteins caused by oxidation of thiol groups during stress, reacting chemically with a wide range of ROS (Noctor et al., 2002). In grapevine, AsA and GSH pools and their adjustments upon stress seem to be variety dependent and under tight control (Carvalho et al., 2014). In cv. "Touriga Nacional" facing oxidative stress the existing AsA and GSH pools assure the cell buffering capacity while in cv. "Trincadeira" AsA and GSH need to be synthesized de novo, leading to a slower response that may be insufficient to maintain the redox pool at working levels (**Table 1**).

### Carotenoids

Carotenoids protect the photosynthetic apparatus against photooxidative damage not only by quenching the triplet states of chlorophyll molecules (Koyama, 1991) but also by scavenging ROS, protecting pigments and unsaturated fatty acids from oxidative damage (Edge et al., 1997). In the grapevine variety "Trincadeira" subjected to heat stress, carotenoids play an important role in leaf ROS scavenging, in tandem with ascorbate and glutathione, the usual first line of antioxidative defense in plants (Carvalho et al., 2014, **Table 1**).

### Proline

The metabolism of proline, including proline oxidation, is extremely important in the response to stress, as it is one of the most widespread osmoprotectants (Kiyosue et al., 1996), increasing in conditions of water deficit, as shown in the grapevine cv. Riesling (Bertamini et al., 2006). Also in grapevine, ROS generated by salinity stress signal the expression of GDH α-subunit, GDH acting as an anti-stress enzyme not only by detoxifying ammonia but also by producing glutamate which is channeled to proline synthesis (Skopelitis et al., 2006). Artificially increased proline levels also lead to the decrease of H2O<sup>2</sup> and malondialdehyde. It was thus suggested that the crosstalk between proline and H2O<sup>2</sup> could play an important role in the response to oxidative stress in grapevine leaves (Ozden et al., 2009, **Table 1**). Proline accumulation increased by two-fold upon salinity stress and by three-fold after water stress, and was accompanied by an increase in transcript abundance of plasma membrane proline transporters and of pyrroline-5-carboxylate synthetase (P5CS), the enzyme that catalyzes the first two steps in proline biosynthesis (Cramer et al., 2007). In parallel, there was an increased transcript abundance of proline dehydrogenase, presumably to enable the removal of excess proline, which can be toxic if allowed to over accumulate (Cramer et al., 2007). Different types of stress can reduce proline levels as it happens in excess boron, leading to an increased lipid peroxidation and APX depletion (Gunes et al., 2006).

### Key Enzymes for Antioxidant Homeostasis

Redox homeostasis comprises the interaction of ROS with antioxidant molecules forming an interface for metabolic and environmental signals, thus modulating the induction of appropriate acclimation processes or cell death programs. In the chloroplasts, a decrease of CO<sup>2</sup> fixation together with an overreduction of the ETC is the foremost source of ROS production during stress; in mitochondria over-reduction of the respective ETC is also a chief mechanism of ROS generation (Yoshida et al., 2007) and in peroxisomes, H2O<sup>2</sup> is produced when glycolate is oxidized to glyoxylic acid during photorespiration (Mittler et al., 2004).

ROS signaling pathways depend upon a strict homeostatic regulation accomplished through antioxidant redox buffering. Antioxidants determine the lifetime and the specificity of the ROS signal or processing products. In this process, enzymes such as SOD, CAT, Prx, APX, and GOR are the key players in antioxidant homeostasis (Carvalho et al., 2006; Vidigal et al., 2013). See **Table 1** for reference values of enzyme activity in different grapevine varieties. A thorough search of the genes coding for these enzymes in grapevine was performed in NCBI (http://www.ncbi.nlm.nih.gov/) and 297 sequences were retrieved, including 109 peroxidases (**Supplementary Table 1**). Functional annotation is still incomplete and many of those sequences are redundant.

Phylogenetic dendrograms of grapevine non-redundant sequences of APX, SOD, CAT, GOR, and Prx gene families were generated based on the members annotated so far of Vitis vinifera, Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa (var. japonica) retrieved from NCBI (**Supplementary Figure 1**). From the analysis of those dendrograms it was observed that V. vinifera has as many isoforms as A. thaliana and also has the highest sequence homology with this species. However, further annotation is still necessary such as in the case of GOR, for which no records of peroxisome and/or mitochondria isoforms are available (**Figure 1**).

### Enzymes of the Water-Water Cycles

Under normal conditions, electrons obtained from the splitting of water molecules at PS II are channeled through the photosynthetic apparatus and transferred to molecular oxygen by PS I. Under stress conditions that decrease CO<sup>2</sup> availability due to stomata closure or increase exposure to continuous excessive light there is an excess of electron transfer toward molecular oxygen, generating O<sup>−</sup> 2 ions in PS I, through the Mehler reaction (Asada, 2006). In this situation, redox homeostasis can be guaranteed in two consecutive steps, the membrane attached copper/zinc superoxide dismutase (CZSOD) which converts O<sup>−</sup> 2 into H2O<sup>2</sup> that is redirected to the ascorbate-glutathione cycle, where it is converted to water. This whole process is referred to as the water–water cycle (Rizhsky et al., 2003) as depicted in **Figure 1**. In "Trincadeira" grapevines submitted to heat stress both CZSOD and FeSOD are induced to scavenge plastidial O<sup>−</sup> 2 and the resulting H2O<sup>2</sup> is scavenged by the MDHAR-GOR branch of the ascorbate-glutathione cycle (Carvalho et al., 2014).

### Catalase

Peroxisomes are subcellular organelles with a single membrane that exist in almost all eukaryotic cells, containing as basic enzymatic constituents CAT and H2O2-producing flavin oxidases (Corpas et al., 2001; **Figure 1**). Despite their simplicity, they perform essential functions (Del Río et al., 2006); namely in the

FIGURE 1 | Localization of reactive oxygen species (ROS) scavenging pathways in different cellular compartments. ROS: <sup>1</sup>O2, hydroxyl radical; H2O2 , hydrogen peroxide; O<sup>−</sup> 2 , superoxide. Water-water cycle and ascorbate-glutathione cycle: APX, ascorbate peroxide; GOR, glutathione

reductase; SOD, superoxide dismutase; CAT, catalase; DHAR, dehydroascorbate reductase; MDHAR, monodehydroascorbate reductase. Peroxiredoxin-mediated alternative water–water cycle: 1CysPrx,

(Continued)

#### Carvalho et al. Antioxidant homeostasis in grapevine

#### FIGURE 1 | Continued

1-cysteine peroxiredoxin; 2CysPrx, 2-cysteine peroxiredoxin; PrxII; Type II-peroxiredoxin; PrxQ, peroxiredoxin Q. ABA, abscisic acid; AOX, alternative oxidase; AsA, ascorbate; Cytc, cytochrome c; DHA, dehydroascorbate; ETC; electron transport chain; Fd, ferredoxin; FTR, ferredoxin-thioredoxin reductase; GLDH, L-galactono-1,4-lactone dehydrogenase; GOX, glycolate oxidase; GPX, glutathione peroxidase;

antioxidative metabolism. They have an essentially oxidative type of metabolism, and great metabolic plasticity, as their enzymatic content varies with the organism, cell/tissue-type and environmental conditions (Baker and Graham, 2002). Upon photooxidative stress the peroxisomal CAT is the most responsive of catalases in grapevine (Carvalho et al., 2011; Vidigal et al., 2013) and it was recently shown that peroxisomal CAT influences Prx activity in the cytosol of N. benthamiana (Vidigal et al., 2014).

### Peroxidases

Peroxidases are a large family of ubiquitous enzymes that have numerous roles in plant metabolism (For review, Passardi et al., 2005), including that of removing the H2O<sup>2</sup> formed as a consequence of stress. In the grapevine genome 109 peroxidases were found (**Supplementary Table 1**). They use different electron donors, such as AsA, in the case of APX in the ascorbateglutathione cycle; glutathione peroxidase (GPX) uses GSH as its reductant and the generically termed "peroxidases" use phenolic compounds (able to use guaiacol as substrate sometimes they are called "guaiacol-peroxidases"). In grapevine, GPX has been implicated in stress responses against Elsinoe ampelina and Rhizobium vitis and GPX is up-regulated in response to abiotic stresses such as drought and salinity (Cramer et al., 2007).

The main role of APX is the scavenging of H2O<sup>2</sup> in the ascorbate-glutathione cycle, keeping its levels tightly controlled in order to maintain redox homeostasis, a similar role as that of GPX (Asada, 2006). Conversely, peroxidases oxidize a large variety of organic substrates and the resulting products are involved in important biosynthetic processes, such as lignification of the cell wall, degradation of IAA, biosynthesis of ethylene, wound healing, and defense against pathogens (Kvaratskhelia et al., 1997).

### The Ascorbate-Glutathione Cycle

The reduction of H2O<sup>2</sup> undertaken by AsA is only possible due to the activity of APX, an enzyme that uses two molecules of AsA to reduce H2O<sup>2</sup> to water. AsA itself is oxidized to monodehydroascorbate (MDHA), an unstable compound that suffers spontaneous disproportionation to AsA and dehydroascorbate (DHA) (Potters et al., 2002). Monodehydroascorbate reductase (MDHAR) regenerates AsA from MDHA, using NADPH as a reducing agent and DHA is reduced to AsA through the action of dehydroascorbate reductase (DHAR), using GSH as the reducing agent. In this reaction GSH is oxidized to GSSG that, in turn is reduced back to GSH using NADPH as reducing potential. This is the ascorbate-glutathione cycle, also called Foyer-Halliwell-Asada cycle (**Figure 1**), where AsA and GSH act together to detoxify H2O<sup>2</sup> in a cycle of oxidation-reduction, without being GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferase; MDA, monodehydroascorbate; NCED, 9-cis-epoxycarotenoid dioxygenase; PSI and II, photosysthem I and II; Q, Coenzyme Q; XO, xanthine oxidase. Numbers in black circles indicate known isoforms in V. vinifera. The question marks indicate uncertainty on the actual number of isoforms, based in NCBI records; the given number is the most likely value.

consumed and using electrons derived from NAD(P)H (Noctor and Foyer, 1998).

In grapevine, function of the ascorbate-glutathione cycle and its contribution to the detoxification process depend upon several factors, namely, the type, intensity and duration of the stress and the genotype under study. This became quite evident in a study comparing two greenhouse-grown genotypes ("Trincadeira" and "Touriga Nacional") subjected to heat stress with previous acclimation to moderate heat stress (Carvalho et al., 2014). The levels of expression of the plastidial SOD genes (CZSOD and FeSOD) were induced, mostly in "Trincadeira," suggesting the scavenge of chloroplast O<sup>−</sup> 2 as a consequence of over-reduction of the ETC, while in "Touriga Nacional" only an increase of O<sup>−</sup> 2 scavenging in the mitochondria (through MnSOD) was observed. H2O<sup>2</sup> accumulation induced the expression of CAT, APX1, and APX3, together with the MDHAR-GOR branch of the ascorbateglutathione cycle in both genotypes. This, together with similar results obtained for micropropagated grapevine subjected to high light (Carvalho et al., 2006) and of plants under viral infection (Sgherri et al., 2013) shows that, upon a severe stress, MDHAR alone is unable of maintaining the AsA pool in the reduced form, thus calling for the function of the whole ascorbate-glutathione cycle. After 3 days of acclimation to high light (Carvalho et al., 2006) or 24 h after the end of heat stress (Carvalho et al., 2014), the MDHAR branch of the cycle can keep the ascorbate pool reduced. However, the activation of the ascorbate-glutathione cycle is not, on its own, a trustworthy indicator of oxidative stress, as its activity can be triggered by differentiation of emerging structures in developing plants (Carvalho and Amâncio, 2002; Carvalho et al., 2006).

### Glutathione S-Transferases

Glutathione S-transferases (GSTs) are enzymes that detoxify cytotoxic compounds by conjugation of GSH to several hydrophobic, electrophilic substrates (Marrs, 1996). Plant GSTs have been intensively studied because of their ability to detoxify herbicides, and several GSTs conferring herbicide tolerance have been characterized in many major crop species. Another plant GST subclass is implicated in stress responses, including those arising from pathogen attack, oxidative stress, and heavy-metal toxicity. In grapevine, the induction of GSTs upon pathogen attack occurs in parallel with that of phenylalanine ammonialyase (PAL) and stilbene synthase (STS) (Aziz et al., 2004). GSTs also play a role in the cellular response to auxins and during the normal metabolism of plant secondary products like anthocyanins and cinnamic acid. In grapevine, 107 GST isoforms were found (**Supplementary Table 1)** and seven genes belonging to the phi class and 57 belonging to the tau class, the two most important classes of plant-specific GSTs (Edwards et al., 2000), are implicated in anthocyanin metabolism during berry development (Zenoni et al., 2010), GST1 and GST4 being implicated in anthocyanin transport to the vacuole (Conn et al., 2008). Also in grapevine, the expression of several GSTs genes was affected by defoliation (Pastore et al., 2013).

### Peroxiredoxins

Prxs catalyze the reduction of H2O2, alkylhydroperoxides, and peroxynitrite to water, alcohols, or nitrite, respectively (for review, Dietz, 2011; **Figure 1**). Prxs are redox sensitive proteins that can undergo reversible oxidation–reduction and as a result, switch "on" and "off " depending on the redox state of the cell. In V.vinifera, under conditions of light and heat stress, Prx activity decreased as a result of the decrease in H2O<sup>2</sup> levels while, under water stress, Prx activity increased, mirroring the increase in H2O<sup>2</sup> (Vidigal et al., 2013).

2CysPrx is the most abundant stromal protein, protecting the photosynthetic apparatus against oxidative stress (König et al., 2003). When in deficiency leads to inhibition of photosynthesis, decrease in chlorophyll and impaired grapevine development (Vidigal et al., 2013). In light of its function in photosynthesis, Vv2CysPrx01 (Vidigal et al., 2013), and Vv2CysPrxB were upregulated in grapevine under light stress while Vv2CysPrxA was down-regulated under similar light stress conditions (Carvalho et al., 2011). These results were different from those obtained in A. thaliana, where an increase in light intensity resulted in little consequence to the expression of 2CysPrxA and 2CysPrxB (Horling et al., 2003). Transcription of 2CysPrxA is induced by H2O<sup>2</sup> and repressed by ABA (Baier et al., 2004). In grapevine several ABA-responsive-genes were consistently up-regulated (Carvalho et al., 2011), which could be an explanation for the discrepancy in Prx expression between these studies. The other possibility could be connected to the dual function of Prx, both in antioxidant defense and in signaling (Dietz, 2003). Vv2CysPrx01 was up-regulated in grapevine under heat and water stress (Vidigal et al., 2013) a result that could be related with the chaperone function of 2CysPrx (Kim et al., 2010) and its role in drought tolerance (Rey et al., 2005).

PrxQ has a specific function in protecting photosynthesis, different from that of 2CysPrx (Lamkemeyer et al., 2006). In grapevine under abiotic stresses, VvPrxQ was either repressed or unresponsive (Vidigal et al., 2013), the same result as after 24 h of high light in in vitro propagated plants (Carvalho et al., 2011). However, in the same work, after 48 h, PrxQ transcripts increased significantly, pointing to a delayed transcription response. PrxQ is responsive to H2O<sup>2</sup> and ABA (Guo et al., 2004), and in grapevine, neither H2O<sup>2</sup> nor ABA increased upon light stress, another explanation for the verified discrepancies.

1CysPrx is a seed specific Prx that is targeted to the cytosol (Dietz, 2011). In grapevine leaves Vv1CysPrx03 is located in the cytosol and is connected to drought and heat tolerance (Vidigal et al., 2013).

PrxIIC was very responsive to light stress in grapevine, with the tendency to increase with time (Carvalho et al., 2011). In A. thaliana, PrxIIE expression was highly induced by light stress (Horling et al., 2003), while in grapevine it was down-regulated (Carvalho et al., 2011; Vidigal et al., 2013). However, the up-regulation of PrxIIE in grapevine under water stress, correlating well with the increase in H2O2, suggests a role in drought tolerance (Vidigal et al., 2013). The expression of the mitochondrial isoform, VvPrxIIF, was unaltered upon high light (Carvalho et al., 2011; Vidigal et al., 2013), in agreement with results obtained in poplar under photo-oxidative conditions or heavy-metal treatments (Gama et al., 2007). Conversely, it was up-regulated in grapevine under heat and water stress, with strong correlation with H2O<sup>2</sup> and ABA (Vidigal et al., 2013) suggesting a role for VvPrxIIF under light independent stress conditions.

Two new possible chloroplast Prx genes were identified in grapevine, VvPrxII-1 and VvPrxII-2 (Vidigal et al., 2013). Transcript levels of VvPrxII-2 showed a strong response to heat stress and an analysis of its promoter region revealed the presence of the ABA-responsive element ABRE. Furthermore, the up-regulation of VvPrxII-2 was positively correlated with ABA concentration, suggesting that this Prx gene may play a role in ABA-mediated heat tolerance (Vidigal et al., 2013) while VvPrxII-1 transcripts were down-regulated under light stress and significantly upregulated under water stress.

### Crosstalk with Hormone Signals

Phytohormones are essential for the ability of plants to adapt to stresses by mediating a wide range of adaptive responses often by the regulation of gene expression mediated by the ubiquitin– proteasome degradation of transcriptional regulators (Santner and Estelle, 2009). One of the key players in the response of plants to abiotic stress, especially when involving water shortage, is ABA. However, other hormones such as cytokinins (CK), salicylic acid (SA), ethylene (ET), and jasmonic acid (JA) play significant roles in keeping cell homeostasis under oxidative stress, and their interactions are schematized in **Figure 2**.

### Abscisic Acid

Drought and high salinity result in strong increases of plant ABA levels, accompanied by major changes in gene expression and in

adaptive physiological responses (Seki et al., 2002; Rabbani et al., 2003). The expression of ABA-inducible genes that leads to stomatal closure, thus reducing water loss through transpiration and eventually restricting cellular growth, occurs promptly upon the sensing of stress (Peleg and Blumwald, 2011). Several loss of function mutants for genes related to de novo ABA synthesis, to ABA receptors and to downstream signaling are now available in several species (Fang et al., 2008; Cutler et al., 2010). In grapevine however, such tools are not available, thus ABA research has been taking place in a more classical approach.

To improve plant water status and decrease leaf temperature of grapevine plants different irrigation regimes are applied in Mediterranean vineyards (Costa et al., 2012). One method of applying regulated irrigation is by partial root-zone drying (PRD). The main rationale to use PRD in grapevine is the action of ABA in modeling stomatal conductance and the demonstration that, by keeping some root areas dry and others wet, the necessary hormonal signals to regulate stomatal conductance are provided by the dry root-zones and the water needed to prevent severe water deficit is delivered by the wet root-zones (Stoll et al., 2000). ABA accumulation depends both on an accelerated ABA biosynthesis under water deficit, and on the rate of ABA catabolism and conjugation, which is quite fast, and is the main factor controlling the disappearance of ABA signal (Jia and Zhang, 1997). Thus, the accumulation of this so-called stress ABA is controlled by a dynamic equilibrium between ABA biosynthesis at the level of NCED (9-cis-epoxycarotenoid dioxygenase) transcription and its catabolism and conjugation.

In berries, the onset of ripening (veraison) when anthocyanin accumulation begins in red varieties, is accompanied by a marked increase in ABA concentration (Deluc et al., 2009; Gambetta et al., 2010) and correlates well with sugar accumulation (Gambetta et al., 2010). ABA has been shown to activate anthocyanin biosynthetic genes and the anthocyanin-synthesis related VvmybA1 transcription factor (Jeong et al., 2004), and to induce the delay of expression of proanthocyanidin biosynthetic genes (VvANR and VvLAR2) (Lacampagne et al., 2009).

### Cytokinins

CK exert an opposite function as ABA, and CK levels decrease upon water shortage (Peleg and Blumwald, 2011). In grapevine the effect of ABA on root growth may be augmented by a reduction in CK concentration in the roots that leads to the enhanced ABA to CK ratios obtained in PRD irrigation cycles (Stoll et al., 2000), since it is known that root growth is inhibited by increased endogenous CK (Werner and Schmülling, 2009). In fact, it is possible to reverse the effects of PRD in stomatal conductance by exogenous application of benzyladenine, that also leads to lateral shoot development (Stoll et al., 2000). Also, when comparing fully irrigated and PRD vines a significant decrease in zeatin and zeatin riboside concentration in shoot tips and axillary buds is observed (Dry and Loveys, 1999).

### Salicylic Acid

SA is a phenolic plant growth regulator, with several physiological and biochemical functions (Raskin, 1992) under normal conditions and, especially, in the response to abiotic stresses, namely heat stress. SA is known to counteract the effects of heat stress by up-regulating the antioxidative system. The treatment of grapevine leaves with SA before, during and after heat stress maintained photosynthesis rates high, chiefly by keeping high levels of Rubisco activation state, and also accelerated the recovery of photosynthesis through effects on PS II function. These effects may be partially related to the presence of a heat shock protein, HSP21, during the recovery period in SA-treated leaves (Wang et al., 2010). Treatment with SA also protects mesophyll cells against cold and heat stress in leaves of young grape plants, affecting Ca2<sup>+</sup> homeostasis, and enzymatic and non-enzymatic components of the ascorbate-glutathione cycle (Wang and Li, 2006). SA treatment also induced the expression of PAL and the synthesis of new PAL protein and increased its activity in grape berries (Wen et al., 2005). PAL is a crucial enzyme of the phenylpropanoid metabolism, catalyzing the formation of trans-cinnamic acid. Its induction results in the accumulation of phenolic acids and flavonoids, thus enhancing the quality of berries.

### Jasmonates

JA and their derivatives are known to play important roles in activating genes coding for proteins involved in the defense against abiotic (drought, salt, and ozone) and biotic (insects and microbial pathogens) stresses. The activity of JA responses can be regulated by antagonistic cross-talk with SA signaling (for review, Balbi and Devoto, 2008). In fact, SA can suppress the JAdependent response to wounding and pathogen or insect attack (Leon-Reyes et al., 2010).

In grapevine, salt stress and biotic defense signaling share common pathways, e.g., the activity of a gadolinium-sensitive calcium influx channel and transient induction of JAZ/TIFY transcripts. Exogenous JA application can rescue growth in saltsensitive Vitis riparia (Ismail et al., 2012). In line of these data, the authors proposed a model where the default pathway is salt stress signaling that is modulated by a parallel signal chain triggered by biotic factors downstream of JA signaling.

JAs are also described as promoting the synthesis and accumulation of the stilbene compound resveratrol in grapevine berries (Tassoni et al., 2005). A transcriptional study of the different berry tissues during development revealed that JA signaling genes are preferentially expressed in the pericarp while JA-biosynthesis genes have differential expressions, lipoxigenase-related genes in the pericarp while the conversion of linoleic acid to jasmonic acid appears to be seed-exclusive (Grimplet et al., 2007). Recently, high levels of expression of both JA and ethylene signaling related genes was reported in berries before veraison (Fasoli et al., 2012).

### Ethylene

ET biosynthesis is induced in response to abiotic stresses and this hormone affects membrane permeability, osmotic potential (sugar and proline accumulation) and the control of cell water potential. Together with H2O2, ET acts as a signaling molecule in the response of grapevine buds to hypoxia, leading to the activation of antioxidative stress genes (Vergara et al., 2012). The oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) to ET is catalyzed by the membrane associated ACC oxidase. ACC oxidase has been reported to increase in response to cold in grapevine (Tattersall, 2006).

### Auxins

Auxins play an important role in fruit development, and the grape berry is no exception. Indole-3-acetic acid (IAA) content is high from anthesis to veraison, then declines to very low levels at maturation (Conde et al., 2007). Amidase (AMI1), responsible for the synthesis of IAA from indole-3-acetamide decreases its levels of gene expression during ripening (Pilati et al., 2007), in tandem with the decline of IAA levels.

Auxins have been implicated in the response to UVacclimation in grapevine, genes belonging to auxin responsive SAUR and Aux/IAA family, auxin response factors and auxin transporter-like proteins are down-regulated in grapevine leaves exposed to low UV-B, supporting evidence for a role in the response to low UV-B fluence light (Pontin et al., 2010).

### Oxidative Stress in *In Vitro* Systems

In vitro systems offer a practical and easily manipulated method for studying oxidative stress. In vitro cultures are usually grown in contained environments with low photon flux density and high relative humidity. Culture media contain high quantities of an organic carbon source and growth regulators, contributing to the development of characteristic features such as abnormal leaf anatomy, poor development of grana (Wetztein and Sommer, 1982) and low photosynthesis rates (Chaves, 1994). Oxidative stress due to photoinhibition is prone to occur upon transplantation to in vivo conditions. The extent in which photoinhibition affects the survival of the plant depends on its physiological status dictated by the prevailing environmental conditions, the efficiency of protective mechanisms against excess energy and the repair processes to restore normal photosynthesis (Krause and Weis, 1991). Nitro-oxidative abiotic stress can also cause damage to in vitro cultures and, in grapevine, procyanidins were shown to have a protective action against the damage caused by peroxisome peroxynitrite thus formed (Aldini et al., 2003).

Immediately after transplantation to ex vitro, in vitro propagated grapevine plants showed severely affected photosynthetic capacity, that recovered after 1 week (Carvalho et al., 2001) as clearly seen in the heat map of **Figure 3**. ROS concentration is maximal on the first 2 days after transfer while chlorophyll fluorescence indicators show symptoms of photoinhibition and the ROS scavenging machinery is activated. Photoinhibition symptoms are less severe when the first stages of in vivo growth are conducted at CO<sup>2</sup> concentrations double the normal atmospheric values and light intensities six-fold higher than in vitro (Carvalho and Amâncio, 2002).

In the early stages of ex vitro growth a stabilization of Rubisco is observed (Carvalho et al., 2005), allowing photosynthesis to regain normal levels. In parallel, the activation of the ascorbateglutathione cycle after 24 h of ex vitro growth helps to maintain cell redox homeostasis and regulates the antioxidative response (Carvalho et al., 2006). When the transcriptome of grapevine

FIGURE 3 | Monitoring changes in antioxidant homeostasis of *in vitro* propagated grapevine plants during the first 7 days of growth in *ex vitro* conditions. <sup>H</sup>2O<sup>2</sup> was quantified inµmol g−<sup>1</sup> FW; O<sup>−</sup> 2 was visualized through nitroblue tetrazolium staining, Fv/Fm (maximum efficiency of PSII photochemistry in dark-adapted leaves), and Y (maximum quantum efficiency of PSII in light adapted leaves) were both measured using a Fluorimager chlorophyll fluorescence imaging system (Technologica Lda. Colchester, UK) and the Fluorchart software to isolate the individual leaves and calculate the values of the parameters, GSH and AsA were both quantified inµmol g−<sup>1</sup> FW; CAT, SOD, APX, GOR, DHAR, and MDHAR expression was quantified through RT qPCR and ABA was quantified in nmol g−<sup>1</sup> DW. The heat map represents the differences between the values measured at the moment of transfer to ex vitro (control) and those monitored for 7 days of ex vitro growth: dark green, very significant increase from the control; light green, significant increase from the control; gray, no significant differences to the control; orange, significant decrease from the control; red, very significant decrease from the control. Values were retrieved from Carvalho et al. (2006) and Vilela et al. (2007).

plants after transplantation was scanned an activation of signaling pathways up to 48 h was reported together with the up-regulation of the protein rescuing mechanism that involves the cooperation of HSP100 and HSP70, two ATP-dependent chaperone systems that remove non-functional and potentially harmful polypeptides deriving from misfolding, denaturation, or aggregation caused by stress (Carvalho et al., 2011). This was an unusually late and time-prolonged reaction when compared with "typical" abiotic stress responses (Mittler, 2006; Cramer, 2010). During this short period, H2O<sup>2</sup> is accumulating and is used as a second messenger to trigger the pathways that are essential for plant survival at this delicate developmental phase (Vilela et al., 2008), as confirmed by the activation of genes related to stress defense pathways, hormones and protein metabolism in the same timeframe (Carvalho et al., 2011).

After overcoming the initial stress of transfer, plants undergo a new cycle of up-regulation of the antioxidative machinery, which reaches a maximum on day 6, and is not accompanied by photooxidative stress symptoms or increase in GSH/AsA pools (Carvalho et al., 2006). This coincides with the protrusion of new roots and the expansion of the first ex vitro leaf and culminates with a peak of ABA and H2O<sup>2</sup> concentration on the seventh day after transfer, both produced in the newly-expanded and functional roots (**Figure 3**; Neves et al., 1998; Vilela et al., 2007). At this point, acclimatization to ex vitro is not yet complete but photooxidative stress is no longer a problem for the growing grapevine plants.

### Response to Biotic Stress

One of the first attempts at "cataloging" biotic stress response genes in grapevine was undertaken with the aid of the Map-Man onthology, adjusted to encompass a few pathways in detail, such as phenylpropanoid, terpenoid, and carotenoid biosynthesis, very responsive upon biotic stress, with a marked effect on wine production and quality (Rotter et al., 2009). The authors describe an overview of transcriptional changes after the interaction of a susceptible grapevine with Eutypa lata, and show that the responsive genes belong to families known to take part in plant biotic stress defense, such as PR-proteins and enzymes of the phenylpropanoid pathway (Rotter et al., 2009).

Several stress response processes are common between biotic and abiotic stresses, exerting either synergistic or antagonistic actions, depending upon the specific stress combination that the plant is facing. One example is the role of dehydrins (DHNs) in the protection of plant cells from drought and also in host resistance to various pathogens. In the genus Vitis, the wild V. yeshanensis is tolerant to both drought and cold, and moderately resistant to powdery mildew due to the precocious induction of DHN1, occurring earlier in drought conditions than in V. vinifera and having more than one up-regulation peak during the infection with Erysiphe necator as compared to V. vinifera (Yang et al., 2012).

ROS signaling seems to have an important role in Plasmopara viticola resistance, as resistant varieties display a specific chronological set of events upon infection, that is not observed in susceptible genotypes, beginning with an increase in O<sup>−</sup> 2 , followed by a hypersensitive response, an increase in peroxidase activity in cells flanking the infection area and finally, an increased accumulation of phenolic compounds (Kortekamp and Zyprian, 2003). Specifically, the peroxidase activity after an infection with P. viticola is strongly correlated with resistance to P. viticola in field plants (Kortekamp and Zyprian, 2003).

In grapevine, the most ubiquitous reaction to fungal infection is the accumulation of phytoalexins. Since the 1970s that it is known that grapevine synthesizes resveratrol in response to fungal attacks (Peter and Pryce, 1977). Viniferins, products of resveratrol oxidation, are also produced in response to biotic and abiotic stresses, and also classified as phytoalexins. These compounds present biological activity against a wide range of pathogens and are considered as markers for plant disease resistance (Pezet et al., 2004b). Resveratrol is synthesized from coumaroyl CoA and malonyl CoA by STS (**Figure 4**). STS is closely related to chalcone synthase (CHS), the key enzyme in flavonoid-type compound biosynthesis leading to the production of chalcones while STS leads to the production of stilbenes (**Figure 4**). Indeed, under certain conditions, such as oxidative stress, the transcriptional response of VvSTS and VvCHS genes appears to be diametrically opposed suggesting that under those conditions, the plant refocuses its metabolism on stilbene biosynthesis, taking precedence over flavonol biosynthesis, as schematized in **Figure 4**, the pathway highlighted in red (Vannozzi et al., 2012).

Resistant grape genotypes artificially inoculated with P. viticola show very high amounts of stilbenes at the site of infection, that actively inhibit the motility of P. viticola zoospores and subsequent disease development (Pezet et al., 2004a). Interestingly, PAL seems to be constitutively expressed in resistant and susceptible genotypes, but was totally repressed in tissues after mock inoculation using the non-host pathogen Pseudoperonospora Cubensis. CHS and STS, however, had their expression increased after inoculation with P. viticola, indicating an activation of the resistance response, in accordance with the increase of stilbenes (Kortekamp, 2006).

The antioxidative response of the grapevine genotype "Trebbiano" when infected by the grapevine fanleaf virus was thoroughly scrutinized. At the early stages of infection, increases in H2O<sup>2</sup> were observed and probably due to enhanced dismutation of O<sup>−</sup> 2 by SOD, whereas, toward the late phase of infection, increases in AsA, GSH, and APX activity might be the reasons for H2O<sup>2</sup> to regain control levels (Sgherri et al., 2013).

Upon infection by necrotrophic pathogens, which need to kill their host cells to gain access to nutrients, an activation of JA-dependent defense mechanisms takes place (Avanci et al., 2010). Many plant pathogens can either produce auxins themselves or manipulate host auxin biosynthesis to interfere with the host's normal developmental processes. In response, plants have evolved mechanisms to repress auxin signaling during infection as a defense strategy, mediated by the accumulation of SA. In grapevine, auxin responsive genes (including SAUR, Aux/IAA, auxin importer AUX1, auxin exporter PIN7) are also significantly repressed in pathogen resistance responses (Wang et al., 2007), supporting the hypothesis that down-regulation of auxin signaling contributes to induce immune responses in plants (Bari and Jonathan, 2009).

### Oxidative Burst

Plant species such as pear, tomato, strawberry, and pineapple show a specific oxidative stress response during fruit development, termed oxidative burst. The respective fruits are themselves named "climacteric." As grapevine is not amongst them, it came somewhat as a surprise when Pilati et al. (2007) reported an oxidative burst in cv. "Pinot Noir" that began at veraison and was characterized by rapid accumulation of H2O<sup>2</sup> and by the modulation of many ROS scavenging enzymes, previously thought not to be up-regulated in this species. This work comprised a thorough transcriptomic analysis of the grape berry in the stages close to veraison and the quantification of H2O2. The

latter increased at the moment of veraison, reaching its maximum 1–2 weeks after, and then decreasing at a slower pace toward ripening. In tandem, transcripts coding APX, GPX, Prxs, Trxs, glutaredoxins, GSTs and metallothioneins were up-regulated, in accordance with the onset of a well-orchestrated antioxidative response. Shortly after, grape's oxidative burst was again reported and associated with high sugar content that impairs photosynthesis in the berries, possibly through ABA signaling (Lijavetzky et al., 2012). It must be referred that high levels of ACC oxidase transcript accumulation have been reported immediately preceding veraison, together with a peak in ACC accumulation and ET emission (Chervin et al., 2004). Proteomics studies also reported an increase in ROS scavenging enzymes toward ripening (Giribaldi et al., 2007; Negri et al., 2008). The subject remained

flavonoid biosynthesis pathway and the lignin biosynthetic pathway. However, under stress the balance between the transcription rates of VvSTS

> wrapped in controversy because other authors did not obtain the same results (Terrier et al., 2005), until Rienth et al. (2014) shed some light onto what might be causing such disparity of results. The authors, in yet another transcriptomic assay of grape berries, found that the oxidative burst occurs markedly during the night, at ripening, following the same trend as sugar transport and phytoalexin synthesis. Together with H2O2, <sup>1</sup>O<sup>2</sup> was also found to increase in chloroplasts together with enzymatic peroxidation of membrane galactolipids (Pilati et al., 2014).

species; UFGT, UDP-glucose:flavonoid 3-O-glucosyl transferase; VvCHS, Vitis vinifera chalcone synthase; VvSTS, Vitis vinifera stilbene synthase.

### Concluding Remarks

Grapevine can be considered a model for fruit species. Several transcriptomic studies are now complementing the existing information on abiotic stress responses of many grapevine varieties, previously described at the physiological level. Knowledge of gene expression patterns points to specific varietal responses and to different levels of stress tolerance, confirming the high phenotypic plasticity of this species. The results obtained so far suggest that some varieties keep redox homeostasis without an apparent boost in their antioxidant pool, just adjusting the activities of antioxidant enzymes and/or the accumulation of antioxidant molecules, while others need to synthesize those antioxidant molecules de novo. The former demonstrate a well-timed and efficient ROS removal and a broad plasticity in adapting to environmental shifts. At the genomic level, for instance, grapevine Prx isoforms are specifically targeted and highly responsive to major abiotic stresses. Examples are the gene expression of cytosol PrxIIE apparently with a role in grapevine drought tolerance, while in other species it was reported as only responding to light; and the identification of two new possible chloroplast Prx genes, VvPrxII-1, and VvPrxII-2, the former down-regulated by light stress and up-regulated by water stress, the latter induced by heat stress in tandem with increased ABA concentration and with an ABRE sequence in its promoter.

Specific development processes can shift redox homeostasis. An obvious example is the oxidative burst in berries, a singular feature occurring in this non-climacteric species during veraison, mostly during the night. Specifically, this metabolic event is accompanied by sugar transport and resveratrol synthesis. Reports from in vitro grapevine systems reveal stress and developmental-related signaling mediated by ROS in growing leaves and roots.

As a whole, the phenotypic plasticity of different grapevine varieties which behave as more tolerant to environmental

### References


aggressions that cause oxidative stress can improve crop yield and quality, and thus the species economic value.

### Acknowledgments

This work was funded by Fundação para a Ciência e Tecnologia (FCT) through CBAA Funding (PestOE/AGR/UI0240/2011) and the post-doc grants SFRH/BPD/85767/2012 and SFRH/BPD/43898/2008 to LC and PV, respectively. This work also benefited from European project KBBE InnoVine (ref. 311775) and the European COST Action FA1106 "QualityFruit."

### Supplementary Material

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fenvs. 2015.00020/abstract

Supplementary Table 1 | Complete list of isoforms for the major antioxidative response genes in grapevine: superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, glutathione peroxidase, glutathione S-transferase, peroxidase, peroxiredoxin and thioredoxin. Accessions were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/) and the gene name, symbol, grape gene accession numbers and gene ID are given.

Supplementary Figure 1 | Dendrogram analysis of APX, SOD, CAR, GOR, and Prx of *V. vinifera*, *Populus trichocarpa*; *Oryza sativa* L. ssp. Japonica and *Arabidopsis thaliana*, with the respective intracellular locations and confidence that the sequences belong within the respective group.


custom-made array and the affymetrix genechip. Mol. Plant 6, 1038–1051. doi: 10.1093/mp/ssr027


sprouting and photo-oxidation in rice. Plant J. 54, 177–189. doi: 10.1111/j.1365- 313X.2008.03411.x


proline synthesis in tobacco and grapevine. Plant Cell 18, 2767–2781. doi: 10.1105/tpc.105.038323


H2O<sup>2</sup> is independent of ABA before the protruding of roots. Plant Cell Rep. 26, 2149–2157. doi: 10.1007/s00299-007-0427-3


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Carvalho, Vidigal and Amâncio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Low-molecular-weight metabolite systems chemistry

#### *Franz Hadacek1 \* and Gert Bachmann2*

*<sup>1</sup> Plant Biochemistry, Faculty of Biology and Psychology, Albrecht-von-Haller Institut, Georg-August Universität, Göttingen, Germany <sup>2</sup> Molecular Systems Biology, Ecogenomics and Systems Biology, Faculty of Life Sciences, Universität Wien, Vienna, Austria*

#### *Edited by:*

*Naser A. Anjum, University of Aveiro, Portugal*

#### *Reviewed by:*

*Naser A. Anjum, University of Aveiro, Portugal Aryadeep Roychoudhury, St. Xavier's College, India Mario Carmelo De Tullio, University of Bari, Italy*

#### *\*Correspondence:*

*Franz Hadacek, Plant Biochemistry, Albrecht-von-Haller Institut, Georg-August Universität, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany e-mail: fhadace@uni-goettingen.de*

Low-molecular-weight metabolites (LMWMs) comprise primary or central and a plethora of intermediary or secondary metabolites, all of which are characterized by a molecular weight below 900 Dalton. The latter are especially prominent in sessile higher organisms, such as plants, corals, sponges and fungi, but are produced by all types of microbial organisms too. Common to all of these carbon molecules are oxygen, nitrogen and, to a lesser extent, sulfur, as heteroatoms. The latter can contribute as electron donators or acceptors to cellular redox chemistry and define the potential of the molecule to enter charge-transfer complexes. Furthermore, they allow LMWMs to serve as organic ligands in coordination complexes with various inorganic metals as central atoms. Especially the transition metals Fe, Cu, and Mn can catalyze one electron reduction of molecular oxygen, which results in formation of free radical species and reactive follow-up reaction products. As antioxidants LMWMs can scavenge free radicals. Depending on the chemical environment, the same LMWMs can act as pro-oxidants by reducing molecular oxygen. The cellular regulation of redox homeostasis, a balance between oxidation and reduction, is still far from being understood. Charge-transfer and coordination complex formation with metals shapes LMWMs into gel-like matrices in the cytosol. The quasi-polymer structure is lost usually during the isolation procedure. In the gel state, LMWMs possess semiconductor properties. Also proteins and membranes are semiconductors. Together they can represent biotransistor components that can be part of a chemoelectrical signaling system that coordinates systems chemistry by initiating cell differentiation or tissue homeostasis, the activated and the resting cell state, when it is required. This concept is not new and dates back to Albert Szent-Györgyi.

**Keywords: primary metabolism, secondary metabolism, redox chemistry, charge-transfer complexes, coordination complexes, semiconductors, biotransistors, chemoelectrical signaling system**

### **INTRODUCTION**

Low-molecular-weight metabolites (LMWMs) are known to us as nutrients, hormones, vitamins, poisons, chemical weapons, spices, perfume aromas, antioxidants, medicines and biopolymer precursors. The question about their raison d'être, especially for secondary LMWMs, is far from satisfactorily answered (Fraenkel, 1959; Hadacek et al., 2011). From focusing on particular aspects, such as central metabolism and chemical defense, the challenge of gaining insights into LMWM systemic functions increasingly becomes more important. All living organisms can synthetize LMWMs albeit not all accumulate them in amounts that are sufficient enough to stimulate attempts of isolation and structure elucidation. In plants they are especially prominent. Plants have evolved a multitude of storage compartments that range from microscopic glandular hairs to prominent lactifers and resin channels (Gershenzon, 2002; Langenheim, 2003)

The big exploratory era of LMWM structure diversity was in the second half of the former century and mainly carried out by organic chemists and pharmacists. In the last two decades, huge screening programs were started by the pharmaceutical industry to identify new antibiotic and drug candidates and when naturally occurring LMWMs from increasingly exotic and difficult-to-access sources did not suffice, synthetic combinatorial chemistry libraries were added. The problem with many identified candidates is and was: activity is usually accompanied by one or several undesired side effects. Consequently, there exists an undeniable challenge to understand their mode of action.

Surviving in a changing environment successfully represents the major challenge to all living organisms. Whereas high stress from the abiotic and biotic environment kills the organism, low stress levels can cause a priming effect. In attempts to understand this idiosyncratic phenomenon, we should perhaps explore the phenomenon of life from a more chemical perspective. The organic chemist Addy Pross suggested that systems biology actually is systems chemistry (Pross, 2012). Accordingly, Section LMWMs in Living Organisms is providing an overview of LMWM chemical structure diversity and recapitulates their basic reaction chemistry. Section LMWM Coordination Chemistry introduces important aspects of coordination chemistry, which extends the chemical exploration to inorganic chemistry. In Section System Chemistry and Bioelectricity we will attempt to outline bioelectricity as an important physiochemical regulatory component and, based on this, in Section Outlook: LMWMs in Chemical Systems Regulated by Electricity propose a concept how LMWMs can contribute to a homeodynamic systems chemistry of living organisms.

### **LMWMs IN LIVING ORGANISMS**

#### **DEFINITION, BIOSYNTHESIS, AND ORGANIC CHEMISTRY**

LMWM include all those organic compounds of biological origin with a molecular weight below 900 Dalton (Macielag, 2011). Confusingly, different terminology is used to address them. Pharmaceutical and medicinal researchers call them natural products, or in case of a proven antibacterial activity, antibiotics. Biologists differentiate between primary or central and secondary metabolites, the former being indispensable for growth and development, and the latter essential to survive in the ecosystem. Ecologists call them allelochemicals if they have been identified them as monitoring components of interactions between organisms. Nutritional scientist use the terms antioxidants or nutraceuticals to denote those LMWMS with more or less proven beneficial health effects. Hormones are universal signaling compounds. Vitamins are, by the majority, plant or microbial metabolites that are required by animals for maintaining of their metabolism. Pathologists and food scientists preferentially use the terms bacterial toxins and mycotoxins to point out bacterial and fungal metabolites that can harm human health if occurring in too high concentrations in our food stuffs. Depending on molecular size and the presence and absence of polar functional groups LMWMs are either volatile or non-volatile. There exist numerous review articles and books on LMWMs that focus on one or more of the above mentioned aspects, the cited ones just representing a subjective selection (Betina, 1989; Gräfe, 1992; Seigler, 1998; Reese et al., 2000; Dixon, 2001; Hadacek, 2002; Crozier, 2006; Hedden and Thomas, 2006; Hartmann, 2007; Bednarek and Osbourn, 2009; Buchanan et al., 2009; Greenstein and Wood, 2011; Bräse et al., 2013).

LMWM structural diversity will be illustrated by selected examples in the on-going text (**Figures 1**–**4**). One of the basic biosynthetic building blocks to secondary LMWMs is a C2 unit, usually a coenzyme A-bonded acetate. Derivatives from this pathway are called polyketides if ring-shaped, or fatty acids, if open chained. A further biosynthetic building block is a C5 unit, isoprene, that can arise either from the mevalonate pathway or from the later discovered deoxyxylulose phosphate pathway. It represents the precursor to the terpenoids, the second large structural class within secondary LMWMs. Amino acids introduce nitrogen into their structures and the amino acids cysteine and methionine sulfur. Aromatic structures can be formed either by the polyketide or the shikimic acid pathway, the latter also being a prerequisite to synthetize aromatic amino acids. More information is available in the literature (Rohmer, 1999; Romeo et al., 2000; Hadacek, 2002; Buchanan et al., 2009; Weng and Noel, 2012; Anarat-Cappillino and Sattely, 2014).

LWMWs are characterized by different combinations of functional groups, unsaturated bonds and/or heteroatoms. These characteristics define their chemical properties. Two-electron transfers underpin chemical reactions that result in the changing of covalent bonds in a LMWM substrate or product. The chemical textbook structures exclusively show structures with covalent bonds, in which one or more pairs of electrons are shared by two atomic nuclei. In inorganic chemistry, by contrast, ionic bonds prevail that are formed between attracting positively and negatively charged ions, in which one or more electrons from one nucleus are removed and attached to another. Oxidoreductions, the classical redox reactions, in which one molecule, the reductant, becomes oxidized, and the other molecule, the oxidant, reduced, are common. According to molecular orbital (MO) theory, the inherent electron transfer reaction occurs from the highest occupied molecular orbital (HOMO) of the reductant (D, donor) to the lowest unoccupied molecular orbital (LUMO) of the oxidant (A, acceptor). Marcus theory predicts that an electron transfer reaction (1) involves a precursor complex that changes into a successor complex resulting in the formation of radicals, highly reactive molecular species, in which an orbital of one of its atoms is occupied by an unpaired electron (Eberson, 1987; Pross, 1995).

$$\rm{D} + \rm{A} \rightarrow \rm{[DA]} \rightarrow \rm{[D^{+} \bullet} \rm{A^{-}]} \rightarrow \rm{D}^{+} + \rm{A}^{-} \tag{1}$$

A combination of various factors, (I) strong D−A pairing, (II) steric interactions that decrease the coupling between D+• and A−•, (III) low D+−A<sup>−</sup> bond strength, and (IV) strong delocalization of D+• and A−• radical centers, creates a one-electron transfer scenario. Such charge-transfers complexes (2) are characterized more aptly by the configuration mixing model (CFM) (Pross, 1985, 1995; Eberson, 1987).

$$\text{D} + \text{A} \leftrightarrow \text{[D}^{+\bullet} + \text{A}^{-\bullet} + \text{DA}\text{]}\tag{2}$$

Charge-transfer complex formation occurs when orbitals of adjacent biomolecules, LMWM and/or proteins, overlap (Szent-Györgyi, 1960, 1968). In protein chemistry, charge-transfer complex formation is viewed as a variant of dipol–dipol interactions. These weak non-covalent bonding forms further include hydrogen bonding, van der Waals forces and hydrophobic interactions. They are generally characterized by locally asymmetric electron distributions (Silverman, 2002). Chargetransfer complexes might also be viewed as a mosaic stone to understand drug-receptor interactions and they are also likely to occur in the gel-like cytosol, the structure of which resembles more a solid than a liquid solution (Doukas, 1975). In attempts to point to potential donor (D) and acceptor (A) atoms that can enter charge-transfer complexes in the illustrated molecule structures, corresponding signatures have been added tentatively into **Figures 1**–**4**. The assignments are based either on known redox chemistry or hints from literature (Szent-Györgyi, 1957, 1960; Doukas, 1975). Charge-transfer complexes facilitate electronic mobility and, as a consequence, shortrange metallic conductivity within molecules. Here we take up Albert Szent-Györgyi's suggestion that conjugated π-electron systems more or less represent electric extension cords because charge-transfer complex formation can induce an electric field (**Figures 1**–**4**). More contemporarily, a USB cable would be appropriate.

### **PRIMARY OR CENTRAL LMWMs**

Primary or central carbon metabolism converts sugars into a wide range of precursors that generate the entire cell biomass by using the shortest possible enzymatic pathways (Noor et al., 2010). **Figure 1** presents exemplary structures of sugars, amino, tricarboxylic (organic), and fatty acids. These metabolites are more or less shared by all extant pro- and eukaryotic organisms with few exceptions; e.g., Archaea possess lipids that are comprised of isoprene chain glycerol ethers instead of fatty acid glycerol esters (De Rosa et al., 1986). Specific combinations of functional element groups in the molecules, alcohols and acids with oxygen, amino groups with nitrogen, and thiol groups with sulfur, occur in the various molecules (**Figure 1**). By far, the amino acids are the most heterogeneous molecule class. **Figure 1** omits nucleobases, which form RNA and DNA.

Central metabolites have been suggested as components of a metabolic chemical system with evolved potential to optimize itself (Pross, 2005, 2012; Shapiro, 2011). Most, tricarboxylic acids being the exception, can serve as building blocks for polymer structures, which not only organize the cell's compartmental structure but also that of tissues of multicellular organisms.

### **HORMONES AND NEUROTRANSMITTERS**

The illustrated hormones (**Figure 2**) represent a group of livesustaining metabolites that can regulate the activities of genes, proteins and other cellular metabolites and thus exert major effects on many physiological and ontogenetic processes within and across tissues (Heyland et al., 2005). Hormones, however, are more specific for particular organismic kingdoms. The illustrated derivatives have been chosen not only to exemplify structural diversity but also to illustrate their occurrence in different living organisms. Proteobacteria use acylated homoserine lactones (AHL, **2.1**) and Actinobacteria butyrolactones, both fatty acid derivatives, for quorum sensing. Both compound classes were shown to facilitate coordination of metabolic activities within

in the text, the arrangement follows common biosynthetic pathways; (A)

extension cord symbols indicate π-electron systems with potential to develop weak localized electromagnetic fields.

bacterial populations (López-Lara and Geiger, 2010). Rhizobia, but also Alpha- and Beta-proteobacteria, can cause the formation of root nodules in legumes. They use lipo-chitooligosaccharidic nodulation (Nod) factors (**2.2**). These glycolipids have been identified as initiators of host plant root hair formation and deformation, intra- and extracellular alkalization, membrane potential depolarization, ion flux changes, nodulin gene expression and formation of nodule primordia (D'Haeze and Holsters, 2002). Trisporic acid (**2.3**) induces spore formation in zygomycete fungi; its mode of action, however, is extracellular similar to that of bacterial acylated homoserine lactones (Gooday, 1978). Interestingly, a prominent plant hormone that stimulates germination, cell differentiation and flowering in plants, gibberellic acid (GA, **2.4**), was identified first as a metabolite of the fungus *Gibberella fujikuroi*(teleomorph *Fusarium moniliforme*). This fungus utilizes it as a toxin to cause bakanae disease in rice seedlings (Curtis and Cross, 1954; Bartoli et al., 2013). Another structurally strikingly similar plant hormone is abscisic acid (ABA, **2.5**), a carotenoid cleavage product (apocarotinoid) that is involved in coordinating responses to various forms of abiotic stress and leaf senescence (Bartoli et al., 2013). Strigolactones, such as strigol (**2.6**), another group of apocarotinoids, are believed to be commonly exuded by plant roots. Originally, it was assumed that these compounds facilitate the establishment of parasitic plant haustoria in host tissues, later the discovery of their involvement in facilitating arbuscular mycorrhizal colonization of plant roots provided a more feasible hypothesis for their existence. Recently, also endogenous signaling roles have been suggested (Waldie et al., 2014). Brassinosteroids (**2.7**) represent a further class of plant growth hormones (Clouse and Sasse, 1998). A volatile plant hormone that can regulate diverse processes is ethylene (**2.8**) (Bleecker and Kende, 2000). Salicylic acid (**2.9**), an aromatic amino acid derivative, and jasmonic acid (**2.10**), a derivative of the unsaturated fatty acid linolenic acid, an oxylipin, represent plant hormones that are involved in resistance against pathogens and herbivores (Fujita et al., 2006). One of the most essential hormones for plant development is the tryptophan derivative auxin (**2.11**), also known as 3-indol-actic acid (Woodward and Bartel, 2005). The majority of plant hormones have been detected also in green, red, and brown algae (Tarakhovskaya et al., 2007).

In animals, the term hormones is reserved for metabolites that are produced by highly specialized endocrine tissues and transported by the circulatory system to their distant targets in the body (Jerzmanowski and Archacki, 2013). Among them, three classes resemble LMWM plant hormones, the amino acidderived, the steroid hormones and the eicosanoids. The latter are not synthetized by specific glands and not well soluble in aqueous solutions and therefore fail to fulfill two major classification criterions for animal hormones; as a result they are designated as hormone-like substances in the literature although, in terms of their evolved functionality, they represent hormones. Other animal hormones, polypeptides and small proteins, are outside of the focus of this review. Eicosanoids are derivatives of arachidonic acid, an unsaturated C20 fatty acid, and comprise prostaglandins (**2.12**) and leukotrienes, both of which are involved in numerous homeostatic functions and inflammation (Funk, 2001). They resemble plant oxylipins, derivatives of the unsaturated C18 fatty acid linolenic acid, and can disturb tissue homeostasis. Epinephrine (adrenaline, **2.13**) and norepinephrine (noradrenaline) are tyrosine derivatives that are secreted by the medulla of the adrenal glands. The former modulates cardiovascular and metabolic response to stress, the latter acts more as a neurotransmitter (Greenstein and Wood, 2011). A further neurotransmitter that occurs in many organisms is acetylcholine (**2.14**) (Preston and Wilson, 2013). The tryptophan derivative melatonin (**2.15**) is a highly interesting hormone; it can be synthetized by Bacteria, Plants, and Animals and modulates circadian rhythms (Hardeland, 2008). Steroid hormones are also involved in stress regulation; cortisol (**2.16**) is produced by the adrenal glands and stimulates gluconeogenesis and activates anti-stress and anti-inflammatory pathways (Greenstein and Wood, 2011). Steroid hormones comprise sex hormones, estrogens, such as estradiol (**2.17**), that regulate menstrual and estrous reproductive cycles, and testosterone (**2.18**), a hormone that occurs in both sexes but acts differently (Greenstein and Wood, 2011). Another steroid hormone, ecdysone (**2.19**), regulates insect developmental transitions (Yamanaka et al., 2013). Notably, ecdysones, can also be synthetized in significant amounts by plants (Williams et al., 1989).

A structural comparison of the various hormones reveals similarities and differences. Some of them are efficient electron donators, some strong acceptors, some both. Some possess metal-like conductivity due to π-electrons, others not. Probably, these different chemical properties not only facilitate diverse interactions with proteins (Doukas, 1975) but may facilitate also crosstalk-like actions between hormones (Pieterse et al., 2009; Spindler et al., 2009).

### **VITAMINS AND ENZYME COFACTORS**

Biochemical reactions require specific metabolites that provide either energy equivalents or electrons. For plants, the term "supportive metabolites" was suggested (Firn and Jones, 2009). Higher animals cannot synthetize them and thus require them as vitamins. The majority of vitamins are enzyme cofactors or precursors of them (Michal, 1999). **Figure 3** exemplifies structures. For example, carotenoids (**3.1**) represent terpenoid pigments that protect chloroplasts from the reactive oxygen species singlet oxygen that can be formed by energy transfer from relaxing chlorophyll pigments (Ramel et al., 2012). All animals that are endowed with the ability of sight absorb, transport and metabolize carotenoids into retinoids (**3.2**) (von Lintig, 2012). Tocopherols (**3.3**) represent further terpenoid antioxidants in the chloroplast and as vitamin E derivatives protect membrane lipids in animals (Denisov and Denisova, 2009). Phylloquinone (**3.4**) is an important electron acceptor in photosystem I and, concomitantly, represents an important antioxidant of the vitamin K group for animals (Asensi-Fabado and Munné-Bosch, 2010). An exception to the rule are the vitamin D forms; ergocalciferol is synthetized from ergosterol and cholecalciferol from cholesterol, the former a triterpene alcohol that confers stability to fungal membranes, the latter to animal membranes. Cholecalciferol is a precursor of calcitriol (**3.5**), which regulates calcium concentrations in the blood (Asensi-Fabado and Munné-Bosch, 2010).

Cobalamine (**3.6**), vitamin B12, represents one of the largest LMWMs; its porphyrine ring forms a coordination complex (see Section LMWM Coordination Chemistry) with the rare transition metal cobalt as central atom. Besides acting as an important cofactor, cobalamine also possesses notable antioxidant activity. Only Archaea and Bacteria can synthetize it (Michal, 1999; Asensi-Fabado and Munné-Bosch, 2010). Other important vitamins include thiamine (**3.7**), vitamin B1, which is important for oxidative decarboxylation and also can act as antioxidant (Michal, 1999; Jung and Kim, 2003); carboxylation reactions depend on biotin (**3.8**); pyridoxal phosphate (**3.9**) is involved in various modifications at the carbon atom 2 of amino acids (Michal, 1999; Ferrier and Harvey, 2014). Ascorbic acid (**3.10**), also known as vitamin C, confers protection against oxidative stress by acting as antioxidant. Furthermore, ascorbic acid can donate electrons to a wide range of enzymes (De Tullio, 2012). The most common cofactor systems in living organisms that are involved in redox reactions comprise NAD+/NADP (mitochondria) (**3.11**), NADP+/NADPH (photosynthesis, pentose phosphate cycle), and FAD/FADH2 (oxidative phosphorylation) (**3.12**). All of them contain the nucleobase adenine as moiety. The former additionally contains nicotine amide, the latter riboflavin. Energy equivalents are provided by ATP (**3.13**) (Torssell, 1993; Buchanan et al., 2009; Ferrier and Harvey, 2014) that also contains an adenine moiety. The biosynthesis of most cofactors is rather complex and difficult to elucidate due to the low available amounts of these LMWMs (Webb et al., 2007).

Comparing the chemical structures of diverse vitamins (**3.1**–**3.13**) with those of hormones (**2.1**–**2.18**), the more diverse polarity is notable; **3.1**–**3.4** are rather unpolar and localized in membranes, **3.5**–**3.13** are definitely more polar and optimized for a cytosolic environment. The presence of negative charges in the phosphoester moieties specifically enhances affinity to proteins. Apart from the latter, the functional groups are similar to those of the hormones and not as uniform as in some central metabolites, such as sugars and tricarboxylic acids. The numerous donor (D) and acceptor (A) sites provide a basis for forming charge-transfer complexes with proteins. Frequent conjugated unsaturated bonds facilitate the local buildup of electric fields.

### **SECONDARY LMWMs**

All LMWM that are deemed dispensable for life-sustaining processes are classified as so-called secondary metabolites, which represent the greatest known LMWM structural diversity by far; their numbers in plants are estimated to exceed 500,000 alone (Mendelsohn and Balick, 1995). Most of them show a highly restricted occurrence, sometimes even limited to a single species. A recently suggested alternative term is "speculative metabolism" (Firn and Jones, 2009). The main difference between Plants and Animals on one hand, and Bacteria and Fungi on the other hand, is that the former accumulate secondary LMWMs in specifically adapted compartments whereas the latter secrete them into their environment (Demain, 1996; Hadacek et al., 2011). Their classifications follows characteristic combinations of biosynthetic building blocks, acetate (C2) or isoprene (C5) units. All organisms, which are capable of synthetizing the aromatic amino acids phenylalanine and tyrosine and possess the enzyme phenylalanine ammonium lyase (PAL), can synthetize cinnamic acid derivatives, the main precursors for aromatic secondary metabolites in photosynthetic Bacteria, Algae and Plants. In heterotrophic organisms, aromatic structures are formed via the polyketide pathway. Combinatorial synthesis that utilizes precursors from various of the mentioned pathways together with variable modification of the base skeletons, which is caused by the low substrate specificity of the involved enzymes, yields the huge structural diversity (Gräfe, 1992; Seigler, 1998; Romeo et al., 2000; Hadacek, 2002; Weng and Noel, 2012). **Figure 4** presents an overview of structures that are mentioned in the ongoing text.

To start with, the flavonoid catechin (**4.1**) and stilbene resveratrol (**4.2**) represent characteristic phenolic cinnamic acid derivatives from plants that can arise from the shikimic acid pathway (Seigler, 1998; Hadacek, 2002). Both are renowned antioxidant constituents of wine grapes (Burns et al., 2000). Small molecules are volatile and characteristic fragrance components of spices, such as anethole (**4.3**) in fennel (Shahat et al., 2011). Bacteria

are also able to produce such volatile metabolites; *p*-cresol (**4.4**) is responsible for the feces odor and a metabolite of colonic bacteria (Smith and Macfarlane, 1996). Another group of antioxidant phenols are coumarins, e.g., esculetin (**4.5**) (Hiramoto et al., 1996). Recent studies have pointed out that esculetin and its methoxylated derivative scopoletin can contribute profoundly to iron uptake capabilities of Arabidopsis (Schmid et al., 2014). This points to complex coordination chemistry that combines organic with inorganic chemistry, and which will be discussed later in more detail (Section LMWM Coordination Chemistry). The anthraquinone aloe emodin (**4.6**) is a polyketide albeit structurally similar to the shikimic acid-pathway derived LMWMs. It possesses both laxative properties and redox chemical activity (Tian and Hua, 2005). Hypericin (**4.7**) is a dimeric anthraquinone that attracted attention because of its phototoxic effects on grazing animals and, out of context with the former activity, potential usage as antidepressant for humans (Barnes et al., 2001). The tyrosine derivative mescaline (**4.8**), a metabolite from the peyote cactus, *Lophophora williamsii*, was used as traditional medicine and hallucinogenic sacrament by North American Indians; recently, a universal redox chemical reaction mechanisms for its effect on the central nervous system has been proposed (Kovacic and Somanathan, 2009). A structurally similar compound, aaptamine (**4.9**) has also been isolated from the sea sponge *Aaptos aaptos* (Nakamura et al., 1982). Morphine (**4.10**) is a prominent opioid analgesic drug from the latex of unripe seedpods of the poppy *Papaver somniferum*. The same compound, however, yielded also positive results in antioxidant assays (Gülçın et al., 2004). Colchicine (**4.11**) is a tropolone alkaloid that is formed from both phenylalanine and tyrosin; it is known to cause lethal poisoning in humans who mistake meadow saffron leaves for wild garlic, but it also proved to be an efficient antioxidant (Modriansky et al., 2002). The glucosinolate sinigrin (**4.12**) is a highly water-soluble metabolite that can be converted into thiohydroximate-*O*-sulfate intermediates. Depending on pH, ferrous iron and the presence of myrosinase interacting enzymes, glucosinolates can be converted enzymatically and non-enzymatically into a variety of volatile degradation products, including isothiocyanates and nitriles amongst others, all of which are characteristic for vegetables of the mustard family (Brassicaceae) (Grubb and Abel, 2006). Both the precursor glucosinolates and their volatile degradation products possess antioxidant activity (Cabello-Hurtado et al., 2012). Amphidinolide N (**4.13**) is a polyketide that is produced by the flat worm symbiotic dinoflagellate *Amphidinium* (Ishibashi et al., 1994). The structure shows no aromatic rings and thus resembles more a terpenoid. This compound class provides predominantly aliphatic structures. Terpenoids can, however, still combine with units from other biosynthetic pathways, e.g., the shikimic acid pathway as in veratridine (**4.14**). This LMWM belongs to a series of highly neurotoxic terpene alkaloids that are synthtized by the Liliaceae s.l. Veratridine and similar compounds efficiently inactivate the regulation of the Na+ channels (Greenhill and Grayshan, 1992). Terpenoids are classified on basis of C5 isoprene unit numbers: Monoterpenes are formed by two, sesquiterpenes by three, diterpenes by four, triterpenes by six and tetraterpenes by eight isoprene units (Seigler, 1998). The latter predominantly comprise chloroplastic pigments with vitamin character (**Figure 3**). Cucurbitacin E (**4.15**) and glycyrrhicic acid (**4.16**) represent two examples with contrasting structures, taste, the former bitter and the latter sweet (Seigler, 1998). One of the most prominent of all diterpene derivatives is taxol, which has gained a reputation in breast cancer chemotherapy; its diterpene precursor, baccatin III (**4.17**), occurs in the stem bark of the American yew tree *Taxus brevifolia* (Wall and Wani, 1995). Bacteria also can synthetize diterpene derivatives; the recently discovered antibiotic platenmycin (**4.18**) represents a notable example (Wang et al., 2006). Drimane sesquiterpenes, such as polygodial (**4.19**), show a restricted occurrence in a few rather unrelated lower and higher plants. Some fungi, however, are also able to synthetize drimane sesquiterpenes (Jansen and de Groot, 2004). If fungal metabolites accumulate in our foods stuffs, especially in the cereal crops maize and wheat, they are designated as mycotoxins. Among the most prominent and deleterious of them we find trichothecene sesquiterpenes. An often mentioned compound is deoxynivaleol (DON, **4.20**), which affects the functioning of ribosomes (ribotoxic stress response) and can cause oxidative stress (Wu et al., 2014). Monoterpenes, in addition to phenylpropenes, represent the major plant odor components; derivatives such as myrcene (**4.21**) occur in especially large amounts in conifer resin. Bark beetles attacking these trees, in some years with devastating consequences, can utilize monoterpenes that are thought to constitute some kind of chemical defense against them as precursors for pheromones, such as ipsdienol (**4.22**). These volatile compounds help bark beetles to coordinate their behavior; some species have been found to be capable of synthetize these monoterpenes *even* by themselves (Seybold et al., 2006). Monoterpens, as all other terpenoid types, can be produced by many different organisms; for example, marine red algae synthetize heavily halogenated derivatives (**4.23**) (Fusetani, 2012). Iridoids, such as aucubin (**4.24**), are irregular monoterpenes and thus not easily recognizable as terpenoids. They occur in the plant families Plantaginaceae, Scrophulariaceae, and Gentianaceae, where they contribute to rapid browning during the drying process and bitter taste; they are, however, also produced by insects, e.g. iridodial (**4.25**) by the ant *Iridomyrmex* (Seigler, 1998). Finally, fatty acids also can act as precursors for secondary metabolites. Polyactylenes have similar chain-like structures with a high proportion of unsaturated double and often also triple bonds albeit without nitrogen. Falcarindiol (**4.26**) and related structures have been identified as neurotoxins and antifungals (Christensen and Brandt, 2006).The fatty acid amide capsaicin (**4.27**) is the pungent principle of red chili, but was also shown to be an antioxidant (Srinivasan, 2014). Volatiles fatty acid derivatives play an important role as intraspecific insect pheromones by facilitating the location of females by males. The first one elucidated was bombykol (**4.28**) that is produced by the domesticated silk moth *Bombyx mori*. Males possess two receptors in adjacent pheromone-sensitive neurons in their antennae, one for bombykol and another, interestingly, for its oxidized form, bombykal (Nakagawa et al., 2005).

The presented secondary LMWM examples illustrate the difficulty of assigning specific structures to specific organisms and specific biological activities to specific structures. One of the more important factors that determines LMWM beneficial and toxic effects is their concentration. Often the same compound can exert beneficial effects in low concentration and toxic in higher, a phenomenon that is known as hormesis (Calabrese, 2005). Generally, organic compounds are not well soluble in aqueous solutions. However, the cytosol is not an aqueous solution but has a mysterious gel-like structure (Pollack, 2001). Its mystery is caused also by its in accessibility to standard analytical methods due to the colloidal matrix structure that is formed by LMWMs and proteins. For certain, strong electron acceptor and donor properties contribute to biological activity, but not all strong electron acceptors are necessarily pro-oxidant toxins and not all strong electron donators antioxidants or hormones. Aromaticity is often connected with biological activity, but not exclusively so. Basically, in terms of functional groups, aromaticity, electron donor and acceptor capabilities, a comparison of hormones, most vitamins and secondary LMWM does not reveal any fundamental differences. Solubility and the potential of forming charge-transfer complexes with protein functional groups may constitute essential factors that affect their biological activity, but not exclusively so.

### **LMWM STRUCTURE–ACTIVITY CONSIDERATIONS**

Comparing the structures of different LMWMs in **Figures 1**–**4**, it becomes apparent that only the different types of primary, basic, or central metabolites, such as amino acids, organic acids, sugars and fatty acids, are characterized by specific combinations of unsaturated bonds and heteroatoms. Others, hormones, vitamins, and secondary LMWMs lack this characteristic. In evolving as components of the general metabolic pathways, central LMWM structures most probably oblige to the specific chemistry that is required of them to contribute accordingly to the metabolic pathways of which they have evolved to be a part of (Bar-Even et al., 2012). Starting from partially enzymatic or nonenzymatic reaction cascades, gene and operon duplication events and gene elongation contributed fundamentally to the evolution of a set of specific enzymes that controls the chemistry of the extant metabolic pathways (Fani and Fondi, 2009).

Other LMWM groups, hormones, vitamins, and coenzymes and secondary LMWMs, however, do not share comparable structural characteristics, similarly as their distribution is not as widespread as that of central metabolites. Their currently attributed functions are also not as clear-cut. For example, several nitrogen-containing secondary LMWM can interact with major neuroreceptors, such as cholinergic, adrenergic, serotonergic and GABAergic neuroreceptors (GABA, γ-aminobutyric acid), and Na+, K+, Cl−, and Ca+-channels. This explains why the intake of larger dosages inevitably causes substantial physiological and psychological disturbances (Wink and van Wyk, 2008). In the past, conversely, certain nitrogen-containing LMWMs were regarded just to serve as simple nitrogen storage intermediaries (Rosenthal, 1982). The currently must broadly accepted concept posits that the major cellular targets of LMWMs are proteins, specifically the three-dimensional structure of proteins, including receptors, enzymes, ion channels, transporters, hormones, transcription factors, regulatory and cytoskeletal proteins. Membrane fluidity and permeability represents a further target area and, last but not least, LMWMs can react directly with both DNA and RNA (Wink and Schimmer, 2010). Non-covalent complex formation, especially that of the charge-transfer type, may contribute as an important mosaic stone to the required specificity of LMWMs (Szent-Györgyi, 1960, 1968; Doukas, 1975). But to obtain more detailed insights into LMWM chemistry, we have to consider oxygen and coordination chemistry, both of which form the boundary between organic and inorganic chemistry.

## **LMWM COORDINATION CHEMISTRY**

**CHEMISTRY OF LIFE**

Predominantly, LMWM chemistry is viewed as organic chemistry. In this section, the focus, however, is set on selected inorganic elements, whose changing availability and adopted utilization has shaped the evolution of living organisms besides organic chemistry. Life started with anaerobic prokaryotes, in which the earliest organic chemistry formed DNA itself in a reductive milieu that required hydrogen input from water. As a result, about 3 billion years (bya) ago, the environment changed to more oxidized conditions. These processes are thought to have facilitated the development of protection mechanisms against oxygen and, subsequently, the development of aerobic prokaryotes. The second most important chemical change in evolution was the appearance of eukaryotic cells, which is assumed to be facilitated by a sequence of events that led to a systematic development of the combined inorganic/organic chemistry in attempts to separate the unavoidable oxidative from the reductive chemistry. Among novel structures we find membranes, the capture of bacteria as organelles (chloroplasts and mitochondria) and the calcium messenger system. During the period of two to one bya less changes occurred. Shortly after one bya, oxygen concentration began rising again leading to unavoidable changes in the environmental chemistry. In parallel, the cellular oxidative chemistry evolved to produce multicellular eukaryotes, the third very important chemical change. Protecting the cellular reductive chemistry from the increasingly diverse oxidative chemistry outside of the cell increased the demand for developing additional compartments, the realization of which was only possible in multicellular eukaryotes. Oxidative extracellular and vesicle chemistry led to crosslinking of connective structures by restricting their movement and creating organs of differentiated cells. Especially copper enzymes helped connective tissues to grow and in the synthesis of many LMWM messengers that facilitated communication between cells and organs. Concomitantly, hydrolytic zinc proteins managed cleavage of these tissues. The same element proved essential in the development of zinc finger transcription factors that help relating hormone information to gene expression. Connective tissues and messengers for the control of the whole organism with outer skin layers enabled the evolution of huge plants and animals. The latter developed fast Na+/K+ exchange currents in nerves, which resulted in the evolution of brains. Both the nerves and the brain utilize the potential of Na+/K+ gradients that have been developed by the earliest cells by excluding Na+. Brain development enabled man finally to understand the chemistry and physics behind it. Between 600 and 400 million years ago (mya), oxygen levels ceased to rise and environmental changes decreased, which implies that no cellular chemical changes occurred from this time point onward and only mutations could change DNA to produce species variants. This topic is covered in much detail by two excellent books (Williams and Fraústo da Silva, 2006; Williams and Rickaby, 2012).

### **OXYGEN AND OTHER REACTIVE SPECIES**

Today's oxygen-rich atmosphere makes oxidative stress unavoidable. Aerobic metabolism and the exposure to various forms of abiotic and biotic stress creates the so-called "Oxygen Paradox." On the one hand, aerobic life's energy supply depends on the reduction of molecular oxygen, on the other hand, oxygen is toxic to life in higher concentrations (Davies, 2000). In tissues, therefore, oxygen concentration usually rises only to one fifth of that which is found in ambient air. One factor that makes oxygen dangerous is its incomplete reduction by accidental oneelectron transfers, which do not yield water but various reactive oxygen species (ROS). These include superoxide anion radical (O•− <sup>2</sup> ), hydrogen peroxide (H2O2) and hydroxyl radical (•OH) (**Figure 5**) (Demidchik, 2015). Another reactive oxygen species is singlet oxygen (1O2), a dangerous byproduct of photosynthesis in plants. Insufficient energy dissipation results in the formation of an excited triplet state chlorophyll that can transfer its energy on

**FIGURE 5 | Redox and coordination chemistry of LMWMs.** Reactive species chemistry is marked in red and coordination chemistry in blue (exception bar graph); subtitles explain the details; chl, chlorophyll; n, variable oxidation states.

ground-state oxygen (3O2) (Triantaphylidès and Havaux, 2009). In attempts of keeping the threat of the high ROS reactivity at minimum, various LMWM antioxidants and enzymes have evolved (Fridovich, 1998; Apel and Hirt, 2004; Halliwell and Gutteridge, 2007).

Besides reactive oxygen species, we also know reactive nitrogen species (RNS), for example nitric oxide (•NO), and sulfur reactive species, for example thiols, disulfides, sulfenic acid derivatives, thio-sulfinates and -sulfonates, and thiyl radicals, the latter presently being less in the research focus compared to RNS (Giles and Jacob, 2002; Gruhlke and Slusarenko, 2012; Groß et al., 2013). The radical •NO can arise by the following routes: (I) by compartment-specific reactions; (II) in chloroplasts and plant mitochondria from nitrite (NO2) reduction by electron transport chain deficient processes (accidental one-electron transfers); (III) in plant peroxisomes from nitrite reduction by xanthine oxidoreductase; (IV) in the plant cytoplasm by nitrite reduction; (V) in the plant apoplast spontaneously at low pH by membrane-bound nitrite and nitrate reductases; and (VI) in mammalian cells nitric oxide synthase (NOS, **Figure 5**) oxidizes the amino acid arginine. The latter enzyme has not yet been identified in plants although being suspected to be involved in most of its formation processes. Nitric oxide can be highly toxic because it can more or less react with ROS, for example with peroxynitrite (**Figure 5**) and also every pro-and antioxidant LMWM (Groß et al., 2013). Furthermore, proteome wide-scale analyses revealed that nitric oxide can nitrosylate sulfur groups besides of cysteine in proteins, which has a fundamental effect on their functions (Astier et al., 2012).

Complex formation, short half-lives and high reactivity characterize the various reactive species of oxygen, nitrogen and sulfur. This supports increasingly the notion that reactive oxygen and nitrogen species signaling, antioxidants and thioredoxin mediated redox regulation just represent parts of the more or less same broad concept. The integration of different redox inputs could represent a system that regulates proteolysis, gene expression and functioning of specific metabolic pathways in a more graded fashion and functions independently of a binary "yes or no" response (De Tullio, 2010). **Figure 5** presents a summary of the various oxygen, nitrogen and sulfur reactive species, and chemical reactions involved in their formation. The various radicals can readily react with each other, as well as with LMWMs, proteins and membranes in the cell (Giles and Jacob, 2002; Halliwell, 2006; Møller et al., 2007; Groß et al., 2013). The ability of some LMWMs to reduce molecular oxygen by one-electron transfers is regarded as the major mechanism contributing to their cytotoxicity (Kappus and Sies, 1981). This pro-oxidant mode of action is utilized by nearly all efficient anti-cancer drugs (Watson, 2013).

### **BIOINORGANIC OR COORDINATION CHEMISTRY OF LMWM**

LMWM contain more or less the same elements as proteins. They can be divided in non-metals and metals. The latter differ from the former in their ability of conducting electricity in the condensed state. Among non-metals we typically find C, N, O, S, P, Cl, as well as H, but also B and Se. The fact that metals of the first two periods possess less than three electrons in their outer scale contributes significantly to their ability to form cations (Mn+). This is especially easy for metals of the groups 1, 2, 3, 12, and 13, among which we find Na, Mg, Al, K, and Ca. Transition metals from the groups 4–11, however, are less prone to behave like this. Fe, the most common transition metal in the Earth's crust, Mn, Cu, Zn, V, and in sea water additionally Co, Ni, and Mo, are among the most abundant and/or available (Fraústo da Silva and Williams, 2001; Williams and Fraústo da Silva, 2006; Crichton, 2008; Ochiai, 2008; Marschner, 2012). **Table 1** presents a compact summary of known biochemical functions of the various elements. Metals can serve as essential catalysts, either in acid/base and/or in electron transfer reactions. Those metals that lack good cation formation properties depend on forming coordination complexes with either organic or inorganic ligands (**Figure 5**). The metal central atom is a Lewis acid and the organic or inorganic ligand a Lewis base. In terms of bond strength, the coordination bond resembles a covalent bond but stability decreases with low pH. Coordination complexes can be of tetrahedral, trigonal, linear, trigonal bipyramid, or octahedral geometry, which means that one central atom can coordinate various ligands. Depending on the nature of the ligand and the stereochemistry of the central atom the standard redox potentials of CuII/Cu, FeIII/FeII, MnIII/MnII, CoIII/CoII can be altered by more than 1.0 V (Crichton, 2008).

Generally we know LMWMs as simple, uncoordinated compounds because in attempts to isolate them from their sources the complexes are usually destroyed. Nearly all LMWMs with oxygen, nitrogen and sulfur can act as Lewis bases and thus participate as ligands in coordination chemistry. Depending on their actual state as uncoordinated molecule or ligand in a coordination complex, LMWMs can act as catalysts. **Figure 5** presents an example: Hydroxyl radicals are among the most aggressive reactive oxygen species with the exclusive ability to trigger chain oxidations on nearly every biomolecule (Voeikov, 2001; Halliwell and Gutteridge, 2007; Demidchik, 2015). The deoxyribose degradation assay allows assessment of their iron-catalyzed formation rate from hydrogen peroxide by quantification of deoxyribose oxidation products as thiobarbituric acid-reactive species. The naphthoquinone juglone, an allegedly allelopathic secondary LMWM of the walnut tree, can form coordination complexes

**Table 1 | Main biochemical functions of elements (Fraústo da Silva and Williams, 2001; Crichton, 2008; Marschner, 2012; Williams and Rickaby, 2012).**


with Fe ions only if iron is not complexed by EDTA. If iron is added as EDTA complex to the reaction mixture, juglone remains a free molecule. **Figure 5** illustrates the dramatic difference between the two scenarios in terms of hydroxyl radical formation (Fenton chemistry) catalysis (Chobot and Hadacek, 2009). The only difference is the presence and absence of EDTA, ascorbic acid is present in identical amounts in all tested concentrations of the two setups. The results from this experiment provide us with a faint idea of the difficile effects ligand identity can exert on metal catalysts. Even in haem iron (**Figure 5**), still two coordination sites remain free to catalytic activity-modifying ligands.

### **ROS CHEMISTRY IN WATER**

One puzzling fact is that half-lives of ROS are assumed to be extremely short in the cytosol, for 1O2 1μs, O•− <sup>2</sup> 1μs, H2O2 1 ms, and •OH 1 ns (Møller et al., 2007). The notion about the potential role of ROS has changed in the last decades. What was originally considered a toxic byproduct is now seen as essential component of cellular information signaling, especially in case of H2O2. In this context, the mechanism how ROS may be efficiently involved in long distance signaling is still under debate. A recent study summarized the problems: whilst H2O2 signaling based on pure diffusion without relaying stations is theoretically possible, the experimentally observed cellular background H2O2 concentrations are too high and enzymatic degradation too slow (Vestergaard et al., 2012). If catalytic formation, enzymatic or non-enzymatic, does not correspond to the observed H2O2 concentrations, the question about their source still remains unanswered.

Research on water has a turbulent history (Pollack, 2013). Actually, the idea that water gets more structured near the freezing point is not a new one (Szent-Györgyi, 1957), and several authors have suggested that this happens to water in the vicinity of hydrophilic and charged surfaces (Ling, 2001; Pollack, 2013). This proposed concept is not generally accepted (Ball, 2008), but the simple experiment that fluorescent dyes change the quality of their emitted light in liquid and freezing water provides us with some food for thought (Szent-Györgyi, 1957). It also creates a novel scenario for ROS (**Figure 5**). Vladimir Voeikov proposes that the formation of reactive oxygen species is more likely to occur in a structured water environment; experimental evidence is provided by ultraweak photon emission studies (Hercules, 1969; Voeikov, 2001, 2006). The possible proposed reactions include the hydrolytic cleavage of a water molecule in hydroxyl and hydrogen radical and the oxidation (burning) of water by molecular oxygen that is entering the aqueous solution by diffusion (**Figure 5**). Furthermore, these reactions are assumed to occur perpetually in an oscillatory mode. One tempting aspect of this proposed concept is that LMWMs can affect the speed of these reactions and thus not only change the intensity but also the frequency of the oscillation (Voeikov, 2001, 2006).

### **SYSTEM CHEMISTRY AND BIOELECTRICITY**

### **SYSTEMS BIOLOGY OR SYSTEMS CHEMISTRY**

During the last decade the term systems biology became more and more prominent. The goals of this emerging field are best described by Denis Noble, one of its pioneers: "Systems biology . . . is about putting together rather than taking apart, integration rather than reduction. It requires that we develop ways of thinking about integration that are as rigorous as our reductionist programs, but different . . . It means changing our philosophy, in the full sense of the term" (Noble, 2006). Undoubtedly, our view on LMWMs would also benefit from such an approach. In Section LMWM Coordination Chemistry we have provided an overview in terms of which chemistry LMWMs can cause. It can happen already on the pre-receptor level; steroid hormones represent an example (Mindnich et al., 2004). Most likely, LMWMs represent an important system component besides proteins and DNA.

In recent years, another new field, systems chemistry has taken shape (Kindermann et al., 2005). It is aimed at understanding the chemical origins of biological organization (Ruiz-Mirazo et al., 2014). As a consequence, Addy Pross suggested to incorporate Darwinian biological theory as replicative chemistry into a more general chemical theory of matter (Pross, 2012). Any chemical system, however, that is part of a biological system requires coordination mechanisms, some kind of cooperativeness. How this cooperativeness can work is perfectly outlined in Albert Szent-Györgyi's book "Bioenergetics": Chemical structures are comprised of letters and dashes, and biochemistry, following chemistry, has excelled similarly in describing structures and reactions by letter-dash-letter symbols (**Figures 1**–**5**). Quantum physics turned atoms into probability densities of electrons that build molecules of fantastic and changing shape. Accordingly, biological phenomena represent subtle changes in the shape that take place in dimensions still unknown to classical chemistry. One problem for quantum physics is that models with more than two electrons create unsurmountable mathematical difficulties. Classical biochemistry assumes that no interaction can take place between two molecules without touching one another but— as Szent-Györgyi points out—manifold interactions can take place through energy bands and the electromagnetic fields, which occur in water and its structure as the matrix of biological reactions (Szent-Györgyi, 1957).

### **BIOELECTRICITY**

At the end of the 18th century electrical phenomena in plants became known a couple of years earlier than in animals, but studies in the former have been eclipsed by those of neurons until recently (Niklas and Spatz, 2012). Electroneutrality requires equal numbers of anions and cations, and even if they differ slightly, an electric potential difference develops. In cells with an approximate radius of 30μm, of membranes lies in the 100 mV range. Cell membranes are semipermeable. In aqueous solutions, all electrically charged molecules exist in form of hydrated ions, and because of the differences in charge and size, their hydration spheres, exclusion zones, they differ in volume. Semipermeable membranes are assumed to use dialysis to restrict the permeability according to the hydrated ion size. A permeation equilibrium constitutes as a result of the osmotic pressure and the electric field potential (**Figure 6A**). Ions that fail permeating a membrane start accumulating on one side of the membrane and accordingly build up an electric charge (Diamond and Wright, 1969; Hille, 2001; Niklas and Spatz, 2012). The resting between the interior and exterior of a biological cell is −40 to −80 mV. If is shifted

#### **FIGURE 6 | Bioelectric system components in living organisms. (A)** Semipermeable membranes develop a resting potential by selectively allowing diffusion of K+ and Cl− until the Coulomb force halts further transfer; **(B)** particles with hydrophilic surfaces stimulate the development of an exclusion zone with negatively charged more ice-like structured water; **(C)** peptides can self-assemble into peptide nanotubes with semiconductor properties (Hauser and Zhang, 2010, with permission); **(D)** protein complexes in photosystem I and II and other protein complexes in the chloroplast can act as light-sensitive and non-light-sensitive diodes

during photosynthesis; **(E)** (1) neurons represent the best explored biotransistors, (2) signaling in the myelin sheath depends on depolarization by Na+ influx; (3) the Na+ channels are regulated by a system combining a membrane capacitor and a channel transistor (Farquhar and Hasler, 2005, with permission); **(F)** biopolymer piezoelectric field electron transistor: voltage in biopolymers, such as membranes, cell walls and their associated mucous matrices, can be affected by LMWM chemical activity (for example, increased metal-catalyzed free radical production) in concert with hydrostatic pressure.

more into the positive (influx of Na+ or Ca+), we speak of depolarization, if is shifted to a more negative value (efflux of K+ and/or influx of Cl−) of hyperpolarization (Hille, 2001; Carlson, 2014). This potential, again, blocks other similarly charged ions and creates an electrostatic resistance to ion and electron transfer. Thus, although there exist no specific high resistance insulator materials in organisms, substantial electric resistance can form. Consequently, electric charges can arise in many biological compartments. If this happens in a serial cascade, the formed charges may reach a quite substantial amplitude, the most spectacular example being the electric organs of fishes, e.g., those of the electric eel, *Electrophorus electricus*, with a of up to 600 V (Gotter et al., 1998).

Gerald Pollack proposed that water that is close to hydrophilic structures, resembles more the honeycomb structure of ice with a ratio of hydrogen:water of 2:1 (**Figure 6B**). Water in this exclusion zone has a negative charge (−1). The adjacent bulk water reacts by forming hydronium ions (H3O+) and thus has a positive charge. Charged entities, such as membranes, proteins, and DNA interact and interface with water. The forming is theoretically available to drive various cellular processes and would also offer an explanation for the negative potential of the cytosol (Pollack, 2013).

### **PIEZOELECTRICITY**

Endothermic reactions depend on energy availability. In this context, we should specifically consider biological piezoelectricity. The piezoelectric effect is described as the accumulation of an electrical charge by application of mechanical stress to a crystal (Martin, 1972), but it is known also to occur in certain solid materials of biological origin. Thus, piezoelectricity can cause changes of the membrane potential, the electrostatic fields of the tissue as well as its form (inverse piezoelectric effect). This can happen also in solid biological structures that maintain their specific inner organization and physicochemistry in living tissues. These structures comprise cell walls made of muramic acid, chitin and cellulose, colloidal mucins, phospholipid bilayers in biomembranes, fascia made of collagen, hyaline cartilage and bones (Fukada, 1984; Kim et al., 2010; Cheng and Qian, 2012). Mucus material and plant and bacterial mucilage can form colloid-like matrices on the surface of intra- and extracellular solid structures, thereby acting as interface to various cell- and tissue-specific liquids, such as cytoplasm, lymph and blood. Likewise, depending on water availability, organs are covered by mucus layers of variable depth, which can lubricate the surface, maintain the functional hydration state, and protect against invading microbes (Leppard, 1995; Boyton, 2002; Evert et al., 2009).

### **METAL-ORGANIC FRAMEWORKS (MOFs)**

The generally accepted notion is that proteins with their surfaces determine the milieu for the majority of chemical reactions. This somehow obscures the fact that LMWMs can form complex ions with a wide range of metals (see Section Bioinorganic or Coordination Chemistry of LMWM), which enables them more or less to act as comparable catalysts to enzymes in terms of potential chemical reactions catalysis though but less on terms of efficiency. A general idea how this can work is illustrated by one of the most-exciting, high profile developments in nanotechnology. Metal-organic frameworks (MOFs) are porous coordination polymers of mucus-like nature that contain metal-containing nodes and organic linker molecules. They are developed currently to serve as drug carriers in form of nanoencapsulators (McKinlay et al., 2010). Owing to their structural regularity and synthetic tunability, considerable hopes are directed at MOFs as platform to hierarchically organize synthetic light-harvesting antennae and catalytic centers to achieve solar energy conversion similarly as in photosynthesis (Zhang and Lin, 2014). Conversely, chloroplastic chlorophyll stacks somehow may be viewed as BioMOFs. LMWM can structure such BioMOFs as organic linker molecules and specifically affect chemical cell processes by regulating substrate availability and catalysis, comparably to and in concert with proteins. Both proteins and BioMOFs cannot avoid being affected by piezoelectric effects.

### **BIOELECTRIC SYSTEM COMPONENTS**

Some cell structures show striking similarities to basic electronic components (**Figure 6**). Some examples are presented in the ongoing text.

### *Biocapacitors*

A capacitor (originally known as a condenser) is a passive twoterminal electrical component used to store energy electrostatically in an electric field (Dorf and Svoboda, 2001). Traditional paper capacitors consist of cellulose layers positioned around a dielectricum in order to store a defined amount of electric charge to facilitate smooth power supply conditioning. A similar structure is found in the intermembraneous space of phospholipid bilayer membranes that surround cell nuclei, chloroplasts, mitochondria and between the cellulose fiber layers of plant cell walls (**Figure 6A**). Biomembranes show the unique feature of displaying phase transitions (melting) in a physiologically relevant regime (Heimburg, 2012).

### *Biosemiconductors*

A semiconductor is a material which possesses electrical conductivity between that of a conductor, for example any metal, and that of an insulator, such as glass or silicon. The conductivity of a semiconductor is augmented by "doping." Doping consists of the addition of electron donators to the insulator. In case of silicon, most commonly coordination complex formation with transition metals of the groups III and V is used. In contrast to metals, the conductivity of semiconductors increases with elevated temperature, and also with increased photon radiation (photovoltaic effect). Mucous colloids with complexed metals or hyaline cartilage can possess semiconductor properties (Sze, 1981). Recently, nanotechnology research revealed that peptides can self-assemble into peptide nanotubes by a mechanism that is not yet understood fully. These peptide nanotubes can be components of metallic/semiconductor-organic frameworks (BioMOFs) (**Figure 6C**) (Amdursky et al., 2010). Consequently, we may assume that peptides can form biosemiconductors *in vivo* too.

### *Biodiodes and biotransistors*

A diode is a serial combination of a doped (n) and an unchanged (p) semiconductor. An applied potential may only result in a current flow if the cathode is placed at the doped part of the diode. Diodes consisting of semiconductors are also sensitive to light by increasing their conductivity, and may emit light when high potentials are applied (light emitting diode, LED). The photosystems in photoautotrophic organisms can be seen as an array of biodiodes (**Figure 6D**).

Transistors are serial combinations of three doped and/or unchanged semiconductor elements (npn, pnp). The complete combination is rendered electrically conductive by applying a potential, placing the cathode at the doped element. The base current is applied at the first (emitter) and the middle (base) part. If this is high enough, conductivity between the first (emitter) and the third part (collector) is established. Thus, in essence a transistor represents an electronic switch depending on charge thresholds.

In biosensor development, different types of biologically sensitive field-effect transistors (BioFET) exist, in which whole cells are used to detect changes in extracellular pH, ion concentration, CO2 production, redox potential and metabolic products such as glucose and lactic acid. A special development represents the "beetle/chip" FET that is used to analyze pheromone perception of insects in electroantennogram studies (Schöning and Poghossian, 2002). The physical principles governing ion flow in neurons resemble electron flow through a metal-oxidesemiconductor field-effect transistors (MOSFET) (**Figure 6E**) (Farquhar and Hasler, 2005).

Apart from neurons, bioelectrical transistor elements have not been investigated intensively so far. The following scenario, however, is feasible: Biopolymer compression (mechanical stress) and/or oxidative (chemical stress) can increase electron availability by piezoelectricity. Mucoproteids with metall–LMWM complexes, especially of iron and copper, can serve as electron accepting "doping" agents. Transition metal-catalyzed electron transfers to molecular oxygen can increase free radicals and other reactive species (Section Oxygen and Other Reactive Species and Bioinorganic or Coordination Chemistry of LMWM). The cytosol is rich in various solutes. Oxidative stress increases LMWM amounts as it triggers antioxidant defenses. As argued by the structured water hypothesis (Ling, 2001; Voeikov, 2001, 2006; Pollack, 2013), structured water zones on hydrophilic and charged surfaces can increase reaction rates of this chemistry by providing more energy due to their battery-like nature and affect the conductivity of a particular tissue region by modulation of the electromagnetic fields in the present biopolymers, such as membrane lipids, cellulose, chitin or collagen and their associated mucous matrices. LMWMs interact with concentrations of reactive species because of their redox chemical properties and affect a multitude of reaction cascades that contribute to the phenomenon of oxidative stress. Such an effect on voltage-sensitive K+ channels is documented and accepted as given in a recent critical review (Sahoo et al., 2014), irrespective of any contributions of structured water to this effect and despite the experimental difficulties. Numerous studies document LMWM effects on various membranous ion channels, amongst others by phenols (Ishimaru et al., 2012), peptides (Maischak et al., 2010), and terpenoid LMWMs (Marrè, 1979; Zimmermann and Mithöfer, 2013). Electrical signals can trigger the same downstream responses as chemical ones, only faster. This has been impressively demonstrated for the wound-induced methyl jasmonate-systemin signal cascade, in which an electrical longdistance signal, probably a system potential (see for more details Section Bioelectric Studies) leads to proteinase inhibitor accumulation as systemic response in damaged tomato plants (Wildon et al., 1992). A substantial component of this signal cascade represent changes in xylem hydrostatic pressure (Farmer et al., 2014). Consequently, the scenario of a "biopolymer piezoelectric field electron transistor (BIOPFET)" as suggested by **Figure 6F** is quite feasible despite of the fact that many details request further exploration and clarification.

### **BIOELECTRIC STUDIES**

Searching the literature for the term "bioelectricity" yields a lot of papers about bioenergy but fewer on physiology. The most cited one dealing with the bioelectricity is a more or less 10 year old review that attempts to clarify several misconceptions that have arisen in connection with physiological bioelectricity (McCaig et al., 2005). The most promising application is the clinical potential of electric field treatment of damaged tissues of epithelia and the nervous system. An important insight from animal studies was that electrical fields exist intra- and extracellularly: (I) Voltage gradients exist within the extracellular spaces underneath the frog skin; (II) disruption of the natural electric fields in amphibians disrupts development; (III) endogenous currents and voltage gradients are present in chick embryos, disrupting them also disrupts development; and (IV) a voltage gradient exists across the neural tube; neuroblasts, the precursor cells of neurons, differentiate in this gradient. Another important insight concerned electrical fields that are generated by healing epithelia, which control the healing process: (I) Rat cornea wound healing is controlled by an electrical field and, specifically—LMWM drugs were shown to affect the cornea's - (Song et al., 2002); (II) epithelial cell proliferation and the cell division axis are regulated by a physiological electrical field; (III) and nerve growth is regulated by an electrical field. Transcellular signals can regulate the spatial expression of genes that control left/right organ symmetry (Levin et al., 2002). Furthermore, supporting data emerged for the notion that intracellular gradients of potential segregate charged proteins within the cytoplasm, a kind of electrophoresis along cell membranes (Jaffe, 1977).

A genetic study on the role of G protein-coupled receptor signaling in originally as chemotactic identified aggregation of the model slime mold *Dictyostelium discoideum* suggested that chemo- and electrotaxis share a similar signaling mechanism (Zhao et al., 2002). However, later studies confirmed this only for the most downstream elements (McCaig et al., 2005). Given the high parallels of electro- and chemotactical phenomena, the lack of shared recognition mechanisms still remains enigmatic.

Whereas in animals only one genuine electrical signal is recognized, the action potential, in plants two more can be found, the variation and system potential (Zimmermann and Mithöfer, 2013). The action potential results from a transient depolarization from the plasma membrane (Davies, 1987). Within plants, they are especially evident in those with rapid (nyctinastic) leaf movement, the best known being touch-me-not (*Mimosa pudica*) (Volkov et al., 2010) and the Venus flytrap (*Dionaea muscipula*) (Volkov et al., 2008).

Variation potentials, sometimes also known as low-wave potentials, represent transient depolarizations of the plasma membrane with variable shape, amplitude and time frame with downstream effects on gene expression (Davies, 1993). They can be elicited by diverse mechanical and physical stimuli, such as heat, wounding, pressure, but also by chemical factors.

Systems potentials, by contrast, reflect a systemic selfpropagating hyperpolarization of the plasma membrane or depolarization of the voltage of the apoplastic colloidal matrix (Zimmermann et al., 2009). The participation of proton pumps in generating these potentials is suggested by triggering activity via the terpenoid LMWM fusicoccin, a toxin of the phytopathogenic fungus *Fusicoccum amygdali* (Marrè, 1979).

Apart from rapid leaf movement, electrical signals have been shown to be involved in plant root-to-shoot communication, fertilization, photosynthesis regulation, gene expression, longdistance signaling in woody plants, and root growth coordination (Fromm and Lautner, 2012). Moreover, experimental evidence documents that action and variation potentials can affect light and dark reactions of photosynthesis as well as respiration in above- and belowground organs (Pavlovic, 2012 ˇ ). Sessile plants never have developed the same degree of neuronal network complexity as scavenging animals even though electricity also constitutes an important component of their signaling that enables them to survive in changing environments. Long-distance signaling suggests the existence of biocircuits, in plants these might be xylem and phloem vascular bundles, in animals connective tissues (fascia), the latter showing functional conformity with the meridians in Traditional Chinese Medicine (Keown, 2014).

### **OUTLOOK: LMWMs IN CHEMICAL SYSTEMS REGULATED BY ELECTRICITY**

Systems, biological and chemical, represent huge crossword puzzles waiting to be resolved and there probably exists only one possible solution that facilitates understanding, a simple unifying concept. Today, discipline fragmentation contributes a lot to impeding us finding it (Ling, 2001; Firn, 2010). Such a possible unifying concept could look like the following one:

The Russian cell physiologist Dimitrii Nasonov described the fundamental phenomenon of the universal cell response, UCR, a biphasic response, resting or activated, to external stimuli (stress). The two states differ in terms of protein structure folding (Nasonov, 1962; Ling, 2001; Matveev, 2010). The resting state is a coherent meta-stable low entropy state with water and K+ bound to proteins and the active state a higher-entropy state because water and K+ are free (Jaeken and Matveev, 2012). The thoughtful reader will now argue that the described cytosolic scenarios contradict the generally favored membrane pump hypothesis (**Figure 6A**). This is true. But in an attempt to provide a synthesis for all the focused chemical detail mechanisms in terms of a chemical system, we have to adopt a view that considers the phenomenon of cooperativeness as a major component of this chemical system. In biological terminology, cooperativeness could be called symbiosis. Consequently, a focus on membrane potentials is perhaps too reductionist.

The second important phenomenon that is inherent to life is replicative chemistry because it facilitates the required organization for the concomitantly running chemical reactions. If an organism loses that, it's unorganized decomposition starts and the chemistry that accompanies the development of diseases resembles that of organic matter decomposition which exactly follows the second law of thermodynamics by increasing the entropy of the involved systems. In his booklet "What is Life," Erwin Schrödinger stated that life feeds on negative entropy (Schrödinger, 1944), or better Gibbs free energy. Coupling energy input from sunlight and exothermic reactions with endergonic reactions that are not entropically favored defines metabolism in living cells.

The requirements for life chemistry have never been summarized more aptly as by the organic chemist Addy Pross: "a self-sustaining kinetically stable dynamic reaction network derived from replication reaction" (Pross, 2012). In his tantalizing book, though, Addy Pross does not offer examples for the chemical reaction network apart from template availability facilitating nucleotide replication as example for autocatalysis. In attempts to find an approach to deal with LMWM chemical complexity, another author merits mentioning, Bruce B. Jarvis. He regards LMWM as molecular communities that self-assemble into structures that can support complexity when a series of interconnected events occur (Jarvis and Miller, 1996). These complexity events "are characterized by participation in complex interlocked cycles involving feedback mechanisms controlled by an elaborate chemical signaling system, a unicellular organism life." Unicellular organisms develop into multicellular, the latter organize themselves in communities, and these yield societies.

At this point we want to look at the signaling system in particular and question the statement that a chemical reaction system can be controlled by chemicals. What do chemical reactions require? Electrons and energy. Both is provided by electricity, fast, and universal. Microorganisms can directly accept electrons from electrodes to reduce carbon dioxide, nitrate, metals, organic acids, protons and oxygen (Lovley, 2011). The bacterium *Geobacter sulfurreducens* masters long-range electrons transport along its pili, so-called microbial nanowires that have the same metallike conductivity as synthetic conducting polymers. Pili networks facilitate biofilm conductivity with supercapacitor and transistor properties (Lovley, 2012). Similarly, proteins and membranes of cellular organelles also represent biotransistors (**Figure 7A**). Both are biopolymers and are doped with oxygen, nitrogen and sulfur functions that can act as electron donors or acceptors or facilitate metal complexation. In membranes, the lipid bilayer acts as insulator but the glycerol esters, sphingolipid and ceramide functions, for example, can contribute to semiconductor properties. LMWMs can form various non-covalent bonds, charge-transfer or coordination complexes, which contribute to the formation of gel-like biopolymers. Similarly to proteins, these gel biopolymers can contain coordination complexes with metals that can act as catalysts of acid–base or electron transfers. Depending on the oxidation/reduction state of contacting functional groups and the electrical field of the biopolymer complex, electrons and energy are either transferred from or to the protein. As a component of a chemoelectrical signaling system (CSS) biotransistors can act

as a local short-distance signaling mechanism. Structured surface water most likely enhances than impedes its functioning. In colloidal matrix organized LMWMs somehow represent "small coins" that an organism is in constant need of in order to maintain its homeodynamics in a changing environment (Kinzel, 1989).

Ultraweak photon emission denotes the low-intensity spontaneous or inducible photon emission that accompanies chemical reactions (electron transfers). It is emitted both by abiotic matter and living cells and tissues, whole organs and organisms. Research on it was mainly carried out in Eastern Europe, but it is known to increase in response to oxidative stress and thought to originate from 1O2, triplet excited states (e.g., carbonyls), peroxynitrite reactions, lipoxygenase activity, haem protein/peroxide reactions and Fenton chemistry, the transition metal catalyzed reduction of H2O2 to •OH (Halliwell and Gutteridge, 2007), as well as in structured water zones close to LMWMs, proteins and other hydrophilic surfaces (Voeikov, 2001, 2006) (**Figure 5**). Vladimir Voeikov suggests that it can be absorbed by aromatic structures that represent a common element in many LMWMs and nucleobases. This property can enable ultraweak photon emission to act as long distance feedback component in the CSS (**Figure 7B**).

This herein proposed concept of a chemoelectrical signaling system (CSS) (**Figures 7A,B**) could serve as a candidate for the self-sustaining kinetically stable dynamic reaction network derived from replication reaction that represents the systems chemistry of a living organisms (Pross, 2012). It also has the potential to regulate its replication by affecting known and hitherto unknown epigenetic control mechanisms of gene expression (Shapiro, 2011). Changes in electrical field intensity and frequency in connected biotransistors could characterize the resting and the active cell state. The CSS can monitor and coordinate the many physiotypes or better physiolomes that cells, tissues and organisms can develop. It provides a concept that can be useful in tackling many still idiosyncratic and enigmatic phenomena in biological sciences. Most of all, it reminds us that we should not loose ourselves in LMWM structural diversity but focus on how the LMWMs cooperate within the CSS they are a part of. Otherwise, we risk not recognizing the forest behind the trees. In the opinion of the authors there exists sufficient compelling evidence for the existence of a CSS in the literature. Last but not least, our conclusions have been already voiced by Albert Szent-Györgyi (Szent-Györgyi, 1960, 1968) and thus are not new. Furthermore, they are aimed at complementing but not at challenging approved paradigms.

### **ACKNOWLEDGMENTS**

We apologize to all authors whose work we could not cite appropriately due to space restrictions. The authors are grateful for constructive comments from three anonymous reviewers. Furthermore, the authors acknowledge the coverage of the publication fee by the Publikationsfonds 2014/2015 of the Georg-August Universität Göttingen.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### *Received: 30 November 2014; accepted: 11 February 2015; published online: 04 March 2015.*

*Citation: Hadacek F and Bachmann G (2015) Low-molecular-weight metabolite systems chemistry. Front. Environ. Sci. 3:12. doi: 10.3389/fenvs.2015.00012*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2015 Hadacek and Bachmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## EPR spectroscopy and its use *in planta*—a promising technique to disentangle the origin of specific ROS

#### *Anja Steffen-Heins <sup>1</sup> and Bianka Steffens <sup>2</sup> \**

*<sup>1</sup> Micro- and Nanostructures in Foods, Kiel University, Kiel, Germany*

*<sup>2</sup> Plant Physiology, Philipps-Universität Marburg, Marburg, Germany*

### *Edited by:*

*Naser A. Anjum, University of Aveiro, Portugal*

#### *Reviewed by:*

*Naser A. Anjum, University of Aveiro, Portugal Preeyaporn Koedrith, Mahidol University, Thailand Goran G. Baci ˇ c, University of ´ Belgrade, Serbia*

#### *\*Correspondence:*

*Bianka Steffens, Plant Physiology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35043 Marburg, Germany e-mail: bianka.steffens@ biologie.uni-marburg.de*

While it is widely accepted that reactive oxygen species (ROS) are common players in developmental processes and a large number of adaptations to abiotic and biotic stresses in plants, we still do not know a lot about ROS level control at cellular or organelle level. One major problem that makes ROS hard to quantify and even to identify is their short lifetime. A promising technique that helps to understand ROS level control *in planta* is the electron paramagnetic resonance (EPR) spectroscopy. Application of the spin trapping method and the spin probe technique by this advanced method enables the quantification and identification of specific ROS in different plant tissues, cells or organelles or under different conditions. This mini review summarizes the knowledge using EPR spectroscopy as a method for ROS detection in plants under different stress conditions or during development. This technique allows disentangling the origin of specific ROS and transient alteration in ROS levels that occur by changes in ROS production and scavenging.

**Keywords: electron paramagnetic resonance (EPR) spectroscopy, reactive oxygen species (ROS), ROS detection, spin probe, spin trap**

### **INTRODUCTION**

Reactive oxygen species (ROS) are derivatives of molecular oxygen. The term "ROS" combines non-radical forms of oxygen such as hydrogen peroxide (H2O2), singlet oxygen (1O2) or ozone (O3), and oxygen-centred radicals such as superoxide anion radicals (O − <sup>2</sup> ) and hydroxyl radicals ( OH). All these kinds of ROS are generated in plants during development or different stresses. The primary ROS is often O − <sup>2</sup> that is produced either by plasma membrane-located NADPH oxidase or in electron transfer chains of mitochondria (Torres et al., 1998; Blokhina and Fagerstedt, 2010; Shapiguzov et al., 2012). ROS such as H2O2 are converted in enzymatic or non-enzymatic steps. All ROS are highly active in terms of oxidative modification of lipids, proteins, DNA and RNA. Also, ROS are indispensable in cellular signaling processes.

ROS are involved in the regulation of many internal plant processes such as growth (e.g., Schopfer et al., 2002) and death of specific cells (e.g., Steffens and Sauter, 2009; Steffens et al., 2011, 2012), to name only two. It is therefore indispensable to find out about ROS levels as well as specific ROS in organs, tissues or even cells, and organelles. ROS are however highly reactive and exhibit very short lifetimes that vary from nanoseconds to seconds. OH, for example, reacts with most organic compounds by electron addition or electron transfer (Renew et al., 2005) and has a lifetime of about 10 ns. O − <sup>2</sup> exhibits a low steady state concentration of around 10−<sup>10</sup> M in different cell or organelle types (Gardner, 2002). The half-life of O − <sup>2</sup> depends on its concentration. At a concentration of 10μM O − <sup>2</sup> exhibits a half-life time of 0.2 ms in water, whereas at a lower concentration of 1μM half-life rises to 20 ms. 1O2 exhibits a lifetime of 2.7μs (Karonen et al., 2014). Effort has been made to develop *in planta* ROS detection methods that are suitable to identify specific ROS and to quantify them in order to understand ROS signaling and ROS level control. These spectrophotometrical techniques, histochemical or live cell imaging approaches have unfortunately tremendous disadvantages; chlorophyll has to be removed from the tissue for histochemical ROS detection. Quantification of ROS is neither possible with histochemical methods nor with the use of small-molecule fluorescent probes (for review see Steffens et al., 2013). Fluorescent probes, however, benefit from their ability to detect ROS in living cells by confocal laser scanning microscopy.

Electron paramagnetic resonance (EPR; also termed electron spin resonance, ESR) spectroscopy is a widely used method for detecting the presence of unpaired electrons, such as ROS. Using the X-band, EPR is the most specific and even sensitive technique to identify, quantify and visualize the short-lived ROS (Baciˇ c et al., ´ 2008). Nevertheless, it is very challenging to monitor ROS successfully in biological systems due to their very low concentration, the enzymatic defense systems and the different compartments of the living cell. A way to make short-lived ROS detectable by EPR is the application of spin traps or spin probes. In this mini review we will focus on these two methods of ROS detection by EPR *in planta*.

### **ROS DETECTION** *IN PLANTA* **BY THE SPIN TRAPPING METHOD**

Spin traps are stable, diamagnetic compounds that form longerlived radical species with transient, very reactive radicals with low half-lives of only 10−<sup>9</sup> to 10−<sup>1</sup> s. The paramagnetic spin adducts are stable for minutes or even hours, accumulate in the tissue and reach a sufficient concentration for detection by EPR (Mojovié et al., 2005). The prerequisites for suitable spin traps are defined by their ability either to exclusively trap one radical species or to lead to different specific signature EPR spectra. The sensitivity of the trapping technique depends on the local spin trap concentration, the concentration of the transient radical, the reaction kinetic to form adducts and the stability of these adducts (Baciˇ c´ et al., 2008). Properties of the spin traps, such as lipophilicity are also crucial for an effective detection of radicals. The trapping technique benefits from the fingerprint spectra of the adducts, allowing identification of the trapped radical species and even quantification by double integration of the whole spectra or the low-field signal by using a calibration curve.

Various radical specific spin traps such as Tiron and 4-POBN are available. Tiron was applied for specific O − <sup>2</sup> detection in microsomal membranes of *Dianthus caryophyllus* or roots of *Triticum spp.* (Mayak et al., 1983; Vylegzhanina et al., 2001; Taiwo, 2008; **Table 1**). 4-POBN solved in ethanol exclusively detects OH (Renew et al., 2005; **Table 1**) by forming a 4-POBN/hydroxyethyl radical adduct generated from oxidation of ethanol by OH. This spin adduct is stable for hours. Renew et al. (2005) used 4-POBN to perform a region-specific OH profiling in roots of *Cucumis sativus* by detecting individual spin adduct spectra in distinct regions of the root. With this EPR spectroscopy analysis, the growth zone of the root was identified as site of OH production (Renew et al., 2005). A couple of studies were done specifically detecting OH with 4-POBN in surrounding medium of growing *Zea mays* coleoptiles (Schopfer et al., 2002; Liszkay et al., 2003) or in roots of *Zea mays* and *Arabidopsis thaliana* (Liszkay et al., 2004; Renew et al., 2005; **Table 1**). In addition, 4-POBN was applicable to analyze OH in single cells of *Oryza sativa* suspension cultures (Kuchitsu et al., 1995) or even in membranes of *Spinacia oleracea* and *Pisum sativum* thylakoids (Borisova et al., 2012). Both spin traps however do not seem to be the best choice in biological systems. The 4-POBN/OH adduct may be converted into a 4-POBN/4-POBN spin adduct during the reaction of peroxidases, whereas Tiron is acidic which decreases intra- and extracellular pH value and may alter O − <sup>2</sup> production (Baciˇ c and Mojovi ´ c,` 2005).

One of the first descriptions of O − <sup>2</sup> detection with the spin trap technique using EPR spectroscopy *in planta* was given by Habour and Bolton (1975). Harbour and Bolton detected O − <sup>2</sup> production in chloroplasts of *Spinacia oleracea* with an O − <sup>2</sup> adduct of DMPO; this spin trap was also used to detect O − <sup>2</sup> in thylakoid membranes about 20 years later ((Hideg et al., 1994); **Table 1**). Since then improvement of spin traps with longer lifetime, less degradation of the spin adducts and a faster reaction kinetic, such as the DMPO analogs DEPMPO, EMPO and BMPO, led to a successful trapping of both, O − <sup>2</sup> and OH (**Figure 1A**). DEPMPO is the phosphorylated analog of DMPO. DEPMPO adducts are stable for 22.3 min and exhibit a lifetime 10 times longer than DMPO adducts. EPR spectroscopy was used to analyze oxygencentered radicals of OH with DEPMPO in apoplastic fluid of *Zea mays* roots (Dragišic Maksimovi ` c et al., 2014 ` ). During growth, cell wall loosening is facilitated by OH. DEPMPO was effectively used to detect ROS in root cells of *Pisum sativum* with EPR and to differentiate between O − <sup>2</sup> and OH (Veljovic-Jovanovi ` c` et al., 2005; Kukavica et al., 2009). Unfortunately, there are four DEPMPO/OOH species, and DEPMPO/OH shows diastereomers (Dikalov et al., 2005), making the identification of radical species more complicated. Both DMPO and DEPMPO lead to the conversion of the O − <sup>2</sup> -adduct into the OH-adduct which underestimates the O − <sup>2</sup> detection (**Figure 1A**). Transformation of DEPMPO occurs at a slower rate. To avoid the problem of transformation, the carboxylated DMPO analog EMPO and an analog with a large butoxycarbonyl group, BMPO, were developed (Baciˇ c´ et al., 2008). Both radical specific spin traps are able to exclusively detect O − <sup>2</sup> (**Figure 1A**). The EMPO/OOH adduct is eight times more stable than the DMPO/OOH adduct. BMPO/OOH adducts are slightly more stable than the EMPO/OOH adducts because of the large butoxycarbonyl group. Other analogs of the DMPO group, such as DPPMPO, DBPMPO, and DEHPMPO, possess a higher lipophilicity and allow measurements in lipophilic media (Baciˇ c et al., 2008 ´ ).

Spin traps specific for 1O2 are TEMP and TMPD. TEMP was used to specifically detect 1O2 in thylakoid membranes of *Spinacia oleracea* (Fischer et al., 2006), and the more hydrophilic spin trap TMPD was used for 1O2 detection in thylakoid and plasma membranes of *Spinacia oleracea*, *Chlamydomonas reinhardtii,* or *Triticum spp.*, respectively (Qiu et al., 1995; Fischer et al., 2007; Yadev et al., 2010). 1O2 is one important reactive species generated under high light conditions in chloroplasts. It is scavenged by tocopherol and plastochromanol in *Arabidopsis thaliana*, as was shown by using a tocopherol cyclase-deficient *vte1* mutant (Rastogi et al., 2014). The spin trap TMPD was used to analyze the production of 1O2 in *Arabidopsis thaliana* under high light conditions at 1000μmol photons m−<sup>2</sup> s <sup>−</sup><sup>1</sup> with EPR spectroscopy. In *vte1* mutant plants 1O2 production was enhanced under high light, as was shown by using EPR spectroscopy (Rastogi et al., 2014). Combining mutant analysis and ROS detection by EPR spectroscopy will help to understand ROS effects and ROS signaling *in planta*.

Although spin traps benefit from their ROS specificity, with some of them detecting exclusively one ROS intermediate, high spin trap concentrations between 10 and 100 mM have to be used to reach an adequate sensitivity (Dikalov et al., 2011). Potential toxic effects, for example inhibition of photosynthesis, might occur at concentrations of more than 25 mM. Spin traps are often solved in ethanol; hence they are unsuited for the use *in planta* or other biological systems. Adducts may be transformed into other products (**Figure 1A**) or they may be reduced by plant metabolites into molecules without EPR activity.

### **SPIN PROBE TECHNIQUE—A BETTER CHOICE FOR ROS DETECTION** *IN PLANTA***?**

To circumvent the drawbacks of spin trapping technique the use of spin probes for ROS detection by EPR spectroscopy is favored. There are two possibilities of the use of spin probes. Commonly used spin probes are, on the one hand, endogenous nitroxides that are reduced by ROS to EPR-silent hydroxylamines. On the other hand, endogenous cyclic hydroxylamines (CHAs) are oxidized by ROS to EPR-active nitroxides (**Figure 1B**). Nitroxide radicals are stable products of CHAs that are much more stable than other known spin adducts. The three types of rings commonly used for nitroxide spin-probes are piperidine, pyrrolidine (e.g., DCP-H; **Table 1**) and doxyl (doxyl stearates). Nitroxides offer different properties and are more or less stable and reactive. In addition,



*(Continued)*

#### **Table 1 | Continued**


*Commonly used spin traps or probes proper to detect ROS in different species, organs, organelles or membrane fractions. If available, rate constants of spin traps or probes toward specific ROS. Characteristics of each spin probe/spin trap summarized. This table gives a broad overview of EPR measurements in planta.*

nitroxides are hydrophilic or lipophilic, charged or neutral and hence applicable to various EPR spin-probing experiments in redox research (Kocherginsky and Swartz, 1995).

Endogenous nitroxides may be reduced by several enzymatic processes such as ascorbate or glutathione relating to the antioxidative status of the organism and therefore to its oxidative status (Valgimigli et al., 2001). A recent study demonstrates that the nitroxide TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) and its derivates react with oxygen-centered radicals under acidic conditions as well (Amorati et al., 2010), being a most effective antioxidant. Spin probes can be reduced by OH and O − <sup>2</sup> without processes analogous to OOH/OH adduct transformation. They exhibit an intense EPR signal allowing quantitative analysis due to the high signal-to-noise ratio. Localization of free radical generation is possible, since spin probes located in or excluded from the membranes are available.

The spin probe technique does however not provide any information to identify specific radical species. Apart from the redox status and ROS detection spin probes offer, via their EPR spectra, information on their mobility and different characteristics of their environment such as viscosity, pH, *p*O2, and temperature (Kocherginsky and Swartz, 1995). Baciˇ c and Mojovi ´ c (2005) ` therefore recommended combining the spin-probe and spin-trap technique to study free radical species produced in biological systems effectively.

CHAs, such as TMT-H and DCP-H, become paramagnetic after oxidation, are EPR-silent and are reduced equimolarly by several ROS into EPR-active nitroxides. The very fast reaction between ROS and hydroxylamine is a major advantage compared with spin traps. For example, the rate constant of the spin trap DMPO to form the O − <sup>2</sup> -adduct DMPO/OOH is 35–75 M−<sup>1</sup> <sup>s</sup> −1 (Dikalov et al., 2002), whereas the rate constant of the CHA TEMPO-H to form the O − <sup>2</sup> -adduct is 103–104 <sup>M</sup>−1s <sup>−</sup><sup>1</sup> (Dikalov et al., 2011). The efficiency of CHAs to detect O − <sup>2</sup> is therefore very high; hence very low concentrations of CHAs are necessary to detect O − <sup>2</sup> , and side effects can be minimized. For example, 1 mM CHAs are sufficient for O − <sup>2</sup> detection whereas concentrations of 10–50 mM of spin traps are needed. One disadvantage is the presence of Cu2<sup>+</sup> and Fe3<sup>+</sup> in biological systems leading to autoxidation of CHAs. This problem is decreased by the use of metal chelators (Dikalov et al., 1999). Since the reaction of CHAs toward ROS is unspecific, control experiments with supplements of ROS-scavenging enzymes, such as superoxide dismutase or catalase, or other non-enzymatic scavengers have to be performed for the identification of specific ROS (Dikalov et al., 2011).

The lipophilic spin probe TMT-H was applied to analyze whether ethylene influences ROS levels in internodes of *Oryza sativa* (Steffens et al., 2013). Using the spin-trapping method showed that ethylene enhances ROS levels in the rice internode. ROS were identified as signals that induce parenchymal cell death resulting in aerenchyma formation in specific regions of the rice internodes (Steffens et al., 2011). The paramagnetic, water-soluble spin probe PTM-TC was used to detect O − <sup>2</sup> via a one-dimensional (1D) imaging method in whole *Arabidopsis thaliana* plants or roots after injury of the apex (Warwar et al., 2011). Negatively charged PTM-TC does not penetrate membranes, is very specific for O − <sup>2</sup> detection and grants a distinct single-line EPR spectrum. After reaction of paramagnetic spin probes with ROS the signal is lost, and the loss of signal indicates the presence of ROS. The spin probe method can be used for *in planta* O − <sup>2</sup> detection with an adequate temporal and spatial resolution. The authors conclude that the wound signal in the *Arabidopsis* root is transmitted at a rate of around 1–3 cm/min. By these high resolution scans, the authors show that the root tip at around 0.7 mm possesses more ROS than the part at around 2.2 mm. In addition, during injury ROS levels change within the whole plant. Leaf injury, for example, results in O − <sup>2</sup> production in roots. This was also shown by the use of the stable spin probe that possesses a relatively sharp and strong signal of around 1 G (Warwar et al., 2011).

### **DISENTANGLING OF SPECIFIC ROS LEVELS AND ROS SIGNALING VIA EPR SPECTROSCOPY IN PLANTS**

Despite the abovementioned issues, EPR spectroscopy is an excellent method for analyzing levels of ROS and for identifying specific ROS. In complex biological systems such as plant cells, compartmentation impedes the possibility of ROS detection and quantification. Fortunately, spin probes of different polarities and charges resulting in different cell permeability are available. These properties allow site-specific ROS detection with a higher sensitivity than nitrone spin traps. This is mainly due to the high reactivity of radicals. The reaction site of radicals and radical spin probes is very close to their generation or solubilisation site (Heins et al., 2007). The compartments in plant cells, in particularly the membranes, are comparable to simple model systems where the compartments act as barriers for stable radicals. It is therefore crucial for an efficient detection to define the solubilisation site of the spin probe close to the site of radical generation.

Detection of different ROS in membrane fractions, such as thylakoids (e.g., Hideg et al., 1994; **Table 1**) and plasma membranes (Qiu et al., 1995; Mojovic et al., 2004 ` ; for details: **Table 1**) have been performed over the years using spin traps. A more sophisticated approach was used to analyze production of ROS in the photosynthetic electron transport chain in chloroplasts under high light with CHAs with different lipophilicities (Kozuleva et al., 2011; Borisova et al., 2012). Even in membrane systems, such as thylakoids, ROS production within or without the thylakoid membranes could be distinguished. As the spin probe TMT-H exhibits a high lipophilicity, O − <sup>2</sup> measurements within thylakoid membranes are possible (Kozuleva et al., 2011; Borisova et al., 2012), while the hydrophilic spin probe DCP-H allows measurement of O − <sup>2</sup> outside the membranes (Kozuleva et al., 2011). At pH 7, DCP-H is negatively charged and hence excluded from membranes. These CHAs are excellent tools for ROS detection with high spatial resolution.

To visualize the distribution of free radicals in tissues or cell compartments with a high spectral resolution, 1D- to 3D-Xband EPR imaging (EPRI) experiments are an excellent choice. The application of spin traps for EPRI experiments *in planta* is limited due to solvent compatibility with living tissue, high concentrations of spin traps needed and a multiple signal spectrum (Warwar et al., 2011). The use of stable exogenous spin probes that possess a relatively sharp and strong signal of around 1 G enable the acquisition of EPRI images (Yan et al., 2008). In particular, the application of 15N spin probes with a lower linewidth and a lower detection limit enhances spatial resolution (Yan et al., 2008). There are few successful 2D- or 3D-spectral-spatial EPRI applications found for herbal foodstuff such as seeds of *Sesamum indicum* (Nakagawa and Hara, 2015), *Piper nigrum* (Nakagawa and Epel, 2014) and *Helianthus annus* (Levêque et al., 2008) and coffee beans (Levêque et al., 2008); however, there are not many examples for *in planta* ROS imaging. Possibly, the different strategies that will be followed to reduce biological responsibility of spin probes in living tissues focusing on narrow EPR lines, tissue-targeting specificity and high stability (Yan et al., 2008) will improve the possibilities for EPRI application *in planta*. The visualization of spatiotemporal intracellular ROS dynamics by time-laps imaging in intact plants, organs, organelles, or even different membrane systems by EPRI would provide new insights into the ROS production, their scavenging and possibly into the ROS signaling during plant development and different stresses occurring in a plants' life.

### **REFERENCES**


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 29 November 2014; accepted: 16 February 2015; published online: 03 March 2015.*

*Citation: Steffen-Heins A and Steffens B (2015) EPR spectroscopy and its use in planta—a promising technique to disentangle the origin of specific ROS. Front. Environ. Sci. 3:15. doi: 10.3389/fenvs.2015.00015*

*This article was submitted to Environmental Toxicology, a section of the journal Frontiers in Environmental Science.*

*Copyright © 2015 Steffen-Heins and Steffens. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*