Functional analysis of glutathione and autophagy in response to oxidative stress Yi Han

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Yi Han. Functional analysis of glutathione and autophagy in response to oxidative stress. Agricultural sciences. Université Paris Sud - Paris XI, 2012. English. ￿NNT : 2012PA112392￿. ￿tel-01236618￿

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UNIVERSITE PARIS‐SUD – UFR des Sciences

ÉCOLE DOCTORALE SCIENCES DU VEGETAL

Thèse Pour obtenir le grade de

DOCTEUR EN SCIENCES DE L’UNIVERSITÉ PARIS SUD

Par

Yi HAN

Functional analysis of glutathione and autophagy in response to

oxidative stress

Soutenance prévue le 21 décembre 2012, devant le jury d’examen :

Christine FOYER Professor, University of Leeds, UK Examinateur Michael HODGES Directeur de Recherche, IBP, Orsay Examinateur Stéphane LEMAIRE Directeur de Recherche, IBPC, Paris Rapporteur Graham NOCTOR Professeur, IBP, Orsay Directeur de la thèse Jean‐Philippe REICHHELD Directeur de Recherche, LGP, Perpignan Rapporteur

ACKNOWLEDGMENTS

It would not have been possible to write this doctoral thesis without the help and support of the kind people around me. It is a pleasure to thank the many people who made this thesis possible.

First of all, I would like to express my deepest sense of gratitude to my supervisor Prof. Graham Noctor who offered his continuous advice and encouragement throughout the course of this thesis. I have been extremely lucky to have a supervisor who cared so much about my work. I thank him for the systematic guidance and great effort he put into training me in the scientific field.

I also want to express my gratitude to the reviewers of my thesis, Dr. Stéphane Lemaire (Institut de Biologie Physico-Chimique, FR), Dr. Jean-Philippe Reichheld (Université de Perpignan, FR), Prof. Christine Foyer (University of Leeds, UK) and Dr. Michael Hodges (Université Paris Sud, FR) for having accepted to evaluate my PhD work.

I would like to thank Dr. Bernd Zechmann, for our collaboration on cytohistochemical analysis of glutathione.

This thesis would not have been possible without the financial support of China Scholarship Council and French Agence Nationale de la Recherche project “Vulnoz” as well as the European Union Marie-Curie project “Chloroplast Signals”.

I am thankful to the former as well as the current colleagues in the lab. My officemate, at the start of my thesis, Guillaume Queval, for always being prepared to advices and help. Thanks to Sejir Chaouch, for teaching many biological measurements, and for the scientific discussion. To Jenny Neukermans for the help and continuous encouragement. To the current colleagues, Amna Mhamdi, a well organized person for the scientific discussion and laboratory management. I am quite happy to work with you and enjoy all our discussions about food, cultures and everything in the past four years. To my Chinese colleague Shengchun Li, and Marie-Sylviane Rahantaniaina for their friendship.

I would like to thank the many people at IBP for their help and support (Patrick Saindrenan, Bertrand Gakière, Dao-Xiu Zhou, Catherine Bergounioux, Gilles Sante, Gilles Innocenti, Sophie Blanchet, Caroline Mauve, Françoise Gilard, Pierre Pétriacq, Edouard Boex-fontvieille…). Also, I am indebted to my many student colleagues at IBP for providing a stimulating and fun environment in which to learn and grow. I am especially grateful to Linda De-bont, Manon Richard, Yuan Shen, Laure Audonnet, Thomas Guerinier, and Laure Didierlaurent.

Lastly, and most importantly, I wish to thank my parents and my wife… 感谢我的妈妈，爸爸，岳父和 岳母，还有我可爱的妻子冯峰，这么多年来，正是你们无私的支持和鼓励，我才能顺利完成我的学 业。我将在以后的工作和生活中尽我最大的努力来回报你们长久以来的支持。

Abbreviations

1 O2: Singlet oxygen 3PGA: 3-Phosphoglycerate 5-OPase: 5-oxoprolinase ABA: Abscisic acid AO: Ascorbate oxidase APX: Ascorbate peroxidase ASC: ascorbate ATG: Autophagy BSO: Buthionine sulfoximine CAT: Catalase CLT: Chloroquinone-like transporter

CO2: Carbon dioxide DCPIP: 2,6-dichlorophenolindophenol DHA(R): dehydroascorbate (reductase) DNA: Deoxyribonucleic acid DTNB: 5,5’-dithiobis-2-nitrobenzoic acid Dpi: day post-inoculation DTT: Dithiothreitol -ECS: -glutamyl cysteine synthetase EDTA: Ethylene diamine tetraacetic acid ER:Endoplasmic reticulum ETI: Effector-triggered immunity FDH: Formaldehyde dehydrogenase G6PDH: Glucose-6-phosphate dehydrogenase GAPDH: Glyceraldehyde-3-phosphate dehydrogenase GC-TOF-MS: Gas chromatography-time of flight-mass spectrometry GDC: Glycine decarboxylase GGC: -glutamyl-cyclotransferases GGP: -glutamyl peptidases GGT: -glutamyl transpeptidase GO: Glycolate oxidase GPX: Glutathione peroxidase GR: Glutathione reductase GRX: Glutaredoxins GSH: Reduced glutathione GSNO(R): S-nitrosylglutathione (reductase) GSSG: Glutathione disulphide GST: Glutathione S-transferase HO•: Hydroxyl radical HPLC: High performance liquid chromatography HR: Hypersensitive response ICS1: Isochorismate synthase 1 JA: Jasmonic acid MAPK: Mitogen-activated protein kinase MDHAR: Monodehydroascorbate reductase MRP: Multidrug resistance-associated protein MV: Methyl viologen NO: Nitric oxide NADP(H): Nicotinamide adenine dinucleotide phosphate (Reduced) NPR1: Nonexpressor of pathogenesis-related genes 1 NTR: NADPH-thioredoxin reductase NR: Nitrate reductase

O2: Oxygen − O2 : Superoxide OPT: Oligopeptide transporter OXI1: Oxidative signal inducible 1 PB: Protein bodies PCD: Programmed cell death PCS: Phytochelatin synthase PE: Phosphatidylethanolamine PI3K: Phosphatidylinositol 3-kinase PR: Pathogenesis-related RBOHs: Respiratory burst oxidase homologues PMS: Phenazine methosulfate RNA: Ribonucleic acid RT-qPCR: Reverse transcription-quantitative real-time polymerase chain reaction ROS: Reactive oxygen species POX: Peroxidase PRX: Peroxiredoxins PSVs: Protein storage vacuoles PTI: Pathogen-associated molecular pattern-triggered immunity roGFP: Reduction-oxidation sensitive green fluorescent protein RuBisco: Ribulose 1,5-bisphosphate carboxylase/oxygenase SA: Salicylic acid SAR: Systemic acquired resistance sid2: Salicylic acid induction-deficient 2 SNP: Sodium nitroprusside SOD: Superoxide dismutase T-DNA: Transferred deoxyribonucleic acid TF: Transcription factors TORC1: Target of rapamycin kinase complex 1 TRX: Thioredoxins VPD: 2-vinylpyridine XO: Xanthine oxidase TABLE OF CONTENTS CHAPTER 1 GENERAL INTRODUCTION

1.1 Reactive oxygen species in plants 1 1.1.1 ROS generating systems 2 1.1.2 Metabolism of ROS 3 1.1.2.1 Non-enzymatic scavenging mechanisms 3 1.1.2.2 Enzymatic metabolism of ROS 4 1.1.3 Functions of ROS in plants 7 1.1.3.1 The role of ROS in abiotic stress 8 1.1.3.2 Regulation of growth and developmental processes by ROS 8 1.1.3.3 Functions of ROS in plant innate immunity 8 1.1.3.4 ROS in cell death 9 1.1.4 ROS signaling network 9 1.2 Glutathione in plants 11 1.2.1 Glutathione synthesis 11 1.2.1.1 Glutathione deficient mutants 12 1.2.1.2 Regulation and overexpression of the glutathione synthesis pathway 12 1.2.2 Glutathione transport 12 1.2.3 Glutathione degradation 14 1.2.4 Metabolic functions of glutathione in plants 15 1.2.4.1 Glutathione S-transferases 15 1.2.4.2 Glutathione reductase 16 1.2.4.3 Glutaredoxins 17 1.2.4.4 Other glutathione-associated metabolic reactions 18 1.2.5 Glutathione in plant development, growth and environmental responses 20 1.2.5.1 Glutathione in the regulation of plant development and growth 20 1.2.5.2 Light signaling 20 1.2.5.3 Biotic interactions, cell death and defence phytohormones 21 1.2.6 Mechanisms of glutathione dependent redox signaling 24 1.2.6.1 Protein S-glutathionylation 24 1.2.6.2 Protein S-nitrosylation and GSNO reductase 25 1.2.6.3 Glutathione redox potential 25 1.3 Autophagy in plants 26 1.3.1 Functions of autophagy in plants 28 1.3.1.1 Functions of autophagy during abiotic stress 28 1.3.1.2 Functions of autophagy during pathogen infection 29 1.3.1.3 Functions of autophagy during growth and seed development 29 1.3.2 Difficulties of monitoring autophagy in plants 30 1.4 Objectives of the work 31

CHAPTER 2 Functional analysis of Arabidopsis mutants points to novel roles for glutathione in coupling H2O2 to activation of salicylic acid accumulation and signaling 2.1 Introduction 34 2.2 Results 37

2.2.1 Genetic blocks over H2O2-triggered glutathione accumulation inhibit cell death and associated pathogenesis responses induced by intracellular oxidative stress 37

2.2.2 Contrasting responses in cat2 cad2 and cat2 gr1 reveal a non-antioxidant role for glutathione in coupling H2O2 to SA- dependent pathogenesis responses 42 2.2.3 Side-by-side comparison of cat2 cad2 with cat2 npr1 46 2.2.4 Complementation experiments reveal non-redundant roles for glutathione and NPR1 in the activation of SA-dependent responses 51 2.3 Discussion 53

2.3.1 Partial impairment of -ECS function confers a genetic block on H2O2-triggered up-regulation of glutathione 53

2.3.2 An essential role for glutathione in activation of H2O2-dependent salicylic acid signaling and related pathogenesis responses 54

2.3.3 Glutathione acts independently of its antioxidant function in transmitting H2O2 signals 55 2.3.4 Glutathione can regulate SA signaling through processes additional to NPR1 56 2.3.5 Multifunctional roles of glutathione status in defence signaling pathways: A model 57

CHAPTER 3 Regulation of basal and oxidative stress-triggered jasmonic acid-related gene expression by glutathione 3.1 Introduction 59 3.2 Results 61 3.2.1 Effects of glutathione content on basal expression of jasmonic acid-associated genes 61 3.2.2 Activation of jasmonic acid signaling by intracellular oxidative stress 63 3.2.3 Modulation of leaf thiol status by the cad2 mutation 63 3.2.4 Comparison of effect of cad2 and npr1 mutations on oxidative stress-triggered JA signalling 65 3.2.5 Effects of complementing mutants with cysteine and glutathione 66 3.3 Discussion 68 3.3.1 Basal expression of the JA pathway is closely correlated with glutathione contents 68 3.3.2 JA-associated gene expression induced by oxidative stress is influenced by glutathione status 68 3.3.3 Glutathione can regulate oxidative stress-triggered activation of JA signaling independent of NPR1 70 3.3.4 Novel roles for glutathione in regulating the JA pathway: a model 71

CHAPTER 4 Analysis of autophagy mutants: interactions with photorespiration and oxidative stress 4.1 Introduction 74 4.2 Results 77

4.2.1 Effects of high CO2 on atg phenotypes and metabolite profiles 78 4.2.2 Impact of oxidative stress on atg-triggered early senescence 80 4.2.3 Impact of atg mutations on cat2-triggered lesions and bacterial resistance 82 4.2.4 Interactions between atg mutations and glutathione 83 4.3 Discussion 84

CHAPTER 5 CONCLUSION AND PERSPECTIVES 5.1 CONCLUSIONS 87 5.1.1 New roles for glutathione in the regulation of defense phytohormone signaling 87 5.1.2 Interactions between autophagy, oxidative stress of peroxisomal origin, and glutathione 88 5.2 PERSPECTIVES 88

5.2.1 Potential mechanisms on the effects of blocking glutathione accumulation in response to enhanced H2O2 availability 88

5.2.1.1 Glutathione oxidation and metabolism in response to H2O2 89

5.2.1.2 Glutathione, H2O2 signaling, and protein thiol-disfufide status 89

5.2.1.3 H2O2, NO, and glutathione 90 5.2.1.4 Glutathione and calcium signaling 91 5.2.1.5 Glutathione and the jasmonic acid pathway 92 5.2.2 Autophagy and oxidative stress 92

CHAPTER 6 MATERIALS AND METHODS 6.1 Plant material and growth conditions 94 6.2 Methods 95 6.2.1 Molecular biology analyses 95 6.2.1.1 DNA extraction and plant genotyping 95 6.2.1.2 RT-qPCR 96 6.2.1.3 CATMA microarray analyses and data-mining 96 6.2.2 Lesion quantification and pathogen tests 97 6.2.3 Cytohistochemical analysis 97 6.2.4 Metabolite analyses 97 6.2.4.1 Assays for ascrobate, glutathione and NADP(H) 97 6.2.4.1.1 Extraction 98 6.2.4.1.2 Quantification of thiols by HPLC 98 6.2.4.1.3 Glutathione measurement by plate reader 98 6.2.4.1.4 Ascorbate 99 6.2.4.1.5 NADP(H) 99 6.2.4.2 Salicylic acid measurement 99 6.2.4.3 Non-targeted GC-TOF-MS analysis 99 6.2.4.4 Peroxide assay 100 6.2.5 Statistical analysis 100 100 REFERENCES 101 ANNEXES Annex 1A Mhamdi et al., 2010 Plant Physiol. Annex 1B Confirmation of the genetic interaction between cat2 and gr1 knockout mutations Annex 2 Noctor et al., 2012 Plant Cell Environ.

CHAPTER 1

GENERAL INTRODUCTION

CHAPTER 1 GENERAL INTRODUCTION

Unlike animals, plants cannot move to a more comfortable location to escape stresses of external origin. Instead, plants have developed coping mechanisms that allow them to withstand environmental fluctuations. Most types of stresses disrupt the metabolic balance of cells, resulting in enhanced production of reactive oxygen species (ROS). It is clear that, over the course of evolution, ROS in plants have been recruited to act as a signal in many biological processes such as growth, the cell cycle, programmed cell death (PCD), and other defence responses to stress. Thus, in addition to their well-documented toxicity, ROS are important actors in cellular and subcellular signaling. There is a close relationship between increased ROS availability and glutathione status, and many glutathione-dependent reactions are involved in oxidative stress-mediated cellular processes. However, while the functions of glutathione as an antioxidant involved in ROS-scavenging and associated detoxification reactions are relatively well described, it is much less clear whether ROS-induced adjustments in glutathione status play important regulatory roles (eg, in ROS signal transmission). One physiological condition in which regulatory roles of glutathione have been studied in plants is biotic stress. Pathogen challenge clearly involves ROS signaling but the interactions between ROS and glutathione in determining responses to biotic stress remain unclear. One important ROS-dependent response to pathogens involves cell death during the hypersensitive response (HR). Emerging evidence implicates autophagic processes in cell death, and also in cellular acclimation to abiotic stress as well as resistance to pathogens, but the relationship between ROS and autophagy is not yet clearly established in plants. As the overall aims of this thesis were to exploit model study systems to investigate interactions between ROS and glutathione, and between ROS and autophagy, the general features and concepts surrounding these factors and processes are described in this first chapter.

1.1 Reactive oxygen species in plants

Molecular oxygen (O2) first began to exist in significant amounts in the earth’s atmosphere over 2.7 billion years ago, an evolutionary event driven largely by the appearance of O2-evolving photosynthetic organisms (Halliwell, 2006). The O2 molecule is a free radical (triplet biradical in the ground state), as it has two impaired electrons that have the same spin quantum number. Although several oxidases are able to catalyze reduction of O2 to H2O2 or water by divalent or tetravalent mechanisms, the spin restriction of the O2 molecule means that it prefers to accept electrons one at a time, leading to the generation of the so-called ROS, which are partially reduced or activated derivatives of oxygen (such as singlet oxygen 1 − ( O2), superoxide (O2 ), hydrogen peroxide (H2O2), and hydroxyl radical (HO•)). In plants ROS are continuously produced as byproducts of various metabolic pathways localized in different cellular compartments (Figure 1; Apel and Hirt, 2004). Adverse environmental conditions lead to an increased production of ROS, resulting in oxidative stress. The evolution of efficient antioxidant systems has most likely enabled plant cells to overcome ROS toxicity and to use these reactive species as signal transducers to regulate diverse physiological processes (Mittler, 2006). These compounds are tightly controlled by a range of enzymatic and non-enzymatic scavenging mechanisms to maintain the cellular redox state which will be discussed in more detail in section 1.1.2. In addition, as well as signaling roles, other key functions for ROS has been identified including the control and regulation of biological processes, such as growth, the cell cycle, PCD, hormone signaling, biotic and abiotic stress responses and development (Mittler et al., 2004).

1 CHAPTER 1 GENERAL INTRODUCTION

1.1.1 ROS generating systems

It is well established that cell organelles such as chloroplasts, mitochondria and peroxisomes are major sources of intracellular ROS production in plant cells through photosynthesis, respiration and photorespiration. ROS are also generated at the extracellular sites such as plasma membrane NADPH-dependent oxidases, or peroxidases and oxidases in the apoplast (Figure 1.1). Localized ROS production in these compartments may trigger different signaling cascades.

Figure. 1.1. Major sites of H2O2 production in photosynthetic cells (scheme taken from Mhamdi et al., 2010b) GO, glycolate oxidase. 3PGA, 3-phosphoglycerate. POX, peroxidase. RuBisCO, ribulose 1,5-bisphosphate

carboxylase/oxygenase. RuBP, ribulose 1,5-bisphosphate. SOD, superoxide dismutase. XO, xanthine oxidase.

In the chloroplast, the reaction centers of photosystems I and II (PSI and PSII) are the major generation sites of ROS since they are found in an environment rich in oxygen, reductants and high-energy − 1 intermediates (Asada, 2006). O2 and H2O2 are formed mainly in PSI, while O2 is notably produced from 3 the triplet ground state ( O2) by photodynamic transfer of energy from excited triplet state chlorophyll such as the P680 reaction center of PSII (Telfer et al., 1994; Hideg et al., 1998).

− As their name implies, peroxisomes are also significant generators of ROS in the form of O2 or H2O2 produced by various oxidases (Mhamdi et al., 2012). Indeed, it has been estimated that in some conditions, these organelles may be the fastest H2O2-generating system in photosynthetic cells (Noctor et al., 2002).

In peroxisomes, H2O2 is notably generated by the enzymatic activity of glycolate oxidase which oxidizes glycolate to glyoxylate, an important reaction in photorespiration (Figure 1.1). Another enzyme that can directly produce H2O2 is acylCoA oxidase, involved in fatty acid oxidation (Nyathi and Baker, 2006). In

2 CHAPTER 1 GENERAL INTRODUCTION

− addition, xanthine oxidase can generate O2 , which in turn is converted to H2O2 by Cu,Zn-superoxide dismutase (SOD; Corpas et al., 2008).

In contrast to mammalian mitochondria, which are well accepted to be a main intracellular source of ROS in animals, mitochondria in photosynthetic cells probably make a relatively minor contribution to overall cellular ROS production (Foyer and Noctor, 2003). In mitochondria, the major sites of ROS production lie in the electron transport chain, especially at the level of Complex I and Complex III (Møller et al., 2001). Mitochondrial terminal oxidases such as cytochrome c oxidase (Complex IV) and the alternative − oxidase, reduce oxygen via tetravalent mechanisms. H2O2 is formed in mitochondria from O2 by Mn-SOD (Finkel and Holbrook, 2000).

Among several types of enzymes which may be required for generating H2O2 in the apoplast, plasma membrane NADPH-dependent oxidases, known as respiratory burst oxidase homologues (RBOHs), have been the subject of intense investigation (Torres and Dangl, 2005; Torres et al., 2006). In plants, RBOH-encoding genes belonged to a small multigenic family (e.g. ten members in Arabidopsis and nine in rice) and contain a multimeric flavocytochrome that is part of a small electron transport chain capable − of reducing extracellular O2 to O2 using NADPH as a cytosolic electron donor. Although much attention has been given to NADPH oxidases, other ROS-producing systems such as peroxidases and oxidases in the apoplast are likely to play a role in ROS signaling in response to different stimuli or developmental signals (Mittler et al., 2004; Bindschedler et al., 2006; Choi et al., 2007; Van Breusegem et al., 2008).

1.1.2 Metabolism of ROS

As noted above, the essential reactions of energy transduction and metabolism involve the production of ROS. Moreover, exposure of plants to adverse environmental conditions such as drought, temperature extremes, pathogen attack, nutrient deficiency, heavy metal and air pollutants can trigger increased ROS production. Thus, plants make use of a sophisticated and complex antioxidant machinery, including enzymatic and non-enzymatic components, to maintain redox homeostasis, and this machinery is likely to be particularly important in stress conditions (Apel and Hirt, 2004; Møller et al., 2007).

1.1.2.1 Non-enzymatic scavenging mechanisms

Numerous components such as tocopherols, flavonoids, alkaloids and carotenoids have been recognized as reductants or energy quenchers that can metabolize ROS. While no enzymatic systems has been found to scavenge singlet oxygen and hydroxyl radical, they are thought to be essentially controlled by avoidance of their production or by reaction with low molecular weight antioxidants such as glutathione, ascorbate, tocopherol and carotenoids (Noctor and Foyer, 1998; Asada, 1999). Indeed, although many metabolites can be oxidized by ROS, the cellular redox buffers ascorbate (ASC) and glutathione (GSH) are two of the major non-enzymatic antioxidant components, although they also function in many other biochemical and physiological processes (Cobbett et al., 1998; Vernoux et al., 2000; Pastori et al., 2003; Ball et al., 2004; Dowdle et al., 2007; Parisy et al., 2007). Both of them are highly reducing and abundant antioxidants found in millimolar (mM) concentrations in plants (Foyer and Noctor, 2011). In addition, both ASC and GSH are reducing co-factors for a range of enzymes that metabolize ROS, as described

3 CHAPTER 1 GENERAL INTRODUCTION

below. Apart from their roles in maintaining redox homeostasis, it has been suggested that some well-known antioxidant metabolites such as glutathione and carotenoids may be involved in oxidative stress signaling (Foyer et al., 1997; May et al. 1998a; Ramel et al., 2012).

1.1.2.2 Enzymatic metabolism of ROS

As noted above, superoxide is formed in several cell compartments and the apoplast, and is metabolized to H2O2 and/or O2. While direct reduction to H2O2 through non-enzymatic systems (e.g., ferredoxin, GSH, ASC) can occur, dismutation via SOD is thought to the most important factor in superoxide metabolism. Several isoforms of SOD with different metal co-factors are found in plants, including Mn-SOD (mitochondria), Fe-SOD (chloroplast) and Cu,Zn-SOD (chloroplast, cytosol, peroxisome, apoplast) (Fink and Scandalios, 2002).

Whether produced directly from O2 by oxidases or indirectly from superoxide by SOD, H2O2 is the most stable ROS. It has therefore attracted considerable attention as a signal molecule. Although H2O2 can oxidize some groups such as protein thiols, it can also be cleaved to the highly reactive hydroxyl radical.

Hence, enzyme systems involved in H2O2 metabolism are particularly important to maintain redox homeostasis and avoid uncontrolled cellular oxidation. Numerous types of enzyme can metabolize H2O2, but these can be divided into two broad classes. The first consists of catalases which, like SOD, catalyze a dismutation reaction and so do not require any other reductant. The second group is more diverse, and is comprised of various peroxidases which reduce H2O2 to water and therefore require a reducing co-factor.

Catalases (CAT), exists in almost all organisms, and possess a high capacity for metabolizing H2O2 through the dismutation of H2O2 to water and O2. In most organisms, CAT are located mainly in the peroxisomes (Zamocky et al., 2008). Three CAT genes have been identified in Angiosperm species studied to date, including tobacco, Arabidopsis, maize, pumpkin and rice (Willekens et al., 1995; Frugoli et al., 1996; Guan and Scandalios, 1996; Esaka et al., 1997; Iwamoto et al., 2000), and a classification system based on the naming of the tobacco genes was introduced by Willekens et al. (1995), with class I CAT designating the major isoform expressed in leaves. According to this grouping, Arabidopsis CAT1, CAT2, and CAT3 correspond to Class III, Class I, and Class II catalases, respectively (Table 1.1). Two are located on chromosome 1 (CAT1, CAT3) and one is located on chromosome 4 (CAT2) based on genome sequencing (Frugoli et al., 1996). The three corresponding amino acid sequences have high similarity. In Arabidopsis, CAT1 is mainly expressed in pollen and seeds. CAT2 is the major form of leaf catalase which is highly expressed in photosynthetic tissue, whereas CAT3 is associated with vascular tissues but also leaves (Hu et al., 2010).

Whereas irreplaceable functions for Arabidopsis CAT1 and CAT3 remain to be described (Mhamdi et al., 2010b), studies of mutants have demonstrated important physiological roles for CAT2 in photorespiration, maintenance of leaf redox homeostasis, and modulation of leaf senescence (Queval et al., 2007; Smykowski et al., 2010). Indeed, the first report of obvious oxidative stress in a catalase-deficient line came from forward genetics studies of a barley mutant isolated via a photorespiratory screen. The mutant has about 10% wild-type leaf catalase activity but shows a wild-type phenotype when grown at high CO2; however, lesions appear when the plant is transferred to air (Kendall et al., 1983). The appearance of

4 CHAPTER 1 GENERAL INTRODUCTION

lesions in this line is preceded by marked accumulation of oxidized glutathione (Smith et al., 1984). This effect is also observed in tobacco transformants in which leaf catalase expression has been markedly decreased by antisense technology (Willekens et al., 1997) or in Arabidopsis cat2 knockouts (Queval et al., 2007), suggesting that accumulation of oxidized glutathione is a conserved response to catalase deficiency in the leaves of plants with C3 photosynthesis. Because of the ease of manipulating oxidative stress symptoms in such lines, they have been used as stress mimic tools to investigate oxidative stress-related signaling pathways (Chamnongpol et al., 1996, 1998; Willekens et al., 1997; Vandenabeele et al., 2004; Vanderauwera et al., 2005; Queval et al., 2007; Chaouch et al., 2010; Mhamdi et al., 2010b). Nevertheless, the functional importance of the changes in glutathione in this signaling have received little attention. A study of double cat2 gr1 mutants, doubly deficient in catalase and glutathione reductase (GR; Mhamdi et al., 2010a; annex 1) revealed that dramatic accumulation of oxidized glutathione was associated with altered phytohormone responses. However, interpretation of these effects is complicated by the simultaneous loss of function of two antioxidative enzymes, reflected in the extreme phenotype of the double mutant when grown in air (Mhamdi et al., 2010a).

Table 1.1. Probable classification of the three catalases found in different plant species (from Mhamdi et al., 2010b).

Class I Class II Class III Tobacco Cat1 Cat2 Cat3 Arabidopsis CAT2 CAT3 CAT1 Maize Cat2 Cat3 Cat1 Pumpkin Cat2 Cat3 Cat1 Rice CatC CatA CatB

As well as catalases, the best described route for H2O2 metabolism is the ascorbate-glutathione pathway in which GSH (indirectly) and ASC (directly) are involved (Figure 1.2). This pathway exists in several cell organelles, including chloroplasts, mitochondria and peroxisomes (Jiménez et al., 1997; Chew et al., 2003).

Figure 1.2. Two of the major pathways for H2O2 metabolism in plants (scheme taken from Foyer and Noctor, 2009). APX, ascorbate peroxidase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, glutathione disulphide; MDHAR, monodehydroascorbate reductase.

5 CHAPTER 1 GENERAL INTRODUCTION

In the ascorbate-glutathione pathway, H2O2 is reduced to H2O by ascorbate peroxidase (APX), an enzyme with a much higher affinity for H2O2 than CAT, leading to oxidation of ASC to monodehydroascorbate

(MDHA; Figure 1.3). As well as APX, which is specific to H2O2 as oxidant, plants also contain several types of other peroxidases that can reduce organic peroxides in addition to H2O2. Among these, peroxiredoxins (PRX) may have an important function alongside APX in H2O2 removal (Dietz et al., 2006). The reduced form of PRX is regenerated from the oxidized form by either a thiol or ASC and the enzymes glutaredoxins (GRX), thioredoxins (TRX) and NADPH-thioredoxin reductase (NTR; Dietz, 2003; Rouhier and Jacquot, 2005; Meyer, 2008; Pérez-Ruiz and Cejudo, 2009). Based on the number of cysteine residues and the catalytic mechanism, in plants PRXs are divided into four sub-classes: 1-Cys PRX, 2-Cys PRX, Prx Q and type II PRX (Dietz, 2003; Meyer, 2008). It has been concluded that 2-cys

PRX could play a significant role in the chloroplast, where peroxiredoxin capacity to reduce H2O2 was reported to be about half that of soluble APX (Dietz et al., 2006). In addition to detoxification of lipid peroxides, glutathione peroxidases (GPXs) are able to remove H2O2. Despite their name, it is likely that GPXs function in vivo with TRX rather than GSH (Herbette et al., 2002; Iqbal et al., 2006). Finally, glutathione S-transferases (GSTs) could contribute to peroxide metabolism. Although the most studied role of these enzymes is in removal of electrophilic xenobiotics by the formation of GS-conjugates, several GSTs can also act as GSH-dependent peroxidases and are able to remove H2O2 (Dixon et al., 2009; Dixon and Edwards, 2010). In plants, GSTs are divided into several groups and some of their possible functions are further discussed in section 1.2.4.1.

Figure 1.3. Part of the complexity of plant H2O2-metabolizing systems (scheme taken from Mhamdi et al., 2010b). APX, ascorbate peroxidase. ASC, ascorbate. CAT, catalase. DHAR, dehydroascorbate reductase. Fdox, oxidized ferredoxin. Fdred, reduced ferredoxin. FTR, ferredoxin-thioredoxin reductase. GPX, glutathione(thioredoxin) peroxidase. GR, glutathione reductase. GRX, glutaredoxin. GSH, glutathione. GSSG, glutathione disulphide. GST, glutathione S-transferase. MDHA, monodehydroascorbate reductase. NTR, NADPH-thioredoxin reductase. PRX, peroxiredoxin. ROOH, organic peroxide. TRXox, oxidized thioredoxin. TRXred, reduced thioredoxin

Unlike catalases, peroxidases require reductant, whether ASC, GSH or other. Maintaining the pools of these reductants requires several other categories of enzyme such as dehydroascorbate reductase (DHAR), GR, and NADPH-generating dehydrogenases (Figures 1.2 and 1.3). Given the high number and diversity

6 CHAPTER 1 GENERAL INTRODUCTION

of H2O2-metabolizing systems, it remains unclear which are the most important. Insight into the interaction between different ROS and ROS scavenging mechanisms has been obtained by studies of double or triple mutants that are defective in key ROS-scavenging enzymes found in different subcellular organelles (Giacomelli et al. 2007; Miller et al. 2007; Mhamdi et al., 2010a) and exploration of crosstalk 1 between distinct ROS such as O2 or H2O2 originating in different compartments (Laloi et al., 2007; Chaouch et al., 2012). These data have not only revealed much apparent redundancy in the ROS-scavenging network, but also suggest that different antioxidant enzymes and different ROS in the same (or different) compartments, mediate specific signals in plant responses to various environmental stimuli and development (Harir and Mittler, 2009).

1.1.3 Functions of ROS in plants

ROS were initially considered as toxic by-products of aerobic metabolism which are tightly regulated by means of antioxidant and enzymatic components of the ROS scavenging pathways (Apel and Hirt, 2004; Møller et al., 2007). However, it is now apparent that they are key components in fundamental plant processes including growth, development, response to biotic and abiotic stresses, as well as PCD. Hence, ROS are important signaling molecules. A simple view of ROS signaling in plants is shown in the model in Figure 1.4.

Figure 1.4. ROS form in plant cells as a consequence of myriad stimuli ranging from abiotic and biotic stress, production of hormonal regulators, as well as cell processes such as acclimation and PCD (scheme taken from Mittler et al., 2004). These active oxygens are produced at a number of intercellular and extracellular sites, including mitochondria, chloroplasts, peroxisomes and apoplast. ROS trigger a series of signal transduction events. The influence of these molecules on cellular processes is mediated by both the perpetuation of their production and their removal by scavenging enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). The location, amplitude, and duration of production of these molecules determine the specificity and rapidity of the responses they elicit.

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1.1.3.1 The role of ROS in abiotic stress

Plants are continually exposed to changes in their environment. Abiotic stresses (eg, ozone, excess light, heavy metal or drought) are perceived by plant cells and induce an acclimatory response through ROS signaling. The most common abiotic stresses lead to increased ROS production from the different subcellular sources through down-regulation of scavenging enzymes or up-regulation of generating systems which dictate the expression pattern of specific sets of genes and the induction of certain acclimation and defence mechanisms (Mittler et al., 2004). For instance, both HsfA4a and HsfA8 were not induced by enhanced peroxisomal H2O2 in a cat2-deficient line but induced by elevated cytosolic H2O2 in an apx1 knockout mutant (Pnueli et al., 2003; Davletova et al., 2005; Vanderauwera et al., 2005).

1.1.3.2 Regulation of growth and developmental processes by ROS

The spatial control of cell growth is a central process in plant development. ROS play an important role in the regulation of growth through their various effects on cell wall elasticity. Unlike the well-known signaling roles in pathogen response (see below), the roles of ROS in development are less well understood. The best studied developmental role for NADPH-derived ROS is in the regulation of tip growth in root hairs and pollen tubes (Foreman et al., 2003; Monshausen et al., 2007; Macpherson et al., 2008; Potocky et al., 2007). However, accumulating evidence indicates that ROS may play a more general role in developmental regulation other than just in tip extension growth. Inhibitor tests indicate that the promotion of maize root elongation may be regulated by NADPH oxidases (Liszkay et al., 2004), and apoplastic ROS accumulation has also been implicated in the involvement of maize leaf development (Rodriguez et al., 2007). Moreover, ROS are involved in abscisic acid (ABA)-mediated stomatal closure, the modulation of cell wall loosening during seed germination, and fruit ripening (Apel and Hirt, 2004, Dunand et al., 2007; Müller et al., 2009a, b).

1.1.3.3 Functions of ROS in plant innate immunity

One of the initial responses observed after pathogen attack is the oxidative burst, which involves a rapid increase in ROS production such as superoxide and H2O2. Several reports in various plant species suggest that both plasmalemma-located NADPH oxidase and extracellular peroxidases contribute to this rapid increase in ROS (Torres et al., 2002, 2005; Bindschedler et al., 2006; Daudi et al., 2012; O’Brien et al., 2012). However, these initial signals at the cell surface/apoplast lead to later downstream adjustments in intracellular redox state that are notably associated with intracellular ROS generated by chloroplasts, mitochondria as well as peroxisomes (Edwards et al., 1991; Volt et al., 2009; Chaouch et al., 2012). The increased ROS may contribute to resistance via several mechanisms. Firstly, invading pathogens may be directly killed by ROS and ROS may mediate the strengthening of the cell wall through H2O2-dependent lignification reactions that confine the pathogen to the infection site (Durner et al., 1997). Secondly, effector-triggered immunity (ETI) is visibly accompanied by the HR in which cell death occurs at the site of pathogen infection, also acting to limit the pathogen growth. ETI is usually linked with ROS accumulation. In addition to roles in triggering HR, it has been suggested that ROS act, together with salicylic acid (SA), to mediate the establishment of systemic acquired resistance (SAR) in the distal uninoculated systemic tissue of a pathogen-infected plant, conferring improved resistance against a broad

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spectrum of pathogens (Fobert et al., 2005). Nonexpressor of pathogenesis-related genes 1 (NPR1) protein is required for SA signaling and it is a key component of SAR regulation (Cao et al., 1994). NPR1 contains an ankyrin-repeat motif and a BTB/POZ domain. The protein can be found in the nucleus, where it functions in SA-dependent PR gene expression. SA-induced changes in cell redox state trigger the reduction of NPR1 disulfide bonds, and NPR1 monomers are subsequently translocated from the cytosol to the nucleus, where they interact with TGA transcription factors to activate the expression of defence genes such as PATHOGENESIS-RELATED 1 (PR1; Després et al., 2003; Tada et al., 2008; Mou et al., 2003). It was proposed that the reduction of disulfide bonds of the oligomeric form of NPR1 could be mediated by TRX (Tada et al., 2008). According to this model, induction of PR gene expression by this pathway involves monomerization of oligomeric NPR1 driven by SA-induced changes in cellular redox state, leading to reduction of two NPR1 cysteine residues (Cys82 and Cys216) by, for example, TRX H5 and/or TRX H3. NPR1 monomers are subsequently translocated from the cytosol into the nucleus, where they induce defence genes by interacting with TGA transcription factors (Després et al., 2003; Mou et al., 2003).

Finally, camalexin, the major recognized phytoalexin in Arabidopsis thaliana, is a low molecular mass secondary metabolite with antimicrobial activity. ROS are also generally involved in camalexin production, as shown by experiments using the oxidative stress-inducing chemical (paraquat) and the oxidative stress mimic mutant, cat2 (Zhao et al., 1998; Chaouch et al., 2010).

1.1.3.4 ROS in cell death

Plants possess active genetic cell death programs that have become integral parts of their growth, development, and reactions with the environment (Lam, 2004). This involves various processes starting as early as embryogenesis. For example, PCD can be observed in the tissues of germinating seeds and during leaf senescence. PCD can also be observed during HR and ozone stress. ROS closely interplay with other signaling components in the regulation of PCD (Overmyer et al., 2005). For example, ROS-induced cell death is influenced by a variety of plant hormones. SA is a key candidate considered to act in concert with ROS to cause HR-like lesions, and ROS-triggered SA accumulation is sufficient to cause lesions, even in the absence of pathogen attack (Danon et al., 2005; Chaouch et al., 2010). Other defence hormones such as jasmonic acid (JA) and ethylene also are involved in ROS-dependent PCD (Fath et al., 2001; de Jong et al., 2002; Overmyer et al., 2000; Moeder et al., 2002; Danon et al., 2005). Besides phytohormones, nitric oxide (NO) operates in a dose-dependent manner, synergistically with

H2O2, in the modulation of HR-like cell death (Delledonne et al., 2001; Zago et al., 2006). In addition, lipidic components such as sphingolipids, oxylipins and phospholipids can interact with ROS to modulate PCD (Liang et al., 2003; Loeffler et al., 2005; Meijer and Munnik, 2003). Metacaspases and metabolites such as myo-inositol have also been implicated in ROS-induced cell death (He et al., 2008; Chaouch and Noctor, 2010).

1.1.4 ROS signaling network

Although specific ROS sensors or receptors has not yet been clearly identified in the plant cell, redox-sensitive proteins and other as yet unknown factors have been proposed to transduce ROS signals

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(Neill et al., 2002; Mittler et al., 2004; Apel and Hirt, 2004; Van Breusegem et al., 2008). To date, NPR1 remains the best example of a redox-sensitive protein clearly associated with ROS signaling. As noted above, TRX may control the NPR1 redox state (Tada et al., 2008). However, recently, other evidence suggests that redox regulation of this protein also involves S-nitrosylglutathione (GSNO; Lindermayr et al., 2010). Besides TRX, PRX have been suggested to act as redox sensors in plants (Dietz, 2008), and such functions could act analogously to the regulation of yAP1 in yeast by peroxidase-dependent oxidation by H2O2 (Delaunay et al., 2002). Genetic screens for mutants impaired in ROS sensing could possibly contribute to revealing some of these mechanisms, though surprisingly few have as yet been identified by such approaches.

Despite the failure to uncover direct “sensors” of ROS in plants, it is apparent that ROS signaling networks with many different signaling components, eg, protein kinases and calcium/calmodulin (Mittler et al., 2011). Roles for mitogen-activated protein kinase (MAPK) cascades in ROS signaling are established. Many different kinases such as MAPKKK MEKK1, MPK3, MPK4 and MPK6 can be activated following ROS accumulation (Teige et al., 2004; Xing et al., 2008; Jammes et al., 2009). The MEKKI pathway is highly activated during abiotic stress, which was suggested to be specifically linked to H2O2-mediated activation of MPK4 (Nakagami et al., 2006; Xing et al., 2008). Two other ROS-responsive MAP kinases, MPK3 and MPK6, are activated by the serine/threonine protein kinase OXI1 (OXIDATIVE SIGNAL INDUCIBLE 1), which has been shown to play a role in downstream ROS signaling (Anthony et al., 2004; Rentel et al., 2004). Recently it has shown that MPK9 and MPK12 function downstream of ROS to regulate ABA-induced stomata closure in guard cells and to activate anion channels (Jammes et al., 2009). In addition, it has been suggested that some phosphatases such as the MAPK phosphatase, AtMKP2, and the tyrosine phosphatase, AtPTP1, have negative effects on MPK activation (Gupta et al., 2003; Lee and Ellis, 2007). A MAPK cascade is also implicated in NO signaling, which synergistically functions with ROS signaling in pathogen defence responses such as HR (Asai et al., 2008).

Apart from the activation of the MAPK cascade in ROS signal transduction, several transcription factors (TF) are affected by ROS availability, including four identified members of the WRKY, Zat, Hsf, and Myb families (Mittler et al., 2004; Davletova et al., 2005; Vanderauwera et al., 2005; Gadjev et al., 2006; Miller et al., 2008). These ROS-sensitive TFs can then lead to changes in gene expression (Vanderauwera et al., 2005; Gadjev et al., 2006).

The intensity, duration and localization of ROS signals are controlled by the complex interaction between ROS generation and ROS scavenging systems (Figure 1.4; Mittler et al., 2004). Many studies of knockout and antisense lines for generating and scavenging systems such as CAT2, APX1, chlAOX, mitAOX, CSD2, 2-cysteine PRX, GR1 and various NADPH oxidases have revealed a strong link between ROS and processes such as growth, development, stomatal responses and biotic and abiotic stress responses (Maxwell et al., 1999; Baier et al., 2000; Torres et al., 2002; Epple et al., 2003; Foreman et al., 2003; Rizhsky et al., 2003; Pnueli et al., 2003; Mittler et al., 2004;; Sagi et al., 2004; Vandenabeele et al., 2004; Mhamdi et al., 2010a; Vanderauwera et al., 2011; Chaouch et al., 2012). These findings demonstrate the complex nature of the ROS gene network in plants and its close integration with key biological processes. Although the redundancy of the ROS network is evidenced by such work, double or triple mutants

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lacking in key ROS network genes can serve as excellent tools to study interactions between different types of ROS generated at different subcellular locations.

1.2 Glutathione in plants

The tripeptide glutathione (GSH) is found in most organisms and is an essential metabolite with multiple functions in plants. Alongside the most widely found form of GSH (-L-glutamyl-L-cysteinyl-glycine) homologs are found in some groups of plants such as homoglutathione (hGSH; -L-glutamyl-L-cysteinyl--alanine) and hydroxymethylglutathione -L-glutamyl-L-cysteinyl-L-serine). In plants, glutathione is the most abundant non-protein thiol, typically accumulating in cells to concentrations of about 1-5 mM.

1.2.1 Glutathione synthesis

In both animals and plants, GSH is synthesized from glutamate, cysteine and glycine by two ATP-dependent steps (Figure 1.5; Rennenberg, 1980; Meister, 1988; Noctor et al., 2002; Mullineaux and Rausch, 2005). Each of the synthetic enzymes is encoded by a single gene in Arabidopsis (May and Leaver, 1994; Ullman et al., 1996). Localization studies in Arabidopsis have demonstrated that the first enzyme, -glutamylcysteine synthetase (-ECS), encoded by GSH1, is located in plastids, whereas the second enzyme, glutathione synthetase (GSH-S), encoded by GSH2, is found in both chloroplasts and cytosol (Wachter et al., 2005).

GSH

GSH-S

-EC Glycine

-EC

Cysteine Glutamate

Figure 1.5. Glutathione biosynthesis (left) and its main features (right)

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1.2.1.1 Glutathione deficient mutants

While knockout mutations in GSH1 cause lethality at the embryo stage (Cairns et al., 2006), T-DNA knockouts for GSH2 show a seedling-lethal phenotype (Pasternak et al., 2008). Less severe mutations in the GSH1 gene, which produce partial decreases in glutathione contents, have contributed appreciably to the study of the functions of glutathione in plants. Of these, the rml1 mutant, which has less than 5% of wild-type glutathione contents, shows the most striking phenotype because it fails to develop a root apical meristem (Vernoux et al., 2000). In other mutants, in which glutathione is decreased to about 25 to 50% of wild-type contents, developmental phenotypes are weak or absent, but alterations in environmental responses are observed. In cad2, lower glutathione is associated with enhanced cadmium sensitivity, whereas rax1 was identified by modified APX2 expression and pad2 shows lower camalexin contents and enhanced sensitivity to pathogens (Howden et al., 1995; Cobbett et al., 1998; Ball et al., 2004; Parisy et al., 2007). Very recently, yet another mutant partly deficient in glutathione because of a mutation in GSH1 has been reported. The zir1 mutant has only about 15% wild-type glutathione levels and was isolated in a screen for mutants that have defects in iron-mediated tolerance to zinc (Shanmugam et al., 2012). As well as genetic approaches, a pharmacological tool that has frequently been used to analyze the functions of glutathione is buthionine sulphoximine (BSO), a specific inhibitor of -ECS (Griffith and Meister, 1979).

1.2.1.2 Regulation and overexpression of the glutathione synthesis pathway

The production of glutathione is regulated at multiple levels, including regulation of cysteine synthesis and GSH1 and GSH2 at the level of transcript accumulation (Xiang and Oliver, 1998; Queval et al., 2009), post-translational regulation of adenosine phosphosulfate reductase (Bick et al., 2001), and post-translational regulation of -ECS (May et al., 1998b; Hicks et al., 2007). Glutathione accumulation is a well known response to enhanced H2O2 availability, and was first described in a barley mutant deficient in catalase (Smith et al., 1984). This response presumably reflects enhanced rates of glutathione synthesis and the major drivers in such situations are probably oxidant-induced increases in the limiting amino acid, cysteine, and post-translational activation of -ECS by oxidation (Queval et al., 2009).

Although the production of glutathione is regulated at multiple levels, plant cells can accommodate marked increases in overall glutathione. Studies using overexpression of -ECS targeted to either the chloroplast or the cytosol increase leaf glutathione by up to four-fold in different plant species (Noctor et al., 1996, 1998; Creissen et al., 1999; Xiang et al., 2001). Recently, considerably greater increases in glutathione have been achieved by overexpression of a bifunctional -ECS/GSH-S from Streptococcus (Liedschulte et al., 2010).

1.2.2 Glutathione transport

Glutathione synthesized in the chloroplast and cytosol is transported between cells and organs. One group of proteins that could be responsible for transport across the plasmalemma is the oligopeptide transporter

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(OPT) family (Figure 1.6). For example, of the nine annotated Arabidopsis OPT genes, OPT6 may play a role in long-distance transport, where it may transport GS-conjugates and GS-cadmium as well as GSH and GSSG (Cagnac et al., 2004).

Several different types of transporter may be important in translocation of glutathione between subcellular compartments. First, direct analysis of 35S-GSH uptake by wheat chloroplasts suggested the existence of components able to translocate glutathione across the inner chloroplast envelope (Noctor et al., 2002). That such transport occurs in vivo is strongly supported by the observation that the wild-type phenotype of gsh2 knockout mutants, which lack the ability to convert -EC to GSH, can be restored by transformation with a construct driving GSH-S expression exclusively in the cytosol (Pasternak et al. 2008). Recently, the chloroquinone-like transporter (CLT) family was described. These proteins are targeted to the plastid envelope and can transport both -EC and glutathione from the chloroplast into the cytosol (Maughan et al., 2010). These transporters are encoded by three genes in Arabidopsis, called CLT1, CLT2 and CLT3. Concerning transport into the vacuole, tonoplast multidrug resistance-associated protein (MRP) transporters of the ATP-binding cassette (ABC) type may act to clear GS-conjugates or GSSG from the cytosol (Figure 1.6; Martinoia et al., 1993; Rea, 1999; Foyer et al., 2001). While the anti-apoptotic factor Bcl-2 is considered as a key component to regulate GSH transport into the nucleus and mitochondria in mammalian tissues (Voehringer et al., 1998), transporters that may regulate the distribution of glutathione between the nucleus and mitochondria remain to be identified.

Figure 1.6. Subcellular compartmentation of glutathione synthesis and glutathione (scheme taken from Noctor et al., 2011).

BAG, Bcl2-associated anathogene. CLT, chloroquinone-like transporter. MRP, multidrug resistance-associated protein. OPT, oligopeptide transporter.

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1.2.3 Glutathione degradation

Rates of glutathione catabolism in Arabidopsis leaves have been estimated to be as high as 30 nmol g-1 FW h-1 (Ohkama-Ohtsu et al., 2008), which can be compared with typical leaf contents of 200-400 nmol.g-1FW. Thus, it is possible that some pools of glutathione could turn over several times in a day. In plants, several pathways of glutathione turnover have been proposed. Among them, four different types of enzymes have been described that could initiate glutathione breakdown (Figure 1.7). Some of these enzymes could use GSH, while others act preferentially on GSSG or other GS-conjugates. Firstly, glutathione or GS-conjugates could be degraded by carboxypeptidase activity (Steinkamp and Rennenberg, 1985), which has been detected in barley vacuoles (Wolf et al., 1996). However, specific genes remain to be indentified.

A second type of enzyme for glutathione catabolism involves the cytosolic enzyme phytochelatin synthase (PCS). Although this enzyme is most associated with polymerization of GSH-derived -EC units to form the metal-complexing phytochelatins (see section 1.2.4.4), it could also play a role in GS-conjugate degradation (Blum et al., 2007, 2010). The third type of enzyme, -glutamyl transpeptidase (GGT), could cleave glutathione to cysteinylglycine and a -glutamyl-amino acid derivative (transpeptidase activity) or free glutamate (peptidase activity). New insights have been gained by analyzing Arabidopsis mutants perturbed in glutathione degradation. In Arabidopsis, GGTs are encoded by four functional genes. The apoplastic enzyme GGT1 is active against GSH, GSSG and GS-conjugates and is prominent in all Arabidopsis tissue except seeds and siliques (Martin and Slovin, 2000; Storozhenko et al., 2002) while another apoplastic GGT2 is mainly expressed in siliques. Unlike the localization in animal cells, GGT1 and GGT2 are probably bound to the cell wall rather than to the plasmalemmma (Martin et al., 2007; Ohkama-Ohtsu et al., 2007a). Besides the extracellular GGTs, intracellular GGT4 (originally called GGT3 in Arabidopsis) is a vacuolar enzyme which is probably involved mainly in the breakdown of GS-conjugates (Grzam et al., 2007; Martin et al., 2007; Ohkama-Ohtsu et al. 2007b). The fourth predicted GGT enzyme, now called GGT3, could be detected in some tissues but is considered unlikely to contribute greatly to GGT activity (Martin et al., 2007; Destro et al., 2010).

It has been suggested that -glutamyl peptides produced by GGT can be further metabolized to 5-oxoproline by -glutamyl-cyclotransferases (GGC) and subsequently 5-oxoprolinase (5-OPase) to produce free glutamate (Ohkama-Ohtsu et al., 2008). No gene has yet been identified for GGC in Arabidopsis but a single gene OXP1 has been identified that likely encodes a cytosolic 5-OPase (Ohkama-Ohtsu et al., 2008). Based on 5-oxoproline accumulation in single oxp1 mutants, and in triple oxp1 ggt1 ggt4 mutants that are deficient in the major GGT activities as well as 5-OPase, it was proposed that the predominant pathway for GSH degradation is cytosolic and initiated by GGC (Figure 1.7), and not vacuolar or extracellular GGT (Ohkama-Ohtsu et al., 2008). GGC is therefore a fourth type of enzyme potentially involved in initiating glutathione degradation, although both the rat and tobacco GGC have been reported to be unable to use GSH (Orlowski and Meister, 1973; Steinkamp et al., 1987).

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Figure 1.7. Possible pathways of glutathione degradation (scheme from Noctor et al., 2012; annex 2). aa, amino acid; Cpep, carboxypeptidase; GGC, -glutamyl cyclotransferase; GGT, g-glutamyl transpeptidase; 5-OP, 5-oxoproline; 5-OPase, 5-oxoprolinase; PCS, phytochelatin synthase; X, S-conjugated compound.

1.2.4 Metabolic functions of glutathione in plants

GSH is oxidized to glutathione disulfide (GSSG) via not only enzymatic reactions catalyzed by DHAR or GSH-dependent peroxidases but also non-enzymatic reactions in which GSH is oxidized by ROS or, chemically, by DHA. The regeneration of GSH mainly is performed by NADPH-dependent GR. In addition to redox cycling, glutathione is involved in many other types of reaction.

1.2.4.1 Glutathione S-transferases

The plant glutathione S-transferases (GSTs; EC 2.5.1.18) are a large and diverse group of enzymes which catalyze the conjugation of electrophilic xenobiotics substrates with glutathione. The glutathione conjugates are then transferred to the vacuole by ATP-dependent GS-X pumps and degraded. In plants, the activity of most GSTs depends on an active site serine, which stabilizes the GS-thiolate anion (Dixon and Edwards, 2010). Based on sequence similarity and gene structure of family members, the monophyletic group of soluble GST super-family is subdivided into evolutionarily distinct classes in plants, each denoted by a Greek letter. Yet another two classes are DHARs and tetrachlorohydroquinone dehalogenase-like (TCHQD) enzymes. In all, the Arabidopsis genome contains a total of 55 GST-encoding genes which can be divided into 8 classes (Table 1.2). While specific nomenclatures have been proposed for these GSTs, much remains to be discovered regarding their functions.

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Table 1.2. Nomenclature of the GST super-family in Arabidopsis (from Dixon and Edwards, 2010) Class Letter code Number of Arabidopsis gene

Phi F 13 Tau U 28 Theta T 3 Zeta Z 2 Lambda L 3 Dehydroascorbate reductase - 4 Tetrachlorohydroquinone dehalogenase-like - 1 Microsomal - 1

As well as their role in catalysing the conjugation and detoxification of herbicides, other GST-associated activities have been proposed including intracellular transport of small molecules such as flavonoids, transient glutathione conjugation to protect reactive metabolites such as porphyrinogens and oxylipins, introduction of sulfur into secondary metabolites such as glucosinolates and camalaxin, and cis-trans isomerisation reactions (Edwards et al., 2000; Wagner et al., 2002; Dixon and Edwards, 2009, 2010; Geu-Flores et al., 2011; Su et al., 2011). Besides these functions, many GSTs have peroxidase or DHAR activity, and therefore function directly as antioxidant enzymes, as described above in section 1.1.2.2 and developed further below.

While ROS or DHA can react chemically with glutathione at significant rates, DHARs, members of the GST superfamily, provide an enzymatic link between GSH and DHA, thus promoting maintenance of ASC at the expense of the glutathione pool. Unlike most other GSTs, DHARs have an active site cysteine instead of serine/tyrosine, so rather than stabilising the thiolate anion of GSH, this residue instead forms a mixed disulfide with GSH as part of the catalytic mechanism (Dixon et al., 2002b). Also, at the protein level, DHARs behave differently from most other GSTs in being expressed as monomers rather than as dimers (Dixon et al., 2002a). They are found in peroxisomes, mitochondria, cytosol, and chloroplast (Chew et al., 2003; Reumann et al., 2009). Arabidopsis has 5 DHAR-like genes of which 3 are transcribed and encode functional proteins, namely DHAR1, DHAR2, and DHAR3. DHAR4 appears to be a pseudogene encoding a full-length but inactive enzyme, while the fifth DHAR corresponds to an untranscribed region encoding an N-terminally truncated, hence inactive enzyme. Several genetic studies show that DHARs play an important role in plants, particularly in conditions of oxidative stress such as ozone, aluminium stress, or pathogen inoculation (Chen and Gallie, 2006; Yoshida et al., 2006; Tamaoki et al., 2008; Vadassery et al., 2009).

1.2.4.2 Glutathione reductase

In most living organisms, NADPH-dependent GR maintains a high cellular GSH:GSSG ratio. GR is a NADPH-dependent dimeric flavoprotein belonging to the pyridine nucleotide-disulfide oxidoreductase group. Although NTR can also reduce GSSG in a TRX-dependent manner (Marty et al., 2009), GR is more efficient than the NTR-TRX system. Two genes encode GR in Arabidopsis. GR2 encodes an

16 CHAPTER 1 GENERAL INTRODUCTION

enzyme that is dual-addressed to plastids and mitochondria, while GR1 encodes a protein that is found in the cytosol and peroxisome (Chew et al., 2003; Kataya and Reumann, 2010).

Arabidopsis knockout mutants for the chloroplast/mitochondrial GR2 are embryo-lethal (Tzafrir et al., 2004), demonstrating the importance of maintaining an appropriate glutathione reduction state in plastids, mitochondria, or both. In contrast, gr1 knockout mutants do not show phenotypic effects, despite a 30-60% reduction in extractable enzyme activity (Marty et al., 2009; Mhamdi et al., 2010a). Genetic analyses have shown that the aphenotypic nature of gr1 mutants results from partial replacement of GSSG regeneration by the cytosol-located NTR-TRX system, though this is not sufficient to prevent significant accumulation of GSSG in the mutant (Marty et al., 2009).

1.2.4.3 Glutaredoxins

GRXs are small oxidoreductases structurally related to thioredoxins and generally considered to be reduced by glutathione. In Arabidopsis, over 30 GRX genes are annotated to encode GRX. They have two major glutathione-dependent biochemical properties: the reduction of disulphide bonds and the binding of iron-sulphur clusters. Normally, GRXs reduce a mixed disulfide between a protein and glutathione or a disulfide bridge via two catalytic mechanisms including monothiol and dithiol dependent catalytic mechanisms (Figure 1.8). In land plants, GRXs can be classified into three distinct subgroups based on amino acid motifs in the active site (Lemaire, 2004; Rouhier, 2010). The activity of the first CYPC-type class, which contain GRXs with C[P/G/S][Y/F][C/S] motifs, include thiol-disulphide exchange, regeneration of PRX and methionine sulphoxide reductase (Rouhier et al., 2002; Rouhier et al., 2006; Zaffagnini et al., 2008; Tarrago et al., 2009; Gao et al., 2010). The second CGFS-type class, which possesses a conserved CGFS active site sequence, could catalyze protein deglutathionylation and are involved in iron–sulphur cluster assembly. While some of these GRX have been characterized biochemically, their in vivo roles remain unclear. In Arabidopsis, GRXs can interact with ion channels and may play a role in response to oxidative stress (Cheng and Hirschi, 2003; Cheng et al., 2006; Guo et al., 2010; Sundaram and Rathinasabapathi, 2010). The third CC-type class, which regroups proteins with CC[M/L][C/S/G/A/I] active sites, is specific to land plants, because of the lack of related sequences in genomes of lower photosynthetic organisms, bacteria and mammals (Lemaire, 2004). Although little information about the biochemistry of the plant specific CC-type GRXs is available, some member of this class are the best studied GRXs in terms of physiological function. Several studies in Arabidopsis have revealed that three GRX forms are involved in plant development and phytohormone responses through interaction with TGA transcription factors (Ndamukong et al., 2007; Li et al., 2009). Notably, GRX480 overexpression was shown to repress the JA signaling marker gene, PDF1.2 (Ndamukong et al., 2007), while ROXY1 and ROXY2 are involved in petal and anther development (Li et al., 2009). In addition, overexpression of ROXY1 in Arabidopsis increased susceptibility to Botrytis cinerea (Wang et al., 2009c).

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Figure 1.8. Catalytic mechanisms of monothiol and dithiol GRXs (scheme from Rouhier et al., 2008).

1.2.4.4 Other glutathione-associated metabolic reactions

As well as the above processses, other glutathione-associated metabolic reactions include phytochelatin synthesis, glyoxylase and formaldehyde metabolism, camalexin production and glucosinolate synthesis.

Glutathione is the precursor of phytochelatins ([-Glu-Cys]nGly), which are required for responses to cadmium and other heavy metals. These compounds are produced from glutathione by PCS, a cytosolic enzyme. In Arabidopsis, there are two genes encoding PCS, of which PCS1 appears to be the most important. As noted in section 1.2.3, PCS1 may play a role in degradation of GS-conjugates as well as phytochelatin synthesis (Rea et al., 2004; Blum et al., 2007, 2010; Clemens and Peršoh, 2009). Phytochelatins sequester the metal to form a complex that is then transported into the vacuole (Grill et al., 1987, 1989; Cobbett and Goldsbrough, 2002; Rea et al., 2004). The importance of sufficient amounts of GSH to confer heavy metal resistance is evidenced by the identification of the glutathione-deficient cad2 mutant which is sensitive to cadmium (Howden et al., 1995; Cobbett et al., 1998). This is in agreement with increased heavy metal resistance by transgenic up-regulation of glutathione synthesis (Zhu et al., 1999a,b). As well as a precursor role in phytochelatin synthesis, glutathione could be involved in heavy metal resistance via its antioxidant function.

Oxo-aldehydes such as methylglyoxal are reactive compounds that may interfere with sensitive cellular compounds. Metabolism of such oxo-aldehydes through the glyoxylase system involves conjugation to GSH, catalyzed by glyoxalase 1 and then the hydrolytic reaction catalysed by glyoxalase 2 liberates the hydroxyacid and free GSH. In several plant species, overexpression of these enzymes increases tolerance to exogenous methylglyoxal and/or salt (Singla-Pareek et al., 2003, 2008; Deb Roy et al., 2008). Apart from its involvement in the catabolism of oxo-aldehydes, GSH may also act in detoxification of formaldehyde via formaldehyde dehydrogenase (FDH) and S-formylglutathione hydrolase. Formaldehyde

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can enter plants through stomata or be produced by endogenous metabolism (Haslam et al., 2002). Both of these enzymes act to oxidize formaldehyde to formic acid, which may then be converted to CO2 or enter C1 metabolism. Genes for both enzymes have been identified in Arabidopsis (Martínez et al., 1996; Haslam et al., 2002; Achkor et al., 2003). However, it seems that one FDH also encodes an enzyme with GSNO reductase activity (GSNOR; Sakamoto et al., 2002; Díaz et al., 2003).

Glutathione has long been implicated in reactions linked to metabolite synthesis (Dron et al., 1988; Edwards et al., 1991). Camalexin (3-thiazol-2-yl-indole)́ is the major phytoalexin with antimicrobial activity that accumulates in Arabidopsis after infection with a range of biotrophic and necrotrophic pathogens (Tsuji et al., 1992; Thomma et al., 1999; Glawischnig, 2007) and following treatment with abiotic factors such as UV-B paraquat, and heavy metal ions (Zhao et al., 1998; Mert-Türk et al., 2003; Bouizgarne et al., 2006; Kishimoto et al., 2006). It is an indole phytoalexin that contains one S atom per molecule and whose thiazole ring is derived partly from cysteine (Glazebrook and Ausubel, 1994). The Arabidopsis pad2 mutant was first identified as deficient in camalexin, with accumulation in the pad2 mutant decreased by 90% compared with wild-type plants following infection with Pseudomonas syringae pv. maculicola strain ES4326 (Glazebrook and Ausubel, 1994). Subsequent genetic studies identified the affected gene in pad2 as GSH1 and the mutation results in GSH contents that during growth in optimal conditions are decreased about 75% relative to wild-type (Parisy et al., 2007). Recent data support the idea that GSH is the sulfur donor required for camalexin biosynthesis, in which GSTF6 catalyzes the conjugation of GSH with indole-3-acetonitrile (IAN), which is then hydrolyzed to Cys-IAN in a process in which the GGT1 and GGT2 as well as the PCS1 have been proposed to be involved (Su et al., 2011). However, Geu-Flores et al. (2011) have argued against a role for GGTs and have presented evidence of the involvement of -glutamyl peptidases 1 and 3 (GGP1 and GGP3) in metabolizing GSH(IAN) conjugation.

In addition to camalexin, the glucosinolates are well-studied sulfur-containing secondary metabolites found in almost all plants of the order Brassicales, and the sulfur donor in their biosynthesis was recently shown to be GSH (Geu-Flores et al., 2011). The first step in the biosynthesis of indole glucosinolates is shared with that of tryptophan (Trp)-derived camalexin. Together with the enzyme myrosinase, glucosinolates are hydrolyzed into various active substances in the presence of water, which serve as defence against generalist-feeding insects (Hopkins et al., 2009). Indeed, the first indication of the involvement of GSH in the biosynthesis of glucosinolates came from the analysis of pad2 mutant plants. The pad2 mutant shows decreased resistance to feeding of the generalist insect Spodoptera littoralis, an effect that is linked to decreased accumulation of glucosinolates (Schlaeppi et al., 2008). This effect is specific to GSH, was not complemented by treatment of pad2 with the strong reducing agent dithiothreitol (DTT). Analysis of the ascorbate-deficient mutant vtc1-1 showed that the effect was not dependent on the ascorbate level, suggesting that it is linked to a requirement for GSH in biosyntheses rather than mediated via generalized changes in antioxidant capacity (Schlaeppi et al., 2008). An indication of the involvement of GSH as direct sulfur donor has come from the de novo engineering of benzylglucosinolate into Nicotiana benthamiana (Geu-Flores et al., 2009). Finally, Geu-Flores et al. (2011) have provided direct genetic evidence of the involvement of GSH as sulfur donor in the biosynthesis of glucosinolates by showing that an Arabidopsis double ggp1 ggp3 mutant has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. Thus, the cytosolic enzymes

19 CHAPTER 1 GENERAL INTRODUCTION

GGP1 and GGP3 seem to metabolize GSH conjugates in the biosynthesis of glucosinolates in Arabidopsis.

1.2.5 Glutathione in plant development, growth and environmental responses

It has long been recognized that GSH is oxidized by ROS as part of the antioxidant barrier that contributes to preventing excessive oxidation of sensitive cellular components. As noted above, however, glutathione is an essential metabolite with multiple functions in plants. Against the background of this complexity, current work is generating new information on the roles of glutathione in influencing the plant developmental processes and their interaction with the environment.

1.2.5.1 Glutathione in the regulation of plant development and growth

Accumulating evidence demonstrate that glutathione status is a key regulator in plant development and growth. The most intriguing data has come from analysis of the phenotypes of glutathione-deficient Arabidopsis mutants, which has shown that glutathione has critical functions in embryo and meristem development (Vernoux et al., 2000; Cairns et al., 2006; Reichheld et al., 2007; Frottin et al., 2009; Bashandy et al., 2010). Analysis of several triple mutants such as ntra nrtb rml1 and ntra ntrb cad2 have established redundancy between glutathione and other thiol systems such as TRX in the regulation of meristem development and growth related-auxin metabolism (Reichheld et al., 2007; Bashandy et al., 2010). Additionally, glutathione synthesis is also required for pollen germination and pollen tube growth in vitro (Zechmann et al., 2011). It has been shown that glutathione depletion by a specific inhibitor BSO in pollen grains causes disturbances in auxin metabolism which lead to inhibition of pollen germination (Zechmann et al., 2011). While the roles of glutathione in the plant cell cycle remain unclear, evidence has been presented that the recruitment of GSH into the nucleus in the G1 phase of the plant cell cycle has a profound effect on the redox state of the cytoplasm and the expression of redox-related genes (Pellny et al., 2009; Diaz-Vivancos et al., 2010a,b). A subsequent increase in the total cellular GSH pool above the level present at G1 is essential for the cells to progress from the G1 to the S phase of the cycle (Diaz-Vivancos et al., 2010a,b).

1.2.5.2 Light signaling

Evidence for the role of glutathione in irradiance-dependent signaling came from the identification of the Arabidopsis rax1 mutant. This line contains less than 50% wild-type glutathione contents, and shows enhanced constitutive expression of the high light-induced gene, APX2 (Ball et al., 2004). Glutathione has also been implicated in the signaling pathways that facilitate acclimation of chloroplast processes to high light (Ball et al., 2004). Exposure to photoinhibitory light can lead to increases in leaf H2O2 levels and to oxidation of the glutathione pool (Mateo et al., 2006; Muhlenbock et al., 2008). The abundance of glutathione or its homologs in leaves does not vary greatly over the day/night cycle. However, young poplar trees show more variation throughout development, with a gradient from young to old leaves (Arisi et al., 1997). The effect of stresses such as drought can also influence the relative abundance of glutathione/homoglutathione in different leaf ranks. Several studies in Arabidopsis and other species point

20 CHAPTER 1 GENERAL INTRODUCTION

to a potential link between growth daylength, light signaling and glutathione status (Becker et al., 2006; Queval et al., 2007; Bartoli et al., 2009). Relationships between glutathione and photoreceptor signaling have been suggested in studies on the arsenic tolerant mutants, ars4 and ars5 (Sung et al., 2007). The ars4 mutation was identified as an allele of phytochrome A (phyA) and caused increased BSO resistance (Sung et al., 2007). The ars5 mutant, which is affected in a 26S proteasome component, had increased levels of glutathione when exposed to arsenic, accompanied by increased GSH1 and GSH2 transcripts (Sung et al., 2009).

1.2.5.3 Biotic interactions, cell death and defence phytohormones

As well as serving as a source of sulfur donor in the biosynthesis of secondary metabolites upon pathogen and insect challenge, it is likely that glutathione is involved in signaling processes during biotic challenge. Exogenous GSH can mimic certain pathogens in activating the expression of defence-related genes such as PR1 (Dron et al., 1988; Wingate, et al., 1988; Senda and Ogawa, 2004; Gomez et al., 2004a). Moreover, accumulation of glutathione is triggered by pathogen infection (Edwards et al., 1991; May et al., 1996). Similar changes have also been reported following exogenous application of the defence-related hormone SA, or biologically active SA analogs (Mou et al., 2003; Mateo et al., 2006; Koornneef et al., 2008). Further evidence that glutathione is involved in defence against biotic stress came from the studies of Arabidopsis mutants altered in GSH synthesis or metabolism. Resistance to fungal and bacteria pathogens is compromised in the glutathione-deficient Arabidopsis mutants pad2, cad2 and rax1, while overexpressing Lycopersicon esculentum -ECS in tobacco enhances resistance to Pseudomonas syringae pv. tabaci (Table 1.3). These observations suggest that there is a critical glutathione status below which the accumulation of pathogen-defence related molecules and consequently disease resistance, is impaired.

The components that link changes in glutathione status to pathogen responses remain to be identified.

Analysis of catalase-deficient tobacco and Arabidopsis have indicated that H2O2 triggered changes in glutathione status accompany the activation of pathogen responses (Willekens et al., 1997; Chamnongpol et al., 1998; Chaouch et al. 2010). Glutathione oxidation and accumulation triggered by increased H2O2 availability in catalase-deficient cat2 is associated with HR-like lesions formation, induced PR genes, camalexin production and increased bacterial resistance as part of a wide spectrum of defence responses (Chaouch et al. 2010). Glutathione is also required for the production of nodules in the association between legumes and nitrogen-fixing rhizobia. These organs contain high levels of both GSH and homoglutathione, and deficiency in these molecules inhibits nodule formation (Frendo et al., 2005; Pauly et al., 2006).

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Table 1.3. Disease resistance phenotypes in glutathione synthesis mutants (- Decreased resistance, + Increased resistance, = No difference relative to Col-0, OE Overexpression)

Pathogen (Lifestyle) Resistance Species lines (mutant/OE) Reference

P. syringae pv. maculicola ES4326 (Virulent bacterium) - Arabidopsis pad2 Glazebrook and Ausubel, 1994

P. syringae pv. tomato DC3000/avrRpm1(Avirulent bacterium) - Arabidopsis cad2/rax1 Ball et al., 2004

P. brassicae (Hemibiotrophic oomycete) - Arabidopsis pad2 Roetschi et al., 2001

H. parasitica (Biotrophic oomycete) - Arabidopsis pad2 Glazebrook et al., 1997

B. cinerea (Necrotrophic fungus) - Arabidopsis pad2 Ferrari et al., 2003

A.brassicicola (Necrotrophic fungus) - Arabidopsis pad2 Van Wees et al., 2003

P. cucumerina (Necrotrophic fungus) - Arabidopsis pad2 Parisy et al., 2007

P syringae pv. tabaci (Virulent bacterium) + Tobacco OE -ECS Ghanta et al., 2011

P. syringae pv. tomato DC3000 (virulent bacterium) = Arabidopsis pad2 Glazebrook and Ausubel, 1994

P. syringae pv. tomato DC3000 (virulent bacterium) = Arabidopsis cad2 May et al., 1996

P. syringae pv. glycinea/avrB (avirulent bacterium) = Arabidopsis cad2 May et al., 1996

P. syringae pv. maculicola ES4326/avrRpt2 (Avirulent bacterium) = Arabidopsis pad2 Glazebrook and Ausubel, 1994

P. syringae pv. maculicola ES4326/avrRpm1 (Avirulent bacterium) = Arabidopsis pad2 Glazebrook and Ausubel, 1994

The phytohormone SA has been implicated as an important signal for the activation of both effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). SA accumulated in response to pathogen infection is synthesized from chorismate, with the key step being catalyzed by isochorismate synthase (ICS1), the enzyme that is inactive in the salicylic acid induction-deficient mutant, sid2 (Wildermuth et al., 2001). Signaling downstream from SA is largely regulated via NPR1. Most of the attention paid to roles of glutathione in pathogenesis responses has focused on NPR1, because this protein is known to be influenced by thiol-disulfide status and SA-dependent PR gene expression can be induced by exogenous GSH (see section 1.1.3.3). The notion that NPR1 function might be linked to glutathione redox state via GRX activity (Mou et al., 2003) has been superseded by a model based on activation by TRX h (Tada et al., 2008), some of which are known to be inducible by oxidative stress and pathogens (Laloi et al., 2004). However, glutathione could be indirectly involved through GSNO-dependent nitrosylation of NPR1 monomers, stimulating reoligomerization, which was postulated to be necessary to prevent protein depletion (Tada et al., 2008).

The identification of NPR1 as a target of S-nitrosylation is another example of the involvement of GSH in plant disease resistance, in which protein S-nitrosylation is considered to be a key post-translational modification. The Atgsnor1 mutant for the enzyme GSNO reductase has compromised resistance to both virulent and avirulent pathogens (Feechan et al., 2005). However, redox regulation of NPR1 and the transcription factors with which NPR1 interacts is turning out to be complex. Four cysteine residues on TGA1 underwent S-nitrosylation and S-glutathionylation after GSNO treatment of the purified protein, while GSNO treatment of Arabidopsis protoplasts caused translocation of an NPR1-GFP fusion protein into the nucleus (Lindermayr et al., 2010). The importance of S-nitrosylation in regulating NPR1 function is in line with an important role for this modification in plant disease resistance.

Although activation of NPR1 involves a reductive change, the initial signal for the events leading to PR gene expression generally involves ROS accumulation. Oxidative bursts can also trigger the HR, in which

22 CHAPTER 1 GENERAL INTRODUCTION

necrotic lesions develop at the site(s) of pathogen infection in plants. While redox changes involved in HR have been most closely associated with events triggered by extracellular ROS production, oxidative stress of intracellular origin can also trigger such effects. It has been proposed that the glutathione redox state is a key determinant of cell death and dormancy (Kranner et al., 2006). According to this concept, an increase in GSSG accumulation or increase in redox potential above a threshold reducing value necessarily causes death. For instance, increases in the total glutathione contents of leaves produced by ectopic -ECS overexpression in the tobacco chloroplast caused accumulation of GSSG and this was associated with lesion formation and enhanced expression of PR genes (Creissen et al., 1999). In addition, the HR-like lesions triggered by intracellular oxidative stress are associated with GSSG accumulation (Smith et al., 1984; Willekens et al., 1997; Chamnongpol et al., 1998; Chaouch et al., 2010). However, based on current understanding on work in catalase-deficient cat2 and related double mutants, changes in glutathione redox potential or GSSG accumulation do not seem to be sufficient to cause cell death. Firstly, HR-like lesions can be abolished by genetically blocking SA synthesis even though this effect is associated with unchanged or greater GSSG accumulation (Chaouch et al., 2010). Secondly, the hyper-accumulation of GSSG in cat2 gr1 does not cause HR-like lesions but seems to repress the SA pathway relative to the single cat2 mutant (Mhamdi et al. 2010a). Thirdly, analyses of double cat2 atrbohF Arabidopsis mutants, which lack both the major catalase and one of the major leaf NADPH oxidase activities, demonstrate that glutathione is much less perturbed than in the cat2 single mutant, yet this is associated with unchanged or even increased lesion formation (Chaouch et al., 2012).

While glutathione status is implicated in SA signaling, glutathione may also modulate signaling through the JA pathway, which is involved in the regulation of development and in responses to necrotrophic pathogens and herbivores and is regulated antagonistically to the SA pathway. Accumulation of JA is triggered by wounding or various types of pathogens, and occurs from linolenic acid in the chloroplast and subsequent synthesis in the peroxisome (Browse, 2009). JA induces genes for glutathione synthesis enzymes and GR (Xiang and Oliver, 1998). Subsequent work has shown that other antioxidative genes can also be activated by JA (Sasaki-Sekimoto et al., 2005). In gr1 mutants lacking the cytosolic/peroxisomal GR, several JA-associated genes are repressed (Mhamdi et al. 2010a). Among the components that could link JA signaling and glutathione are GRXs and GSTs. Overexpression of the SA-inducible protein GRX480 represses JA-triggered induction of the defensin, PDF1.2 (Ndamukong et al., 2007). At the same time as relaying part of the SA-dependent signaling, NPR1 antagonizes JA signaling (Spoel et al., 2003), and this antagonism was abolished by treatments with a glutathione biosynthesis inhibitor, BSO, suggesting that SA-JA interactions may be mediated by transient changes in the redox status (Koornneef et al., 2008). Based on these data, glutathione could act to repress JA signaling through SA-dependent induction of NPR1 and GRX480, both of which interact with TGA transcription factors. Other possible effects of glutathione could occur through the regulation of JA synthesis or excess accumulation of intermediates like 12-oxophytodienoic acid (OPDA). Several GSTs may catalyze formation of GS-oxylipin conjugates based on JA- or oxylipin-dependent gene expression patterns (Yan et al., 2007; Mueller et al., 2008) and the peroxidatic or conjugation activities of GSTs against electrophilic metabolites (Wagner et al., 2002; Davoine et al., 2006). Roles for GS-conjugates in JA metabolism receive support from the observation that ggt4 mutants lacking an enzyme likely to be important in GS-conjugate degradation (section 1.2.3) were found to accumulate a GS-conjugate of the JA synthesis precursor, OPDA, during incompatible interactions with bacteria (Ohkama-Ohtsu et al.,

23 CHAPTER 1 GENERAL INTRODUCTION

2011).

1.2.6 Mechanisms of glutathione dependent redox signaling

Thiol-disulfide exchange reactions are a fundamental mechanism of regulation enzyme activity in plants (Buchanan and Balmer, 2005). While thiol-disulfide redox signaling through the chloroplast TRX system was well established by the end of the 1970s, attention has been focused on glutathione-linked redox signaling in plants only more recently (Foyer et al., 1997; May et al., 1998a). The following discussion will outline factors that could link glutathione to modification of target protein activity.

1.2.6.1 Protein S-glutathionylation

Glutathione is involved in a post-translational modification called glutathionylation. Protein S-glutathionylation involves the formation of a stable mixed disulphide bond between glutathione and a protein cysteine residue (Figure 1.9). Described protein targets include enzymes involved in primary metabolism such as TRXf, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycine decarboxylase (GDC; Michelet et al., 2005; Zaffagnini et al., 2007; Palmieri et al., 2010). TRX of the f type is glutathionylated on a cysteine residue found outside the active site, decreasing its activity against target proteins such as plastidial GAPDH, while another TRX-independent isoform of GAPDH is inhibited by glutathionylation (Michelet et al., 2005; Zaffagnini et al., 2007). In the mitochondria, GDC has been shown to be sensitive to oxidative stress (Taylor et al., 2002), and is inhibited by thiol modifications, including S-glutathionylation (Palmieri et al., 2010). However, the in vivo mechanisms underlying S-glutathionylation remain to be established. The mechanisms of S-glutathionylation may involve GRX-catalysed thiol-disulphide exchange between protein thiol groups and GSSG, conversion of thiol groups to thiyl radicals or sulphenic acids, or be GSNO mediated (Dixon et al., 2005; Holtgrefe et al., 2008; Gao et al., 2009; Palmieri et al., 2010). The reverse reaction, deglutathionylation, could be mediated by certain class I and class II GRX. From a signaling perspective, GRXs activities could regulate the status of protein thiols through reactions involving disulfides or mixed disulfides (Figure 1.9).

Figure 1.9. Some possible glutathione linked signaling mechanisms (scheme taken from Foyer and Noctor, 2011). GR, Glutathione reductase; GRX, Glutaredoxin; GS-R, Glutathione S-conjugate; GSNOR, GSNO reductase; GST, glutathione S-transferase; NO; Nitric oxide; R, small organic molecule; SH, sulfhydryl (thiol) group; SSG, glutathionylated protein Cys residue, TRX, Thioredoxin.

24 CHAPTER 1 GENERAL INTRODUCTION

1.2.6.2 Protein S-nitrosylation and GSNO reductase

As well as S-glutathionylation, S-nitrosylation of protein Cys residues may be mediated by GSNO in plants (Lindermayr et al., 2005, 2010; Romero-Puertas et al., 2008; Lindermayr and Durner, 2009). S-nitrosylation is a post-translational modification involving the reversible covalent binding of NO to the cysteine residue of proteins to form S-nitrosocysteine (SNO). GSNO is thought to be the physiological NO molecule that mediates direct protein S-nitrosylation. Inducible NO-producing enzymes in plants remain to be clearly described, but GSNOR may be an important enzyme that can regulate SNO homeostasis via the breakdown of GSNO into GSH and ammonia (Sakamoto et al., 2002; Barroso et al., 2006; Díaz et al., 2003). This enzyme has also been implicated in the regulation of cell death, pathogen resistance and other functions such as thermotolerance (Feechan et al., 2005; Lee et al., 2008; Chen et al., 2009). It is not yet clear whether changes in glutathione status can affect GSNO availability or the probability of either protein S-glutathionylation and S-nitrosylation. In Arabidopsis many potential protein candidates carrying cysteine residues have already been reported such as NPR1, SA-binding protein 3, GAPDH, GDC and type II metacaspase 9 (Tada et al., 2008; Wang et al., 2009a; Leitner et al., 2009; Palmieri et al., 2010). S-nitrosylation of type II PRX has been proposed to play a role in ROS signaling (Romero-Puertas et al., 2007). As described above, S-nitrosylation is thought to act in opposition to disulphide reduction by TRX in the regulation of NPR1 (Tada et al., 2008). While the monomerisation reaction of NPR1 was shown to be catalysed TRX, oligomerisation is facilitated by GSNO (Tada et al., 2008). However, the accumulation of NPR1-GFP fusion protein in the nucleus was significantly increased when Arabidopsis mesophyll protoplasts were treated with the physiological NO donor, GSNO (Lindermayr et al., 2010). In the nucleus, NPR1 interacts with the transcription factor TGA1 and binds to the promoter region to active PR gene expression (Després et al., 2003). In vitro studies using GSNO have shown that S-nitrosylation of both NPR1 and TGA1 favoured DNA binding and TGA1 stability (Lindermayr et al., 2010). Thus, S-nitrosylation may play a crucial role in regulating SA mediated plant innate immunity.

1.2.6.3 Glutathione redox potential

A key factor governing interactions between glutathione and protein targets could be redox potential. Genetic support for the physiological importance of glutathione-dependent disulphide reduction comes from the observations that the NADPH-TRX and glutathione systems play overlapping roles in development (Reichheld et al., 2007; Marty et al. 2009; Bashandy et al., 2010). It is possible that in some conditions changes in glutathione redox potential could influence the mechanisms that contribute to changes in the TRX redox potential and thus the biological activity of TRX targets. The actual glutathione redox potential is related to [GSH]2:GSSG. Thus, unlike many other redox couples (e.g. NADP+/NADPH), the glutathione redox potential depends on and can be influenced by absolute concentration as well as by changes in GSSG relative to GSH (Mullineaux and Rausch, 2005; Meyer, 2008). Even if the GSH:GSSG ratio remains unchanged, decreases in glutathione concentration alone will lead to an increase in redox potential (less reducing). It has been experimentally confirmed that the cytosolic redox potential as detected by reduction-oxidation sensitive green fluorescent protein (roGFP) was more positive in cad2 in which glutathione is decreased to about 30% of wild-type contents (Meyer et al., 2007). Moreover, increases in the cytosolic but not plastidial redox potential in clt1 clt2 clt3

25 CHAPTER 1 GENERAL INTRODUCTION

mutants, lacking the chloroplast envelope glutathione transporters, are also consistent with a depleted glutathione pool in the cytosol but not the chloroplast (Maughan et al., 2010). In addition, physiological and environmental factors might cause shifts in glutathione redox potential in vivo. For example, the cytosolic glutathione redox potential has been shown to increase in response to wounding (Meyer et al. 2007). Whole tissue GSH:GSSG ratios can be markedly affected in catalase deficient mutants in conditions favouring high rates of H2O2 production (Smith et al., 1984; Willekens et al., 1997; Rizhsky et al., 2002; Queval et al., 2007, 2009), and this is a key observation underlying the studies reported in Chapters 2 and 3.

1.3 Autophagy in plants

As sessile organisms, intracellular recycling is an important mechanism that is critical for the responses of plants to adverse environmental conditions. A key intracellular degradation process during intracellular remodeling, senescence and nutrient starvation is vacuolar autophagy, whereby cytoplasmic components are degraded in the vacuole to provide raw materials and energy for growth, and also to eliminate damaged or toxic components, for the maintenance of essential cellular functions. The term ‘autophagy’ comes from the Greek words ‘phagy’ meaning eat, and ‘auto’ meaning self. The basic autophagy process is conserved among eukaryotes from yeast to animals and plants (Bassham, 2007; Yang and Klionsky, 2009; Mehrpour et al., 2010). While several of types of autophagy have been described in many species, two types of autophagy, called microautophagy and macroautophagy, are found in plants (Figure 1.10; Bassham et al., 2006). Microautophagy involves the formation of a small intravacuolar vesicle called an autophagic body by invagination of the tonoplast, thus engulfing cytoplasmic components. On the other hand, macroautophagy is the most extensively studied in plants and is mediated by a special organelle termed the autophagosome (Yoshimoto, 2012). The core marker for autophagy is the double membrane-bound autophagosome in plants into which, during macroautophagy, bulk cytosolic constituents and organelles are sequestered. The outer membrane of the autophagosome then fuses with the vacuolar membrane and thus delivers the inner membrane structure and its cargo, namely the autophagic body, inside the vacuole for degradation by vacuolar hydrolases (Figure 1.10), and the products are exported from the vacuole to the cytoplasm for reuse. The discussion here will focus on the plant macroautophagy pathway, which hereafter is referred to as autophagy.

Figure 1.10. The microautophagic and macroautophagic processes in plants (scheme from Yoshimoto, 2012).

26 CHAPTER 1 GENERAL INTRODUCTION

In plants, autophagic structures have been observed, largely based on morphological observation by electron microscopy, since the late 1960s (Villiers, 1967; Matile and Moor, 1968; Matile and Winkenbach, 1971; Matile, 1975; Marty, 1978; Van der Wilden et al., 1980). However, the underlying molecular mechanisms of autophagy have only recently been begun to be identified. Progress in understanding the mechanistic basis of autophagy has been greatly facilitated by studies of yeast autophagy. The initial studies of most genes functioning in autophagy pathways were done via mutagenesis studies in the yeast, Saccharomyces cerevisiae. Hence, the identification of the ATG genes in yeast led to molecular analysis of autophagy in higher eukaryotes. Since the first autophagy (ATG) genes were identified in yeast in 1993 (Tsukada and Ohsumi, 1993), the number of identified yeast autophagy-related genes has increased to 32 (Barth et al., 2001; Klionsky et al., 2003; Kanki et al., 2009; Nakatogawa et al., 2009; Okamoto et al., 2009). These genes can be divided into several functional groups: the ATG1 protein kinase complex; the phosphatidylinositol 3-kinase (PI3K) complex specific for autophagy; the ATG9 complex; and two ubiquitin-like conjugation systems, leading to AG8 lipidation and ATG12 protein conjugation (Table 1.4). The knowledge gained from yeast research has greatly facilitated the identification of homologous genes that are required for autophagy in plants. Indeed, the genes essential for the central autophagic machinery consist of 18 ATG genes in yeast. Although 4 homologues of the 18 yeast ATG genes have not yet been identified in plants, approximately 30 Arabidopsis ATG genes have been identified on the basis of their sequences (Table 1.4; Doelling et al., 2002, Hanaoka et al., 2002, Xiong et al., 2005). It has thus been suggested that Arabidopsis as well as other crop species such as rice and maize may share the core autophagic machinery with yeast (Su et al., 2006; Ghiglione et al., 2008; Chumg et al., 2009; Shin et al., 2009; Xia et al., 2011; Kuzuoglu-Ozturk et al., 2012).

Table 1.4. Arabidopsis Atg homologs (from Yoshimoto, 2012).

a T-DNA insertional knock-out mutants of these genes have been published. b Unpublished data. TORC1, TOR kinase complex 1; PI(3)P, phosphatidylinositol 3-phosphate; PE, phosphatidylethanolamine.

27 CHAPTER 1 GENERAL INTRODUCTION

1.3.1 Functions of autophagy in plants

Although morphological studies provided initial insights into the structural components and characteristics of the process in plants several decades ago, these results provided only static images of autophagy, and the physiological roles of plant autophagy remain largely unknown. The recent identification of Arabidopsis homologs of most of the core ATG genes has allowed studies of many autophagy-defective plants with impaired ATG genes (Bassham et al., 2006; Hayward et al., 2009). As with other systems such as yeast and animals, autophagy in plants has essential roles not only during starvation, but also during development, senescence, and in response to different abiotic stresses as well as pathogens (Bassham, 2007; Bassham, 2009; Hayward and Dinesh-Kumar, 2011). Even under favorable environments, basal autophagy has an important housekeeping function by clearing damaged or unwanted cytoplasmic components for recycling and to provide a source of amino acids, lipids and sugars for basic cellular processes.

1.3.1.1 Functions of autophagy during abiotic stress

Autophagy is induced under nutrient starvation such as carbon or nitrogen deprivation, which is one of most studied abiotic stresses (Doelling et al., 2002; Hanaoka et al., 2002; Thompson et al., 2005; Xiong et al., 2005; Phillips et al., 2008). During nutrient starvation, the breakdown of damaged or unwanted cell materials such as bulk protein, carbohydrates and lipids through autophagy degradative pathways becomes crucial to replenish the supply of carbon and nitrogen needed for survival and new development. The early morphological studies demonstrated that autophagic structures are often observed under nutrient-limiting conditions in plant cells, suggesting a nutrient recycling function for plant autophagy. Indeed, when Atatg mutant plants are grown under nitrogen- and carbon-depleted or limiting conditions, autophagy defective plants display accelerated starvation-induced chlorosis and stunted growth, most likely because autophagy is required for nutrient remobilization to allow temporal adaptation of plant cells to nutrient starved conditions (Doelling et al., 2002; Hanaoka et al., 2002; Thompson et al., 2005; Xiong et al., 2005; Phillips et al., 2008). It has been recently confirmed that autophagy is required for nitrogen remobilization (Guiboileau et al., 2012). It is well established that the target of rapamycin (TOR) complex integrates nutrient signaling and associates with the ATG1 complex to inhibit the activity of autophagy in yeast and mammals (He and Klionsky, 2009). As well as the interaction between TOR and ATG1 as the primary molecular switch for starvation-induced autophagy in yeast and mammals, it has also been shown that in plants TOR may function as a negative regulator of autophagy (Liu and Bassham, 2010). In addition, autophagy has been shown to be a rather general response to a variety of abiotic stresses (Bassham, 2007). When treatment of plants with H2O2 or methyl viologen (MV) results in severe oxidative stress, autophagy is quickly induced. The autophagy-defective AtATG18a knockdown transgenic plants are hypersensitive to MV treatment and accumulate higher levels of oxidized proteins, implying that the defence mechanism of autophagy is to degrade oxidized components (Xiong et al., 2007). Similar effects have also been observed in the rice Osatg10 mutant (Shin et al., 2009). Moreover, autophagy is essential for plant tolerance to drought and salt stresses (Liu et al., 2009; Kuzuoglu-Ozturk et al., 2012). These results suggest a protective role for autophagy in transporting oxidized proteins or organelles into the vacuole for degradation in plant cells. However, it remains unknown whether this transport is selective. Additionally, AtTSPO, an ABA-induced protein, has been shown to be degraded by

28 CHAPTER 1 GENERAL INTRODUCTION

the autophagy pathway, implying the involvement of autophagy in responses to ABA in plants (Vanhee et al., 2011).

1.3.1.2 Functions of autophagy during pathogen infection

Conflicting results have been reported on the relationship between autophagy and cell death in plants. It has been reported that autophagy can either restrict or promote cell death upon pathogen infection (Hayward and Dinesh-Kumar, 2011; Hofius et al., 2009; Yoshimoto et al., 2009; Lenz et al., 2011a,b; Lai et al., 2011; Wang et al., 2011). The roles of autophagy may vary depending on the type of pathogen and the age of the plant. Plant pathogens are often divided into two types based on their lifestyles: necrotrophic and biotrophic. Necrotrophic pathogens derive nutrients from dead or dying cells, whereas biotrophic pathogens feed on living host tissue for survival (Glazebrook, 2005). In Arabidopsis, infection with the necrotrophic fungal pathogen Botrytis cinerea induces the expression of autophagy genes and formation of autophagosomes (Lai et al., 2011). Autophagy defective mutants exhibit susceptibility to the necrotrophic fungal pathogen Botrytis cinerea and Alternaria brassicicola (Lai et al., 2011; Lenz et al., 2011b). These results suggest that autophagy functions in a pro-survival role during necrotrophic pathogen infection. However, the role of autophagy upon biotrophic pathogen infection is open to debate. When plants are infected with tobacco mosaic virus (TMV) pathogen, the execution of HR cell death is not compromised, but spreads to throughout the infected leaves and even into the healthy uninfected tissue of the BECLIN1/ATG6-silenced tobacco plants (Liu et al., 2005). Similar results have been also confirmed in Arabidopsis ATG6 RNAi lines and atg5 mutants using the avirulent pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 (avrRPM1; Patel and Dinesh-Kumar, 2008; Yoshimoto et al., 2009). These results suggest that autophagy functions to restrict the spread of HR cell death, thus serving a pro-survival role. By contrast, it was found that cell death is suppressed and resistance is increased in atg7 and atg9 knockout mutants when challenged with the avirulent pathogen Pst DC3000(avrRps4; Hofius et al., 2009). Furthermore, several atg knockout mutants (atg2, atg5, atg7 and atg10) which are defective in autophagy exhibited enhanced resistance to the powdery mildew Golovinomyces cichoracearum (Wang et al., 2011). Other biotrophic pathogens including the virulent bacterial strain PstDC3000 and avirulent Hyaloperonospora arabidopsidis have recently been used in resistance tests in ATG-defective mutant plants (Hofius et al., 2009; Lenz et al., 2011b). The ATG-defective plants all showed enhanced resistance to both of these biotrophic pathogens (Hofius et al., 2009; Lenz et al., 2011b). These different results indicate that autophagy may have both a pro-death role and pro-survival functions upon challenge with biotrophic pathogens. It has been proposed that the contrasting results observed during biotrophic pathogen challenge could be due to the differences of plant age and growth conditions (Hayward and Dinesh-Kumar, 2011; Liu and Bassham, 2012), and the exact roles of autophagy in plant innate immune response remain to be elucidated.

1.3.1.3 Functions of autophagy during growth and seed development

Although a lot of autophagy studies have been focused on stress related conditions, the functions of autophagy under normal growth conditions have also been investigated (Slavikova et al., 2005; Inoue et al., 2006; Yano et al., 2007; Yoshimoto et al., 2009). Here, it is important to note that plants may maintain a basal level of housekeeping autophagy to eliminate damaged materials, which are continually generated

29 CHAPTER 1 GENERAL INTRODUCTION

even under favorable growth conditions (Bassham et al., 2006). As well as slower growth, almost all of the autophagy-defective mutants have an early senescence phenotype under nutrient-rich conditions, suggesting that autophagy has a pro-survival function in development (Doelling et al., 2002; Hanaoka et al., 2002; Patel and ; Thompson et al., 2005; Xiong et al., 2005; Qin et al., 2007; Phillips et al., 2008; Dinesh-Kumar, 2008; Yoshimoto et al., 2009). It is interesting that premature senescence is genetically blocked by inactivation of the SA synthesis or signaling pathways in atg5 sid2 and atg5 npr1 double mutants, and that this is accompanied by decreased H2O2 (Yoshimoto et al., 2009). This raises an interesting question regarding the relationship between autophagy and H2O2 during SA related leaf senescence. As discussed above, autophagy could be involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis (Xiong et al., 2007). It is also possible that autophagy may function selectively to remove specific H2O2-generating organelles such as chloroplasts and peroxisomes. However, the underlying mechanisms remain to be fully investigated, and the relationship between autophagy and H2O2 generated intracellularly is a focus of Chapter 4 of this study.

During senescence, plants could recycle nutrients from senescing leaves to newly forming organs such as developing seeds. Because autophagy is involved in recycling cytoplasmic materials, it is understandable that it functions during seed development, which is a process of extensive nutrient remobilization. Although no obvious defect in seed formation was observed in Arabidopsis atg mutants in under normal growth conditions, several ATG genes are up regulated during seed maturation and desiccation (Angelovici et al., 2009). More recently it has been shown that autophagy is required for seed filling (Guiboileau et al., 2012). Seed storage proteins are synthesized in the endoplasmic reticulum (ER) and form protein bodies (PB) during seed development, and accumulate in protein storage vacuoles (PSVs) which are degraded to support the growth of newly forming organs in plants (Ibl and Stoger, 2012). Indeed, it has been reported that intact PB is transported to the vacuoles by autophagy pathway in wheat (Levanomy et al., 1992). Furthermore, after being assembled in the ER, zeins, the prolamin storage protein in maize, were recently found to be delivered from ER to aleurone PSVs in atypical prevacuolar compartments by an atypical autophagy pathway (Reyes et al., 2011).

1.3.2 Difficulties of monitoring autophagy in plants

As mentioned above, autophagic processes were identified by electron microscopy in early plant studies. More recently, a more reliable marker used to monitor plant autophagy has been developed, ie, monitoring of the ATG8 protein. ATG8 is integrated into the phagophore during the early stages of autophagosome formation. ATG8 is covalently attached to the lipid phosphatidylethanolamine (PE) to produce ATG8-PE that is bound to autophagic membranes via its lipid moiety. Hence, plants engineered to express ATG8-GFP reporter constructs have been particularly informative. Autophagic vesicles can be easily visualized in plant cells using ATG8-GFP by fluorescence confocal microscopy (Yoshimoto et al., 2004; Thompson et al., 2005; Chung et al., 2010). Some of these studies have revealed that structures observed in plants sometimes seem to be protein aggregates other than autophagosomes. Such aggregation often occurs when proteins are overexpressed in plant cells and it has therefore been considered that autophagic flux should be confirmed using biochemical approaches (Chung et al., 2010). ATG8-GFP fusions are transported into the vacuole as a consequence of autophagy, and accumulation of free GFP released from the GFP-ATG8-PE conjugate during breakdown of autophagic bodies in the

30 CHAPTER 1 GENERAL INTRODUCTION

vacuole can be monitored biochemically by immunoblotting with an anti-GFP antibody (Chung et al., 2010; Suttangkakul et al., 2011). This accumulation is almost completely blocked in autophagy-deficient mutants. Furthermore, concanamycin A or protease inhibitors such as E64D have also been used to monitor the autophagic process in combination with the tagging of ATG8 in plant cells. Because autophagic bodies are short lived in the vacuole of plant cells, their stability and thus detection is greatly improved by prior incubation of plants with inhibitors of vacuolar degradation (Yoshimoto et al., 2004; Thompson et al., 2005; Ishida et al., 2008). In addition, LysoTracker red or monodansylcadaverine has also been used to monitor acidic autophagosomes in plants as well as in animals (Comtento et al., 2005; Liu et al., 2005; Patel et al., 2008). However, these dyes should be used cautiously because of their potential to cross identify other acidic bodies in the tissue (Yoshimoto, 2012).

1.4 Objectives of the work

In our laboratory, a conditional photorespiratory mutant cat2 has been used as a stress mimic tool to assess the importance of components that could be important in oxidative stress signaling. Oxidative stress occurring in cat2 is derived from enhanced H2O2 availability through photorespiration and can be prevented by growth at high CO2 or low light (Queval et al., 2007). During growth of cat2 in air in long days (but not short days), intracellular oxidative stress triggers SA-dependent responses including SA accumulation, PR gene expression, HR-like cell death and increased resistance to pathogens, effects that are all accompanied by the oxidation and accumulation of glutathione (Chaouch et al., 2010). However, it remains unclear whether glutathione oxidation and accumulation is required for intracellular oxidative stress-mediated SA-dependent pathogenesis responses and, more generally, there is little direct in vivo evidence that glutathione plays a signaling role downstream of H2O2. To analyze these questions, novel and double triple mutants were constructed, and the results obtained are presented in Chapter 2.

A second aim of this work arose from the observation that JA-associated gene expression was repressed in gr1, an effect that was reinforced in cat2 gr1, which has a strongly perturbed leaf glutathione pool. These effects point to possible links between the JA pathway and glutathione status (Mhamdi et al., 2010a; annex 1). However, the repression of JA signaling gene expression is complicated by the extreme phenotype of cat2 gr1, which has severe oxidative stress. Hence, it is unknown whether the previously described repression of JA signaling is linked to glutathione status rather than GR activity, especially in an oxidative stress context. In Chapter 3, several of the mutants reported in Chapter 2 are exploited to analyze this specific question and the role of glutathione in interplay between SA and JA pathways.

Leaf senescence has been considered to be linked to enhanced oxidative stress. Indeed, senescence can be induced by a range of stresses such as salt, drought and pathogens (Dwidedi et al., 1979; Dhindsa et al., 1981; Bohnert et al., 1995; Balazadeh et al., 2010). One signaling molecule that could be important in the regulation of senescence is H2O2. The roles of antioxidant enzymes such as CAT and APX, which metabolize H2O2, are therefore of particular interest, and it has been reported that the onset of leaf senescence is associated with lower expression and activity of CAT2 (Smykowski et al., 2010). Furthermore, it has been proposed that autophagy may function in degrading oxidized proteins to confer tolerance to oxidative stress (Xiong et al., 2007). Indeed, several autophagy mutants such as atg2 and

31 CHAPTER 1 GENERAL INTRODUCTION

atg5 exhibit an early senescence phenotype which is associated with enhanced oxidative stress as well as the activation of SA pathway (Yoshimoto et al., 2009). Chapter 4 reports an analysis of possible interplay between photorespiration, oxidative stress and autophagy in the regulation of senescence and SA-dependent lesion formation by phenotypic and other analyses of double mutants including cat2 atg2, cat2 atg5 and cat2 atg18a.

32 CHAPTER 2

Functional analysis of Arabidopsis mutants points to novel roles for glutathione in coupling H2O2 to activation of salicylic acid accumulation and signaling

A modified version of this chapter has been provisionally accepted for publication as the following original paper:

Han Y., Chaouch S., Mhamdi A., Queval G., Zechmann B. and Noctor G. (2012). Functional analysis of Arabidopsis mutants points to novel roles for glutathione in coupling H2O2 to activation of salicylic acid accumulation and signaling. Antioxidants & Redox Signaling

CHAPTER 2

Summary

Through its interaction with H2O2, glutathione is a candidate for transmission of signals in plant responses to pathogens but identification of signaling roles is complicated by its antioxidant function. Using a genetic approach based on a conditional catalase-deficient Arabidopsis mutant, cat2, this study aimed to establish if glutathione plays an important functional role in the transmission of signals downstream of

H2O2. For this, glutathione content or antioxidative capacity was independently modified in an H2O2 signaling background. Introducing the cad2 or allelic mutations in the glutathione synthesis pathway into cat2 blocked H2O2-triggered glutathione oxidation and accumulation. While no effects on NADP(H) or ascorbate were observed, and H2O2-induced decreases in growth were maintained, blocking glutathione modulation antagonized salicylic acid (SA) accumulation and SA-dependent responses. Other novel double and triple mutants were produced and compared with cat2 cad2 at the levels of phenotype, expression of marker genes, non-targeted metabolite profiling, accumulation of SA, and bacterial resistance. The effects of the cad2 mutation on H2O2-triggered responses were distinct from those produced by mutations for GLUTATHIONE REDUCTASE1 (GR1) or NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), and were linked to compromised induction of ISOCHORISMATE SYNTHASE1 (ICS1) and ICS1-dependent SA accumulation. This study of new double and triple mutants allowed us to infer previously undescribed regulatory roles for glutathione, in which this compound acts in parallel to its antioxidant role to allow increased intracellular H2O2 to activate SA signaling, a key defence response in plants.

Résumé

Grâce à ses interactions avec le H2O2, le glutathion se présente comme un candidat pour jouer des rôles dans la transmission de signaux lors des interactions plantes-pathogènes. Cependant, l’identification de tels rôles est compliquée par la fonction antioxydante de glutathion. En utilisant une approche génétique basée sur cat2, un mutant d’Arabidopsis conditionnel déficient en catalase, cette étude visait à établir si le glutathion joue des rôles de signalisation en aval du H2O2. Pour ce faire, la teneur en glutathion ou sa capacité antioxydante ont été modifiées, d’une manière indépendante, dans le fond génétique cat2. L’introduction de la mutation cad2 ou d’autres mutations alléliques pour la voie de synthèse de glutathion produit un blocage sur l’oxydation et l’accumulation de glutathion H2O2-dépendante qui sont autrement observées chez le mutant cat2. Alors que les pools de NADP(H) et d’ascorbate restent inaffectés, et la diminution de la croissance causée par le H2O2 sont maintenues au niveau cat2 chez cat2 cad2, le blocage de la réponse du glutathion produit un effet antagoniste sur l’accumulation de l’acide salicylique (SA) et des effets SA-dépendants. D’autres nouveau doubles et triples mutants ont été générés et comparés avec cat2 cad2 au niveau de leurs phénotypes, de l’expression de gènes marqueurs, de profils métaboliques non-ciblés, de l’accumulation du SA, et de la résistance aux bactéries. Ces analyses ont révélé que les effets de la mutation cad2 chez cat2 sont distincts de ceux produits par des mutations pour la GLUTATHION REDUCTASE1 (GR1) ou la NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), et sont liés à une induction attenuée de l’ISOCHORISMATE SYNTHASE 1 (ICS1) et l’accumulation de SA ICS1-dépendante. L’étude de ces doubles et triples mutants nous permet de conclure à des rôles nouveaux pour le glutathion, qui semble agir en parallèle à son rôle antioxydant pour coupler le H2O2 intracellulaire à la signalisation par le SA, une réponse de defénse clé chez les plantes.

33 CHAPTER 2

2.1 Introduction

As potent signal molecules, reactive oxygen species (ROS) play important roles in transmitting information during responses of both plant and animal cells to the environment. Since the seminal studies that established key roles for ROS in plant-pathogen interactions (Doke, 1983; Apostol et al., 1989; Levine et al., 1994), primary redox-linked events have been considered to be extracellular or plasmalemma-located, mediated primarily by NADPH oxidases, peroxidases, or other enzymes (Torres et al., 2002, 2005; Bindschedler et al., 2006; Daudi et al., 2012; O’Brien et al., 2012). However, these initial signals at the cell surface/apoplast lead to later downstream adjustments in intracellular redox state (Edwards et al., 1991; Vanacker et al., 2000; Volt et al., 2009) that are notably associated with thiol-dependent activation of the cytosolic protein, NON EXPRESSOR OF PATHOGENESIS RELATED GENES1 (NPR1). Reduction of NPR1 is required to link part of the salicylic acid (SA) signaling pathway to induction of genes such as PATHOGENESIS-RELATED1 (PR1; Mou et al., 2003).

One of the key players governing intracellular redox state is the thiol/disulfide compound, glutathione. Several reports have used single mutants or transformants deficient in glutathione in studies of pathogenesis responses (May et al., 1996; Ball et al., 2004; Parisy et al., 2007; Höller et al., 2010; Dubreuil-Maurizi et al., 2011; Baldacci-Cresp et al., 2012). While some of these have described effects of altered glutathione status, it remains unknown where glutathione acts in the signaling chain and how it interacts with ROS-dependent events. Interpretation of the role of glutathione is complicated by its antioxidant function which depends on regeneration of the thiol form, GSH, from the disulfide form, GSSG, by glutathione reductase (GR). The predominant concept of glutathione function is as a negative regulator that acts to oppose or limit H2O2 signals. In terms of thiol-dependent signaling functions in pathogenesis responses, the NPR1 pathway remains by far the best studied (Koornneef et al., 2008; Ghanta et al., 2011; Maughan et al., 2010). However, although exogenous glutathione can induce PR1 expression, NPR1 reduction has been reported to be mediated through thioredoxin-dependent systems (Tada et al., 2008). Adding to these uncertainties are recent studies that have revealed the complexity both of NPR1 redox regulation (Lindermayr et al., 2010) and of potential functional overlap between cytosolic thiol/-disulfide systems (Reichheld et al., 2007; Marty et al., 2009; Bashandy et al., 2010).

Enhanced oxidation of glutathione, accompanied by increases in the total pool, is a well-documented response in plants subjected to treatments such as ozone, cold, pathogens, or SA (Edwards et al., 1991; Sen Gupta et al., 1991; May et al., 1996; Vanacker et al., 2000; Bick et al., 2001; Gomez et al., 2004b; Koornneef et al., 2008). Photorespiratory catalase-deficient plants are model systems in which such changes are particularly evident (Smith et al., 1984; Willekens et al., 1997; Rizhsky et al., 2002; Queval et al., 2007). The interest of these systems is that they allow the role of H2O2 signaling to be studied specifically and controllably, because the endogenous signal can be manipulated easily by external conditions (Dat et al., 2001; Mhamdi et al., 2010b). When conditions allow active photorespiration, increased H2O2 availability in the Arabidopsis knockout cat2 mutant triggers accumulation of glutathione followed by induction of pathogenesis responses in the absence of pathogen challenge (Queval et al., 2007; Chaouch et al., 2010). The well-defined changes in glutathione in catalase-deficient plants are useful as a readout of altered redox state, but their functional impact is unclear. It remains to be established whether they play any role in coupling H2O2 to downstream defence responses or are rather an

34 CHAPTER 2

accompanying, passive response of the cell to enhanced H2O2.

Increases in H2O2 in catalase-deficient plants are much less apparent and reproducible than those observed in glutathione (Mhamdi et al., 2010b), raising the possibility that some of the signals downstream of intracellular H2O2 may be mediated by changes in glutathione status. Altered glutathione status in catalase-deficient plants or in response to stress presumably reflects an increased load on reductant-requiring antioxidant pathways. This is underscored by analysis of Arabidopsis gr1 knockout mutants for one of the two GR-encoding genes. While GR1 deficiency does not in itself lead to an oxidative stress phenotype, the gr1 mutation greatly enhances stress in the cat2 background, showing that glutathione-dependent antioxidative pathways are increasingly solicited when catalase activity is compromised (Mhamdi et al., 2010a).

By exploiting cat2 as a model H2O2 signaling background, evidence was recently reported that intracellular oxidative stress interacts with specific NADPH oxidases to determine activation of salicylic acid (SA) accumulation and SA-dependent pathways. The atrbohF mutant lacking expression of a specific NADPH oxidase shows compromised SA accumulation and resistance to virulent bacteria, and the atrbohF mutation also attenuates cat2-triggered SA accumulation and induced resistance (Chaouch et al., 2012). Intriguingly, the clearest indicator of redox interactions between the cat2 and atrbohF mutations was not H2O2 itself but the status of glutathione: attenuation of SA contents and SA-dependent responses was correlated with decreased accumulation of glutathione in cat2 atrbohF compared to cat2 (Chaouch et al., 2012).

The study presented in this chapter uses the cat2 mutant as the basis for a targeted analysis of the role of glutathione status in transmitting H2O2 responses. This mutant was chosen because it is a well-defined conditional redox signaling system in which H2O2 provokes oxidative modulation of the glutathione pool accompanied by activation of SA signaling and associated pathogenesis-related responses. Relationships between enhanced H2O2 production, the response of glutathione, and the activation of the SA pathway were explored to answer the following specific questions. (1) Does glutathione play a specific role in

H2O2 signaling, independent of other potentially redundant thiol systems? (2) To what extent is any such role dependent on glutathione status, rather than glutathione redox turnover in an antioxidant function? (3) What is the relationship between glutathione status and NPR1 function in transmitting signals downstream of H2O2? To examine these questions, mutant lines available in Arabidopsis that have decreased glutathione or salicylic acid, or loss of GR1 or NPR1 function, were used (Table 2.1). By functional analysis of the effects of these specific mutations in the cat2 H2O2 signaling background, previously unidentified roles for glutathione in controlling oxidative stress-triggered SA signaling are inferred.

35 CHAPTER 2

Table 2.1. Details of mutant lines used in this study. The principal characteristics of the lines are given, as well as the reference(s) in which the mutant and/or mutation was first reported or characterized. ______cat2 T-DNA knockout for CATALASE2. Conditional photorespiratory oxidative stress mutant (Queval et al., 2007). Constitutive activation of salicylic acid

pathway under oxidative stress conditions (Chaouch et al., 2010). Wild-type phenotype when grown under high CO2. gr1 T-DNA knockout for GLUTATHIONE REDUCTASE1. Wild-type phenotype in optimal conditions due to back-up NADPH-thioredoxin system for GSSG reduction (Marty et al. 2009), but gr1 shows altered responses to oxidative stress (Mhamdi et al., 2010a). cad2 Deletion mutation in GSH1 gene leading to loss of two amino acids in -GLUTAMYLCYSTEINE SYNTHETASE. Identified in a screen for enhanced cadmium sensitivity, cad2 has about 30% wild-type leaf glutathione contents (Howden et al., 1995; Cobbett et al., 1998). Wild-type phenotype in optimal growth conditions. rax1 Point mutation in GSH1 gene leading to an amino acid substitution in -GLUTAMYLCYSTEINE SYNTHETASE. Identified in a screen for altered high light responses, rax1 has about 30-40% wild-type leaf glutathione contents (Ball et al., 2004). Wild-type phenotype in optimal growth conditions. pad2 Point mutation in GSH1 gene leading to an amino acid substitution in -GLUTAMYLCYSTEINE SYNTHETASE. Identified in a screen for decreased phytoalexin (camalexin) contents, pad2 has about 20-25% wild-type leaf glutathione contents (Parisy et al. ,2007). Wild-type phenotype in optimal growth conditions. sid2 Point mutation in ISOCHORISMATE SYNTHASE 1. Identified in a screen for decreased induction of salicylic acid (Nawrath and Métraux 1999). Allowed ICS1 to be identified as major source of pathogen-induced salicylic acid production in Arabidopsis (Wildermuth et al., 2001). In response to bacterial challenge, salicylic acid stays close to basal uninduced Col-0 levels or below. Wild-type phenotype in optimal growth conditions. npr1 Point mutation in NONEXPRESSOR OF PATHOGENESIS RELATED GENES 1. Identified in a screen for mutants that fail to induce pathogenesis-related PR genes (Cao et al., 1994). The NPR1 protein was subsequently shown to be thiol-regulated, with reduction allowing the protein to move into the nucleus from the cytosol to induce PR genes (Mou et al., 2003). Wild-type phenotype in optimal growth conditions.

______

36 CHAPTER 2

2.2 Results

Plants deficient in the major leaf catalase show accumulation of glutathione that is conditional on H2O2 production through photorespiration (Queval et al., 2007). When cat2 is grown in air, glutathione accumulation accompanies the appearance of lesions which initially develop after about two weeks growth then spread to cover about 15% total rosette area within the following week. This is associated with induction of SA-dependent PR genes and enhanced resistance to bacteria. All these H2O2-triggered effects in cat2, except glutathione accumulation, can be reverted by the sid2 mutation (Chaouch et al., 2010), which blocks SA synthesis through the ICS1-dependent pathway (Wildermuth et al., 2001). During growth at high CO2, where photorespiratory glycolate oxidase activity is negligible, cat2 shows no signs of oxidative stress and is phenotypically indistinguishable from the wild-type, Col-0. However, when plants grown first at high CO2 are transferred to air, glutathione in cat2 accumulates to similar levels to those observed in cat2 grown in air from seed. This effect is followed by initiation of pathogenesis-related responses about five days after transfer to air and their continued development during subsequent days (Chaouch et al., 2012). These features allow cat2 to be used as a conditional model in which the roles of glutathione in defence signaling triggered by intracellular oxidative stress can be examined either during growth in air from seed or by the induced stress that follows the transfer of plants from high CO2 to air. As reported in this study, both conditions allow activation of the SA pathway in the cat2 mutant. However, studying responses after transfer from high CO2 to air is particularly useful for double mutants in which the interpretation is complicated by their extreme phenotypes when they are grown from seed in air. Thus, for some experiments, transfer from high CO2 condition was preferred as a protocol for inducing oxidative stress. However, the principal effects of glutathione on the SA pathway here were observed using both experimental protocols.

2.2.1 Genetic blocks over H2O2-triggered glutathione accumulation inhibit cell death and associated pathogenesis responses induced by intracellular oxidative stress

When cat2 plants were grown in non-stress conditions (at high CO2), leaf glutathione remained at wild-type levels and reduction state (about 95% reduced). Within 4h after transfer to air from high CO2, the reduction state in cat2 fell from 95% to 61% reduced (Figure 2.1A). Glutathione oxidation continued in cat2 on subsequent days, and the pool was more than 50% oxidized 4d after transfer. This oxidation was accompanied by a more than 2-fold increase in the total pool. The cat2-triggered oxidation and accumulation of glutathione were blocked by the cad2 mutation: in cat2 cad2, glutathione remained about 90% reduced, and was only slightly increased above cad2 levels after 4d exposure to air (Figure 2.1A). In contrast to the effects of the cat2 and cad2 mutations on glutathione, two major intracellular redox pools that glutathione donates electrons to (ascorbate) or receives electrons from (NADPH) were much less affected. Ascorbate reduction status was above 80% in all lines while NADP(H) pools were constant at between 50 and 60% reduced. Thus, when plants are grown in conditions permissive for photorespiratory

H2O2 production, the cat2 mutation triggers a response of the glutathione pool that is quite specific and introduction of the cad2 mutation produces a specific block on this response.

Studies of plants deficient in catalase grown in different conditions suggest that the glutathione pool is in close correspondence to the predicted H2O2 availability (Queval et al., 2007; Mhamdi et al., 2010b). It has

37 CHAPTER 2

4h 4d

A 1200 1200 bd a c bd a c 800 800 FW ) -1 400 400

(nmol.g Leaf glutathione glutathione Leaf 0 0

8 8 aa a a aa a a 6 6 FW) -1 4 4

2 2 (µmol.g

ascorbate Leaf 0 0

40 40 aaaa aaaa 30 30 FW) -1 20 20

(nmol.g 10 10 NADP(H) Leaf 0 0 cat2 cat2 cat2 cat2 cad2 cad2 cad2 cad2 Col-0 Col-0 Col-0 Col-0

cat2 cad2 cat2 cat2 cad2 cat2 cat2 cad2 cat2 cad2 cat2 4h 4d 1000 1000 B 750 750

FW)

-1 b 500 c 500 b a aaa 250 a 250

(nmol.g a b

Peroxide content 0 0

cat2 cat2 cat2 cat2 Col-0 Col-0 Col-0 Col-0 cat2 gr1 cat2 gr1 cat2 gr1 cat2 gr1 cat2 cat2 cad2 cat2 cad2 cat2 cat2 cad2 cat2 cad2 cat2

Figure 2.1. Oxidant and antioxidant capacity in plant lines with modulated glutathione contents. (A) Leaf contents of NADP(H), ascorbate, and glutathione in Col-0, cad2, cat2 and cat2 cad2. White bars, reduced forms. Black bars, oxidized forms. (B) Leaf peroxide contents in Col-0, cat2, cat2 cad2 and cat2 gr1.

Plants were grown at high CO2, where the cat2 mutation is silent, then transferred to air to initiate oxidative stress in the cat2 backgrounds. Samples were taken 4h and 4d after transfer. Data are means ± SE of at least three biological repeats. Different letters indicate significant difference at P<0.05 for leaf contents. Note that x-axis legends to parts A and B are different.

38 CHAPTER 2

been previously reported that H2O2 itself (measured as in situ staining or extractable H2O2) is minor or undetectable in cat2, even when plants are clearly undergoing oxidative stress (Chaouch et al., 2010, 2012; Mhamdi et al., 2010a). In the present study, extractable peroxides in cat2 were re-examined and compared with levels found in cat2 cad2 at two time-points after transfer to oxidative stress conditions. For comparison, the cat2 gr1 double mutant was included, in which loss of GR1 function exacerbates oxidative stress compared to cat2 (Mhamdi et al., 2010a). At 4d after transfer from high CO2 to air, no difference was apparent between any of the lines, suggesting that any excess H2O2 had been efficiently metabolized at this point (Figure 2.1B). At 4h after transfer, however, a 60% increase in peroxides relative to Col-0 was detected in cat2 and this increase was slightly more pronounced in the cat2 gr1 double mutant (Figure 1B). In cat2 cad2 at this early time-point, peroxides were intermediate between Col-0 and cat2. Together, these findings show that when placed in air, the cat2 background causes an accumulation of H2O2 that is transient and relatively minor, suggesting that other, reductive systems are able to replace the major leaf catalase in efficiently metabolizing photorespiratory H2O2 when CAT2 function is lost. Its marked, progressive in cat2 implicates glutathione as a significant component in these reductive systems, and the cat2 cad2 mutant offers an interesting system to establish the functional significance of

H2O2-triggered adjustments in glutathione.

To examine how the cad2 mutation affects the subcellular distribution of glutathione in cat2 in oxidative stress conditions, a specific antibody was exploited to quantify glutathione pools in different compartments (Zechmann et al., 2007; Zechmann and Müller, 2010). In agreement with the increase in total tissue glutathione (Figure 2.1A), immunogold labelling was significantly increased in cat2 compared to Col-0 (Figure 2.2A). This increase was mainly due to enhanced signal in chloroplasts, vacuoles, and the cytosol (Figure 2.2B). All these effects were annulled by the cad2 mutation. In cat2 cad2, glutathione was decreased in all measured compartments especially the chloroplast, cytosol, nucleus, and peroxisomes (Figure 2.2). From multiple images of photosynthetic mesophyll cells, subcellular glutathione concentrations were estimated in the three lines (see Materials and methods for details). They ranged from 1-10 mM in Col-0 and cat2, whereas concentrations in all compartments except the mitochondria did not greatly exceed 1 mM in cat2 cad2 (Table 2.2). ______

Table 2.2. Subcellular glutathione concentrations in Col-0, cat2, and cat2 cad2. Plants were grown as described for Figure 2.1. Values are in mM and were calculated from relative gold labelling densities as described in Materials and methods based on mean values of multiple counts of different cells (n>20 for peroxisomes and vacuoles, and n>60 for all other cell compartments). ______

Col-0 cat2 cat2 cad2

Cytosol 4.8 7.4 0.8 Nuclei 7.5 9.0 1.2 Chloroplasts 1.1 2.7 0.1 Mitochondria 10.3 10.1 7.5 Peroxisomes 5.4 6.0 0.8 Vacuole 0.04 0.5 0.03 ______

39 CHAPTER 2

Col-0 4 cat2 A B * 3

2 * 1 * * 0 mutant/Col-0)

2 -1 MCNPx CyV 1

0 -1 * * cat2 -2 -3 Fold-change in glutathione labeling labeling glutathione in Fold-change relative to Col-0 (log Col-0 to relative * * * * -4 cat2 cad2

Figure 2.2. Effects of the cad2 mutation on subcellular distribution of glutathione in response to oxidative stress. (A) Transmission electron micrographs showing subcellular distribution of gold-labelled glutathione in cat2 cad2 photosynthetic mesophyll cells of Col-0, cat2 and cat2 cad2. The bars indicate 1 µm. (B) Relative quantitation of glutathione labelling in different subcellular compartments. Plants were grown as described for Figure 1. Values show log fold change compared to Col-0. C, chloroplast. CW, cell wall. Cy, cytosol. M, mitochondria. N, nucleus. Px, peroxisome. St, starch. V, vacuole. *Significant difference from Col-0 at P<0.05.

The effect of genetically blocking glutathione accumulation on H2O2-triggered pathogenesis responses was first examined by growing plants in air from seed. This analysis revealed that the cad2 mutation markedly affected the lesion formation that is spontaneously triggered in cat2 in the absence of pathogen challenge and the associated induced resistance to subsequent bacterial challenge (Figure 2.3). PR gene expression, which is spontaneously activated alongside lesion formation in cat2, was also decreased in cat2 cad2, and the cad2 mutation decreased cat2-triggered resistance to bacteria to wild-type levels

(Figure 2.3). An effective block over H2O2-induced glutathione accumulation and decreased lesion extent were also observed in double mutants carrying allelic mutations in the same glutathione synthesis gene as cad2 (Figure 2.4). The decrease in lesion extent was in good agreement with the severity in glutathione deficiency, with rax1, the weakest allele, producing a slightly weaker effect than cad2 or pad2 (Figure

2.4). In contrast to their effects on H2O2-triggered lesion extent, none of the secondary mutations reverted

40 CHAPTER 2

the decreased rosette size in cat2 (Figure 2.4). Thus, blocking H2O2-triggered up-regulation of glutathione deficiency led to down-regulation of lesion spread without producing general effects on the cat2 oxidative

6 A Col-0 cad2 C s ) a 4

ACTIN2 transcript 2 b (rel.

PR1 cc 0 cat2 cat2 cad2 D 6

) 4 a

ACTIN2 transcripts 2 b (rel. cc PR2 0

26 B a E 9 ) h

-2 a aa b 13 6 b cfu cm

cc 10 Lesions og (l

ND ND Bacterial growt area) rosette (% 0 3

l-0 cat2 cat2 cad2 cad2 Col-0 Co cat2 cad2 cat2

cat2 cad2

Figure 2.3. Effect of the secondary cad2 mutation on cat2-induced phenotype, PR gene expression, and bacterial resistance. (A) Phenotype of plants grown in air from seed. Photographs and samples were taken 21 days after sowing. Bars indicate 1 cm. (B) Lesion quantification in the different genotypes as a percentage of the total rosette area. ND, not detected. Values are means ± SE of at least 12 plants as in (A). (C) and (D) PR transcripts quantified by qPCR in the four genotypes, plants as in (A). (E) Growth of virulent P.syringae DC3000 in the four genotypes. stress phenotype. Of the three alleles, cad2 is the best characterized and is intermediate in its effects on glutathione contents (Parisy et al., 2007; Figure 2.4). Because of this, the cat2 cad2 line was chosen for a detailed examination of the processes underlying the role of glutathione in linking intracellular H2O2 to induction of pathogenesis responses, with the aim of answering the following specific questions: How is the effect of blocking glutathione accumulation related to an antioxidant function? And how is it related to the SA pathway and/or NPR1 function?

41 CHAPTER 2

600 Col-0 pad2 a 400 a a a

(mg) 200 Fresh weight Fresh b b b b cad2 rax1 0

24 a

18

b 12 cat2 cat2 pad2 c c Lesions 6

(% rosette area) rosette (% dddd d ND ND ND ND 0 1200 a cat2 cad2 cat2 rax1 900

FW) -1 600 ` b c

(nmol.g 300 e d Leaf gl utathione h g f 0

cat2 rax1 pad2 cad2 Col-0 cat2 rax1

pad2 cat2 cat2 cad2

Figure 2.4. Confirmation of effects of genetic deficiency in glutathione on H2O2-triggered lesion formation using allelic mutants. The rax1 and pad2 mutations (Ball et al., 2004; Parisy et al., 2007) were introduced into the cat2 background by crossing and compared with cat2 cad2. Bars in photographs indicate 1 cm. For fresh weight, values are means ±SE of ten to twenty-eight plants. Values are means ± SE of three biological repeats. For lesion formation, values are means ± SE of fifteen plants. For glutathione, white bars indicate GSH and black bars, GSSG. Letters indicate significant difference at P<0.05.

2.2.2 Contrasting responses in cat2 cad2 and cat2 gr1 reveal a non-antioxidant role for glutathione in coupling H2O2 to SA-dependent pathogenesis responses

Spreading lesions and associated responses in cat2 can be abolished by introducing the sid2 mutation (Chaouch et al., 2010). To check whether the decreased lesion extent in cat2 cad2 occurs through the

42 CHAPTER 2

same ICS1-dependent pathway, a cat2 cad2 sid2 triple mutant was produced. Growth of cat2 backgrounds under conditions promoting lesion formation in cat2 (continuous oxidative stress in air) revealed that, like cat2 sid2, cat2 cad2 sid2 showed no detectable lesion formation (Figure 2.5).

0021 Lesion formation (%)

Col-0 cad2 cat2

900Lesion formation (%)

cat2 cad2 cat2 sid2 cat2 cad2 sid2

Figure 2.5. The cat2 cad2 sid2 triple mutant shows that residual H2O2-induced lesions in cat2 cad2 are dependent on the ICS1 pathway. Photographs show plants after 21 d growth from seed in air. The bar indicates 1 cm. Lesion quantification was performed on 12 plants for each genotype.

Because partial glutathione deficiency does not affect growth or biomass production in the cat2 mutant, effects of the cad2 mutation on cat2 responses may not be related to changes in oxidative stress intensity. This hypothesis was tested by comparing cat2 cad2 phenotypes and SA dependency with those observed in cat2 gr1, in which loss of GR1 function exacerbates oxidative stress in cat2, as reflected by an extreme dwarf phenotype and dramatic accumulation of oxidized glutathione (Mhamdi et al., 2010a). To analyze the dependence of responses on SA and glutathione accumulation, the sid2 and cad2 mutations were introduced into cat2 gr1.

To enable a meaningful comparison of the interactions between the different mutations, plants were initially grown at high CO2, where the cat2 mutation is silent. In this condition, cat2-dependent phenotypes and, in particular, the extreme phenotype of cat2 gr1, are annulled. Accordingly, in high CO2 growth conditions, no phenotypic difference was observed between any of the lines (data not shown). After transferring plants to air, the phenotypes of sid2, gr1 and cad2 were indistinguishable from Col-0 and none of these four genotypes presented lesions on the leaves (Figure 2.6). In contrast, the cat2 mutation induced lesions that first became visible after 5 to 6d, spreading to become very apparent after 10d in air (Figure 2.7A). Similar to its effects on cat2 grown in air from seed, the cad2 mutation restricted lesion spread while the sid2 mutation abolished it completely so that no lesions were visible in either cat2 sid2 or cad2 cad2 sid2 (Figure 2.7A). All lines in which both cat2 and gr1 were present showed rapid

43 CHAPTER 2

onset of leaf bleaching, which was severe after 4d exposure to air, irrespective of whether or not the sid2 or cad2 mutations were additionally present (Figure 2.7A). Unlike the more slowly forming, persistent lesions observed in cat2 and, residually, in cat2 cad2, the phenotypes of cat2 gr1 backgrounds were transient and the bleaching had largely disappeared within 10 days after transfer to air (Figure 2.7A). Although cat2 gr1 and cat2 gr1 sid2 dramatically accumulated oxidized glutathione, the phenotype induced by the combination of cat2 and gr1 mutations did not require this accumulation: introduction of the cad2 mutation into cat2 gr1 restricted glutathione contents well below cat2 levels yet reversible leaf bleaching was still observed (Figure 2.7A and 2.7B). Thus, the rapid-onset bleaching associated with loss of glutathione recycling capacity appears to be associated with an antioxidant function rather than glutathione status per se and does not require ICS1-dependent SA accumulation. This phenotypic response is therefore quite distinct from that produced by modulating glutathione status through the cad2 mutation.

Transfer to air from high CO2

4 days 10 days

1.2 Glutathione

0.8 FW

Col-0 Col-0 -1

0.4

g µmol

0.0

gr1 sid2 cad2

sid2 sid2 Col-0

Figure 2.6. Phenotypes and glutathione contents in Col-0, sid2, gr1 and cad2 mutants following transfer gr1 gr1 to air after three weeks growth from seed at high

CO2. Bars in the photographs indicate 1 cm. For glutathione, white bars show GSH, black bars GSSG. Leaf glutathione contents in the different lines. White bars, GSH. Black bars, GSSG. Values are means ± SE of 3 cad2 cad2 plants, sampled 4d after transfer to air.

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A B

Time after transfer to air from high CO2 Glutathione 4 days 10 days GSH GSSG 5 cat2 4

1

cat2 cat2 0 5 cat2 cad2 4

1

cat2 cad2 cat2 cad2 0

5 glutathione contentLeaf (µmol g cat2 sid2

4

1

cat2 sid2 cat2 sid2 0 5 cat2 cad2 sid2

4

1 cat2 cad2 sid2 cat2 cad2 sid2 0 5 -1 cat2 gr1 FW) 4

1

cat2 gr1 cat2 gr1 0 5 cat2 cad2 gr1 4

1

cat2 cad2 gr1 cat2 cad2 gr1 0 5 cat2 gr1 sid2 4

1

cat2 gr1 sid2 cat2 g r1 sid2 0

Figure 2.7. Blocking glutathione accumulation produces distinct effects on H2O2-triggered phenotypes to those produced by decreasing glutathione-dependent antioxidative capacity. (A) Phenotypes in cat2 cad2 and cat2 gr1 induced by onset of oxidative stress in cat2 and comparison with lines additionally carrying the sid2 mutation in the SA synthesis pathway. Plants were grown for 3 weeks at high CO2 then transferred to air to induce oxidative stress in the cat2 backgrounds. Photographs were taken 4d and 10d after transfer of plants to air. Scale bars indicate 1 cm. Bleaching in gr1 genotypes is indicated by arrowheads. (B) Leaf glutathione contents in the different lines. White bars, GSH. Black bars, GSSG. Values are means ± SE of 3 plants, sampled 4d after transfer to air.

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Several recent publications have implicated myo-inositol in SA-dependent responses (Meng et al., 2009; Chaouch and Noctor, 2010; Donahue et al., 2010). In cat2, decreases in myo-inositol occur prior to SA accumulation and are necessary for induction of SA-dependent responses as these can be prevented simply by treatment with exogenous myo-inositol (Chaouch and Noctor, 2010). In both cat2 and cat2 cad2, 4d after transfer from high CO2 to air, myo-inositol was decreased and this was associated with decreases in galactinol, which is a product of myo-inositol (Figure 2.8). In contrast, the bleaching phenotype of cat2 gr1 and cat2 gr1 cad2 was associated with much less marked effects on these compounds. These observations provide further evidence (1) that myo-inositol metabolism is important in linking intracellular H2O2 to downstream SA responses, and (2) that loss of glutathione antioxidant recycling function entrains an SA-independent stress response that is distinct from the SA-dependent pathway operating in cat2 and cat2 cad2.

0.3 0.8 Galactinol myo-Inositol a 0.2 a ab abc ab bc b b c 0.4 b 0.1 d d d c Relative units Relative 0.0 0.0 gr1 gr1 cat2 cat2 cad2 cad2 Col-0 Col-0 cat2 gr1 cat2 gr1 cat2 cad2 cat2 cad2 cat2 cad2 gr1 gr1 cat2 cad2 Figure 2.8. Effects of cat2 and secondary cad2 and gr1 mutations on leaf myo-inositol and galactinol contents.

Samples were taken 4d after transfer of plants grown at high CO2 to air, and metabolites were measured by GC-MS (means ± SE of three biological repeats). Different letters show significant difference at P < 0.05.

2.2.3 Side-by-side comparison of cat2 cad2 with cat2 npr1

To establish whether the effect of glutathione deficiency on pathogenesis responses in cat2 is related to impaired NPR1 function, double cat2 npr1 mutants were produced and analyzed in parallel with cat2 cad2. The npr1 mutation did not affect glutathione status in either Col-0 or cat2 backgrounds (Figure. 2.9A). It did, however, decrease lesion formation in cat2, though less markedly than did cad2 (Figure 2.9B). The triple cat2 cad2 npr1 line showed similar glutathione contents and lesion formation to those observed in cat2 cad2 (Figure 2.9). Thus, H2O2-induced alterations in glutathione status were independent of the presence or absence of functional NPR1 in both cat2 and cat2 cad2 backgrounds. None of the mutations affected bacterial growth, when samples were taken immediately after inoculation (data not shown) but clear differences were observed in growth in samples taken at two days post inoculation (Fig. 2.9C). Like cad2, npr1 annulled cat2-induced resistance: both cat2 cad2 and cat2 npr1 showed Col-0 resistance levels (Fig. 2.9C). However, cat2 npr1 still showed higher resistance than npr1.

46 CHAPTER 2

1200 a A a 900

FW) -1 600

b b

(nmol.g 300 glutathione Leaf e c d 0

B 20 a 15 b 10

Lesions c c d dd (% rosette area) rosette (% 5

NDND ND 0

9 C a a ) b -2 b b b c

cfu cm 6 10 (log growth Bacterial

3

cat2 cat2 npr1 npr1 cad2 cad2 Col-0 Col-0

cat2 npr1 cat2 cat2 npr1 cat2 cat2 cad2 cat2 cad2

cat2 cad2 npr1 cad2 cat2 npr1 cad2 cat2

Figure 2.9. Comparison of lesions, glutathione contents, and bacterial resistance in cat2 cad2 and cat2 npr1. (A) Leaf glutathione contents. White bars, GSH. Black bars, GSSG. Values are means ± SE of 3 plants. (B) Lesion quantification as a percentage of the total rosette area. ND, not detected. Values are means ± SE of at least 12 plants.

(C) Growth of virulent P.syringae DC3000 in all seven genotypes. Plants were grown at high CO2 for 3 weeks to prevent any cat2 phenotype, and inoculations were performed on plants 7d after transfer to air to induce oxidative stress in cat2 backgrounds. No difference in bacterial growth was observed between the genotypes at 0 hpi.

47 CHAPTER 2

A B

cat2 sid2 36 9 7 cat2 cad2 Col-0 cad2 npr1 cat2 cad2 cat2 npr1 cat2

2 9 common metabolites cat2

1 Signal in cat2

Ratio relative to 0 n.d. n.d. l npr1 d n e e e e e e i a n n s s d to n c h i i o o i i i a c c s e n p u s n ib o r o effect ic o h t le e m R n e n p tr a a T r o o r h y s u h y c r I P R T u T p l o G c lu -g SA

cat2 sid2 37 8 3 cat2 npr1

3 8 common metabolites cat2 2

1 Signal in cat2 Ratio relative to 0 d e i e d d d e e n c n i i i n n i a i c c c i i a a a r n n c in e a i c c o cad2 l g r r i i c S e A a n r li r t A o u a h u a l c M T effect g u L o l t G e -k α

cat2 cad2 14 2 9 cat2 npr1

2 2 common metabolites

cat2 1 Signal in cat2

Ratio relative to 0

d e i n c i a n o ic e n r o h c T u l G

-1.5 +1.5

Figure 2.10. GC-TOF-MS analysis of the impact of cad2 and npr1 mutations on H2O2-dependent metabolite profiles.

(A) Heatmap showing hierarchical clustering of all detected metabolites. Values were centered-reduced prior to clustering analysis. (B) Comparison of metabolite profiles in cat2 cad2 and cat2 npr1 with those previously reported for cat2 sid2 (Chaouch et al., 2010). The values in the Venn diagrams indicate the number of metabolites that were significantly different in each double mutant relative to cat2. Overlapping sections indicate the number of metabolites that showed same-direction significant effects in cat2 sid2 and cat2 cad2 (top), cat2 sid2 and cat2 npr1 (middle), and cat2 cad2 and cat2 npr1 (bottom). For each comparison, the graphs show values (normalized to cat2) plotted for common metabolites in each comparison using the same color coding as in the Venn diagrams. Red type is used for the only two metabolites affected similarly by all three secondary mutations. Plants were grown from seed in air in long days and sampled 23 days after sowing. Three biological repeats were analyzed for each genotype.

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In a first approach to analyze whether the effects of blocking glutathione up-regulation were linked to impaired NPR1 function, comparative GC-TOF-MS analysis of the two double mutants was performed. By generating information on about 100 different compounds, this technique produces a metabolic signature for intracellular oxidative stress in cat2 which shows substantial overlap with that induced by bacterial pathogens (Chaouch et al., 2012). A key feature of the cat2 metabolic signature is accumulation of a wide range of metabolites in an SA-dependent fashion, ie, most of the signature is annulled in cat2 sid2 double mutants (Chaouch et al., 2010).

Both cad2 and npr1 mutations affected cat2-triggered metabolite profiles, but in very different ways (Figure 2.10A). Comparison of the profiles shown in Figure 2.10A with those previously described for cat2 sid2 sampled in the same conditions revealed that cat2 npr1 and cat2 cad2 recapitulated different parts of the cat2 sid2 profile (Figure. 2.10B). For this comparative analysis, only metabolites that were detected both in the earlier study of cat2 sid2 (Chaouch et al., 2010) and the present one were included. For example, 17 metabolites were significantly different from cat2 in cat2 npr1 but only 11 of these were also detected in the previous study. Of these 11, the sid2 and npr1 mutations significantly affected eight in the same-direction (Figure 2.10B, middle). Using the same approach, only two metabolites that were detected in this study and the earlier one (gluconic acid and threonine) were affected similarly by the cad2 and npr1 mutations in the cat2 background. These two compounds are among those strongly induced in cat2 and in response to bacterial infection (Chaouch et al., 2012) and the induction of both was significantly decreased by sid2, cad2 or npr1 mutations (Figure 2.10B). Six other metabolites were affected similarly in cat2 sid2 and cat2 npr1 (Figure 2.10B, middle) whereas the response of seven metabolites in cat2 was unaffected by npr1 but decreased by both cad2 and sid2 mutations (Figure 2.10B, top). These seven compounds included several metabolites associated with pathogenesis responses, including tryptophan, putrescine, and the glucosylated form of SA.

Targeted quantification of free and total SA by HPLC showed that both accumulated in cat2 and that their accumulation was markedly enhanced by the npr1 mutation (Figure 2.11A). In cat2 npr1, total SA accumulated to three times the values in cat2. This hyper-accumulation of SA is consistent with effects of the npr1 mutation in other mutants showing constitutive induction of SA-dependent pathogenesis responses (Shirano et al., 2002; Zhang et al., 2003). In marked contrast to the effect of npr1, the cad2 mutation substantially decreased SA accumulation (Figure 2.11A). SA levels in the triple cat2 cad2 npr1 mutant were similar to the cat2 single mutant (Figure 2.11A). Thus, the cad2 mutation decreased SA accumulation in both the cat2 and cat2 npr1 backgrounds, providing further evidence that the principal effect of glutathione deficiency on H2O2-triggered pathogenesis responses is mediated via a different route than impairment of NPR1 function.

49 CHAPTER 2

6 A

5 a

FW) b -1 2 c (µg g 1 eee d acid salicylic Free 0

120 a 90

FW) -1 40 bb

(µg g 20 c salicylicTotal acid ddd 0 cat2 npr1 cad2 Col-0

cat2 npr1 cat2 cat2 cad2 cat2

npr1 cat2 cad2 B 0.4 ICS1 0.3

0.2

0.1

ACTIN2 to relative T ranscript abundance 0.0 6 8 10 12 14 16 18 20 22 24 26 Time (days)

Figure 2.11. Analysis of salicylic acid (SA) and ICS1 transcripts in double and triple mutants. (A) Free and total SA in cat2, cad2, npr1 and double and triple mutants. Different letters indicate significant difference at P<0.05. Plants were grown in air and samples taken 21 days after sowing. (B) Time course of ICS1 transcript levels in Col-0 (black circles), cat2 (white circles) and cat2 cad2 (triangles). Plants were grown as in (A). Time indicates days after sowing. All values are means ± SE of three biological replicates.

Because of the decreased SA accumulation in cat2 cad2, the expression of ICS1, the key enzyme in the production of SA in response to pathogens (Wildermuth et al., 2001) was analysed, during continuous growth of plants in air from seed. ICS1 expression showed a marked transient increase in cat2 (Figure 2.11B), with the initial increase correlating with the onset of lesions, which begin to be visible after about

50 CHAPTER 2

16 days growth. The induction of ICS1 was strongly damped in cat2 cad2 (Figure 2.11B), in agreement with the decreased lesions (Figure 2.3) and SA contents (Figure 2.11A).

2.2.4 Complementation experiments reveal non-redundant roles for glutathione and NPR1 in the activation of SA-dependent responses

Experiments in which SA content and ICS1 and PR1 transcripts were quantified after transfer to high CO2 following prior growth in air confirmed that both were significantly less accumulated in cat2 cad2 compared to cat2 (Figures 2.12A and 2.12B). To examine whether SA accumulation in cat2 cad2 could be restored by glutathione, plants were treated with GSH or GSSG. Both forms induced SA above basal levels in Col-0, with GSSG being the more effective of the two (Figure 2.12A). In cat2 cad2, in which the cat2 mutation should activate H2O2 signaling, GSH was most effective, restoring total SA to about 60% of cat2 levels (Figure 2.12A). This effect was not observed in cat2 cad2 sid2, showing that it was dependent on functional ICS1. We therefore investigated the effects of glutathione supplementation on ICS1 expression. Restoration of SA accumulation in cat2 cad2 by added GSH was associated with enhanced induction of both ICS1 and PR1 genes (Figure 2.12B). Further, GSSG effectively induced ICS1, even in the absence of an H2O2 signal (Col-0), although GSSG was much less effective than GSH in inducing PR1. The glutathione treatments also promoted lesion formation in cat2 cad2.

To establish whether down-regulation of SA-dependent responses in cat2 cad2 was linked to attenuated SA accumulation or impaired NPR1 function, the effects of SA supplementation on PR1 expression were examined in the different lines. In Col-0, npr1, and cad2, PR1 expression was very low in the absence of added SA (Figure 2.13). SA treatment strongly induced PR1 in Col-0 but not significantly in npr1. In cad2, SA was able to induce PR1 but to lower levels than in Col-0 (Figure 2.13). SA treatment of cat2 showed that PR1, already strongly induced in the absence of added SA, was induced a further 2-fold (Figure 2.13). In cat2 npr1, PR1 was induced in the absence of SA, though to a lower level than in cat2 (Figure 2.13). This suggests that an NPR1-independent pathway of PR gene induction can operate in the cat2 npr1 background, as previously reported during SA-dependent interactions with certain pathogens (Rate et al., 1999; Shah et al., 2001; Liu et al., 2010a). While SA was unable to further induce PR1 in cat2 npr1, PR1 was induced by SA about 8-fold in cat2 cad2, so that transcripts were intermediate between cat2 –SA and cat2 + SA (Figure 2.13).

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1.5 A ab 1.0 a ab FW) a -1 0.5 b

(µg g c c d d d Free salicylicFree acid 0.0 30 a 20 b

FW) -1 10

(µg g (µg c cd cd cd

salicylicTotal acid e f f f 0

cat2 Col-0 Col-0 Col -0 cat2 cad2 cat2 cad2 cat2 cad2 at2 cad2sid2 cat2 cad2cat2 sid2 cad2cat2 sid2 c

Mock GSH GSSG

B 0.12 ICS1 a ab a 0.08 ab ACT IN2 b 0.04 c c relative to relative

abundance Transcript 0.00

1.8

a PR1 a 1.2

ACT IN2 b 0.6 b b

to relative

rncitabundance Transcript cc 0.0 cat2

Col -0 Col-0 Col -0 t2 cad2 cat2 cad2 cat2 ca cad2 cat2 Mock GSH GSSG

Figure 2.12. Glutathione complementation of salicylic acid (SA) accumulation and related gene expression in cat2 cad2.

Plants were grown at high CO2 for 3 weeks to prevent any cat2 phenotype, then transferred to air to induce oxidative stress in cat2 backgrounds. From the first day after transfer to air, rosettes were treated once daily by spraying with water, 1 mM GSH, or 1 mM GSSG. Samples were taken after 9 days. (A) Free and total SA contents in Col-0, cat2, cat2 cad2, and cat2 cad2 sid2. (B) ICS1 and PR1 expression in Col-0, cat2, and cat2 cad2. Values are means ± SE of 3 biological replicates. Different letters indicate significant difference at P<0.05.

52 CHAPTER 2

9 9 9

6 6 6 ACTIN2

3 3 3

transcript abundance to relative

PR1 0 0 0 +-+-+-+- -++ -+-

Col-0 npr1 cad2 cat2 cat2 cat2 cat2 npr1 cad2 cad2 npr1

Figure 2.13. Comparison of PR1 inducibility by salicylic acid (SA) in npr1, cad2, cat2, and double and triple mutants. Plants were grown in air from seed for 20 days after sowing and then sprayed either (-) with water (mock) or (+) with 0.5 mM SA and samples were taken 24h later. Values are means ± SE of 3 biological replicates.

2.3 Discussion

A substantial body of work shows that glutathione is a multifunctional metabolite with diverse functions in plant defence and antioxidative metabolism. Because signaling roles of glutathione might be mediated by several types of reactions, many of which could be dependent on functionally redundant proteins encoded by quite large gene families, establishing the role of specific glutathione-dependent components is a formidable task. This study investigated the role of glutathione-dependent processes in H2O2 signaling by seeking to genetically abrogate oxidation-triggered accumulation of glutathione.

2.3.1 Partial impairment of -ECS function confers a genetic block on H2O2-triggered up-regulation of glutathione

The cad2, rax1 and pad2 mutations in the single gene encoding the first committed enzyme of glutathione synthesis (-ECS) produce constitutive partial decreases in glutathione (Cobbett et al., 1998; Ball et al.,

2004; Parisy et al., 2007). Here, we show that these mutations also block H2O2-triggered up-regulation of glutathione. In Arabidopsis, the -ECS protein is located in the chloroplast (Wachter et al., 2005). There is little evidence that accumulation of glutathione in cat2 is linked to enhanced synthesis of -ECS protein (Queval et al., 2009). Based on current knowledge, the major player is post-translational activation of -ECS by disulfide bond formation (Hicks et al., 2007; Gromes et al., 2008). However, the factors that mediate oxidation of -ECS thiols in vivo remain to be identified. Unlike the wild-type enzyme present in cat2, the mutant -ECS does not appear to be oxidatively activated in cat2 cad2. However, whereas in cat2 cad2 glutathione remained close to cad2 values, in cat2 gr1 glutathione accumulated to much higher levels than in cat2 (Figure 2.7). Thus, glutathione stayed highly reduced and below Col-0 levels in cat2 cad2 while a highly oxidized glutathione pool in cat2 gr1 was associated with dramatic accumulation. These observations are consistent with a model for -ECS regulation in which GSSG produced from GSH

53 CHAPTER 2

oxidation allows activation of the enzyme to up-regulate glutathione synthesis. This receives support from glutathione contents in cat2 cad2 gr1, which were intermediate between cat2 cad2 and cat2 (Figure 2.7).

Indeed, the chloroplast was among the cellular compartments that showed the highest H2O2-triggered glutathione accumulation in cat2 (Table 2.2). Since the accumulated glutathione in cat2 is almost all in the disulfide form (Figures 2.1 and 2.9), it is highly likely that in cat2 the chloroplast is enriched in GSSG but that this enrichment does not occur in cat2 cad2.

In all three double mutants blocked in glutathione accumulation, including cat2 rax1 carrying the weakest allele, the overall leaf glutathione pool remains highly reduced in conditions that permit increased H2O2 availability (Figure 2.4). In cat2 cad2 gr1, a limited accumulation of glutathione was accompanied by significant oxidation relative to cat2 cad2 (Figure 2.7). From a cellular point of view, these striking observations suggest that the plant cell glutathione redox system is configured so that not only does oxidation drive enhanced accumulation of total glutathione but also decreased contents inhibit glutathione oxidation. This two-way interaction may be important in setting appropriate conditions for cellular signaling. The underlying factors remain unclear but could include, for example, affinities of the enzymes that oxidize GSH to GSSG or the differences in glutathione compartmentation in cat2 and cat2 cad2. Whatever the underlying causes, glutathione contents are known to change during development (Queval et al., 2007) and in response to factors such as sulfur nutrition (Nikiforova et al., 2003). A strong interplay between concentration and redox state could influence the outcome of redox-dependent responses to external stresses in different circumstances.

2.3.2 An essential role for glutathione in activation of H2O2-dependent salicylic acid signaling and related pathogenesis responses

Alongside up-regulation of glutathione, the cat2 mutation induces SA and a wide range of SA-dependent responses in an ICS1-dependent manner (Chaouch et al., 2010). Using non-targeted metabolite profiling and targeted analysis of recognized defence compounds, it was recently reported that SA-dependent responses triggered by intracellular H2O2 in cat2 show considerable similarity with responses to pathogenic bacteria (Chaouch et al., 2012). Thus, intracellular H2O2 generated by the peroxisome-located photorespiratory glycolate oxidase closely mimics redox processes involved in biotic stress. Several studies suggest that down-regulation of catalase plays some role in pathogenesis responses (Mhamdi et al., 2010b; Volt et al., 2009) while the analysis of glycolate oxidase mutants provides further indications of roles for peroxisomally produced H2O2 in pathogenesis responses (Rojas et al., 2012). The potential relevance of redox-triggered events in cat2 is underscored by effects observed in double mutants in which cat2 responses are modulated by loss of function of recognized players in pathogenesis (Chaouch et al., 2010, 2012). The present study provides direct evidence that glutathione is a key player linking increased

H2O2 to downstream phytohormone signaling. Blocking up-regulation of glutathione in cat2 antagonizes

H2O2-triggered SA accumulation, expression of SA-dependent marker genes, and induced resistance to bacteria.

The residual SA-linked responses in cat2 cad2 are completely annulled in cat2 cad2 sid2, suggesting that glutathione status modulates the efficiency of H2O2-triggered signaling through the ICS1-dependent pathway. This conclusion is supported by the attenuated induction of the ICS1 gene in cat2 cad2

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compared to cat2 (Figure 2.11). Like -ECS, ICS1 is a chloroplast enzyme that could potentially be redox-regulated. However, post-translational control of ICS1 by thiol-disulfide status has been discounted on the basis of the protein’s structural features (Strawn et al., 2007). Our data reveal that the expression of

ICS1 may be under redox regulation through glutathione-dependent processes downstream of H2O2. The intermediates in this signaling pathway remain to be identified.

2.3.3 Glutathione acts independently of its antioxidant function in transmitting H2O2 signals

A central premise of this study was that H2O2-triggered modulation of glutathione (as a result of the antioxidant function of glutathione) might be perceived by the cell as a signal. To test this hypothesis, the aim was to manipulate glutathione status in an H2O2 signaling system, without affecting overall cellular antioxidant capacity. That the block over glutathione up-regulation in cat2 cad2 and allelic lines achieves this objective is evidenced by the following observations. First, introduction of pad2, cad2, or rax1 mutations does not affect the oxidative stress-dependent decreased growth phenotype of cat2. Second, the block over H2O2-triggered glutathione accumulation in cat2 cad2 is quite specific, and not accompanied by increased oxidation of ascorbate or NADPH, or by enhanced accumulation of peroxides. Third, blocking the glutathione synthesis pathway decreases rather than increases cat2-dependent lesion spread. Fourth, and most crucially, the ICS1-linked effects observed in cat2 cad2 are quite distinct from the responses to exacerbated oxidative stress produced by knocking out both CAT2 and GR1.

In addition to the ICS1-independent phenotypes of cat2 gr1, the contrasting effects of cad2 and gr1 mutations on the cat2 response are underscored by analysis of metabolite markers for the SA-dependent pathway. Entrainment of SA-dependent responses in cat2 requires decreased myo-inositol (Chaouch and Noctor, 2010). In cat2 cad2, as in cat2, this compound and the related metabolite galactinol were decreased (Figure 2.8), even though the cat2-triggered SA response was attenuated in the double mutant. This indicates that glutathione status exerts its effects on the SA pathway downstream of myo-inositol.

Together with previous analyses of cat2, it suggests a model in which H2O2 decreases myo-inositol concentrations, an effect that then allows SA accumulation through glutathione-dependent processes. Despite the enhanced oxidative stress in cat2 gr1 and cat2 cad2 gr1, both these compounds remained at levels close to wild-type, suggesting that decreases in myo-inositol are not simply related to oxidative stress intensity.

Together, these data further illustrate that SA-dependent lesions in cat2 are not a simple consequence of oxidative stress intensity. Rather, they are the result of an H2O2-initiated programmed response whose intensity is modulated by glutathione. Elucidation of events underlying the intriguing reversibility of the SA-independent phenotypes in cat2 gr1 backgrounds will require further study, although it is perhaps worth noting that Arabidopsis lines deficient in both CAT2 and APX1 show induction of novel protective mechanisms that are not observed in lines deficient in only CAT2 or APX1 (Vanderauwera et al., 2011). Failure of cat2 gr1 to induce the SA-dependent phenotype observed in cat2 perhaps suggests that an optimal level of oxidation is required to entrain effective induction of pathogenesis responses. Beyond a certain threshold intensity, SA-dependent pathogenesis programme are no longer activated. Thus, even when triggered by a single type of ROS (H2O2 in this study), intracellular oxidative stress can drive several distinct phenotypic outcomes.

55 CHAPTER 2

2.3.4 Glutathione can regulate SA signaling through processes additional to NPR1

NPR1 is by far the best characterized thiol-dependent protein involved in pathogenesis responses, and can be activated by glutathione addition to cells or leaves (Mou et al., 2003; Gomez et al., 2004a). Pathogens and SA can both cause adjustments in leaf glutathione pools (Edwards et al., 1991; May et al., 1996; Vanacker et al., 2000; Mateo et al., 2006; Koornneef et al., 2008). However, the precise nature of the redox factors controlling NPR1 in vivo is still open to debate. While h-type thioredoxins, which are known to be induced during pathogenesis responses (Laloi et al., 2004), were shown to perform the reductive activating step (Tada et al., 2008), a recent study has revealed the complexity of NPR1 regulation (Lindermayr et al., 2010).

As well as abolishing H2O2-triggered increases in chloroplast glutathione, the cad2 mutation prevented increases in cytosolic and nuclear concentrations (Figure 2.2; Table 2.2). Based on current knowledge, this effect might be predicted to compromise NPR1 function. Indeed, the NPR1 pathway is clearly functional in cat2, as the npr1 mutation produced the following effects in cat2 npr1: impaired lesion spread relative to cat2, partial loss of PR gene expression, a complete loss of PR1 inducibility by exogenous SA, and a characteristic metabolite signature, including hyper-accumulation of SA. Consistent with previous studies of plants with altered cytosolic glutathione status (Maughan et al., 2010), exogenous SA induced PR1 less effectively in cad2 than in Col-0. However, if the effect of the cad2 mutation on cat2 responses occurred exclusively through partial or complete loss of NPR1 function, it would be predicted that cat2 cad2 should show same-direction responses to cat2 npr1. In fact, of the cat2 npr1 features listed above, the only one shared by cat2 cad2 was decreased lesion spread relative to cat2. PR1 expression was even lower in cat2 cad2 than in cat2 npr1 and was inducible by exogenous SA. Crucially, blocking glutathione accumulation in cat2 cad2 produced a metabolite signature that was distinct from that observed in cat2 npr1. These distinct effects were most striking for SA contents: if effects of the cad2 mutation were purely explainable in terms of loss of NPR1 function, cat2 cad2 should be expected to have higher SA contents than cat2 (as in cat2 npr1). In fact, the opposite is observed. Thus, it seems that glutathione plays a regulatory role in SA signaling additional to NPR1. Indeed, the similar glutathione status in cat2 and cat2 npr1 provides little evidence for feedback between NPR1 and glutathione.

Hyper-accumulation of SA in cat2 npr1 was associated with H2O2-triggered NPR1-independent PR1 expression and bacterial resistance. The operation of this NPR1-independent pathway seems itself to be dependent on glutathione, because cat2 cad2 npr1 showed similarly low resistance (Figure 2.9C) and PR1 expression to npr1 (Figure 2.13), showing that the cad2 and npr1 mutations act additively to annul part of the cat2-induced resistance responses. Again, this suggests that blocking up-regulation of glutathione impairs H2O2-triggered pathogenesis responses via effects that are distinct from those of the npr1 mutation. A key difference between cat2 npr1 and cat2 npr1 cad2 is that the hyper-accumulation of SA in the former is not observed in the latter. Together with differences in SA accumulation in cat2 and cat2 cad2, this suggests that glutathione plays an important role in H2O2 signaling at the level of induction of SA itself.

56 CHAPTER 2

2.3.5 Multifunctional roles of glutathione status in defence signaling pathways: A model

The observations reported in this study reveal that H2O2-triggered changes in glutathione status are not merely a passive response to oxidative stress. Rather, they suggest that modulation of glutathione status is required to link increases in intracellular H2O2 production to activation of the ICS1-dependent SA pathway. This function would operate within the context of dynamic modulation of glutathione that involves initial oxidation leading to downstream reduction during some pathogenesis responses (Vanacker et al., 2000; Mou et al., 2003; Koornneef et al., 2008). While a potential role for glutathione in the reductive phase has been described at the level of NPR1, results presented in this chapter provide the first direct evidence that initial oxidative events necessary for SA accumulation require changes in glutathione status. The down-regulation of the SA pathway in cat2 cad2, in which glutathione stays highly reduced, suggests that these oxidative events involve a decrease in the GSH:GSSG ratio (Figure 2.14). Failure to produce an appropriately oxidized glutathione status would then inhibit initiation of the pathway, explaining our observation that introducing the cad2 mutation antagonizes SA accumulation, PR1 induction and induced resistance in both cat2 and cat2 npr1 (Figure 2.14). When glutathione oxidation is accompanied by excessively severe oxidative stress, as in cat2 gr1 backgrounds, an alternative response to the SA pathway is entrained.

Initiation and control of Downstream reductive Signal readout oxidative signaling signaling

Thioredoxins/ Glutathione

GSH/ ICS1 SA SA-dependent H O NPR1 2 2 GSSG accumulation gene expression

Cell death NPR1- independent Resistance pathway

Figure 2.14. Glutathione status is a key player linking intracellular H2O2 to activation of the salicylic acid pathway.

Oxidation by H2O2 modulates glutathione status. This oxidative modulation is part of the signal network required for optimal ICS1-dependent SA accumulation that then leads to activation of NPR1 function through reductive processes (Mou et al., 2003), to which glutathione may also contribute.

Both pathogenesis responses and glutathione status are influenced by sulfur nutrition (Höller et al., 2010; Király et al., 2012). More generally, photoautotrophism means that redox modulation is a central player in linking energy and nutritional status to appropriate stress outcomes within the complex metabolic network and plastic developmental program of plants (Noctor, 2006; Foyer and Noctor, 2009). Glutathione status may be an important factor to take into account in attempts to understand and optimize pathogenesis responses in plants growing in natural environments with variable nutrition.

57

CHAPTER 3

Regulation of basal and oxidative stress-triggered jasmonic acid-related gene expression by glutathione

This chapter is presented as a slightly modified version of a paper submitted for publication by the following authors:

Han Y., Mhamdi A., Chaouch S. and Noctor G. Regulation of basal and oxidative stress-triggered jasmonic acid-related gene expression by glutathione. Submitted to Plant, Cell & Environment

CHAPTER 3

Summary As one of the components that determine protein thiol-disulfide status, emerging concepts view glutathione as potentially important in cellular signaling. While several studies have indicated interactions between phytohormone signaling and glutathione in biotic stress responses, effects of glutathione could reflect several processes. In chapter 2, the results showed that glutathione accumulation may play an important role in linking oxidative stress to salicylic acid (SA) dependent signaling. Antagonistic interactions between SA and jasmonic acid (JA) are well described in plants. Most attention has been focused on the thiol-regulated protein NPR1. This chapter reports evidence for potentially important NPR1-independent roles for glutathione in setting signaling strength through the jasmonic acid (JA) pathway. First, it is shown that basal expression of JA-associated genes in the absence of stress is correlated with leaf glutathione content. Second, analyses of an oxidative stress mutant, cat2, reveal that up-regulation of the JA pathway triggered by intracellular H2O2 signals requires accompanying glutathione accumulation. Genetically blocking this accumulation largely annuls H2O2-induced expression of JA-linked genes, and this effect can be rescued by exogenously supplying glutathione. Through a comparison of JA-linked gene expression in double mutants in which oxidative stress occurs in the absence of either glutathione accumulation or NPR1 function, evidence is provided that the regulation of H2O2-triggered JA signaling by glutathione can be mediated by factors additional to NPR1. Hence, this study provides new information that implicates glutathione as a factor determining basal JA gene expression and points to novel glutathione-dependent control-points that regulate JA signaling in response to oxidative stress.

Résumé Vu son role en tant que composante determinant le statut thiol-disulfure, le glutathion est potentiellement important dans la signalisation cellulaire. Plusieurs études ont indiqué l’existence d’interactions entre la signalisation phytohormonale et le glutathion lors des réponses des plantes aux stress biotiques, mais ces interactions pourrait avoir lieu grâce à plusieurs processus dont les détails restent à élucider. Dans le chapitre 2, nous avons vu que l’accumulation de glutathion pourrait jouer un rôle pour relier le stress oxydatif à la signalisation dépendant de l’acide salicylique (SA). Les interactions antagonistes entre SA et acide jasmonique (JA) sont bien documentées chez les plantes, notamment au niveau de la protéine régulatrice, NPR1. Ce chapitre met en évidence des rôles NPR1-indépendants pour le glutathion dans la détermination de l’intensité de signlisation JA. D’abord, l’expression basale de gènes associés à la voie JA est corrélée avec les teneurs en glutathion. Ensuite, des analyses du cat2, un mutant « stress oxydant », revèlent que l’induction de la voie JA déclenché par le H2O2 nécessite une accumulation de glutathion. Introduisant un blocage génétique de cette accumulation chez cat2 diminue significativement l’induction

H2O2-dépendante des gènes associés au JA, un effet qui peut être surmonté en fournissant du glutathion éxogène à la plante. Une comparaison de l’expression génique associée au JA dans des doubles mutants chez lesquels le stress oxydant a lieu en absence de l’accumulation du glutathion ou bien en absence de la fonction NPR1, suggère que la régulation de la signalisation JA déclenchée par le glutathion pourrait être médiée par d’autres facteurs que NPR1. Donc, les résultats impliquent le glutathion comme facteur déterminant l’expression basale de la voie JA et indiquent l’existence de nouveaux points de régulation glutathion-dépendants qui régulent la signalisation JA en réponse au stress oxydant.

58 CHAPTER 3

3.1 Introduction

As a major determinant of cell redox status, glutathione is a key player in stress responses (Noctor et al., 2012). In addition to its accepted antioxidant function mediated by the thiol form, GSH, it influences defence, secondary metabolism and regulation of protein activity through conjugation reactions to metabolites or to protein cysteine residues (Rouhier et al., 2008; Schlaeppi et al., 2008; Su et al., 2011). Glutathione contents are influenced by light and sulphur nutrition, and adjustments in glutathione status can be triggered by a variety of stresses (Bick et al., 2001; Gomez et al., 2004b; Höller et al., 2010). As discussed in the preceding chapters, attention has been paid to roles of glutathione in biotic stress, for several reasons. First, such stresses are known to modulate glutathione status (Edwards et al., 1991; Vanacker et al., 2000; Mou et al., 2003; Koornneef et al., 2008). Second, alterations in responses to pathogens have been described in some studies of mutants with modified glutathione contents or reduction (May et al., 1996; Ball et al., 2004; Parisy et al., 2007; Mhamdi et al., 2010a; Dubrueil et al., 2011). Third, NONEXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), a major player in signal transmission linked to some pathogenesis responses, is known to be regulated by modulation of thiol status (Mou et al., 2003; Tada et al., 2008; Lindermayr et al., 2010). However, the exact role of glutathione status in regulating NPR1 is not clearly established, because pathogen-induced thioredoxins (TRX) have also been implicated (Laloi et al., 2004, Tada et al., 2008). Other important glutathione-sensitive regulatory proteins may await description, and, generally, much remains to be elucidated on how glutathione impacts on pathogenesis-related signaling.

As described in Chapter 2, activation of pathogenesis responses in cat2 is correlated with perturbations in leaf glutathione, is followed by activation of SA-dependent processes and pathogen resistance, and the

H2O2-triggered up-regulation of the SA pathway is glutathione-dependent. Jasmonic acid (JA) is another important defence hormone and its signaling strength is known to be negatively controlled by SA (Leon-Reyes et al., 2010). There is some evidence that glutathione also interacts with the JA pathway. Genes encoding glutathione synthesis enzymes and glutathione reductase (GR) are induced by JA (Xiang and Oliver, 1998) and gr1 mutants with decreased leaf GR capacity show repression of the JA pathway in both basal and oxidative stress contexts (Mhamdi et al., 2010a). Glutathione has also been implicated in the interplay between SA and JA pathways at the level of NPR1, which acts to relay part of the negative control of JA signaling by SA (Spoel et al., 2003; Koornneef et al., 2008). Because of the role of glutathione in linking H2O2 to activation of the SA pathway (Chapter 2), and the known antagonism of SA and JA pathways, the aim of the work presented in this chapter was to explore whether blocking cat2-triggered glutathione accumulation has any impact on JA signaling and, if so, whether any such impact is mediated by NPR1 (Figure 3.1). By analysis of the glutathione-deficient cad2, the oxidative stress signaling system cat2 and the double mutants cat2 cad2 and cat2 npr1, evidence is provided that glutathione status is a determinant of the abundance of transcripts associated with JA synthesis, signaling and downstream processes, and that this effect appears to occur through novel components additional to NPR1.

59 CHAPTER 3

SA accumulation

IC S1 NPR1

Oxidation and induction of glutathione ? GSH/ JA-dependent H2O2 gen e expression GSSG

Figure 3.1. Possible hypothesis for the interaction among glutathione, SA and JA in the H2O2 signaling background.

Since glutathione status impacts on H2O2-triggered SA signaling, and SA is known to repress JA signaling, key questions addressed in this chapter are whether glutathione status has any effect on the JA pathway and, if so, whether these can be explained purely in terms of glutathione-dependent effects on SA signaling via ICS1 and/or NPR1. ICS1, Isochorismate synthase. NPR1, Nonexpressor of pathogenesis-related genes 1.

60 CHAPTER 3

3.2 Results

3.2.1 Effects of glutathione content on basal expression of jasmonic acid-associated genes

To investigate effects of partial glutathione deficiency on phytohormone-linked pathways, microarray analysis of the cad2 mutant was performed. The data revealed that multiple genes associated with phytohormone signaling were differentially expressed in cad2 compared to Col-0. Of these genes, most are annotated as being associated with JA and/or wounding. A suite of genes down-regulated in cad2 included genes involved in JA synthesis and activation (LOX3, LOX4, OPR3, JAR1), seven members of the jasmonate/ZIM domain family of JA-induced JA signaling repressors (JAZ1, JAZ2, JAZ3, JAZ6, JAZ7, JAZ9, JAZ10), and transcription factors and other genes known to be induced by wounding (Table 3.1).

Table 3.1. Genes associated with JA/wounding showing significantly repressed transcript levels in cad2.

Locus Description cad2/Col JA/Wounding At1g01060 Late elongated hypocotyl 1 (LHY1) -1.03 JA At1g01470 LATE EMBRYOGENESIS ABUNDANT 14 (LEA14) -1.14 Wounding At1g17420 Lipoxygenase_ lipoxygenase 3 (LOX3) -2.35 JA At1g18710 Myb family transcription factor (MYB47) -1.59 JA At1g19180 Jasmonate-zim-Domain Protein 1 (JAZ1) -1.67 JA At1g28480 Glutaredoxin family protein (GRX480) -1.13 JA At1g43160 Related to AP 2.6 (RAP2.6) -2.11 JA At1g52890 Arabidopsis NAC Domain Containing Protein 19 (ANAC019) -1.72 JA At1g70700 Jasmonate ZIM-Domain protein 9 (JAZ9) -1.65 JA At1g72450 Jasmonate ZIM-Domain protein 6 (JAZ6) -1.62 JA At1g72520 Lipoxygenase 4 (LOX4) -2.16 JA At1g74430/40 Arabidopsis thaliana MYB domain protein 95 (MYB95) -1.58 JA At1g74950 Jasmonate ZIM-Domain protein 2 (JAZ2) -1.55 JA At1g80840 Pathogen induced-transcription factor (WRKY40) -1.29 JA At2g06050 12-oxophytodienoate reductase (OPR3) -2.09 JA At2g27690 Oxygen binding/fatty acid (omega-1)-hydroxylase_CYP94C1 -1.78 JA At2g29440 glutathione S-transferas 24 (GSTU6) -1.30 JA At2g34600 Jasmonate ZIM-Domain protein 7 (JAZ7) -2.04 JA At2g46370 Jasmonate resistant 1 (JAR1) -2.32 JA At2g46510 ABA-inducible BHLH-type transcription factor (AIB) -1.11 JA At3g10985 Arabidopsis wound-induced protein 12 -1.05 Wounding At3t17860 Jasmonate ZIM-Domain protein 3 (JAZ3) -1.17 JA At3g23250 MYB Domain protein 15 (MYB15) -1.51 JA At3g50260 Cooperatively regulated by ethylene and jasmonate 1 -1.20 JA At4g37260/70 MYB Domain protein 73 (MYB73); -1.02 JA At5g05730 Jasmonated-induced defective lateral root 1 (JDL1) -0.87 JA At5g06870 Polygalacturonase inhibiting protein 2 (PGIP2) -1.08 JA At5g13220 Jasmonate ZIM-Domain protein 10 (JAZ10) -2.52 JA At5g47220 Ethylene-responsive element-binding factor 2 (ERF2) -2.18 JA At5g47240 Arabidopsis thaliana nudix hydrolase homolog 8 (ATNUDT8) -1.19 Wounding At5g53750 CBS domain-containing protein -1.49 JA At5g54170 Polyketide cyclase/dehydrase and lipid transport family protein -1.70 JA At5g60890 Myb-like transcription factor (MYB34) -1.08 JA At5g67300/10 Myb domain protein 44 (MYB44); Member of CYP81G -1.48 JA Microarray analyses and data processing were performed as in Mhamdi et al. (2010a). Only genes that were significantly repressed in independent biological replicates of cad2 RNA dye-swapped with Col-0 RNA are included. The full data set is available at http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_expce.pl?experiment_id=256

61 CHAPTER 3

To further investigate the relationship between glutathione and JA, we examined how the levels of transcripts of two JA signaling marker genes (PDF1.2, VSP2) responded to glutathione deficiency in two available allelic mutants (cad2, rax1) or in Col-0 treated with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO). Irrespective of the genetic background, the results revealed a close correlation between leaf glutathione content and expression of the two JA marker genes (Figure 3.2).

A 400 Glutathione 300 a

FW) -1 200

Leaf content Leaf content (nmol g b b 100 c d 0

B 0.0008 PDF1.2a 0.0006 a a

ACTIN2 0.0004

bbb 0.0002

relative to Transcript abundance 0.0000

C 0.12 VSP2 a

0.08

bc ACTIN2 b 0.04 cc

relative to Transcript abundance 0.00 Col-0 0.2 0.5 cad2 rax1

BSO (mM)

Figure 3.2. The basal level of jasmonic acid-associated gene expression is closely correlated with glutathione contents. (A) Total glutathione contents. (B) PDF1.2a transcripts. (C) VSP2 transcripts. Col-0 and the two allelic glutathione-deficient lines, cad2 and rax1, were grown on agar for two weeks. The glutathione synthesis inhibitor, buthionine sulfoximine (BSO), was supplied to Col-0 plants in the medium at the indicated concentration. All values are means ± SE of three biological repeats. Different letters indicate significant differences at P<0.05.

62 CHAPTER 3

3.2.2 Activation of jasmonic acid signaling by intracellular oxidative stress

Because of its role as an antioxidant, glutathione status is likely to become particularly important during enhanced production of ROS, in particular, H2O2 (Mhamdi et al., 2010b). To explore relationships between glutathione, intracellular oxidative stress, and JA signaling, the cat2 mutant was used. This line is a conditional oxidative stress signaling mutant which, when exposed for several days to air and moderate light, shows H2O2-activated oxidation and accumulation of glutathione, predominantly as GSSG (Queval et al., 2007; Mhamdi et al., 2010a). To establish whether intracellular oxidative stress in cat2 activates the JA pathway, four genes were selected for qRT-PCR analysis in cat2 and Col-0. Two of the genes encode enzymes involved in JA synthesis (LOX3, OPR3), one encodes the JA signaling protein JAZ10, and the last is a JA/wounding marker gene (VSP2). Inducing oxidative stress by transferring cat2 from high CO2 to air caused accumulation of transcripts of all four genes in a time-dependent manner: while transcript abundance remained at Col-0 levels 4d after transfer to air, induction of several-fold was observed 8d after transfer (Figure 3.3).

0.10 0.015 LOX3 * JAZ10 * 0.08 0.010 ACTIN2 ACTIN2 0.005 0.01 * * 0.00 0.000

0.30 0.06 OPR3 0.20 * VSP2

0.10 0.04 *

0.05 0.02

* * Transcript abundance relative to to Transcript relative abundance

to relative Transcript abundance 0.00 0.00 Col-0 cat2 Col-0 cat2

Figure 3.3. Time-dependent induction of jasmonic acid-associated gene expression in the cat2 mutant.

White bars, 4d after transfer from high CO2 to air. Black bars, 8d after transfer. *Significant cat2/Col-0 difference at P<0.05.

3.2.3 Modulation of leaf thiol status by the cad2 mutation

To establish the functional importance of glutathione modulation in oxidative stress-triggered expression of JA-associated genes, the the cat2 cad2 double mutant described in Chapter 2 was analysed. As shown in Chapter 2, the cad2-linked block on cat2-triggered glutathione accumulation prevented the oxidative up-regulation of glutathione that was otherwise observed in cat2. To explore this effect further,

63 CHAPTER 3

leaf thiols were determined by HPLC. Profiling of soluble thiols showed that, in agreement with previous studies (Queval et al., 2009), glutathione and its precursors, cysteine and -glutamylcysteine (-EC), were all accumulated in cat2 to about two- to three-fold Col-0 levels (Figure 3.4). In cad2, carrying a mutation in the enzyme that converts cysteine to -EC, glutathione and -EC levels were lower than in Col-0 while cysteine contents were increased. These effects are consistent with previous studies of cad2 (Cobbett et al., 1998). Introducing the cad2 mutation into cat2 showed that the cad2 mutation overrode the main features of the cat2 effect on thiol profiles. These were essentially similar in cat2 cad2 and cad2, except that cysteine contents were increased in cat2 cad2 to even higher levels than in cat2, and reached about 5-fold Col-0 levels (Figure 3.4). Cys-gly, which is a product of glutathione degradation by -glutamyl transpeptidases, was detected at low levels in Col-0 but accumulated to significantly higher levels in cat2 (Figure 3.4). In line with the low glutathione levels in cad2 and cat2 cad2, cys-gly was below the limit of detection in these lines (Figure 3.4).

15

a

FW) 10 -1

5 Cys-Gly Leaf cys-gly cys-gly Leaf (nmol.g bc c ND ND 0

1200 a GGT 900 FW) -1 600 b Glutathione 300 (nmol.g d c Leaf glutathione 0

GSH-S 21 a

FW) 14 -EC -1

 b -Glu-Cys 7 Leaf c c (nmol.g

0 cad2 75 -ECS a

FW) 50 -1 b b Cys 25 a + (nmol.g cysteine Leaf 0 cat2

cat2 cat2 + cad2 cad2 Col-0 Col-0 Sulfate cat2 cad2 cat2 cad2 cat2 Figure 3.4. Profiling of free thiols in cad2, cat2, and cat2 cad2. The scheme on the right indicates a simplified version of the glutathione synthesis pathway and the first step of glutathione breakdown. -ECS, -glutamylcysteine synthetase. GGT, -glutamyl transpeptidase. GSH-S, glutathione synthetase. Different letters indicate significant difference at P < 0.05. ND, not detected.

64 CHAPTER 3

3.2.4 Comparison of effect of cad2 and npr1 mutations on oxidative stress-triggered JA signaling

The well-documented antagonism between SA and JA pathways is considered partly to depend on NPR1 function (Spoel et al., 2003; Koornneef et al., 2008). The data in Figure 3.3 in this chapter and Figure 2.3 in the previous chapter show that both pathways can be activated in parallel following the glutathione oxidation and accumulation that occurs in cat2 within a few days of transferring to oxidative stress conditions. Based on current concepts, disabling NPR1 function in cat2 npr1 should allow oxidative stress to hyper-induce JA-associated genes. Similarly, if any effects of blocking glutathione accumulation on the oxidative stress-induced JA pathway are mediated by down-regulation of SA accumulation or defective NPR1 function (or both), the cad2 mutation should act in a qualitatively similar manner to npr1, ie, the effects of the two secondary mutation on JA-associated genes should be in the same direction. qRT-PCR analysis showed that the npr1 mutation did not significantly affect stress-induction of the two genes involved in JA synthesis but that JAZ10 and, particularly, VSP2, were more induced than in cat2 (Figure 3.5). The response of JAZ10 and VSP2 in cat2 npr1 is consistent with previous studies implicating NPR1 in SA-mediated repression of the JA pathway (Spoel et al., 2003; Leon-Reyes et al., 2010). In stark contrast to effects observed in cat2 npr1, the cad2 mutation did not cause enhanced induction of JA-associated genes. Rather, expression of all four genes remained close to Col-0 levels in cat2 cad2 (Figure 3.5). Hence, both the SA and JA pathways were down-regulated in parallel in cat2 cad2 relative to cat2 (Figure 3.5; Chapter 2).

1.2 3 LOX3 JAZ10 1.0 cat2 * 2

0.8

cat2 1 0.2 * cat2 * 0.0 0

1.2 OPR3 20 cat2 VSP2 15 * 0.8

10

to relative Fold change 0.4 * 1 cat2

* 0.0 0 Col-0 cat2 cat2 Col-0 cat2 cat2 npr1 cad2 npr1 cad2

Figure 3.5. Differential regulation of cat2-triggered induction of jasmonic acid-associated gene expression by cad2 and npr1 mutations. Effects of cad2 and npr1 mutations on gene expression were examined at 8d after transfer. The dotted line shows expression level in cat2, which is set to a value of 1 and indicated by arrows. *Significant difference of double mutants from cat2 at P<0.05.

65 CHAPTER 3

3.2.5 Effects of complementing mutants with cysteine and glutathione

Because the cad2 mutation not only prevents cat2-triggered accumulation of glutathione but also further promotes cysteine accumulation, effects of exogenous supplying either cysteine or glutathione on the cat2 phenotype and expression of the JA pathway were examined. The aim of these experiments was to establish whether increased cysteine (as occurs in cat2 cad2) contributes to the effects of the cad2 mutation on cat2-triggered expression of the JA-associated genes. In Col-0, neither cysteine nor glutathione treatments affected rosette phenotype but both produced some induction of the JA-associated genes (Figure 3.6). Treating cat2 with cysteine or glutathione did not markedly affect lesion spread or expression of the JA pathway (Figure 3.6). Thus, these observations provide no evidence that the effects of the cad2 mutation observed in cat2 cad2 are linked to increased cysteine.

Mock Cys GSH 0.18 ) a a 0.12 a

transcripts b 0.06 b (rel. ACTIN2(rel. c LOX3 Col-0 Col-0 Col-0 0.00 0.50 a ) a a 0.25 b

transcripts c cat2 cat2 cat2 d (rel. ACTIN2(rel.

OPR3 0.00 Figure 3.6. Effects of externally supplied cysteine or 0.026 glutathione on rosette phenotype and jasmonic a

) a a acid-related gene expression in Col-0 and cat2. 0.013 transcripts Plants were grown at high CO2 for three weeks to ACTIN2 b c b

prevent any cat2 phenotype, then transferred to air to (rel. 0.000 JAZ10 0.50 induce oxidative stress in cat2 backgrounds. From the a first day after transfer to air, rosettes were treated once ) a 0.25 daily by spraying with water, 1 mM cysteine, or 1 mM a transcripts GSH. Photographs show plants eight days after transfer ACTIN2 b c b (rel. (rel.

VSP2 0.00 to air. Bars indicate 1 cm. Transcripts were measured at the same point after spraying leaves with water, cysteine, cat2 cat2 cat2 Col-0 Col-0 Col-0 or glutathione. Different letters indicate significant Mock Cys GSH difference at P < 0.05.

To test the second possibility – that the effects of the cad2 mutation are caused by the block on glutathione accumulation – we examined whether induction of the four genes could be rescued by supplying glutathione exogenously to cat2 cad2. As shown in Figure 3.7, the antagonistic effect of the cad2 mutation on cat2 responses could largely be overcome by supplying glutathione. Exogenous glutathione, particular in the form of GSH, allowed all four transcripts in cat2 cad2 to approach cat2 levels. GSH or GSSG also stimulated expression in Col-0, albeit to a lesser extent (Figure 3.7).

66 CHAPTER 3

0.12 LOX3

0.08 a ab b ab 0.04 cd c d c 0.00 0.2 OPR3 a a a a 0.1 a

ACTIN2 b b

0.0 0.02 JAZ10

a

0.01 b

c c c d d 0.00

Transcript abundance relative to 1.2 VSP2 a a 0.8 bc b

c 0.4 d d

0.0

cat2 Col-0 Col-0 Col-0

cat2 cad2 cat2 cad2 cat2 cad2

Mock GSH GSSG

Figure 3.7. Complementation of jasmonic acid-related gene expression in cat2 cad2 by glutathione. Plants were grown and treated as described for Figure 3.6. GSSG was supplied at 1 mM. Different letters indicate significant difference at P < 0.05.

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3.3 Discussion

Glutathione has long been recognized as a key player in oxidative stress metabolism in plants. It can act as a general reductant, notably in direct metabolism of ROS or to maintain the reduced form of ascorbate (Foyer and Noctor, 2011). Because this role involves oxidation of GSH to GSSG, and because glutathione can interact with protein cysteines, several authors have proposed that changes in glutathione status could be part of oxidative signaling networks (Foyer et al., 1997; May et al., 1998a; Meyer et al., 2007; Noctor et al., 2012). According to such hypotheses, ROS-triggered changes in glutathione would be important in activating downstream signaling pathways. However, direct evidence that glutathione plays such a role is scarce. It was shown in Chapter 2 that genetic modulation of glutathione status antagonizes activation of the SA pathway by intracellular oxidative stress.. Because of the close interplay between SA and JA signaling, present study used cat2 cad2 and other genetic tools to examine possible roles for glutathione in regulating JA signaling.

3.3.1 Basal expression of the JA pathway is closely correlated with glutathione contents

Previous studies of mutants deficient in glutathione point to roles in responses to pathogens and wounding, although the underlying processes remain to be defined (Ball et al., 2004; Parisy et al., 2007; Schlaeppi et al., 2008). Data included in one of these studies pointed to slight repression of wounding genes in the pad2 mutant (Schlaeppi et al., 2008), though this effect was much less clear than those shown here in Table 3.1 and Figure 3.2. In the light of subsequent annotation of the Arabidopsis genome, examination of a previous study of cad2 and rax1 based on a more limited analysis of the transcriptome (about 8500 genes; Ball et al., 2004) also reveals that some JA- or wounding-related genes were among defence genes affected by glutathione deficiency. It is important to note that growth conditions such as irradiance or photoperiod could influence the extent to which glutathione affects JA signaling. The effects observed here were found to be dependent on day length: when the cad2 transcriptome was analyzed in short day conditions, repression of JA-linked genes was much less evident (data not shown). The data point to a role for glutathione-dependent processes in regulating basal expression of the JA pathway in the standard conditions used throughout this study (growth in long day conditions). This is evident both from the high number of JA genes down-regulated in cad2 (Table 3.1) and the close correlation between JA marker gene expression and glutathione content (Figure 3.2). Thus, glutathione status, which can be influenced by several abiotic factors (eg, irradiance, mineral nutrition, heavy metal stress), may be influential in setting signal strength through the JA pathway in response to biotic stress. Further work is required to elucidate the mechanisms, although it is intriguing that a glutaredoxin (GRX480) and a glutathione S-transferase (GSTU6), both of which have been previously implicated in JA signaling, were among the genes down-regulated in response to partial glutathione deficiency (Table 3.1).

3.3.2 JA-associated gene expression induced by oxidative stress is influenced by glutathione status

Induction of both SA and JA signaling by the cat2 mutation in a time-dependent manner following transfer from high CO2 to air at moderate irradiance shows that changes in intracellular redox state driven by enhanced availability of photorespiratory H2O2 can trigger activation of multiple defence hormone pathways in parallel. The potential relevance of redox-triggered events in cat2 is underscored by our

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previous reports on metabolite profiles and the effects of introducing mutations for recognized players in pathogenesis into the cat2 background (Chaouch et al., 2010, 2012). A well characterized response to catalase deficiency is accumulation of glutathione, mainly as GSSG (Chapter 2). Blocking this response through the cad2 mutation largely prevents the up-regulation of JA-associated genes that is observed in cat2 (Figue 3.5), indicating that glutathione-dependent processes link intracellular oxidative stress to activation of the JA pathway.

Thiol profiling of cat2 cad2 shows that the cad2 mutation produces an effective block in oxidation-triggered glutathione accumulation at the level of -ECS, the first dedicated enzyme of glutathione synthesis (Figure 3.4). Thus, under conditions of active photorespiration, the cat2 mutation drives accumulation of thiols associated with glutathione metabolic pathways, ie, cysteine, -EC, glutathione, and cys-gly. This suggests that enhanced availability of intracellular H2O2 activates both cysteine and glutathione synthesis, and that the accumulation of glutathione also activates glutathione turnover through enzymes such as GGTs. Transcript data suggests that up-regulation of cysteine and glutathione synthesis in cat2 is closely associated with the chloroplast, while immunolabelling analyses revealed that the ensuing enrichment of glutathione occurs in most subcellular compartments (Queval et al., 2009, 2011). Production of cys-gly in cat2 could occur though the action of GGTs located in the vacuole or apoplast (Grzam et al., 2007; Ohkamu-Ohtsu et al., 2007a,b). Based on marked accumulation of GSSG in the vacuole (Queval et al., 2011), our working hypothesis is that this compartment could be an important site of glutathione turnover in cat2.

In cat2 cad2, the block over cat2-triggered glutathione accumulation is reflected in decreased glutathione is almost all measured subcellular compartments (Table 2.2 in Chapter 2). The present analysis shows that the secondary cad2 mutation also decreases -EC and cys-gly in cat2 cad2 to cad2 levels, observations that are consistent with restraint at the level of -ECS and thus limitation of glutathione synthesis and subsequent turnover. However, it seems that oxidative activation of cysteine synthesis may still occur in cat2 cad2 because cysteine accumulates to at least two-fold cat2 levels, ie, about five times the level in Col-0. As discussed in Chapter 2, it seems unlikely that effects of blocking glutathione accumulation could be ascribed simply to possible changes in oxidative stress intensity because (1) leaf peroxides, NADP(H), ascorbate, and rosette growth are largely unaffected compared to cat2, and (2) the phenotype of cat2 cad2 is quite distinct from cat2 gr1 double mutants, in which oxidative stress is clearly exacerbated compared to cat2. However, the possibility remains that differences in gene expression between cat2 and cat2 cad2 could reflect accumulated cysteine in the latter, rather than decreased glutathione. The complementation experiments provide little evidence for this. First, providing additional cysteine to cat2 (to mimic the high cysteine contents of cat2 cad2) did not decrease JA-associated gene expression (Figure 3.6). Second, restoration of cat2-triggered JA-associated gene expression by supplying glutathione to cat2 cad2 (to mimic conditions in cat2; Figure 3.7) suggests that the factors underlying the differences in JA transcripts between cat2 and cat2 cad2 are associated with glutathione rather than cysteine. Nevertheless, we cannot definitively rule out some influence of differences in cysteine, or the intriguing possibility that the cell may possess mechanisms that sense changes in the glutathione synthesis/metabolism pathways rather than glutathione status per se or glutathione status alone.

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3.3.3 Glutathione can regulate oxidative stress-triggered activation of JA signaling independent of NPR1

NPR1 is by far the best characterized thiol-dependent protein involved in pathogenesis responses. A recent study indicates that NPR1 can interact with leaf concentrations of both glutathione and ascorbate in the regulation of ozone-dependent induction of both SA and JA pathways (Brosché and Kangasjärvi, 2012). The NPR1 protein appears to be involved in responses observed in cat2, because previously documented NPR1-dependent responses are impaired in cat2 npr1, notably decreased induction of SA-dependent PR genes (Cao et al., 1994) and hyper-accumulation of SA (Figure 2.11 in Chapter 2), an effect that is known to occur in the single npr1 mutant challenged with certain pathogens (eg, Rate et al., 1999). However, as described in the previous chapter, comparisons of effects of secondary npr1 and cad2 mutations on cat2-triggered SA signaling suggest that glutathione-dependent processes play regulatory roles additional to NPR1 in regulating SA accumulation (Chapter 2). This is consistent with the decreased SA accumulation in cat2 cad2, an effect that is opposite to the very marked accumulation of SA in cat2 npr1.

In conditions where both SA and JA signaling are induced by oxidative stress, as in cat2, the decreased accumulation of SA produced by the cad2 mutation should mitigate SA-dependent restraint of JA signaling. Similarly, if the cad2 mutation negatively affects activation of NPR1, the intensity of this restraint would also be predicted to decrease. Based on these notions, any effects of the cad2 mutation on cat2-triggered induction of JA genes would be expected to be positive. In fact, the opposite is observed when glutathione accumulation is blocked: the cad2 mutation down-regulates JA-associated gene expression at the same time as down-regulating the SA pathway. Indeed, the contrast between effects of cad2 and npr1 mutations on cat2 responses is striking (Figure 3.5). Disabling NPR1 function had no effect on two JA synthesis genes while allowing markedly increased expression of JAZ10 and VSP2. That all four genes were down-regulated in cat2 cad2 relative to cat2 and, even more so (for JAZ10 and VSP2) relative to cat2 npr1, is not consistent with an action of glutathione at the level of NPR1. This does not rule out roles for glutathione in mediating repression of JA signaling through this previously described mechanism, but it does imply the existence of other glutathione-dependent processes in the regulation of the JA pathway.

Previous studies have drawn attention to roles of GRX480 in mediating certain aspects of the interaction between SA and JA pathways (Ndamukong et al., 2007). GRX480 encodes a member of the largest sub-class of plant glutaredoxins, proteins that are likely important in linking glutathione and protein cysteine status (Rouhier et al., 2008). This class of glutaredoxins is specific to plants (Lemaire, 2004). Their biochemical activities remain to be characterized, although at least some members are known to interact with transcription factors (Ndamukong et al., 2007; Wang et al., 2009c). GRX480 is inducible by SA and its overexpression represses the JA marker gene, PDF1.2, although knockout mutants do not show altered PDF1.2 expression, possibly because of gene redundancy (Ndamukong et al., 2007). While GRX480 and potentially redundant similar genes could play some role in the effects we describe here, other factors are probably at work because (1) cat2 cad2 shows repression of SA signaling as well as JA signaling; (2) our observations suggest a generalized effect of glutathione on JA-associated genes, not just PDF1.2; and (3) this effect appears to be apparent even in basal, non-induced conditions (Table 3.1 and

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Figure 3.2), where SA signaling is only weakly or not activated and GRX480 expression is decreased.

3.3.4 Novel roles for glutathione in regulating the JA pathway: a model

Based on the above discussion, it is possible that novel glutathione-dependent components involved in JA signaling exist (Figure 3.8). Two possibilities could explain the dual effect of glutathione on signaling through the SA and JA pathways in response to oxidative stress. First, it is possible that glutathione status acts to modulate a master switch that governs the oxidative activation of both pathways (Figure 3.8, dotted line). A second scenario would involve regulation of both pathways in parallel (Figure 3.8, dashed lines). Our analysis of the SA pathway implicates glutathione in regulating SA accumulation at the level of ICS1 expression (Chapter 2; Figure 3.8). Whatever the glutathione-dependent process(es) involved in the initial activation of the JA pathway, interplay between the SA and JA pathways mediated by other redox-linked factors such as thiol-regulation of NPR1 or induction of GRX480 could act downstream to determine the relative signaling strength of these two key defence hormones.

H2O2

Oxidation and induction of glutathione

GSH/ GSSG

? ICS1

ICS1 ?

JA-dependent SA

gene expression accumulation NPR1

Figure 3.8. An NPR1-independent role for glutathione in modulating oxidative stress-triggered JA-associated gene expression.

Oxidation by H2O2 modulates glutathione status. This oxidative modulation is part of the signal network required for optimal ICS1-dependent SA accumulation that then leads to activation of NPR1 function through reductive processes (Mou et al., 2003), to which glutathione may contribute. Through a pathway that is parallel to SA activation (dashed lines) or simultaneously induced by a master switch (dotted lines), glutathione modulation also links H2O2 to induction of JA signaling. The impact of glutathione on JA-related gene expression, which is also apparent in non-induced (basal) conditions, operates alongside restraint of JA signaling by the SA pathway, eg, through reductive activation of NPR1 (Koornneef et al., 2008).

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It is possible that the regulatory influence of glutathione is mediated by direct control of protein thiol-disulfide status through redox potential (Meyer et al., 2007). This notion receives some support from the observation that plants lacking expression of GR1 also show repression of the JA pathway (Mhamdi et al., 2010a), albeit less strongly than in cad2. Both gr1 and cad2 should have an increased glutathione redox potential compared to Col-0, ie, glutathione should be less reducing (Meyer et al., 2007; Marty et al., 2009). In cat2 gr1 double mutants, JA-associated genes are strongly repressed, as in cat2 cad2 (Mhamdi et al., 2010a). Although the interpretation of effects in cat2 gr1 is complicated by a much more extreme phenotype than in cat2 cad2, it is possible that repression of the JA pathway reflects mechanisms that are common to the two double mutants. In cat2 gr1, the absence of GR1 function and the consequent, marked accumulation of GSSG are expected to increase the glutathione redox potential relative to cat2. It is more difficult to predict differences in glutathione redox potential between cat2 cad2 and cat2 because the double mutant has a more reduced leaf glutathione pool than cat2 (favouring a lower redox potential) but the total glutathione pool is lower (favouring a higher redox potential). Direct measurements using redox-sensitive GFP could be informative here, though such technologies are currently difficult to apply to photosynthetic leaf mesophyll cells, in which cat2 effects are expected to predominate.

As well as effects on signaling proteins that could be mediated by glutathione redox potential or S-glutathionylation reactions, other possible mechanisms are related to glutathione S-transferase activities. Conjugation reactions have been implicated in JA metabolism (Ohkamu-Ohtsu et al., 2011). Because of the large number of genes encoding proteins such as GRXs and GSTs, and possible gene redundancy, further work is required to elucidate the mechanistic details of what seem to be multiple roles of glutathione in modulating defence hormone-linked responses. Understanding these mechanisms may be relevant to understanding and optimizing the biotic stress responses of plants growing in a variable environment that can lead to marked changes in tissue glutathione status.

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CHAPTER 4

Analysis of autophagy mutants: interactions with photorespiration and oxidative stress

CHAPTER 4

Summary Autophagy involves degradation of cytoplasmic materials and may be important in plants during nutrient starvation, senescence, pathogenesis responses or under oxidative stress conditions. While much remains to be elucidated, over 30 genes have been implicated in autophagic processes in plants. Autophagy has been considered to play a positive role in nutrient recycling under conditions of carbon or nitrogen limitation, but an early senescence phenotype is still observed in atg mutants under optimal conditions. The aim of this study was to use previously identified atg mutants to analyze possible interactions between autophagy, photorespiration and oxidative stress using the cat2 and cad2 lines described in previous chapters. Growth of atg2, atg5 and atg18a mutants at high CO2 showed that the accelerated-senescence of these lines was enhanced compared to growth in air. Data on metabolic profiling and glutathione indicated that this effect was related to intracellular oxidative stress. However, introducing the cat2 mutation into atg mutants delayed the atg-triggered senescence, suggesting that other ROS-generating pathways contribute to this process. On the other hand, induced pathogen resistance in cat2 seems to be compromised by atg2 and atg18a mutations, while SA-dependent lesion formation was not different in cat2 compared to any of the three double cat2 atg mutants. Additionally, preliminary data are presented on a defect in the development of young leaves in high CO2 in atg2 cad2 double mutants with decreased glutathione. Taken together, these results may indicate that oxidative stress originating from excess peroxisomal H2O2 can act antagonistically to the programs induced in atg mutants and that oxidative stress in these mutants is caused by other cellular sources of ROS.

Résumé L’autophagie a été impliquée dans la dégradation de composantes cytoplasmiques. Ce processus pourrait jouer des rôles importants chez les plantes dans des conditions comme une carence nutritive, la sénescence, les réponses aux pathogènes, ou encore les stress oyxdants. Alors que nos connaissances restent limitées, plus de 30 gènes ont été impliqués dans les processus d’autophagie chez les plantes. L’autophagie jouerait un rôle positif lors du recyclage nutritif dans des conditions de limitation en carbone ou en azote. Cependant, un phénotype de sénescence précoce est observé dans des mutants atg cultivés dans des conditions optimales. Cette étude avait pour but d’utiliser des mutants atg déjà identifiés afin d’analyser des interactions éventuelles entre autophagie, photorespiration et stress oxydant grâce aux lignées cat2 et cad2 décrites dans les chapitres précédents. La culture de mutants atg2, atg5 et atg18a à fort CO2 a montré que leurs phénotypes de sénescence accélérée sont plus marqués par rapport à ceux observés lors de la culture dans l’air. Des profilages métaboliques et des données sur le glutathion indiquent que ces effets sont associés à une condition de stress oxydant. Cependant, la mutation cat2 retarde la sénescence atg-dépendante, suggérant que d’autres voies de production de ROS contribuent à ce processus. En revanche, la résistance aux pathogènes induite chez cat2 semble être compromise par les mutations atg2 et atg18a, alors que la formation de lésions SA-dépendantes est essentiellement similaire chez cat2 et les trois doubles mutants cat2 atg. De plus, des données préliminaires içi présentées indiquent un défaut de développement de jeunes feuilles dans les doubles mutants atg2 cad2 cultivées à fort CO2. Dans leur ensemble, ces résultats indiqueraient qu’un stress oxydant déclenché par un excès de

H2O2 peroxysomal pourrait agir d’une manière antagoniste aux programmes induits dans les mutants atg et que le stress oxydant dans ces mutants serait causé par des ROS provenant d’autres sources cellulaires.

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4.1 Introduction

Autophagy is an intracelluar macromolecule degradation process that delivers unwanted cell materials into the vacuole/lysosome for recycling of needed nutrients (Liu and Bassham, 2012; Pérez-Pérez et al., 2012). Upon the induction of autophagy, cytoplasmic constituents and organelles are engulfed into double-membraned autophagosomes, which deliver this cargo into the vacuole for further degradation. Although there have been reports of autophagy-like processes in plants since the early 1960s, the molecular mechanism is best described via mutagenesis studies in yeast (Tsukada and Ohsumi, 1993; Thumm et al., 1994; Barth et al., 2001). However, most of the autophagy-related (ATG) genes are well conserved in plants (see Chapter 1), suggesting that the core molecular basis of autophagy is similar between plants and yeast. For instance, the ATG1/13 kinase complex initiates autophagosome formation in response to nutrient demands sensed by an assemblage of upstream regulators in yeast. Under nutrient-rich conditions, activation of the Target Of Rapamycin (TOR) complex represses the ATG1/ATG13 kinase by the hyperphosphorylation of the ATG13 subunit which in turn prevents complex assembly (Kamada et al., 2010). During starvation, TOR is inhibited and, consequently, ATG13 is no longer phosphorylated. ATG1 can associate with the ATG13 subcomplex, subsequently leading to autophagy induction through several steps (Nakatogawa et al., 2009; Kamada et al., 2010). It has been shown that the ATG1/ATG13 kinase complex in plants plays a key role in inducing autophagy under nutrient-limiting conditons, as in yeast (Suttangkakul et al., 2011). Furthermore, the defect in autophagy was also observed in many Arabidopsis atg knockout lines (Bassham et al., 2006).

Over the past decade, our physiological understanding of autophagy and its molecular mechanisms in plants has greatly increased. While most attention has focused on the functions of autophagy in nutrient starvation in plants (Doelling et al., 2002; Hanaoka et al., 2002; Thompson et al., 2005; Xiong et al., 2005; Phillips et al., 2008), Arabidopsis atg mutants are also hypersensitive to salt and drought stresses (Liu et al., 2009). Moreover, oxidative stress triggered by exogenous H2O2 or methyl viologen (MV) treatment can induce autophagy and ATG18a RNAi lines are more sensitive to oxidative stress, a sensitivity that is accompanied by enhanced accumulation of oxidized proteins relative to wild-type (Xiong et al., 2007). Based on this study, plant autophagy has been proposed to play an important role in the degradation of oxidized proteins as well as nutrient recycling under stress conditions (Xiong et al., 2007). Although autophagy has been suggested to be involved in resistance to oxidative stress in plants (Xiong et al., 2007; Liu et al., 2009), the precise function of autophagy and whether plant cells also use autophagy as a defence against oxidative stress triggered by specific source of ROS in different organelles remains unclear. Besides the impact of autophagy in abiotic stresses, accumulating evidence points to a key role of autophagy in the regulation of plant immune responses (Liu et al., 2005; Hofius et al., 2009; Hayward and Dinesh-Kumar, 2011). For instance, atg5 or atg18a mutations not only result in an increased resistance to the virulent bacterium to P. syringae DC3000, but also increase susceptibility to infection with the fungus Alternaria brassicicola (Lenz et al., 2011b). This suggests that autophagy could play differential roles in responses to invasion of biotropic and necrotrophic pathogens.

Besides studies of the physiological function of autophagy in stress conditions, autophagy could also play a housekeeping role to remove unwanted cellular compounds generated by basal metabolism or to remobilize nutrients, even under optimal growth conditions. For example, almost all Arabidopsis

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autophagy mutants are able to grow from seed to seed except for ATG6 T-DNA lines (which are lethal) but many of them such as atg2, atg5, and atg10 display slower growth phenotype under nutrient-rich conditions (Liu and Bassham, 2012; Fujiki et al., 2007; Harrison-Lowe and Olsen, 2008). Several atg mutants such as atg2 and atg5 have an early senescence phenotype even under favorable conditions, and this is linked to the NPR1-dependent SA signaling pathway (Yoshimoto et al., 2009). As for the mechanism of how autophagy negatively regulates premature senescence, one possibility is that autophagy may be selectively capable of removing specific ROS-generating organelles such as mitochondrion and peroxisomes in plants as observed in yeast (Sakai et al., 2006; Kanki et al., 2009; Pérez-Pérez et al., 2012). However, the underlying mechanism of organelle-specific autophagy remains to be identified in plants. It is also possible that the induction of oxidative stress and related senescence could be caused by failure to recycle oxidized proteins and lipids, leading to the accumulation of damaged and toxic compounds.

While ROS have been implicated in senescence, the relationships between plant senescence and oxidative stress are not yet clear. Indeed, leaf senescence has been particularly associated with enhanced H2O2 availability (Smykowski et al., 2010; Wu et al., 2012), which could be a signal involved in the regulation of senescence. As discussed in Chapter 1, plants have developed numerous antioxidant systems to metabolize H2O2 (Mhamdi et al., 2010b), and changes in the potency of some of these systems may be influential in the regulation of senescence. While there are three CAT genes in Arabidopsis, CAT2 is highly expressed in leaf peroxisomes and is the major form of the enzyme in photosynthetic tissues (Queval et al., 2007). It has been shown that leaf catalase activity can be decreased by a variety of stresses including salt, heat, cold and biotic stress (Volk and Feierabend, 1989; Hertwig et al., 1992; Dorey et al., 1998). Recently, it has been suggested that decreased CAT2 expression and activity is linked to the onset of leaf senescence, via regulation of the intracellular H2O2 content (Smykowski et al., 2010). Precocious senescence and a higher level of H2O2 are no longer observed in a gbf1 mutant that was reported to lack senescence-associated down-regulation of CAT2.

In the second half of my thesis studies, I undertook to begin an analysis of interactions between peroxisomal-triggered oxidative stress and autophagy. For this, three published autophagy mutants were selected (atg2, atg5, and atg18a) and crossed with cat2 which, as we have seen in Chapter 2 and 3, is a useful system to study the importance of photorespiratory H2O2 signaling in plant function (Mhamdi et al.,

2012). To analyze interactions between photorespiration and autophagy, I also examined whether CO2 level affects responses in the three atg mutants. In addition, because the cat2 mutant activates SA-dependent signaling (Chaouch et al., 2010; Chapter 2), and some of the atg mutants have also been implicated in regulating this pathway, I wanted to analyze whether cat2-dependent lesion formation was affected by secondary atg mutations. The phenotypic analysis of interactions between cat2 and atg was performed in several growth conditions, because the cat2 mutant is highly conditional. When grown in air in long days (as for the studies reported in Chapters 2 and 3), the cat2 mutation triggers SA-dependent responses and lesion formation. These responses are absent when cat2 is grown in short days, even though oxidative stress is still apparent. This property allows cat2 to be used as a system to analyze effects of oxidative stress in the presence or absence of induction of the SA pathway (Figure 4.1). Because the oxidative stress observed in cat2 in air is absent when photorespiration is repressed by growth at high CO2, the effects of induced oxidative stress can be studied by transferring mutants grown

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initially at high CO2 to air, which also allows induction of the SA pathway specifically after transfer to air in long days but without the decreased growth in cat2 grown in air from germination (Figure 4.1). Finally, because of the roles of glutathione reported in the two previous chapters, I introduced the cad2 mutation into atg2 (the mutant that shows the most striking phenotype of the three atg selected for study). These double mutants were obtained in the final year of the thesis and their functional analysis is as yet preliminary. This chapter reports some of the results already obtained through phenotyping and metabolite profiling.

Short days, air High CO2 Long days, air

Oxidative stress No oxidative stress Oxidative stress Decreased growth Wild-type phenotype Decreased growth No lesions SA accumulation Lesions

Induced oxidative Induced oxidative stress stress WITHOUT SA accumulation decreased growth Lesions

(no lesions) WITHOUT decreased growth

Figure 4.1. Summary of the conditional nature of the cat2 mutant, showing its dependence on CO2 level and on growth daylength.

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4.2 Results

T-DNA mutants for atg2, atg5, and atg18a obtained from seed banks were genotyped to identify plants homozygous for the mutations (Figure 4.2A). Homozygotes were crossed with cat2 and double homozygotes identified in the F2 generation (Figure 4.2A). The atg2 and atg5 mutations cause knockout of the expression of the corresponding genes (Inoue et al., 2006; Yoshimoto et al., 2009; Lenz et al., 2011b). RT-PCR analysis of atg18a showed that transcripts of ATG18a were undetectable in this line (Figure 4.2B).

atg5

atg2

0 0 ‐ A ‐ cat2 atg5 Col atg2 cat2 Col 121212 121212

8a atg18a atg5

atg2 atg1 0

0 ‐ ‐ atg18a cat2 Col cat2 cat2 cat2 cat2 Col 121212 1212121212

RT‐PCR B Figure 4.2. Genotyping and expression analysis of atg 0 0 0 ‐ ‐ ‐ mutants. Col Col Col atg18a atg18a atg18a (A) PCR analysis of homozygous single atg2, atg5, and

atg18a T-DNA mutants and after crossing into cat2 to ATG18a produce double mutants. For each gene, lane 1 indicates

primer combinations to amplify the wild-type gene and

lane 2 primer combinations to amplify gene-specific

fragments using a T-DNA-specific primer.

(B) RT-PCR verification of the absence of corresponding

ACTIN2 transcript in atg18a.

Primer combinations are given in Table 6.2 (Chapter 6)

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4.2.1 Effects of high CO2 on atg phenotypes and metabolite profiles

As a first step to analyzing interacttions between photorespiration and autophagy, single atg mutants were -1 grown in short days (8h light/16h dark), either at high CO2 (3000 µL.L ) or in air. Short day conditions were chosen because this greatly retards flowering in Arabidopsis, enabling effects on leaf senescence to be studied without this complicating process. In agreement with previous reports (Xiong et al., 2005; Yoshimoto et al., 2009), phenotypic analysis revealed that all three atg mutants showed a phenotype of accelerated senescence on older leaves when they were grown in air. This phenotype was first observed after about 6 weeks growth in air (Figure 4.3A), a stage at which no senescence was observed in Col-0.

However, appearance of senescent leaves occurred significantly earlier in atg mutants grown in high CO2 such that this phenotype was already apparent after 5 weeks growth in this condition (Figure 4.3A and 4.3B). Similar to growth in air, no phenotypic symptoms of senescence were observed in Col-0 at high

CO2. The early senescence phenotype was most evident for atg2, and was correlated with decreased rosette size (Figure 4.3C).

A 30 30 30 atg2 * atg5 atg18a 20 20 20 * * * 10 10 10 * * * (% rosette area) Senescent areas 0 0 0 * 3456 3456 3456 Weeks after sowing Weeks after sowing Weeks after sowing B

2

High CO Col-0 atg2 atg5 atg18a

Air

Col-0 atg2 atg5 atg18a

C 12 900 8 * * * * 600 * * * * * 4 300

Fresh weight (mg)Fresh weight 0

Rosette diameter (cm) 0

atg5 atg2 atg2 atg5 atg5 atg2 atg2 atg5 Col-0 Col-0 Col-0 Col-0 atg18a atg18a atg18a atg18a High CO Air 2 High CO2 Air

Figure 4.3. Phenotypes of atg2, atg5 and atg18a grown in high CO2 and air for five weeks in short day conditions.

(A) Development of senescent yellowing in mutants grown in high CO2 (black circles) or in air (white circles) for six weeks in short days. *Significant difference at P<0.05 (n = 6-10 different plants of each line). (B) Photographs of plants after five weeks growth in the two conditions. (C) Rosette diameter (left) and fresh mass (right) after five weeks growth in the two conditions. *Significant difference at P<0.05 (n = 12 or more different plants of each line).

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To gain further insight into the underlying metabolic features of this phenotype, non-targeted metabolite profiling was performed using GC-TOF-MS (Figure 4.4). Hierchical clustering of all detected compounds showed that different metabolic patterns were obtained in high CO2 and air for the wild-type, Col-0 (Figure 4.4A). Whereas the atg mutations did not radically alter profiles in air, atg2 and atg18a strongly impacted on the metabolite profiles observed at high CO2. A large sub-cluster of compounds showed higher contents in these two mutants specifically at high CO2 (Figure 4.4A, bottom half of heatmap). Metabolites that were significantly different from Col-0 were identified (Figure 4.4B). In agreement with the phenotype (Figure 4.3), the highest number of significantly different metabolites was detected in atg2 and then atg18a (Figure 4.4B). A clear interaction with CO2 was observed, because the number of signficant metabolites was lower in air (Figure 4.4B). This pattern was exemplified by isoleucine (Figure

4.4C), which was one of many amino acids that were increased in abundance in atg2 in high CO2 but less so in air. Comparison with metabolite profiles for cat2 (Chapter 2) indicated substantial overlap with the profile observed in atg2. Of 26 compounds that were significantly different between Col-0 and atg2, almost two-thirds also showed the same significant difference between cat2 and Col-0 (Figure 4.4D). These metabolites included gluconic acid, which is the most markedly accumulatedcompound in cat2 (Chaouch et al., 2012). Indeed, of the 17 common metabolites that were induced in cat2 and atg2 (Table 4.1), seven were among the most strongly accumulated compounds in response to both oxidative stress and bacterial challenge (Chaouch et al., 2012). A B High CO Air High CO Air 2 2 2518a2518a2518a2518a Metabolite 2,4‐OHbutanoic acid 2‐hydroxyglutaric acid Alanine Aminoadipic acid atg2 atg18a Col-0 atg18a Col-0 atg5 atg2 atg5 Arabinose b‐alanine b‐sitosterol Citramalic acid Ethanolamine Fructose GABA Galactono‐1,4‐lactone Galactinol Galactose Gluconic acid Glycerate Homoserine Isoleucine Lysine Malate Mannose O‐acetylserine Phenylalanine Phosphate Isoleucine Putrescine Raffinose Rhamnose Ribose Serine Shikimic acid Sucrose Tetradecanoic acid Threonine Threonic acid Tyrosine Tyramine Uracil -2.0 +2.5 Valine Xylose

C Isoleucine D 0.006 atg2 cat2 * 0.004 0.002 9 17 32 intensity peak Relative 0.000 atg2 atg2 Col-0 Col-0 CO2 Air

Figure 4.4. Metabolite profiling of Col-0 and atg mutants grown at high CO2 and air. (A) Heatmap display of all data after center-reduction. (B) Significantly increased (red) or decreased (green) metabolites in each mutant grown in air or at high CO2 after t tests against Col-0 in the same condition (P<0.05). (C) Graph to illustrate typical high CO2-specific response in atg2. The position of the selected metabolite on the heatmap is indicated in (A). (D) Overlap between atg2 metabolite profile and oxidative stress-triggered profiles described for the catalase-deficient mutant, cat2 (Chapter 2). Note that the analysis only considers metabolites that were detected in both series of experiments.

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Table 4.1. Overlapping metabolites induced by atg2 and cat2 mutations. Metabolites were identified by t test at P<0.05 (n = 3 biologically independent samples) and numbers give mean fold-change relative to Col-0 sampled in the same conditions. *Among metabolites that most strongly accumulate in response to both oxidative stress and bacterial challenge (Chaouch et al., 2012).

atg2/Col-0 cat2/Col-0 Gluconic acid * 12.2 159.9 Threonic acid 4.2 2.8 Isoleucine * 4.2 2.6 2-Hydroxyglutaric acid * 3.5 8.8 2,4-Dihydroxybutanoic acid 3.5 4.2 Ribose * 3.1 3.6 Phenylalanine * 2.6 2.0 Malic acid 2.4 3.1 Valine 2.3 2.4 Shikimic acid 2.1 1.6 Putrescine * 2.0 2.8 Lysine 1.9 1.8 Rhamnose 1.8 2.3 β-alanine 1.8 1.8 Glyceric acid 1.4 2.0 Serine 1.3 3.5 Threonine * 1.3 2.3

4.2.2 Impact of oxidative stress on atg-triggered early senescence

To analyze possible effects of oxidative stress on the atg phenotype, all three mutants were crossed with cat2. First, as for the experiments described above, the double mutants were analyzed in short-day conditions. Under this growth regime, cat2 shows oxidative stress in air but not at high CO2, and oxidative stress that occurs in air is not associated with activation of the SA pathway and SA-dependent responses (Figure 4.1; Queval et al., 2007; Chaouch et al., 2010). No obvious effect of the cat2 mutation on the early senescence phenotype was observed when the plants were grown at high CO2. This is unsurprising because the cat2 mutant is phenotypically silent in this condition. When plants were grown to an age of six weeks in air, at which point early senescence is apparent in atg single mutants (Figure 4.3), no senescence was observed in cat2 atg double mutants (Figure 4.5B). However, this observation is somewhat difficult to interpret because of the general effects of the cat2 mutation on rosette growth when plants are grown in air from seed (Figure 4.1). Single atg2 and double cat2 atg2 mutants were therefore grown for four weeks in high CO2, at which point early senescence is initiated in atg2 (Figure 4.3), and then transferred to air to induce oxidative stress in cat2 genotypes and grown for a further week. This analysis revealed that whereas the atg2 single mutant develops obvious senescence on the outer leaves, this phenotype was less apparent in cat2 atg2 (Figure 4.5C).

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A

Col-0 atg2 atg5 atg18a

cat2 cat2 atg2 cat2 atg5 cat2 atg18a

B

Col-0 atg2 atg5 atg18a

cat2 cat2 atg2 cat2 atg5 cat2 atg18a

C 20 *

10 Col-0 atg2

(% rosette area) Senescent areas ND ND 0

cat2 atg2 Col-0 cat2 cat2 atg2

atg2 cat2

Figure 4.5. Interactions between peroxisome-generated oxidative stress and atg mutations during growth in short days in high CO2 and air.

(A) Growth in high CO2 for five weeks (no oxidative stress in cat2). (B) Growth in air for six weeks (oxidative stress in cat2)

(C) Growth in high CO2 for four weeks then in air for one week (one week of oxidative stress in cat2). *Significant difference between atg2 and cat2 atg2 at P<0.05 (n = 8 different plants).

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4.2.3 Impact of atg mutations on cat2-triggered lesions and bacterial resistance

Previous studies of cat2 have established that the lesion formation is SA-dependent (Chapter 2) and does not occur in short day growth conditions (Figure 4.1; Queval et al., 2007). Introduction of atg mutations into cat2 does not promote lesions in short day conditions (Figure 4.5). We therefore compared the impact of atg mutations on cat2 phenotypes in long days, conditions that are permissive for cat2-triggered SA accumulation and spreading SA-dependent lesions. This analysis revealed little effect of any of the atg mutations on lesion formation in cat2, whether lesions were induced during growth from seed in air

(Figure 4.6A) or following transfer to air after initial growth at high CO2 (Figure 4.6B). Because the cat2 mutation induces enhanced resistance to bacteria, the influence of the atg mutations on this response was tested. While the phenotypes of atg5 and atg18a were associated with a similar level of induced resistance to that observed in cat2, the atg2 and atg18a mutations both annulled the cat2-triggered resistance (Figure 4.7).

Lesions A (% rosette area) 30

20 Col-0 atg2 atg5 atg18a 10

0

cat2 cat2 atg5 cat2 cat2 atg2 cat2 atg5 cat2 atg18a cat2 atg2 cat2 atg18a

B Lesions (% rosette area) 30

20 Col-0 atg2 atg5 atg18a 10

0

cat2 cat2 cat2 atg2 cat2 atg5 cat2 atg18a cat2 atg2 cat2 atg5 cat2 atg18a

Figure 4.6. Effect of secondary atg mutations on cat2-triggered formation of salicylic acid-dependent lesions. (A) Growth for three weeks in air in long days to produce constitutive induction of pathogenesis responses in cat2

(B) Growth for three weeks in high CO2 in short days, then eight days in air in long days to trigger pathogenesis responses in cat2

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0 dpi 2 dpi 7 7 a a a a atra growth Bacterial (l ) 6 b b b 6 og -2 b 10

cm cfu

cfu cm 5 5

10 -2 ) (log aa aaa aaa Bacterial growth 4 4

3 3

cat2 cat2 atg2 atg5 atg2 atg5 Col-0 Col-0 atg18a atg18a cat2 atg2 cat2 atg5 cat2 cat2 atg5 cat2 atg2 cat2 cat2 atg18a cat2 cat2 atg18a cat2 Figure 4.7. Effect of secondary atg mutations on Pst resistance induced by the cat2 mutation. Plants were grown as for Figure 4.6B to induce salicylic acid accumulation and Pst resistance in cat2. Different letters indicate significant difference at P<0.05.

4.2.4 Interactions between atg mutations and glutathione

Assay of ascorbate and glutathione as redox markers showed that none of the atg mutations affected ascorbate contents but that glutathione was increased slightly in all mutants grown at high CO2 and also in atg2 grown in air. Thus, there seems to be an overall correlation between early senescence phenotypes (Figure 4.3) and leaf glutathione content (Figure 4.8). To test the possible influence of glutathione, the cad2 mutation was crossed into atg2 (Figure 4.9). As in the cat2 background (Chapters 2 and 3), the cad2 mutation effectively decreased glutathione in cat2 atg2 (Figure 4.9C) but did not greatly affect rosette mass (Figure 4.9B). The cad2 mutation also seemed to have little effect on the early senescence phenotype observed in atg2 (Figure 4.9A). However, interpreting this result is complicated because the interaction between atg2 and cad2 seemed to produce a defect in leaf development, which was observed in the double mutant grown at high CO2 but not in air (Figure 4.9A).

4 0.8 A B 3 0.6 * * * FW) FW) -1 -1 2 0.4 *

1 (µmol.g 0.2 (µmol.g Leaf ascorbate Leaf Leaf glutathione

0 0.0 atg5 atg2 atg5 atg2 atg2 atg5 atg5 atg2 Col-0 Col-0 Col-0 Col-0 atg18a atg18a atg18a atg18a High CO Air High CO Air 2 2

Figure 4.8. Ascorbate and glutathione in atg mutants grown in high CO2 or air. (A) Ascorbate. White bars, reduced form. Black bars, dehydroascorbate. (B) Glutathione. White bars, GSH. Black bars, GSSG. *Significant difference at P<0.05 (n = 3 biological repeats).

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Col-0 cad2 atg2 atg2 cad2 A 2

High CO Col-0 cad2 atg2 atg2 cad2

Air

B C 1000 0.8

FW)

* * -1 500 * 0.4 (mg) * Fresh weight Fresh (µmol.g 0 Leaf glutathione 0.0

atg2 atg2 atg2 atg2 cad2 cad2 cad2 cad2 Col-0 Col-0 Col-0 Col-0 atg2 cad2 atg2 cad2 atg2 atg2 cad2 atg2 cad2 atg2 High CO Air 2 High CO2 Air

Figure 4.9. Phenotype and leaf glutathione contents in atg2 cad2 double mutant.

(A) Phenotypes of plants grown for five weeks in short days at high CO2 or in air. (B) Fresh weight after five weeks growth. *Significant difference at P<0.05 (n = 10 different plants). (C) Leaf glutathione. White bars, GSH. Black bars, GSSG.

4.3 Discussion

Cytoplasmic components are recycled and remobilized by autophagy process (Guiboileau et al., 2012). It is therefore clear that autophagy functions in nutrient remobilization during senescence. If autophagy is the only intracellular process for degradation in senescing leaves, a delayed senescence phenotype should be predicted when the autophagic process is defective. Intriguingly, however, loss of ATG functions causes an early senescence phenotype, indicating that activation of autophagy-independent pathway(s) may function in recycling cytoplasmic materials from old leaves to remobilize nutrients when autophagic process is impaired. Indeed, many amino acid and sugar related compounds are significantly increased in atg2 and atg18a mutants relative to Col-0, and the analyses reported here show that this effect, together with the early senescence phenotype, is much stronger when plant are grown at high CO2.

It has been reported that autophagy can confer tolerance against oxidative stress in plants such as salt and drought, suggesting that autophagy plays some role in the regulation of ROS metabolism (Liu et al., 2009; Shin et al., 2009; Péréz-Péréz et al., 2012). Indeed, after treatment with methyl viologen, which produces

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ROS in chloroplasts or mitochondria in plants, ATG18a RNAi lines display a more chlorotic phenotype relative to Col-0 (Xiong et al., 2007). In addition, atg2 and atg5 mutants were reported to suffer oxidative stress, even in the absence of external stress (Yoshimoto et al., 2009). Inversely, exogenous H2O2 and MV could rapidly induce autophagy (Xiong et al., 2007). Moreover, tolerance of oxidative stress is tightly related with completion of life cycle. Several aspects of the present results suggest that the early senescence occurring in atg2 in high CO2 may be associated with intracellular oxidative stress. First, most of the significantly accumulating metabolites identified in atg2 were also observed to respond similarly in cat2, which triggers intracellular oxidative stress through the photorespiratory pathway (Figure 4.4D).

Second, glutathione accumulation was observed in atg mutants in high CO2 (Figure 4.8). These observations indicate that the atg2 mutation is indeed associated with intracellular oxidative stress but that this is unlikely to be linked to enhanced availability of photorespiratory H2O2 generated in the peroxisomes, because both the phenotypic and metabolomics responses were stronger at high CO2. Based on this logic, we propose that other ROS-generating systems are associated with intracellular oxidative stress in atg2. While it has been proposed that down-regulation of CAT2, leading to enhanced availability of ROS, is a contributor to leaf senescence (Zimmermann et al., 2004; Smykowski et al., 2010), other ROS-producing compartments such as chloroplasts and mitochondria contribute to the intracellular oxidative stress signaling network. Furthermore, transcriptomic profiles of cat2 provide relatively little evidence that senescence programs are activated by loss of catalase function (Queval et al., 2007; Mhamdi et al., 2010b). Indeed, the cat2 mutation did not accelerate early senescence phenotypes of atg mutants; in fact, in atg2 transferred from high CO2 to air, loss of CAT2 function seems to oppose the spread of senescence. This observation could point to negative interactions between photorespiratory

H2O2 and senescence, and maybe suggests that ROS of different origins have differential effects on autophagy-regulated senescence.

Autophagy has been proposed to be involved in the regulation of pathogenesis responses as well as SA-dependent senescence (Hofius et al., 2009; Yoshimoto et al., 2009; Lenz et al., 2011b; Wang et al., 2011). Both atg5 and atg18a mutants show increased resistance to virulent bacterial PstDC3000 and increased SA during this bacterial challenge (Lenz et al., 2011b). As in cat2 (Chaouch et al., 2010), the atg5 mutation was also able to trigger some SA accumulation in the absence of pathogen challenge (Yoshimoto et al., 2009). However, phenotypic observation revealed little effect of atg mutations on cat2 triggered SA-dependent lesion formation (Figure 4.6). In fact, the atg2 and atg18a mutations compromised cat2-triggered induced resistance to PstDC3000 when plants were inoculated at 7 days after transfer from high CO2 to air (Figure 4.7). One possibility is that the potential activation of senescence programs in atg2 and atg18a after 3 weeks growth at high CO2 has an antagonistic effect on cat2-triggered enhanced pathogen resistance. Further experiments to determine pathogen resistance and SA levels using different inoculation times, as well as different pathogens and growth conditions (Figure 4.1), could be informative.

One developmental process in which autophagy could be particularly important is recycling materials from old leaves to allow the development of young leaves. Glutathione is involved in the regulation of sulphur metabolism (Noctor et al., 2012) and also in meristem control (Vernoux et al., 2000; Reichheld et al., 2007; Bashandy et al., 2010). It is therefore intriguing that the atg2 cad2 double mutants exhibit a defect in the development of young leaves in high CO2 (Figure 4.9). While it is not clear why this effect

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should be observed at high CO2 but not in air, it could indicate that glutathione may be degraded by autophagy-independent pathways to provide amino acids for biosynthesis, or act through a regulatory function involving redox control or roles as a sulfur donor in key metabolic pathways. The high

CO2-specific interaction between cad2 and atg2 was observed in two independent experiments, but will require further study both to confirm the effect and to investigate the underlying causes.

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CONCLUSION AND PERSPECTIVES

CHAPTER 5 CONCLUSION AND PERSPECTIVES

5.1 CONCLUSIONS

Numerous studies have demonstrated that H2O2 plays an important role in plant pathogen responses and cell death as a signal, but much remains to be elucidated concerning the details of the underlying processes. In particular, the importance of H2O2-driven changes in cell thiol-disulfide remains to be clearly described. While the glutathione pool is an important component of cell thiol-disulfude status, and is crucial as an antioxidant, its physiological function(s) in oxidative signaling remain(s) largely unknown. Autophagy has also been implicated in processes such as PCD and senescence partly through the modulation of H2O2 metabolism. In this work, we adopted a reverse genetics approach to studying the interaction of glutathione and autophagy with increased H2O2 availability in the regulation of pathogenesis-linked responses (Chapters 2 and 3) and senescence (Chapter 4) in Arabidopsis thaliana. A useful genetic system to test above interactions is the catalase-deficient Arabidopsis mutant, cat2 (Queval et al., 2007). This line is of interest because intracellular H2O2 production occurs in peroxisomes through a physiologically relevant pathway (photorespiration) and it can be easily controlled by growth conditions such as ambient CO2 level and light intensity. Previous work of the laboratory showed that glutathione oxidation and accumulation in the cat2 mutant precedes or accompanies the activation of SA-dependent pathogenesis-associated responses and bacterial resistance (Chaouch et al., 2010). While other work from the laboratory recently reported that down-regulation of SA responses triggered by intracellular H2O2 in cat2 arbohF double mutants are associated with altered glutathione status (Chaouch et al., 2012), it remained unclear how important this altered statusis in linking phytohormone signaling to oxidative stress.

5.1.1 New roles for glutathione in the regulation of defence phytohormone signaling

In Chapter 2 of this thesis, through a direct genetics-based approach in which -ECS activity is compromised, it was shown that blocking glutathione oxidation and accumulation largely annulled cat2-triggered SA responses. We suggested that these effects are difficult to explain in terms of potentially altered oxidative stress intensity when the response of glutathione is blocked because (1) leaf peroxides, NADP(H), ascorbate, and rosette growth are largely unaffected compared to cat2, and (2) the phenotype of cat2 cad2 is quite distinct from cat2 gr1 double mutants, in which oxidative stress is clearly exacerbated compared to cat2. Furthermore, side by side comparison of cat2 cad2 with cat2 npr1, in which thiol-regulated NPR1 function is disabled, revealed little overlap between responses in the two double mutants, and indicates that glutathione oxidation and accumulation in cat2 play a signaling role in coupling enhanced H2O2 availability to SA-dependent responses through the activation of ICS1-derived SA accumulation upstream of NPR1.

JA is another defence phytohormone which is negatively controlled by SA and this antagonistic interaction is considered to be at least partly mediated by NPR1. In Chapter 3, by further analysis of the genetic tools reported in Chapter 2, it was shown that, while the cat2 mutation triggers up-regulation of JA-associated gene expression in parallel to activation of SA pathway, genetically blocking glutathione accumulation attenuates JA-associated gene expression triggered by cat2 mutation, decreasing expression of the JA pathway to close to Col-0 levels. Contrasting responses in the transcript abundance of JA associated genes in cat2 cad2 and cat2 npr1 reveal that the regulation of H2O2-triggered JA signaling by

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glutathione can be mediated by NPR1-independent pathway(s). Overall, the work presented in the Chapters 2 and 3 of this thesis points to new roles for glutathione-dependent processes in linking oxidative stress to downstream SA and JA hormone signaling and the perspective arising from the main parts of the study are discussed below, in section 5.2.

5.1.2 Interactions between autophagy, oxidative stress of peroxisomal origin, and glutathione

As noted in Chapter 4, it has been shown that the function of autophagy is associated with the regulation of senescence associated cell death. Early senescence phenotype occurs in several autophagy-deficient mutants and is dependent on SA signaling, which is associated with increased H2O2 (Yoshimoto et al., 2009). In addition, CAT2 expression is down-regulated at the onset of leaf senescence (Zimmermann et al., 2004). However, given the close association of leaf CAT2 with photorespiration, decreased expression of this enzyme is in line with an overall increase in photosynthetic competence as leaves undergo a transition from classical carbon-producing source organs to become a source of remobilized nutrients for reproductive organs. The specific importance of peroxisomally-sourced H2O2 and CAT2 expression in regulating senescence therefore remains to be established.

The analysis reported in Chapter 4 confirmed the early senescence phenotypes described by other groups, but also reveal that the early-senescence phenotypes of atg mutants are enhanced when plants are grown at high CO2. A comparison of metabolite profiles with those observed in cat2 suggests that the senescence phenotypes may involve some of the same processes as those elicited by intracellular oxidative stress. However, the phenotypes observed when atg mutants are crossed with cat2 provide little evidence that enhanced oxidative stress of peroxisomal origin promotes these senescence processes; in fact, it seems that the cat2 mutation has an antagonistic effect, acting rather to delay the development of the atg phenotypes. The lack of induction of a generalized senescence programme in cat2 has been previously discussed (Queval et al., 2007; Mhamdi et al., 2010b). While the cat2 mutation can clearly induce SA-dependent lesions (Chapter 2), it is also possible that in certain conditions or genotypes, ROS act as a pro-life signal or that SA-dependent HR-like cell death caused by excess intracellular H2O2 is in opposition to senescence programmes. A study of cat2 atg double mutants in conditions promoting SA-dependent HR-like lesions (long days) also generated little evidence that the corresponding ATG genes play an important non-redundant role in controlling this response. Clearly, the results of Chapter 4 will need to be extended by further analyses, and some perspectives are discussed at the end of this chapter.

5.2 PERSPECTIVES

5.2.1 Potential mechanisms on the effects of blocking glutathione accumulation in response to enhanced H2O2 availability

While the genetic analysis reported in Chapters 2 and 3 provide clear evidence for potential roles of glutathione status in transmitting signals downstream of H2O2, the details of the underlying mechanisms remain to be described. The double and triple mutants described in these chapters could be very useful tools for further analyses based on cellular and biochemical approaches, twinned with the production of

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other mutants to genetically test the importance of specific proteins involved in glutathione metabolism or glutathione-dependent reactions. Some of these possibilities are now discussed.

5.2.1.1 Glutathione oxidation and metabolism in response to H2O2

Glutathione in cat2 is about 2-3 fold higher than in Col-0, and accumulates essentially in the form of GSSG (Chapters 2 and 3). Exogenous GSSG treatment has some positive effects both on SA and JA associated gene expression, indicating that GSSG produced from H2O2-triggered GSH oxidation may act as an indirect signal of H2O2 levels to transmit information to activate downstream phytohormone responses. A role for GSSG is possible based on the fact that the cad2 and allelic mutations not only block glutathione accumulation in cat2 but also cause the glutathione pool to stay close to the highly reduced values observed in Col-0. The reasons underlying this surprising observation are not yet clear. A key uncertainty relates to the mechanism of GSSG production in cat2, an issue related to the interdependence of ascorbate and glutathione pools in reductive metabolism of H2O2 (Foyer and Noctor, 2011). When catalase is deficient, APX may be the major enzyme that is engaged to deal with the extra H2O2; in this case, GSH oxidation to GSSG could occur through DHA reduction, either chemically or catalyzed by DHAR. However, several other types of enzyme could catalyze GSSG production from GSH, independent of the ascorbate pool. These questions are currently being analyzed by production of cat2 double mutants in the laboratory.

Cys-gly, which is a product of glutathione degradation by -glutamyl transpeptidases (GGT), accumulated to significantly higher levels in cat2 relative to Col-0 (Chapter 3), suggesting that the marked accumulation of glutathione in cat2 stimulates not only glutathione neosynthesis but also turnover. In plants, GGT1 is probably the major apoplastic enzyme for glutathione degradation (Martin et al., 2007; Ohkama-Ohtsu et al., 2007a). Moreover, analyses of compartmentation of glutathione were performed using in situ immunolabelling (Chapter 2), which can be used to detect glutathione in different organelles of Arabidopsis leaf and root cells (Zechmann et al., 2008). Glutathione accumulation in cat2 occurs in most organelles, but the increase relative to Col-0 is strongest in the vacuoles (Queval et al., 2011; Chapter 2), where another GGT (GGT4) is specifically located. To explore the influence of these pathways, mutations for GGT1 and GGT4 have been crossed with cat2 and the characterization of double mutants is underway in the laboratory. Concerning glutathione accumulation in the vacuole, multi-drug resistance associated proteins (MRPs) are the primary candidates to perform transport from the cytosol (Lu et al., 1998). Indeed, several mrp genes were reported to be up-regulated in microarray studies of cat2 (Mhamdi et al., 2010a; Queval et al., 2012). To analyze whether any of the encoded proteins play an important role in glutathione homeostasis during oxidative stress, several double cat2 mrp mutants have been produced and these lines are currently being studied.

5.2.1.2 Glutathione, H2O2 signaling, and protein thiol-disfufide status

Lesion formation and assaociated pathogenesis responses in cat2 do not seem to be caused simply by widespread, indiscriminate, irreversible cellular damage, because they can be prevented simply by blocking SA synthesis (Chaouch et al. 2010). Nevertheless, with the notable exceptions of two genes identified in the singlet oxygen-accumulating flu mutant (Wagner et al., 2004), classical genetic screens

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have failed to identify “H2O2 sensors”. This may reflect chance or limitations in experimental design. In this respect, secondary mutagenesis of the cat2 mutant could produce interesting results, and a search for cell death revertants in chemically mutagenized cat2 is currently in progress in the laboratory of Frank

Van Breusegem (Ghent, Belgium). An alternative possibility to explain unidentified “H2O2 receptors” is that H2O2 signaling is mediated by multiple reactions operating in parallel. If so, the data shown here in Chapters 2 and 3 suggest that these reactions are influenced by glutathione: the down-regulation of both SA and JA signaling when glutathione synthesis is blocked point to new thiol-sensitive signals, in addition to the previously described NPR1 protein. Given the possible involvement of glutathione in influencing protein thiol-disulfide status, such reactions could involve oxidation of sensitive protein thiol groups that form intra- or intermolecular disulfides or mixed disulfides with glutathione as the redox potential of the free glutathione pool becomes more positive. Hence, it would be interesting to examine the response in cat2 and cat2 cad2 of green fluorescent proteins that are sensitive to the glutathione redox potential (roGFP; Jiang et al., 2006; Meyer et al., 2007). While reliable data of this type would be informative, they would not demonstrate that the glutathione redox potential is the factor mediating the signaling. Applying this approach in cat2 and derived mutants is complicated by the need to measure the signal with accuracy in underlying leaf mesophyll cells, where the initial H2O2 trigger is expected to occur in CAT2-deficient lines. Because of technical difficulties, most studies to date have used roGFP in non-photosynthetic epidermal cells. The stomatal guard cells, epidermal cells that are photosynthetic, could be an interesting compromise to obtain interesting data here. A comparison of glutathionylated proteins in cat2 and cat2 cad2 could identify possible candidates involved in the signaling cascade. Here, it is important to consider quantitative aspects as the effects of glutathionylation on the biological activity of a protein may be progressive and gradual, rather than all or nothing. Such an approach is envisaged in collaboration with S Lemaire (Paris, France).

5.2.1.3 H2O2, NO, and glutathione

Although the individual roles of NO and H2O2 in plant defence against pathogens is well established, the two molecules interact to activate the HR and pathogen resistance (Delledonne et al., 1998, 2001). Indeed, the NO donor sodium nitroprusside (SNP) provokes cell death in catalase-deficient tobacco leaves exposed to moderate light stress (Zago et al., 2006). Work over recent years has established S-nitrosoglutathione (GSNO) as an important reservoir of NO that is likely a physiologically important reagent involved in modifying protein thiol groups through either S-nitrosylation or S-glutathionylation (eg, Lindermayr et al., 2010). During the first year of my PhD study, I performed experiments seeking to examine the effect of NO on responses in the cat2 mutant. However, treatment with exogenous SNP under standard growth conditions did not accelerate or enhance lesions in cat2. One explanation is that increased H2O2-triggered intracellular NO induction is sufficient for execution of HR-like responses or that exogenously supplied SNP may not adequately mimic endogenous NO production. The response of endogenous NO to oxidative stress in cat2 remains to be established. Because of technical difficulties with the most commonly used techniques for NO detection, I did not attempt to resolve this question. Despite their widespread use for over a decade in plants, the limitations of in vivo studies using NO-generating agents and NO detection are becoming increasingly recognized. Unfortunately, well-defined genetic resources for studies of NO also remain limited. Although a nitric oxide synthase

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(NOS) has been identified in the green algae Ostreococcus tauri, the exact source of NO in higher plants has not yet been elucidated (Foresi et al., 2010).

Despite doubt over the existence of a specific NOS in plants, three proteins potentially involved in NO production or metabolism have been described, including nitrate reductase (NR), GSNO reductase

(GSNOR), and nitric oxide associated 1 (NOA1). With the aim of exploring interactions between H2O2, glutathione and NO, I introduced mutations for some of these systems into cat2. So far, functional analysis remains preliminary, although it seems that the cat2 noa1 double mutant shows much decreased lesion formation compared to cat2 (Y. Han, unpublished results). However, like the noa1 single mutant, cat2 noa1 shows a pale green phenotype (compared to Col-0 and cat2). Indeed, other authors have concluded that impaired NO production in noa1 is indirectly due to the impact of normal chloroplast function (Flores-Pérez et al., 2008; Gas et al., 2009). Furthermore, it has been reported from quantitative proteomic analyses that several photorespiratory enzymes were down-regulated in noa1 RNAi mutants, and that these lines also show decreased chlorophyll content and photosynthetic capacity (Liu et al., 2010b). Additionally, treating the double cat2 noa1 mutant with SNP did not restore the cat2-triggered lesion phenotype (Y. Han, unpublished results). Hence, we cannot discount the possibility that decreased lesion formation in cat2 noa1 results from the attenuation of oxidative stress intensity through decreased photorespiratory flux rather than the function of NO signaling per se.

The cytosolic enzyme, NR, which is required to catalyze the first step of nitrate assimilation, is encoded by two genes (NIA1 and NIA2) in Arabidopsis. This enzyme can also contribute to the reduction of nitrite to NO. Thus, the triple mutant cat2 nia1 nia2 is presently under construction although, like the noa1 mutation, possible effects on plant nutritional or energy status could complicate the interpretation of any observed effects. Mutants for GSNOR would seem to be particularly interesting to further explore roles for NO-linked processes in the observations reported in Chapters 2 and 3. Given our results in cat2 cad2, it would be interesting to establish whether the effects of blocking glutathione accumulation are mediated by impaired GSNO levels and associated S-nitrosylation processes. Moreover, GSNOR is a key player in regulating S-nitrosylation, because the enzyme catalyzes the NADH-dependent reduction of GSNO to S-aminoglutathione, which then decomposes to free GSH and ammonia or other compounds (Sakamoto et al., 2002; Barroso et al., 2006; Díaz et al., 2003). In Arabidopsis, GSNOR appears to be a cytosolic protein encoded by a single gene, GSNOR1 (Martínez et al., 1996). A double mutant cat2 gsnor1 is currently being produced and characterized in the laboratory by Dr. A Mhamdi.

5.2.1.4 Glutathione and calcium signaling

One of the earliest steps in the plant response to many pathogens involves an increase in cytosolic calcium levels. Close interaction have been described between intracellular oxidative stress and cytosolic calcium in response to pathogen attack. Indeed, increases in cytosolic calcium can be accompanied by activation of SA-dependent responses as well as the production of ROS (Ma et al., 2009). Calcium-dependent calmodulin binding to the CBP60g protein has been shown to be essential for its function in SA signaling (Wang et al., 2009a). It has been also reported that cytosolic calcium concentrations and SA-dependent PR1 gene expression can both be modulated by glutathione content, suggesting a possible interaction between calcium signaling and glutathione in the regulation of

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SA-dependent responses (Gomez et al., 2004a). Interestingly, at least one glutamate receptor identified in plants is sensitive to glutathione and may be involved in linking glutathione contents to calcium flux into the cytosol (Qi et al., 2006). Additionally, catalase activity may be activated by calmodulin binding in the presence of calcium (Yang and Poovaiah, 2002). Finally, at least some glutaredoxins have been identified that may interact with ion channels (Cheng and Hirschi, 2003). In view of these elements, and the data presented in Chapters 2 and 3, it would be interesting to study the effects of crossing cat2 with mutants for candidate genes involved in linking glutathione status to ion channel activity or for transporters that could link H2O2 signaling to changes in calcium concentrations.

5.2.1.5 Glutathione and the jasmonic acid pathway

SA generally antagonizes JA signaling in plants by processes that are at least partly dependent on NPR1. In addition, SA antagonism of JA signaling was abolished by the glutathione biosynthesis inhibitor, BSO, suggesting that glutathione status plays a key role in the crosstalk between SA and JA signaling (Koornneef et al., 2008). Results shown in Chapter 3 strongly suggest the existence of novel glutathione-dependent processes that can regulate JA signaling independently of NPR1. While the data shown in Chapter 2 indicate that glutathione is somehow involved in determining the accumulation of SA itself in response to oxidative stress, we do not yet know whether effects on the JA pathway are linked to changes in JA accumulation. This question is currently under investigation. Inversely, it could be interesting to analyze glutathione contents and glutathione-associated gene expression when JA synthesis mutants are introduced into the cat2 background. Futhermore, it has been reported that overexpression of GRX480 completely blocked the induction of the JA marker gene PDF1.2 by MeJA in an NPR1-independent manner (Ndamukong et al., 2007). Interestingly, in our microarray analysis of cad2, expression of GRX480 was modulated in cad2 in a similar way to other genes associated with JA function. Hence, genetic and physiological characterizations of the effect of the GRX480 mutation in the cat2 background will possibly give more information on the interaction of glutathione with hormone signaling. As well as GRX480, potentially redundant genes encoding this type of glutaredoxin could play some role in the effects. On the other hand, recent studies have pointed to functional overlap between glutathione and cytosolic NADPH-dependent thioredoxin reductase (NTR) system in the control of developmental processes (Reichheld et al., 2007; Marty et al., 2009; Bashandy et al., 2010). It would be interesting to investigate interactions between glutathione and NTR systems in H2O2 signaling (e.g. by collaborations with JP Reichheld, Perpignan). Finally, it could also be interesting to examine whether some of the effects observed in the Chapters 2 and 3 are relevant to agriculturally important species such as barley and rice in which catalase deficient mutants are available (Kendall et al., 1983; Lin et al., 2012). For example, work in our laboratory has confirmed that the barley catalase mutant shows visible lesion formation and accumulation and oxidation of glutathione that is very similar to effects observed in cat2 (J. Neukermans, unpublished results). Analysis of such plants could be of interest, for example, to test effects of blocking glutathione accumulation using pharmacological approaches.

5.2.2 Autophagy and oxidative stress

The data shown in Chapter 4 remain somewhat preliminary, and several obvious complementary analyses are apparent. Because the onset of the early senescence phenotype in the atg mutants seems to be

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accelerated in high CO2 compared to air, it would be useful to investigate the underlying processes more closely using molecular markers such as selected senescence associated genes (eg. SAG12) in the two conditions. It has been reported that early senescence phenotypes in atg mutants are dependent on the SA pathway (Yoshimoto et al., 2009). Therefore, it would be interesting to understand whether the delayed senescence phenotype in double mutants of cat2 atg in short days is linked to compromised SA levels. As well as analysis of SA levels, this could include crosses with the sid2 mutant (Chapter 2). Additionally, although peroxisomal H2O2-triggered oxidative stress seems to have a delaying effect on the atg senescence phenotypes, other potential ROS-generating systems could interact with ATG functions to determine this response, perhaps in antagonistic ways to the cat2 mutation. For instance, ascorbate peroxidase 1 (APX1) and AtRbohF could be players in oxidative stress-associated leaf senescence, and have been suggested as key actors in the regulation of intracellular oxidative stress signaling (Vanderauwera et al., 2011; Chaouch et al., 2012). It would be interesting to produce double mutants such as atg2 apx1 and atg2 atrbohF to further investigate possible interactions of ROS with atg-triggered senescence and the location and source of oxidative stress with which ATG interact.

It should be noted that although the early senescence phenotypes of atg mutants have been linked to SA, they are morphologically quite distinct from the SA-dependent lesions triggered by the cat2 mutation in long days, which are very similar to HR-like processes triggered by certain pathogens. Although the cat2 HR-like phenotype was not affected by the atg mutations, decreased resistance to PstDC3000 in cat2 atg2 and cat2 at18a relative to cat2 was observed, suggesting that some of SA-dependent pathogenesis responses activated in cat2 could be regulated by the autophagic process. Hence, SA levels and SA-dependent marker genes should be examined in these conditions. Moreover, time course analyses of resistance to other pathogens as well as PstDC3000 in atg single and double mutants will be useful to extend our understanding of the role of autophagy in oxidative stress-triggered pathogen resistance.

Very preliminary results indicate that combining atg2 and cad2 double mutants affects leaf development at an early stage, when plants are grown at high CO2. One possibility is that this reflects a role for glutathione in the regulation of nutrient remobilization. Indeed, glutathione is well known to be a form of intercellular transport of organic sulfur in plants. Further investigation of this interaction would require genetic confirmation by crossing atg2 with other allelic glutathione-deficient mutants such as pad2 and rax1.

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CHAPTER 6

MATERIALS AND METHODS

CHAPTER 6 MATERIALS AND METHODS

6.1 Plant material and growth conditions

The Arabidopsis mutant lines used in this study were all in the Columbia genetic background and are listed in Table 6.1. Double and triple mutants were produced by crossing. After verification of double heterozygosity in F1 plants, F2 individuals were genotyped to identify double homozygotes. Genotyping of the new mutants produced for the analyses described in Chapters 2 and 3 is depicted in Figure 6.1. Functional analysis was performed on F3 plants.

A

cat2 CAT2 CAT2 CAD2 cad2 CAD2 cat2 cat2 CAT2 CAD2 cad2 cad2

624 bp 600 bp 507 bp 600 bp 400 bp 362 bp 400 bp WP MP WP MP WP MP 200 bp 200 bp 123 bp

Genotyping at CAT2 locus Genotyping at CAD2 (GSH1) locus

B C sensCAT2 + revCAT2

sensCAT2 + LB1

sensCAD2 + revCAD2 + Genotyping at NPR1 locus Digestion

sensGR1 + revGR1

500 bp sensGR1 + LB1 250 bp sensSID2 + revSID2 + Digestion

npr1 Col-0 Col-0

cat2 npr1

cat2 gr1 sid2 gr1 cat2 cat2 cad2 gr1 cad2 cat2 cat2 cad2 sid2 cat2 cad2 npr1 cat2 cad2

Figure 6.1. Genotyping of double and triple mutants. (A) Genotyping of cat2 cad2 mutant. MP, mutant primer pair. WP, wild-type primer pair. (B) Genotyping at the NPR1 locus to obtain cat2 npr1 and cat2 cad2 npr1 mutants. (C) Genotyping of other triple mutants by crossing cat2 cad2 with cat2 sid2 (Chaouch et al., 2010) and cat2 gr1 (Mhamdi et al., 2010a).

94 CHAPTER 6 MATERIALS AND METHODS

Table 6.1. Arabidopsis thaliana Col-0 background mutant lines used within this study. AGI, Arabidopsis gene identifier.

Line AGI code Origin Single mutant cat2 At4g35090 Salk_057998; Queval et al., 2007 pad2 At4g23100 Point mutant; Parisy et al., 2007 cad2 At4g23100 Deletion mutation; Cobbett et al., 1998 rax1 At4g23100 Point mutation; Ball et al., 2004 gr1 At3g24170 Salk_060425; Mhamdi et al., 2010a sid2 At1g74710 Point mutation; Wildermuth et al., 2001 npr1 At1g64280 Point mutation; Cao et al., 1994 atg2 At3g19190 Salk_076727; Inoue et al., 2006 atg5 At5g17290 Sail_129B07; Inoue et al., 2006 atg18a At3g62770 Gabi_651D08; Lenz et al., 2011b Double mutants cat2 pad2 New construction cat2 cad2 New construction cat2 rax1 New construction cat2 gr1 Mhamdi et al., 2010a cat2 sid2 Chaouch et al., 2010 cat2 atg2 New construction cat2 atg5 New construction cat2 atg18a New construction atg2 cad2 New construction Triple mutant cat2 cad2 gr1 New construction cat2 cad2 sid2 New construction car2 gr1 sid2 New construction cat2 cad2 npr1 New construction

Unless mentioned otherwise, seeds were directly sown on soil in square 7 cm pots and incubated for 2 d at 4ºC in the dark. Plants were grown in a controlled-environment growth chamber in a 16h (long days) or 8h (short days) photoperiod and an irradiance of 200 μmol m-2 s-1 at leaf level, 20ºC day/18ºC night temperature, 65% humidity, and given nutrient solution twice per week. The CO2 concentration was -1 -1 maintained at 400 μL L (air) or 3000 μL L (high CO2). For buthionine sulfoximine (BSO) treatment Col-0 and the two allelic glutathione-deficient lines, cad2 and rax1, were grown on agar in 0.5x Murashige-Skoog medium without sugar for two weeks at an irradiance of 40 μmol m-2 s-1. BSO was supplied to Col-0 plants in the medium at the indicated concentration. Samples were rapidly frozen in liquid nitrogen and stored at -80ºC until analysis. For complementation experiments, plants were grown on soil at high CO2 as described above. On transfer from high CO2 to air, rosettes were sprayed once daily with 1 mM cysteine, 1 mM GSH or 1 mM GSSG. Unless otherwise stated, data are means ± SE of at least three independent samples from different plants.

6.2 Methods

6.2.1 Molecular biology analyses

6.2.1.1 DNA extraction and plant genotyping

Leaf DNA was extracted in 400 μL extraction buffer (200 mM Tris pH7.5, 250 mM NaCl, 25 mM EDTA and 0.5%SDS) using a bead shaker. The obtained solution was centrifuged for 10 min at 10 000g at 4ºC. 200 μL supernatant was mixed well with 200 μL isopropanol and centrifuged for 10 min at room temperature. The pellet was washed with 1 mL 70% ethanol (v/v), dried and then redissolved in 100 μL

95 CHAPTER 6 MATERIALS AND METHODS

TE buffer (10 mM Tris pH 8.0, 1mM EDTA). For T-DNA insertion lines, leaf DNA was amplified by PCR using primers specific for left T-DNA borders (Table 6.2). Zygosity was analyzed by PCR amplification of leaf DNA. Homozygote of the point/deletion mutations was established using restriction length polymorphism (Table 6.2).

6.2.1.2 RT-qPCR

Total RNA was extracted with TRIzol (Invitrogen) following the manufacturer’s instructions. RNA quality and concentration were determined by gel electrophoresis and estimated using a nanodrop spectrophotometer at 260 nm respectively. Reverse transcription and first-strand cDNA synthesis were performed using the SuperScript III First-Strand Synthesis System (Invitrogen). qPCR was performed according to Queval et al. (2007). Primer sequences are listed in Table 6.2.

Table 6.2. DNA primers and restriction endonucleases used in this study

N° Oligonucleotide Sequence Restriction enzyme Technique 1 ForCAT2 5’-CCCAGAGGTACCTCTTCTTCTCCCATG-3’ Genotyping 2 RevCAT2 5’-TCAGGGAACTTCATCCCATCGC-3’ Genotyping 3 ForPAD2 5’-CTGACTTTGCGCCTTTTCAT-3’ DdeI Genotyping 4 RevPAD2 5’-TGCGAGACAGAGTCATTGGT-3’ Genotyping 5 ForCAD2 5’-AGCATTTCACTTGAACCTGG-3’ BsI1 Genotyping 6 RevCAD2 5’-TCCTTGTCAGTGTCTGTCC-3’ Genotyping 7 ForGR1 5’-TTCTGGGGAAACATTCTCCTCTTGC-3’ Genotyping 8 RevGR1 5’-GAGGTAACTAGTGCCTGCAATATGACACAC-3’ Genotyping 9 ForSID2 5’-GCTCTGCAGCTTCAATGC-3’ Tru9I Genotyping 10 RevSID2 5’-CGAAGAAATGAAGAGCTTGG-3’ Genotyping 11 ForNPR1 5’-CTCGAATGTACATAAGGC-3’ NlaIII Genotyping 12 RevNPR1 5’-CGGTTCTACCTTCCAAAG-3’ Genotyping 13 ForATG2 5’-GCACTTTCCATCAGCTACTCG-3’ Genotyping 14 RevATG2 5’-CATTCGAGGTTCTGGCCTAAC-3’ Genotyping 15 ForATG5 5’-GCTAATTGCACAAAGCTTACCTC-3’ Genotyping 16 RevATG5 5’-TGATATGCCTAACATCGTCCAC-3’ Genotyping 17 ForATG18A 5’-AACCCTAATCCCGATTCCAC-3’ Genotyping 18 ForATG18A 5’-ACATGGACCGTTCCTTTGTC-3’ Genotyping 19 Lb1 5’-TGGACCGCTTGCTGCAACTCTC-3’ Genotyping 20 ForACTIN2 5’-CTGTACGGTAACATTGTGCTCAG-3’ RT-qPCR 21 RevACTIN2 5’-CCGATCCAGACACTGTACTTCC-3’ RT-qPCR 22 ForPR1 5’-AGGCTAACTACAACTACGCTGCG-3’ RT-qPCR 23 RevPR1 5’-GCTTCTCGTTCACATAATTCCCAC-3’ RT-qPCR 24 ForPR2 5’-TCAAGGAGCTTAGCCTCACC-3’ RT-qPCR 25 ForPR2 5’-CGCCTAGCATCCCGTAGC-3’ RT-qPCR 26 ForICS1 5’-TTGGTGGCGAGGAGAGTG-3’ RT-qPCR 27 RevICS1 5’-CTTCCAGCTACTATCCCTGTCC-3’ RT-qPCR 28 ForPDF1.2A 5’-CCAAACATGGATCATGCAAC-3’ RT-qPCR 29 RevPDF1.2A 5’-CACACGATTTAGCACCAAAGA-3’ RT-qPCR 30 ForVSP2 5’-TACGACTCCAAAACCGTGTG-3’ RT-qPCR 31 RevVSP2 5’-GACGGTGTCGTTCTTTAGGG-3’ RT-qPCR 32 ForLOX3 5’-GTGGCCGGAGTTATCAACC-3’ RT-qPCR 33 RevLOX3 5’-GGGACGTAGCCACCGTAAG-3’ RT-qPCR 34 ForOPR3 5’-GGCTCAAAGCTCGCTTACC-3’ RT-qPCR 35 RevOPR3 5’-ACTCCCTTGCCTTCCAGACT-3’ RT-qPCR 36 ForJAZ10 5’-CATCGGCTAAATCTCGTTCG-3’ RT-qPCR 37 RevJAZ10 5’-CGGTACTAGACCTGGCGAGA-3’ RT-qPCR

6.2.1.3 CATMA microarray analyses and data-mining

Microarray analysis was performed using the CATMA arrays containing 24,576 gene-specific tags

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corresponding to 22,089 genes from Arabidopsis (Crowe et al., 2003; Hilson et al., 2004) plus 1,217 probes for microRNA genes and putative small RNA precursors (information available at http://urgv. evry.inra.fr/projects/FLAGdb++). Samples of approximately 200 mg fresh weight were collected from plants after 4 days transfer when plant were initially grown at high CO2 3 weeks. To determine differentially expressed genes, a paired t test was performed on the log ratios, assuming that the variance of the log ratios was the same for all genes. The raw P values were adjusted by the Bonferroni method, which controls the family wise error rate (with a type I error equal to 5%) in order to keep a strong control of the false positives in a multiple comparison context (Ge et al., 2003). Differentially expressed genes were considered to be those with a Bonferroni P ≤ 0.05, as described by Gagnot et al. (2008). Only genes that showed same direction statistically significant changes in biologically independent replicates were considered as statistically significant. The full dataset is available at http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_expce.pl?experiment_id=256.

6.2.2 Lesion quantification and pathogen tests

Percentages of lesion areas were quantified using IQmaterials software. Growth of virulent Pseudomonas syringae pv. tomato strain DC3000 was assessed 48 h post-inoculation according to Chaouch et al. (2010). Three of the middle leaves from 5 to 7 different plants of each genotype were inoculated using a 1-mL syringe without a needle with virulent Pst DC3000 in a medium titer of 5 x 105 colony-forming units mL-1. Leaf discs (0.5 cm2 each) were taken for analysis either immediately (0h) or 48 h later. Four to six samples were made by pooling two leaf discs from different inoculated leaves. Bacterial growth was assessed by homogenizing leaf discs in 400 μL of H2O, plating appropriate dilutions on soild LB medium containing rifampicin (100 μg/mL) and kanamycin (25 μg/mL), and counting colony numbers after 3 days.

6.2.3 Cytohistochemical analysis

Sample preparation for cytohistochemical investigations was performed as previously described in detail (Zechmann et al., 2007; Queval et al., 2011). Immunolocalization of glutathione on samples prepared in Orsay was performed by Dr Bernd Zechmann (University of Graz, Austria) according to Zechmann and Müller (2010). Subcellular concentrations of glutathione from immunogold labelling densities were estimated according to Queval et al. (2011). Concentrations for each genotype were based on global leaf GSH + GSSG contents measured in Col-0, cat2, and cat2 cad2. The amount of glutathione in each compartment (nmol g-1 FW) was obtained by multiplying the leaf glutathione contents by the measured fractional contribution of each compartment to the overall gold label. From these values, concentrations were calculated using sub-cellular volumes estimated in leaf sections of each genotype. Measurements of the percentage volume for each compartment were estimated according to Queval et al. (2011). Subcellular volumes for each cell compartment were finally calculated per g fresh weight from the percentage volumes based on a mesophyll volume per leaf mass of 773 μL g-1FW (Winter et al., 1994).

6.2.4 Metabolite analyses

6.2.4.1 Assays for ascorbate, glutathione and NADP(H)

97 CHAPTER 6 MATERIALS AND METHODS

Oxidized and reduced forms of ascorbate, glutathione and NADP(H) were assayed using a plate-reader method previously described in Queval and Noctor (2007), as described in detail below. Total glutathione (GSH+GSSG) was also measured in acid extracts, along with other non-protein thiols, by HPLC-fluorescence.

6.2.4.1.1 Extraction

Ascorbate, dehydroascorbate (DHA), reduced glutathione (GSH), glutathione disulfide (GSSG) and NADP+ were extracted into 0.2 mM HCl while NADPH was extracted in 0.2 M NaOH. To measure ascorbate, glutathione, thiols and NADP+, approximately 100 mg of leaf tissue was ground in liquid nitrogen with a pestle and mortar and then extracted into 1 mL of 0.2 mM HCl. The homogenate was centrifuged at 16 000g for 10 min at 4ºC. An aliquot of 0.5 mL of the supernatant was neutralized to pH 4.5-5 for assaying thiols, glutathione and ascorbate. Another 0.2 mL of the same supernatant was incubated in boiling water for 1 min, rapidly cooled and neutralized to pH 6.5-7 for assaying NADP+. To measure NADPH, parallel leaf material was extracted as for NADP+ except that the extraction solution was 0.2 M NaOH and the heated supernatant aliquot was neutralized with 0.2 mM HCl to a final pH 7-8.

6.2.4.1.2 Quantification of thiols by HPLC

Thiols were measured by HPLC separation of bimane derivatives followed by fluorescence detection, as described in Queval and Noctor (2007). Following neutralization, 0.2 mL extract supernatant was added to 0.1 ml of 0.5 M Ches (pH 8.5) and 20 µL of 10 mM DTT. After incubation at room temperature for 30 min to reduce disulfide forms, all thiols were derivatized by the addition of 20 µL of 30 mM monobromobimane. The mixture was incubated in the dark at room temperature for 15 min, and then the reaction was stopped by the addition of 0.66 mL of 10% (v/v) acetic acid. The derivatized mix was centrifuged at 10,000g for 10 min, and 0.9 mL supernatant was filtered through 0.2 µm mesh into autosampler vials. Vials were loaded into the HPLC, and 50 µL from each vial was injected onto the column by the HPLC autosampler. Bimane derivatives were separated by isocratic elution with 10% methanol and 0.25% acetic acid (pH 4.3) at a flow rate of 0.8 mL/min and a column temperature of 40 °C. Bimane derivatives of Cys, Cys-Gly, -EC and glutathione can be identified and quantified according to this protocol. Peaks were identified by reference to standards and were quantified according to solutions of mixed standard using quadratic curve-fitting available within the Waters Empower software.

6.2.4.1.3 Glutathione measurement by plate reader

This method relies on GR-dependent reduction of DNTB by GSH monitored at 412 nm and is used to measure either total glutathione (GSH + GSSG) or GSSG (Tietze, 1969). Specific assay of GSSG was done by pre-treatment of extract aliquots with 2 μL of 2-vinylpyridine (VPD), which efficiently complexes GSH (Griffith, 1980). To measure total glutathione, the mixture contained 100 μL of 0.2 M

NaH2PO4, 10 mM EDTA (pH 7.5), 10 μL 10 mM NADPH, 10 μL 12 mM DTNB, 10 μL of neutralized extract and 60 μL water. After shaking, the reaction was started by addition of 10 μL GR. GSSG measurement was based on the same principle except that extract aliquots were incubated with VPD for

98 CHAPTER 6 MATERIALS AND METHODS

30 min at room temperature and were centrifuged twice for 15 min at 4ºC before assay. The increase in absorbance at 412 nm was monitored for 5 min and all assays were done in triplicate for each extract. Rates were converted to quantities of glutathione using standard curves generated by assay of known concentration in the same plate.

6.2.4.1.4 Ascorbate

Ascorbate was measured by the absorbance at 265 nm that is specifically removed by ascorbate oxidase (AO). The reduced form was measured in untreated acid extract aliquots, while total ascorbate (DHA + ascorbate) were measured following pre-incubation of aliquots with DTT to reduce DHA to ascorbate. To assay ascorbate, the buffer contained 100 μL 0.2 M NaH2PO4 (pH 5.6), 40 μL extract and 55 μL H2O. The absorbance at 265 nm was read before and 5 min after adding 0.2 U of AO. Total ascorbate was measured after incubation of 100 μL neutralized extract with 140 μL 0.12 M NaH2PO4 (pH7.5), 10 μL 25 mM DTT for 30 min at room temperature. Both of above assays were done in triplicate for each extract. According -1 -1 to a standard extinction coefficient of 14 mM cm , absorbance changes in A265 were converted to quantities of ascorbate (Queval and Noctor, 2007).

6.2.4.1.5 NADP(H)

The assay involves the phenazine methosulfate (PMS)-catalyzed reduction of dichlorophenolindophenol (DCPIP) in the presence of glucose-6-phosphate (G6P) and glucose-6-phosphate dehydrogenase (G6PDH). NADP+ and NADPH are distinguished by preferential dectruction in acid or base. To measure NADP+ and NADPH, triplicate 20 μL aliquots of neutralized extract (acid extraction for NADP+ and basic extraction for NADPH) were added to plates which contained 100 μL HEPES, 2 mM EDTA (pH 7.5), 20 μL 1.2 mM DCPIP, 10 μL 20 mM PMS, 10 μL 10 mM G6P, and 30 μL water. After shaking, the reaction was started by addition of 10 μL G6PDH. The decrease in absorbance at 600 nm was monitored for 5 min. Pyridine nucleotide contents were calculated using standard curves generated simultaneously by assay of standard solutions on the same plate.

6.2.4.2 Salicylic acid measurement

Salicylic acid (SA) was extracted as described in Langlois-Meurinne et al. (2005). Radio-labelled SA was added to each sample as an internal standard. After extraction, samples were dried in a Speed-Vac. One series of dried samples were used to analyze free SA while another series of dried samples were subjected to acid hydrolysis to assay total SA. SA was assayed using fluorescence detection by High Performance Liquid Chromatography (HPLC). Identification and quantification was performed by comparison of peaks with SA standards. Extraction yields of radioactively marked SA were estimated using a scintillation counter.

6.2.4.3 Non-targeted GC-TOF-MS analysis

Non-targeted metabolite profiling was performed by gas chromatography-time of flight spectrometry (GC-TOF-MS) on triplicate biological repeats as in Noctor et al. (2007), except that a mix of alkanes was

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included during derivatization to aid metabolite identification. Compounds identified by retention index were confirmed by reference to mass spectra libraries. Peak area was quantified based on specific fragments and was corrected on the basis of an internal standard (ribitol) and sample fresh weight. Data display was performed using TMEV4 software. Peak areas were mean-centered and reduced by dividing by the standard deviation across all samples before hierarchical clustering and production of “heat-maps”. To identify significantly different metabolites, peak areas normalized to ribitol and fresh weight were subject to ANOVA (P < 0.05) in TMEV4 or pair-wise t tests in Microsoft Excel.

6.2.4.4 Peroxide assay

Peroxides were measured by luminol luminescence according to Queval et al. (2008). Tissue samples of 50 mg fresh weight were ground to a fine powder in liquid nitrogen and then extracted into 1 mL of 0.2 N HCl. The homogenate was centrifuged for 10 min at 10,000g. The extract was neutralized to pH 5.6 with 0.2 M NaOH. To avoid interference from ascorbate, an aliquot (50 mL) was treated with ascorbate oxidase, and the treated aliquot was added to 0.5 mL of 0.2 M NH3 (pH 9.5), and 0.05 mL of 0.5 mM luminol (prepared in 0.2 M NH3, pH 9.5), vortexed rapidly, and injected into a luminometer. The reaction was started by adding 100 mL of 0.5 mM K3Fe(CN)6 (in NH3), and luminol chemiluminescence was monitored for 2 s. To check the linearity of this assay, a standard curve of H2O2 was done for each experiment, and assay standards and extracts were assayed in triplicate.

6.2.5 Statistical analysis

Unless stated otherwise, the statistical analysis of data was based on Student’s t-tests. Calculations were performed on a minimum of three independent data sets, assuming two sample equal variance and a two-tailed distribution. Unless otherwise indicated, significant difference is expressed using t-test at P < 0.05.

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ANNEX 1A

Mhamdi et al., 2010 Plant Physiol.

ANNEX 1A. Publication by Mhamdi et al. (2010) Plant Physiol. 153: 1144–1160.

This article first reported the cat2 gr1 mutant that was used in Chapter 2 of this study. My main contribution to results shown in this paper was the production and characterization of an allelic double mutant to confirm the cgeneti interaction between cat2 and gr1 knockout mutations (data shown in Annex 1b).

Arabidopsis GLUTATHIONE REDUCTASE1 Plays a Crucial Role in Leaf Responses to Intracellular Hydrogen Peroxide and in Ensuring Appropriate Gene Expression through Both Salicylic Acid and Jasmonic Acid Signaling Pathways1[C][W][OA]

Amna Mhamdi, Jutta Hager, Sejir Chaouch, Guillaume Queval2, Yi Han, Ludivine Taconnat, Patrick Saindrenan, Houda Gouia, Emmanuelle Issakidis-Bourguet, Jean-Pierre Renou, and Graham Noctor* Institut de Biologie des Plantes, UMR8618 CNRS, Universite´ de Paris Sud, 91405 Orsay cedex, France (A.M., J.H., S.C., G.Q., Y.H., P.S., E.I.-B., G.N.); De´partement de Biologie, Faculte´ des Sciences de Tunis, Universite´ de Tunis El Manar, 2092 Tunis, Tunisia (A.M., H.G.); and Plant Genomics Research Unit, Unite´ de Recherche en Ge´nomique Ve´ge´tale, 91057 Evry cedex, France (L.T., J.-P.R.)

Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductase (GR), which is encoded by two genes in Arabidopsis (Arabidopsis thaliana; GR1 and GR2). This study addressed the role of GR1 in hydrogen peroxide (H2O2) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H2O2 signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione (GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven by H2O2. Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines were dependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicating glutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H2O2-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H2O2 availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes, and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSG accumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but there is little or no evidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays a crucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis GR gene or by thiol systems such as the thioredoxin system.

Thiol-disulfide exchange plays crucial roles in pro- redox signaling, and it is principally mediated by tein structure, the regulation of enzymatic activity, and thioredoxin (TRX) and glutathione reductase (GR)/ glutathione systems (Buchanan and Balmer, 2005; Jacquot et al., 2008; Meyer et al., 2008). Arabidopsis 1 This work was supported by the French Agence Nationale de (Arabidopsis thaliana) lines identified in independent la Recherche-GENOPLANTE program Redoxome, by the Tunisian screens for alterations in heavy metal tolerance, mer- Ministry of Higher Education and Scientific Research and the istem function, light signaling, and pathogen resis- Universite´ de Paris Sud XI (to A.M.), by the French Centre National de la Recherche Scientifique (to S.C.), and by the Chinese Scholarship tance have been shown to harbor mutations in the Council (to Y.H.). gene encoding the first enzyme of glutathione synthe- 2 Present address: Department of Plant Systems Biology, Flanders sis (Cobbett et al., 1998; Vernoux et al., 2000; Ball et al., Institute for Biotechnology, and Department of Plant Biotechnology 2004; Parisy et al., 2007). Studies on the rml1 mutant, and Genetics, Ghent University, 9052 Gent, Belgium. which is severely deficient in glutathione synthesis, * Corresponding author; e-mail [email protected]. define a specific role for glutathione in root meristem The author responsible for distribution of materials integral to the function (Vernoux et al., 2000). However, shoot meri- findings presented in this article in accordance with the policy stem function is regulated in a redundant manner by described in the Instructions for Authors (www.plantphysiol.org) is: cytosolic glutathione and TRX (Reichheld et al., 2007), Graham Noctor ([email protected]). providing a first indication for functional overlap [C] Some figures in this article are displayed in color online but in black and white in the print edition. between these thiol-disulfide systems in plant devel- [W] The online version of this article contains Web-only data. opment. [OA] Open Access articles can be viewed online without a sub- Modifications of cellular thiol-disulfide status may scription. be important in transmitting environmental changes www.plantphysiol.org/cgi/doi/10.1104/pp.110.153767 that favor the production of oxidants such as hydrogen

Ò 1144 Plant Physiology , July 2010, Vol. 153, pp. 1144–1160, www.plantphysiol.org Ó 2010 American Society of Plant Biologists Glutathione Reductase in H2O2 Responses

peroxide (H2O2; Foyer et al., 1997; May et al., 1998; ascorbate, down-regulation of cytosolic GR capacity Foyer and Noctor, 2005). The glutathione/GR system should produce a similar effect to APX deficiency. is involved in H2O2 metabolism by reducing dehy- Two genes are annotated to encode GR in plants. Pea droascorbate generated following the (per)oxidation (Pisum sativum) chloroplast GR was the first plastidial of ascorbate (Asada, 1999). This pathway is one way in protein shown also to be targeted to the mitochondria which H2O2 reduction could be coupled to NADPH (Creissen et al., 1995). Dual targeting of this protein oxidation, with the first reaction catalyzed by ascor- also occurs in Arabidopsis (Chew et al., 2003), where bate peroxidase (APX) and the last by GR, although the plastidic/mitochondrial isoform is named GR2. ascorbate regeneration can occur independently of re- The second gene, GR1, is predicted to encode a cyto- duced glutathione (GSH) through NAD(P)H-dependent solic enzyme. In pea, cytosolic GR has been well cha- or (in the chloroplast) ferredoxin-dependent reduc- racterized at the biochemical level (Edwards et al., tion of monodehydroascorbate (MDAR; Asada, 1999). 1990; Stevens et al., 2000), but the functional signifi- Additional complexity of the plant antioxidative cance of the enzyme remains unclear. Underexpres- system has been highlighted by identification of sev- sion or overexpression studies in tobacco and poplar eral other classes of antioxidative peroxidases that (Populus species) have reported significant effects of could reduce H2O2 to water. These include the TRX modifying chloroplast GR capacity (Aono et al., 1993; fusion protein CDSP32 (Rey et al., 2005) and several Broadbent et al., 1995; Foyer et al., 1995; Ding et al., types of peroxiredoxin, many of which are themselves 2009). Less evidence is available supporting an impor- TRX dependent (Dietz, 2003). Plants lack animal- tant role for cytosolic GR. In insects, GSSG reduction type selenocysteine-dependent glutathione peroxi- can also be catalyzed by NADPH-TRX reductases dase (GPX), instead containing Cys-dependent GPX (NTRs; Kanzok et al., 2001), and it has recently been (Eshdat et al., 1997; Rodriguez Milla et al., 2003). De- shown that Arabidopsis cytosolic NTR can function- spite their annotations as GPX, these enzymes are now ally replace GR1 (Marty et al., 2009). thought to use TRX rather than GSH (Iqbal et al., 2006). Therefore, key outstanding issues in the study of However, H2O2 could still oxidize GSH via peroxidatic redox homeostasis and signaling in plants are (1) the glutathione S-transferases (GSTs) and/or glutaredoxin importance of GR/glutathione in H2O2 metabolism (GRX)-dependent peroxiredoxin (Wagner et al., 2002; and/or H2O2 signal transmission and (2) the specific- Jacquot et al., 2008). Metabolism of H2O2 may occur ity of GSH and TRX systems in H2O2 responses. In this through APX or through ascorbate-independent GSH study, we sought to address these questions by a peroxidation, both of which potentially depend on GR genetically based approach in which the effects of activity, as well as through TRX-dependent pathways. modified H2O2 and glutathione were first analyzed in The relative importance of these pathways remains parallel in single mutants and then directly through unclear. the production of double mutants. This was achieved The major sites of intracellular H2O2 production in using gr1 insertion mutants and a catalase-deficient most photosynthetic plant cells are the chloroplasts Arabidopsis line, cat2, in which intracellular H2O2 and peroxisomes (Noctor et al., 2002). Despite this, drives the accumulation of GSSG in a conditional Davletova et al. (2005) demonstrated a crucial role for manner (Queval et al., 2007). Our study provides in cytosolic APX1 in redox homeostasis in Arabidopsis. vivo evidence for a specific irreplaceable role for GR1 This finding implies that the cytosolic ascorbate- in H2O2 metabolism and signaling. glutathione pathway is important in metabolizing H2O2 originating in other organelles, thus marking out this compartment as a key site in which redox signals are RESULTS integrated to drive appropriate responses. Cytosolic Characterization of gr T-DNA Mutants metabolism of H2O2 could be key in setting appropri- ate conditions for thiol-disulfide regulation through While T-DNA insertions in the coding sequence of cytosolic/nuclear signal transmitters such as NPR1 dual-targeted chloroplast/mitochondrial GR2 are and TGA transcription factors (Despre´s et al., 2003; embryo lethal (Tzafrir et al., 2004), homozygous gr1 Mou et al., 2003; Rochon et al., 2006; Tada et al., 2008). mutants were readily obtained. Reverse transcription Because peroxisomal reactions can be a major pro- (RT)-PCR confirmed the absence of GR1 transcript, ducer of H2O2, a related issue that remains unresolved while total extractable GR activity was decreased by is to what extent reductive H2O2 metabolism overlaps 40% relative to ecotype Columbia (Col-0; Supplemen- with metabolism through catalase, which is confined tal Fig. S1). Despite these effects, repeated observa- to peroxisomes and homologous organelles. Intrigu- tions over a period of 5 years showed that the mutation ingly, double antisense tobacco (Nicotiana tabacum) produced no difference in rosette growth rates from lines in which both catalase and cytosolic APX were Col-0 in either short days (SD) or long days (LD) or in down-regulated showed a less marked phenotype flowering timing and leaf number (data not shown). than single antisense lines deficient in either enzyme Thus, our analysis is in agreement with the report of alone (Rizhsky et al., 2002). This observation implies Marty et al. (2009) that absence of cytosolic GR activity that if the major role of the GR-glutathione system is caused a measurable decrease in leaf GR activity but indeed to support reduction of dehydroascorbate to that this did not cause phenotypic effects.

Plant Physiol. Vol. 153, 2010 1145 Mhamdi et al.

Comparative Analysis of Glutathione- and ated via glutathione should also be observed in gr1, H2O2-Dependent Changes in Gene Expression even in the absence of the oxidative stress that drives GSSG accumulation in cat2 (Fig. 1A). gr1 Because mutants are aphenotypic in typical As previously reported, cat2 showed a wild-type growth conditions, this study analyzed the potential phenotype at high CO2. The consequences of the cat2 role of the enzyme in H2O2 metabolism and signaling. mutation for gene expression and phenotype in air are First, we performed parallel microarray analysis of strongly influenced by growth photoperiod (Queval GSSG-accumulating gr1 and the catalase-deficient mu- et al., 2007). Thus, measurements of transcripts and tant cat2, which conditionally accumulates GSSG trig- antioxidant were performed after transfer of gr1 and gered by photorespiratory H O production (Queval 2 2 cat2 from high CO2 to air in both SD (8-h photoperiod) et al., 2007). To compare effects on transcript abun- and LD (16-h photoperiod). In both photoperiods (and dance while minimizing possible interference of long- at high CO2), the glutathione reduction state was lower term developmental effects due to oxidative stress in in gr1 than in Col-0 (Fig. 1B). In cat2, however, this cat2 grown from seed in air, plants were sampled factor was only lower than in Col-0 after transfer to air, following initial growth at high CO2, where the cat2 where it was decreased below the values observed mutation is silent (Queval et al., 2007). This experi- in gr1. In both mutants, leaf ascorbate contents were mental design was chosen to allow comparison of much less affected than glutathione. The only signif- the effects of modified glutathione status in gr1 with icant difference was observed in ascorbate reduction similar H2O2-induced effects triggered in cat2 after state in cat2 in LD (Supplemental Fig. S2). transfer from high CO2 to air. Our rationale was that Analysis of microarray data was performed by while H2O2 may modify gene expression through a considering as being differentially expressed genes number of signaling mechanisms, any effects medi- with a Bonferroni P # 0.05, as described by Gagnot

Figure 1. Comparison of H2O2- and glutathione- regulated changes in gene expression. A, Scheme depicting experimental strategy. B, Glutathione reduction states (100 GSH/total glutathione) in

Col-0, gr1, and cat2 in high CO2 or in air in 8-h (SD) or 16-h (LD) growth photoperiods. Different letters indicate significant difference between genotypes at P , 0.05. The numbers show the GSH to GSSG ratios. Data are means 6 SE of five to seven independent extracts. C, Overlap in significantly induced or repressed genes in cat2 or gr1 following induction of glutathione oxida- tion in cat2 after transfer from high CO2 to air. [See online article for color version of this figure.]

1146 Plant Physiol. Vol. 153, 2010 Glutathione Reductase in H2O2 Responses et al. (2008). An additional selection criterion was Sixteen of the 58 gr1-sensitive genes are annotated as introduced by considering only genes that showed phytohormone associated. In all cases, induction or statistically significant same-direction change in repression of these genes was dependent on daylength both biological replicates. The analysis showed that context (Table I). A putative monoxygenase with sim- H2O2-regulated gene expression (in cat2) was highly ilarity to salicylic acid (SA)-degrading enzymes was daylength dependent (Fig. 1C). More genes were sig- among the induced genes, whereas other differentially nificantly induced in cat2 transferred to air in SD than expressed genes in gr1 were associated with auxin, in LD, whereas the reverse was true for significantly gibberellin, abscisic acid, and ethylene function (Table repressed genes. Fewer genes were affected in gr1, but I). The most striking impact of the gr1 mutation was on effects were also daylength conditioned. Of genes the expression of genes involved in jasmonic acid (JA) induced in gr1 in SD, only three were also induced synthesis and signaling. Of the 18 genes repressed in LD, together with another four LD-specific genes. in gr1 in LD, eight have been shown to be early The number of repressed genes in gr1 in LD was JA-responsive genes (Yan et al., 2007). These genes en- similar to that in SD (Fig. 1C), but of these, only two coded, among others, LIPOXYGENASE3 (LOX3), were common (Supplemental Table S2). Thus, as for MYB95, GSTU6, and two of the 12 JASMONATE/ ZIM DOMAIN (JAZ) proteins recently characterized genes regulated by an H2O2 signal, gene expression dependent on a drop in glutathione reduction state as JA-inducible repressors of JA signaling (Staswick, was strongly determined by photoperiod context. Of 2008; Browse, 2009). All but one of the JA-dependent the 58 genes showing significantly modified expres- genes repressed in gr1 in LD showed the same response sion in response to a mild perturbation of glutathione in the GSSG-accumulating cat2 mutant (Table I). redox state in gr1, most showed significant same- Two induced stress-associated genes and four JA- direction changes in at least one cat2 replicate (Sup- dependent genes that showed similar responses in gr1 plemental Table S2). It should be noted that Figure 1C and cat2 were selected for quantitative PCR analysis. provides a conservative estimate of overlap between This confirmed the induction in gr1 of the cold-regulated gr1 and cat2 transcriptomes because only genes that COR78 in SD and of the cadmium-responsive At3g14990 showed statistically significant same-direction changes in both daylength conditions (Fig. 2). For the JA- in all four dye-swap repeats (two gr1/Col-0 and two associated genes, quantitative PCR revealed that in addi- cat2/Col-0) are considered. tion to repression in gr1 and cat2 in LD, three of the four Several genes whose expression was modified in gr1 genes (LOX3, MYB95,andJAZ10) were also induced in encoded stress-related proteins, notably cadmium-, SD (Fig. 2). Thus, GR1-dependent glutathione status salt-, and cold-responsive genes, as well as two GSTs influences the expression of genes involved in JA synthe- (GSTU7 and GSTU6) and three multidrug resistance- sis and signaling in a daylength-dependent manner. associated proteins (Supplemental Table S2). This is consistent with the roles of glutathione in redox ho- Genetic Analysis of GR1 Function in Responses to H O meostasis, heavy metal resistance, cold acclimation, 2 2 and metabolite conjugation and transport (Cobbett To directly explore the role of GR1 and glutathione et al., 1998; May et al., 1998; Kocsy et al., 2000; Wagner status in the H2O2 response, the gr1 mutation was et al., 2002; Gomez et al., 2004). crossed into the cat2 background. Preliminary analysis

Table I. Annotated phytohormone-associated genes showing significantly modified transcript levels in gr1 Gene Identifier Name 8-h Days 16-h Days Hormone At3g50970a XERO2 Induced – Abscisic acid At4g15760 MO1 Induced – SA At4g23600a CORI3 (JR2) Induced – JA At5g05730 ASA1 Induced – Ethylene At1g74670a Unknown protein Repressed – GA At5g54490 PBP1 Repressed – Auxin At5g61590 ERF B3 member Repressed – Ethylene At5g61600 ERF B3 member Repressed – Ethylene At1g17420a LOX3 – Repressed JA At1g74430/40a MYB95 – Repressed JA At2g24850 TAT3 – Repressed JA At2g29440a GSTU6 – Repressed JA At2g34600a JAZ7 – Repressed JA At2g38240a 2OGOR – Repressed JA At4g15440a HPL1 – Repressed JA At5g13220a JAZ10 – Repressed JA aSignificant same-direction response in cat2 in the same condition.

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of F2 seeds grown in air failed to identify double cat2 gr1 homozygotes. Thus, F3 seeds from a cat2/cat2 GR1/ gr1 genotype were germinated and grown at high CO2, where, as noted above, the cat2 mutation is phenotyp- ically silent because photorespiratory H2O2 produc- tion is largely shut down. Genotyping of these seeds identified several plants with cat2/cat2 gr1/gr1 geno- types, suggesting selection against cat2 gr1 double homozygotes in the presence of photorespiratory H2O2 production (air) but not in its absence (high CO2). When F4 seeds obtained from F3 cat2 gr1 double homozygotes identified at high CO2 were sown on agar plates in air, germination was similar to Col-0, gr1, and cat2, but growth was severely compromised in all cat2 gr1 plants from the cotyledon stage onward (Fig. 3A). Although cat2 gr1 was able to grow on soil at moderate irradiance, all plants with this genotype showed a dwarf rosette phenotype that was much more severe than that observed in cat2 (Fig. 3B). The cat2 gr1 phenotype was completely reverted by growth at high CO2 (Fig. 4A). Together with the aphenotypic nature of the single gr1 mutant, this observation indicates that the functional importance of GR1 is highly correlated with conditions of in- creased intracellular H2O2 availability. To further in- vestigate this point, we analyzed the phenotypic responses of cat2 gr1 grown at high CO2 after transfer to air in either SD or LD. Lesions developed in cat2 at 5 to 6 d after transfer to LD but did not develop in SD (data not shown). Four days after the transfer to LD, few or no lesions were apparent on cat2 leaves (Fig. 4B). By contrast, extensive bleaching was observed on cat2 gr1 leaves as early as 2 d after the transfer to air in LD (Fig. 4B). In both photoperiods, photorespiratory H2O2 induced slower growth in cat2, and this effect was exacerbated by the absence of GR1 function (Fig. 4C). Remarkably, however, bleaching was limited or absent in cat2 gr1 transferred to air in SD. To verify the conditional genetic interaction between the cat2 and gr1 mutations, a second allelic gr1 T-DNA line was obtained and crossed with cat2. F2 plants from this cross were grown at high CO2 and then transferred to air in LD, and lesions were quantified and plants were genotyped (Supplemental Fig. S3). Neither F1 CAT2/cat2 GR1/gr1 double heterozygotes grown in air nor any F2 plants grown at high CO2 showed any apparent phenotype (data not shown). Of 93 F2 plants transferred from high CO2 to air in LD, seven displayed readily visible lesions within 4 d (Supplemental Fig. S3). Genotyping confirmed that all plants showing substantial lesions were cat2/cat2 gr1/ gr1 double homozygotes, and lesion quantification and rosette fresh mass distinguished these plants from the eight other genotypes (Supplemental Fig. S3). Figure 2. Verification of selected genes repressed or induced in gr1 by quantitative RT-PCR. Data are means 6 SE of two independent extracts. * P , 0.1, ** P , 0.05, *** P , 0.01. Microarray Analysis of the H2O2-GR1 Interaction

To identify H 2O2-regulated genes whose expression is dependent on GR1, transcript profiling of cat2 and cat2 gr1 was performed after transfer of plants grown at high

1148 Plant Physiol. Vol. 153, 2010 Glutathione Reductase in H2O2 Responses

Figure 3. Germination and growth phenotype of cat2 gr1 double mutants. A, Germination and growth on agar. B, Plants grown on soil from germination in a 16-h/8-h day/night regime. Aster- isks indicate significant differences between mutants and Col-0, while pluses indicate significant differences between cat2 and cat2 gr1. FW, Fresh weight. [See online article for color version of this figure.]

CO2 to air. In view of the above phenotypic observations In the cat2 single mutant, the predominant effect of and the daylength-dependent transcript profiles for cat2 H2O2 on genes involved in JA synthesis and signaling and gr1 single mutants (Fig. 1; Supplemental Table S2), was induction in SD and/or repression in LD. This the analysis was performed after transfer to both SD and daylength-dependent regulation was strongly modu- LD. A full list of significantly different genes and signal lated by the absence of GR1 function in cat2 gr1. Of the intensities is given in Supplemental Table S3. 47 JA-associated transcripts whose abundance was Clustering analysis of significantly different tran- significantly modified in cat2 and/or cat2 gr1 relative scripts delineated two major patterns of H2O2-induced to Col-0, only 10 did not show differential expression gene expression (Fig. 5). The first was largely com- between the two lines in at least one photoperiod posed of genes that were induced by photorespiratory (Supplemental Table S4). The overwhelmingly pre- H2O2 in cat2 and cat2 gr1 (cluster 1), while the second dominant effect of the gr1 mutation in the cat2 back- comprised genes that responded in a daylength- ground was to cause or enhance repression (e.g. LOX2, dependent manner (cluster 2). The most striking im- JMT1, JAZ3), to oppose induction in SD (e.g. RAP2.6, pact of the gr1 mutation in the cat2 background was JAZ10, AOS), or both (LOX3, TAT3, OPR3). to annul or impair the induction of genes by photo- respiratory H2O2 in SD, an effect exemplified by a Profiling of Glutathione and Associated Gene Expression subcluster of genes in cluster 2. Of 62 genes in this in cat2 gr1 subcluster, 33 are annotated as involved in JA- or wounding-dependent responses (Taki et al., 2005; Yan Consistent with the absence of phenotype at high et al., 2007; http://www.arabidopsis.org) and are CO2 (Fig. 4A), cat2 glutathione status was similar to the highlighted yellow in Figure 5. Data mining of all wild type (95% GSH), whereas in cat2 gr1 the gluta- significantly different genes using the above data sets thione pool was only about 70% reduced (i.e. similar to revealed that, in all, 47 genes inducible by JA or the gr1 single mutant in air or at high CO2; Fig. 6A; wounding, or encoding proteins involved in JA syn- Supplemental Fig. S2). These observations are consis- thesis, conjugation, or signaling, were differentially tent with the silent nature of the cat2 mutation at high expressed in cat2 and/or cat2 gr1 (Supplemental Table CO2 and with a constitutive oxidation of glutathione S4). Twenty-five of these were among 35 genes recently in the gr1 genotype. The impact of the gr1 mutation shown to be induced by wounding in a JA-dependent on glutathione in conditions of increased H2O2 was manner (Yan et al., 2007), while among the others were assessed under the same conditions as for the micro- established JA-associated genes such as LOX2, LOX3, array analysis. A time course following transfer to air OPR3, JAZ1, JAZ3, JAZ7, JAZ9, JAR1, and JMT1. revealed that leaf glutathione (1) became progressively

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Figure 4. Rescue of a wild-type phe- notype in cat2 gr1 at high CO2, and rapid daylength-dependent induction of leaf bleaching following transfer to air. A, Plants were germinated and grown for 25 d at high CO2 (3,000 mL 2 L 1). FW, Fresh weight. B, Plants trans- ferred from high CO2 to air in either SD or LD conditions. False-color imaging of lesions (red) are shown under each photograph. C, Rosette fresh weights of plants transferred to air in either SD or LD. Histograms show means 6 SE of at least six plants. Black asterisks indicate significant differences between mutant and Col-0, and red asterisks indicate significant differences between cat2 and cat2 gr1:**P , 0.05, *** P , 0.01.

oxidized and accumulated in cat2; (2) became much Five glutathione-associated genes showed a different more rapidly oxidized and accumulated more strongly expression pattern (Fig. 6B, bottom) and were all found in cat2 gr1; and (3) was most oxidized and most within cluster 2 shown in Figure 5. While one GRX strongly accumulated in cat2 gr1 in SD. The changes (At5g18600) showed a tendency to repression in all in glutathione involved a fall in the GSH to GSSG ratio conditions, repression was only significant in cat2 gr1 in to below 1 in cat2 and cat2 gr1, with the lowest ratio SD. GSTU5 and GSTU6 were both induced in cat2 but observed in cat2 gr1 in SD (Fig. 6A). not in cat2 gr1 in SD and were repressed in both ge- Mining of the transcriptome data for glutathione- notypes in LD (Fig. 6B). The gr1 mutation in the cat2 associated genes showed that modified glutathione background also produced repression of GGT1 and status in cat2 and cat2 gr1 was associated with the GRX480, specifically in LD. Thus, the expression pat- induction of genes encoding three GSTs of the tau terns of these five genes were similar to those of JA- class (GSTU7, GSTU8, GSTU19)andtwoofthephi dependent genes (Fig. 5; Supplemental Table S4). In class (GSTF2, GSTF8), two multidrug resistance- contrast to the differential expression of these glutathione- associated proteins (MRP2, MRP14), a glutathione/ dependent genes, the expression of cytosolic TRXs and thioredoxin peroxidase (GPX6), a glyoxylase I family associated genes involved in thiol-disulfide exchange protein, two members of the GRX family, and a GRX- reactions was little or not affected in either the cat2 or like expressed protein (Fig. 6B). These genes were cat2 gr1 genotype (Supplemental Table S5). equally or more strongly induced in cat2 gr1 com- pared with cat2, although the effect depended on Analysis of Oxidative Stress and the daylength. In no case did the gr1 mutation oppose Ascorbate-Glutathione Pathway induction of these genes in cat2,whichwereallfound to be grouped in cluster 1 on the heat map shown in Catalase-independent pathways of H2O2 metabo- Figure 5. lism depend on ascorbate, which is considered to be

1150 Plant Physiol. Vol. 153, 2010 Glutathione Reductase in H2O2 Responses

Figure 5. Comparison of gene expres- sion in cat2 and cat2 gr1 following

transfer from high CO2 to air in either 8-h (SD) or 16-h (LD) growth condi- tions. Hierarchical clustering was per- formed using MultiExperimentViewer software. The exploded heat map at the right shows a subcluster of 62 genes that includes 33 JA- or wounding- associated genes (highlighted yellow).

partly regenerated by glutathione, while NADPH is Queval et al., 2008). In this study, we used three required for reduction of GSSG. Despite this, the different techniques to assess H2O2 or reactive oxygen effects of gr1 and cat2 mutations on glutathione pools species (ROS) levels in cat2 and cat2 gr1. In situ were not associated with marked perturbation of leaf staining using 3,39-diaminobenzidine did not produce ascorbate pools, which remained more than 80% re- evidence for generalized accumulation of H2O2 in gr1, duced in all plants. In LD conditions, ascorbate was cat2,orcat2 gr1 leaves in SD or LD (Supplemental Fig. decreased in cat2 and cat2 gr1, although this small S5A). Likewise, assays of extractable peroxides using effect was only statistically significant for cat2 (Fig. luminol chemiluminescence did not find increased 7A). Like ascorbate, the NADP reduction state was not contents in cat2 or cat2 gr1 in either daylength condi- greatly affected in any of the samples (Fig. 7B). The tion (Supplemental Fig. S5B). Semiquantitative ROS only significant effects were observed in cat2 gr1, visualization within the mesophyll cells in vivo also which in SD had more NADPH than Col-0 and in revealed that the cat2 mutation caused only minor LD less NADP+. Because of these effects, the most increases in dichlorofluorescein fluorescence (Fig. 8). reduced NADP(H) pools were observed in cat2 gr1. The cat2 gr1 double mutant showed an appreciably Assay of lipid peroxidation products using thiobarbi- stronger fluorescence signal, but this effect was spe- turic acid also showed no significant effect between cific to plants in SD (Fig. 8). any of the samples in either condition (Fig. 7C). To further assess the impact of the mutations on the Potential problems that must be taken into account ROS-antioxidant interaction, we measured the extract- in measuring H2O2 include low assay specificity, in- able activities and expression of APX, catalase, GR, terference, and extraction efficiency (Wardman, 2007; and dehydroascorbate reductase (DHAR), the enzyme

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Figure 6. Glutathione contents and glutathione-related gene expression in cat2 and the cat2 gr1 double mutant. A, Time course of changes in percent- age glutathione reduction and total glutathione in the different lines fol- lowing transfer from high CO2 to air. Black circles, Col-0; white circles, cat2; white triangles, cat2 gr1. Top, 100 3 GSH/total glutathione (the num- bers indicate GSH to GSSG ratio for the final time points). Bottom, total gluta- thione (GSH + 2 GSSG). Error bars that are not apparent are contained within the symbols. FW, Fresh weight. B, Ex- pression values of significantly differ- ent glutathione metabolism transcripts in cat2 and cat2 gr1 relative to Col-0. Black bars, cat2; white bars, cat2 gr1. The top two panels show genes that were induced, while the bottom panel shows genes that were significantly repressed in at least one sample type.

linking glutathione and ascorbate pools. The only 9C). Induction of APX1 in cat2 was accompanied by one of these enzyme activities that was significantly induction of a cytosolic DHAR (DHAR2), although this different between Col-0 and gr1 was GR (Fig. 9A). effect was stronger in SD than in LD and was not Similarly, the gr1 mutation in the Col-0 background associated with a marked increase in the overall ac- produced no significant change in transcripts other tivity of this enzyme. Compared with Col-0, effects in than GR1 (Fig. 9C). Decreased catalase activity in cat2 cat2 gr1 were similar to those observed in cat2 (i.e. was associated with an increase in APX activity in both induction of APX1 in both SD and LD, an increase in conditions (Fig. 9A). Increased APX activity was ac- extractable APX in the two conditions, and induction companied by induction of transcripts for APX1, en- of DHAR2 transcripts, particularly in SD; Fig. 9). coding the cytosolic isoform (Davletova et al., 2005). Of Overall, the presence of the gr1 mutation in Col-0 or eight APX transcripts present on the chip, only APX1 cat2 backgrounds did not greatly affect the responses was significantly increased in cat2, and as for APX of these antioxidative systems relative to the respective activity, this effect was similar in both SD and LD (Fig. controls. Despite the oxidation of the glutathione pool

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in cat2 gr1. Leaf SA contents were similar in all samples in SD, although slightly decreased in cat2 gr1, whereas in LD H2O2-induced accumulation was observed in cat2 but not in cat2 gr1 (Fig. 10A). The gr1 mutation also decreased basal SA levels in the Col-0 background in LD (Fig. 10A). We investigated the response of gr1 to infection with the virulent bacterium Pseudomonas syringae pv tomato strain DC3000 (Fig. 10B). Bacterial growth in gr1 leaves was intermediate between that observed in Col-0 and the npr1-1 mutant, in which cytosolic redox- modulated NPR1 function is lacking (Mou et al., 2003). In agreement with the data of Figure 10A, increased sensitivity to bacteria in gr1 was associated with lower total SA levels than in Col-0 (Fig. 10B).

DISCUSSION Recent data have highlighted the functional over- lap between glutathione and TRX systems (Reichheld et al., 2007; Marty et al., 2009). A key question, therefore, concerns the specificity of cytosolic GR- glutathione and NTR-TRX functions. Glutathione has been proposed to play a role in H2O2 signaling (Foyer et al., 1997; May et al., 1998). Here, we present a targeted, genetically based study of such a role by using cat2 and gr1 mutants in combination.

GR1PlaysaNonredundantRoleinH2O2 Metabolism Figure 7. Leaf ascorbate, NADP(H), and thiobarbituric acid-reactive and Signaling substances (TBARS) in the four genotypes placed in SD (left) and LD (right) in air following growth at high CO2. A, Ascorbate (white bars) In this study, the cat2 mutant was used both as a and dehydroascorbate (black bars). B, NADP+ (white bars) and NADPH reference GSSG-accumulating H2O2 signaling system (black bars). C, TBARS. For A and B, numbers above each bar indicate and as a genetic background in which to directly percentage reduction states [100 ascorbate/(ascorbate + dehydroascor- explore the GR1-H O interaction. Because daylength- bate) in A and 100 NADPH/(NADP+ + NADPH) in B]. Samples were 2 2 dependent effects have been described for redox sig- taken 4 d after transfer to air. All data are means 6 SE of three independent leaf extracts. Asterisks within blocks indicate significant naling during pathogen and oxidative stress responses differences from Col-0 values in the same condition: * P , 0.1. FW, (Dietrich et al., 1994; Karpinski et al., 2003; Queval Fresh weight. et al., 2007; Vollsnes et al., 2009), we analyzed the importance of GR1 in Col-0 and cat2 backgrounds in two growth daylengths. in cat2 and its very marked oxidation in cat2 gr1 (Fig. Our analysis of gr1 in the Col-0 background confirm 6A), neither genotype showed compensatory induc- the results of Marty et al. (2009) that GR1 is not tion of GR2 transcripts, encoding the chloroplast/ required for growth in optimal conditions. However, mitochondrial isoform (Fig. 9C). GR1 plays an important role when intracellular H2O2 production is increased and during pathogen chal- SA Signaling and Pathogen Reponses lenge. The effect of gr1 on the cat2 phenotype contrasts with results in which plants were exposed to exoge- Given the observed effects on JA-associated genes nous H2O2 present in agar, where no difference was (Fig. 5; Table I; Supplemental Table S4) and the oppo- found between gr1 and Col-0 phenotypes, even at sition between JA signaling and SA-dependent path- highly challenging H2O2 concentrations (Marty et al., ways (Dangl and Jones, 2001; Koorneef et al., 2008), we 2009). This suggests that the site of H2O2 production is analyzed SA and expression of SA marker genes in crucial. Enhanced H2O2 availability in cat2 leaves gr1, cat2,andcat2 gr1 in both daylength conditions. placed in air occurs intracellularly through the pho- SA-dependent PR genes (PR1, PR2) were both signif- torespiratory enzyme, glycolate oxidase, which is icantly induced in cat2 in LD but less strongly or not located in peroxisomes. In these conditions, redox at all in SD (Fig. 10A). Induction of SA-dependent perturbation in cat2 produces similar effects to those PR genes in cat2 in LD was antagonized by the gr1 observed in stress conditions, notably oxidation and mutation, producing significantly lower expression accumulation of glutathione and changes in the abun-

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Figure 8. In vivo visualization of ROS using dichlorofluorescein fluorescence. Leaf mesophyll cells of plants grown at high CO2 and then transferred to air for 4 d in SD or LD were imaged in intact tissue by confocal microscopy. Each pair of images shows the same area measured for green dichlorofluorescein fluorescence (left) and red chlorophyll autofluorescence (right). Repre- sentative examples from three independent ex- periments are shown. Magnification was the same for all samples. [See online article for color version of this figure.]

dance of many stress-related transcripts. GR activity However, our data show that although growth arrest has been detected in pea leaf peroxisomes, and it is was linked to glutathione perturbation in both day- likely that GR1 is found in Arabidopsis peroxisomes as lengths, leaf cells can tolerate extreme perturbation of well as in the cytosol (Jime´nez et al., 1997; Kaur et al., intracellular thiol-disulfide state without undergoing 2009). Therefore, it is possible that GR1 is important in bleaching (Fig. 5). Analysis of ascorbate, thiobarbituric peroxisomal ascorbate- and/or glutathione-dependent acid-reactive substances, and ROS also provided little H2O2 metabolism. At least one (APX3) and possibly as evidence that oxidative stress was stronger in LD many as three (APX3, APX4, APX5) Arabidopsis APXs compared with SD. are targeted to peroxisomes or the peroxisomal mem- The dramatic perturbation of glutathione pools in brane (Narendra et al., 2006), but the corresponding cat2 gr1 compared with cat2 provides direct evidence transcripts were not significantly induced in gr1, cat2, for the role of GR1 in intracellular H2O2 metabolism. or cat2 gr1 (Fig. 9). Most of the antioxidative genes that Analysis of ROS suggests that H2O2 accumulation in were induced in cat2 and cat2 gr1 encoded cytosolic cat2 is localized and/or minimized by metabolism enzymes, including APX1, DHAR2, and MDAR2 through ascorbate- and/or glutathione-dependent (Supplemental Table S3). Our analysis thus points to pathways. Little or no detectable increase in H2O2 in tight coupling between H2O2 produced in the perox- cat2 compared with the wild type is consistent with isomes and cytosolic antioxidative systems. previous studies of catalase-deficient tobacco and The dramatic accumulation of GSSG in cat2 gr1 barley (Hordeum vulgare) lines (Willekens et al., 1997; placed in air shows that GSH regeneration through Noctor et al., 2002; Rizhsky et al., 2002). Although alternative pathways is rapidly exceeded under con- certain antioxidative enzymes and transcripts were ditions of enhanced intracellular H2O2 availability. To up-regulated in cat2 and cat2 gr1, these effects were at our knowledge, the accumulation of GSSG in cat2 gr1 least as marked in SD as in LD. ROS signal intensity 2 (up to 2 mmol g 1 fresh weight; Fig. 6A) exceeds was increased in cat2 gr1 in SD, but increases were less previously reported values for this compound in leaf evident in this genotype in LD (Fig. 8), conditions in tissues. Further work is required to identify the com- which bleaching occurred (Fig. 5). One explanation of partments in which such marked accumulation of the requirement of LD conditions for leaf bleaching is GSSG occurs. Analyses of the gr1 single mutant in that other daylength-linked signals are required in air and the cat2 gr1 double mutant at high CO2 show addition to oxidative stress intensity. that mild perturbation of the glutathione pool is not The phenotype of cat2 gr1 in LD contrasts with sufficient to produce marked phenotypic effects, while observations of tobacco plants deficient in both cyto- effects produced by the more severe perturbation of solic APX and catalase, which showed an ameliorated glutathione in cat2 gr1 in air were dependent on phenotype compared with parent lines (Rizhsky et al., growth daylength. Lesions were not observed in cat2 2002). One explanation of the different effects of GR gr1 in SD, even though these plants showed the most and APX deficiency in catalase-deficient plants is that dramatic GSSG accumulation and the lowest GSH to some proportion of H2O2 is metabolized through GSSG ratios. Oxidized glutathione pools are consid- GR-dependent but ascorbate-independent pathways. ered to be among the cellular factors underlying Hence, as well as GSH oxidation by dehydroascorbate, dormancy and cell death (Kranner et al., 2002, 2006). glutathione perturbation in cat2 may occur through

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Figure 9. Major antioxidative enzyme activities and transcript levels in Col-0, gr1, cat2, and cat2 gr1 after transfer to air in SD (gray backgrounds) or LD (white backgrounds). A, Enzyme activities. The x axis numbers refer to Col-0 (1), gr1 (2), cat2 (3), and 2 2 2 2 cat2 gr1 (4). Units are mmol mg 1 protein min 1 (catalase) and nmol mg 1 protein min 1 (other enzymes), and values are means of three independent extracts. Asterisks indicate significant differences from Col-0 at P , 0.05. B, Simplified scheme of catalase-

and ascorbate-dependent H2O2 metabolism. C, Abundance (log2 scale, relative to Col-0) of corresponding transcripts for which probes were present on the CATMA array. Asterisks indicate transcripts that showed significant same-direction differences from controls in both biological replicates. Where no bar is apparent, transcripts were almost identical in abundance in the mutant and the corresponding Col-0 control. enzyme-catalyzed peroxidation of GSH (e.g. catalyzed daylength. This observation suggests that modulation by certain GSTs, as discussed further below). Although of redox-linked gene expression by growth daylength increases in APX activities in cat2 and cat2 gr1 were is not restricted to oxidative stress or is a secondary associated with induction of APX1 (Fig. 9), several consequence of stress-induced phenotypes. glutathione-associated genes with potential peroxida- Fewer genes were affected in gr1 than in cat2. Genes tive functions were also induced in these lines (Fig. 6). affected in cat2 but not gr1 could reflect H2O2 signaling that occurs independently of changes in glutathione (Fig. 1A). The difference could also be related to the stronger H2O2 and Glutathione Modulate the Expression of Specific Glutathione-Associated Genes in a perturbation of the glutathione pool in cat2 (Fig. 1B). If Daylength-Dependent Manner the difference can be partly explained by weaker changes in glutathione in gr1, then the overlap in tran- Although there was no evidence of oxidative stress scriptomes we report could provide a minimum esti- or phenotypic effects in the gr1 single mutant, tran- mate of the importance of glutathione in H2O2 signaling scriptomes in this line partly overlapped with that of Many TRX-linked components play roles in oxida- the H2O2 signaling reference system, cat2. Both tran- tive stress (Dietz, 2003; Rey et al., 2005; Pe´rez-Ruiz scriptomes were found to be highly dependent on et al., 2006), but none of these transcripts was induced

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Figure 10. Pathogen-associated responses in GR- deficient lines. A, PR gene expression and SA contents in Col-0, gr1, cat2, and cat2 gr1 in two photoperiods after transfer from high CO2 to air. Top, 8-h days; bottom, 16-h days. Data are means 6 SE of two to three biological samples. FW, Fresh weight. B, Bacterial resistance and SA contents in Col-0 and gr1 grown under standard conditions in air. Asterisks indicate significant differences be- tween mutants and Col-0 (* P , 0.1, ** P , 0.05, *** P , 0.01), while pluses indicate significant differences between cat2 and cat2 gr1 (+ P , 0.1, ++ P , 0.05, +++ P , 0.01).

by increased H2O2 availability in cat2 or cat2 gr1.A Analysis of GRX function is also complicated by the lack of response was also observed for chloroplast number of genes, notably due to a large subclass that is ferredoxin-TRX reductase subunit genes and for the specific to higher plants. At least 30 GRX genes have two cytosolic NTRs and cytosolic TRXs (Supplemental been described in Arabidopsis (Lemaire, 2004; Meyer Table S5). The only TRX-linked gene that was signif- et al., 2008). Based on active-site sequences, these are icantly induced by H2O2 was GPX6, and this effect was subclassified into CPYC, monothiol CGFS, and CC- largely independent of both daylength and the gr1 type GRX, with the last subclass being specific to land mutation (Fig. 6). plants (Lemaire, 2004). Although at least one CGFS In contrast to the lack of response of TRX systems, GRX is implicated in oxidative stress (Cheng et al., H2O2-orgr1-driven perturbation of glutathione triggered 2006), no CPYC or CGFS GRXs showed differential changes in the abundance of transcripts of several expression in gr1, cat2,orcat2 gr1 in either photoperiod glutathione-associated genes, notably GSTs and GRXs. condition (Supplemental Table S5). All differentially Analysis of GST functions is complicated by the num- expressed GRX genes observed in our analysis belong ber of genes in plants, notably due to the presence of to the CC-type subclass, consisting of 20 identified large plant-specific phi and tau subclasses, as well as members that are thought to encode cytosolic proteins. substrate overlap between the different enzymes. Our The functions of most CC-type GRXs remain obscure, transcript profiling analysis showed that H2O2 modi- but two have been implicated in the regulation of fied transcript levels for two phi class (GSTF2, GSTF8) gene expression by interacting with TGA transcription and three tau class (GSTU7, GSTU8, GSTU19) en- factors. GRX480 overexpression represses the JA zymes and that in all cases, these effects were further marker gene, PDF1.2, while ROXY1 is required for modulated by daylength and/or the gr1 mutation. petal development (Ndamukong et al., 2007; Li et al., Type F GSTs exhibit both conjugase and peroxidase 2009). The predicted protein encoded by the GRX-like activities, with GSTF8 showing considerable activity sequence (Fig. 6B) has an active-site CCMS motif that against cumene hydroperoxide (Wagner et al., 2002; is shared with nine annotated GRX proteins, and Dixon et al., 2009). As well as GSTFs, some GSTUs, BLAST analysis revealed that the protein has 58% to including GSTU8, show quite high peroxidase activity 60% amino acid identity with five of these CCMS (Dixon et al., 2009). GRXs. The H2O2- and/or gr1-dependent changes in 1156 Plant Physiol. Vol. 153, 2010 Glutathione Reductase in H2O2 Responses

CC-type GRXs suggest that the encoded proteins may 2002; Ndamukong et al., 2007; Yan et al., 2007; Mueller play roles in the redox regulation of gene expression. et al., 2008; Li et al., 2009), it is possible that several GSTs and CC-type GRX could be involved in linking GR1 Is Required for SA Responses and for Expression glutathione to JA signaling. of Genes Involved in JA Signaling Interestingly, the glutathione-deficient pad2 mutant is compromised in insect resistance (Schlaeppi et al., The cat2 mutation causes accumulation of SA and 2008). Glutathione status is modulated by various induction of SA responses in LD but not SD (Fig. 10). stresses, notably biotic challenge, while sulfur defi- GR1 function appears to be required for optimal SA ciency, which can induce JA synthesis genes (Hirai production, as this was compromised in the gr1 single et al., 2003), is an important determinant of glutathione mutant relative to Col-0 and in cat2 gr1 relative to cat2 concentration. As well as the influence of glutathione (Fig. 10). Furthermore, gr1 showed decreased resis- status, our data also underline the potential importance tance to bacteria, while induction of PR genes in cat2 of daylength context in modulating redox-triggered gr1 was compromised compared with cat2. As well as signaling through the JA pathway. effects on SA production itself, the gr1 mutation could interfere with SA-dependent gene expression by al- tered redox regulation of NPR1 (Mou et al., 2003; Tada CONCLUSION et al., 2008). Because loss of GR1 function in an H2O2 signaling context produces accelerated lesion forma- While TRX- and glutathione-dependent pathways tion, the enzyme appears to be required both to restrict have overlapping functions in plants (Reichheld et al., leaf bleaching and to enable the induction of SA and 2007; Marty et al., 2009), this study shows that GR1 PR genes. plays specific roles in intracellular H2O2 metabolism, Several antioxidative genes can be induced by JA, in daylength-linked control of phytohormone gene including genes encoding enzymes of glutathione expression, and in certain responses to biotic stress. synthesis (Xiang and Oliver, 1998; Sasaki-Sekimoto The transcriptomic patterns we report also point to et al., 2005). Recent reports implicate glutathione- glutathione- and TRX-specific pathways in redox sig- associated components in JA signaling (Ndamukong naling, with relatively little cross talk between the two et al., 2007; Koorneef et al., 2008; Tamaoki et al., pathways at the level of gene expression. 2008). In both Col-0 and cat2 backgrounds, the gr1 Two nonexclusive hypotheses could explain how mutation affected a suite of JA-associated genes in a the GR-glutathione system influences H2O2-linked daylength-dependent manner. We observed induction gene expression. In the first, glutathione turnover of JA genes in cat2 and gr1 single mutants in SD but would influence H2O2 concentration through its anti- repression in all mutant genotypes in LD. This may oxidative function. Most of our observations do not indicate (1) an optimum glutathione redox status for suggest that this is the main cause of the gr1-linked induction of JA genes and (2) daylength-dependent changes in gene expression, although in situ visuali- signals that operate to modulate the impact of changes zation of ROS suggests that it could contribute to in glutathione status on JA signaling. The daylength- differences between cat2 and cat2 gr1, particularly in and gr1-modulated expression of several GRX and SD. In the second hypothesis, glutathione status GSTs suggests that these components may functionally would be a key part of H2O2-triggered signal trans- link glutathione and JA signaling. Such components duction. This hypothesis receives support from several could include GRX480 (Ndamukong et al., 2007), of our observations, notably the partial overlap be- GSTU8-dependent formation of glutathione-oxylipin tween cat2 and gr1 transcriptomes, but further work is conjugates (Mueller et al., 2008), and GSTU6, which is required to confirm the role of glutathione status per se. among the JA-dependent genes that are rapidly in- duced by wounding (Yan et al., 2007). In our analysis, similar expression patterns were observed for these MATERIALS AND METHODS glutathione-associated genes and for a suite of JA- dependent genes. Other genes that link glutathione Plant Material and JA may include GSTU7, GSTU19, and MRP2, Arabidopsis (Arabidopsis thaliana) mutant lines carrying T-DNA insertions which were induced most strongly in cat2 gr1 in SD in the GR1 gene (At3g24170) were identified using insertion mutant informa- and less strongly in cat2 and cat2 gr1 in LD. These tion obtained from the SIGnAL Web site (http://signal.salk.edu), and seeds were obtained from the Nottingham Arabidopsis Stock Centre (http://nasc. genes are up-regulated by phytoprostane or 12-oxo- nott.ac.uk). The default gr1 line was gr1-2 (SALK_060425). A second gr1 phytodienoic acid treatment (Mueller et al., 2008). insertion mutant (SALK_105794), named gr1-1 by Marty et al. (2009), was used Several of the glutathione-associated genes shown to confirm the phenotypic effects of the cat2 gr1 interaction. After identifica- in Figure 6 are rapidly induced by methyl jasmonate, tion of homozygotes, all further analyses were performed on plants grown from 12-oxo-phytodienoic acid, phytoprostane, or wound- T3 seeds. The cat2 line was cat2-2 (Queval et al., 2007), now renamed cat2-1. ing (Supplemental Fig. S6). Based on JA- or oxylipin- dependent expression patterns, the activities of GSTs Identification of Homozygous gr1 Insertion Mutants against electrophilic metabolites, and interactions of Leaf DNA was amplified by PCR (30 s at 94°C, 30 s at 60°C, 1 min at 72°C, CC-type GRX with transcription factors (Wagner et al., 30 cycles) using primers specific for left T-DNA borders and the GR1 gene

Plant Physiol. Vol. 153, 2010 1157 Mhamdi et al.

(Supplemental Table S1). Fragments obtained were sequenced to confirm the (Invitrogen). ACTIN2 transcripts were measured as a control. cDNAs were insertion site. Zygosity was analyzed by PCR amplification of leaf DNA, and amplified using the conditions described above for analysis of genomic DNA RT-PCR analysis of GR1 transcripts was performed using gene-specific except that the number of cycles was 50. Quantitative RT-PCR was performed primers (Supplemental Table S1). according to Queval et al. (2007). Gene-specific primers are listed in Supple- mental Table S1. Plant Growth and Sampling

Seeds were incubated for 2 d at 4°C and then sown either on agar or in soil Enzyme Assays, Metabolite, and ROS Analysis in 7-cm pots. Plants were grown in a controlled-environment growth chamber 2 2 at the specified photoperiod and an irradiance of 200 mmol m 2 s 1 at leaf Extractable enzyme activities were measured as described previously level, 20°C/18°C, 65% humidity, and given nutrient solution twice per week. (Veljovic-Jovanovic et al., 2001). Oxidized and reduced forms of glutathione, 21 21 The CO2 concentration was maintained at 400 mLL (air) or 3,000 mLL ascorbate, and NADP were measured by plate-reader assay as described by (high CO2). Samples were rapidly frozen in liquid nitrogen and stored at Queval and Noctor (2007). SA was measured according to the protocol of 280°C until analysis. Unless otherwise stated, data are means 6 SE of three Langlois-Meurinne et al. (2005). Thiobarbituric acid-reactive substances were independent samples from different plants, and significant differences are assayed and calculated as described by Yang et al. (2009). In situ visualization expressed using t test at P , 0.1, P , 0.05, and P , 0.01. of ROS was performed using dichlorodihydrofluorescein-diacetate (DCFH2- DA) by a protocol modified from Oracz et al. (2009). Leaves were vacuum

infiltrated twice for 15 min at room temperature with DCFH2-DA, carefully Pathogen Tests rinsed, and kept in the dark. DCF fluorescence at 510 to 550 nm was visualized on a confocal microscope, simultaneously with red chlorophyll autofluores- The virulent Pseudomonas syringae pv tomato strain DC3000 was used for 2 cence, using argon laser excitation at 488 nm. resistance tests in a medium titer of 5 3 105 colony-forming units mL 1. Whole leaves of 3-week-old plants grown in a 16-h photoperiod were infiltrated Microarray data from this article will be deposited at the Gene Expression using a 1-mL syringe without a needle. Leaf discs of 0.5 cm2 were harvested Omnibus and can be consulted at CATdb (http://urgv.evry.inra.fr/cgi-bin/ from inoculated leaves at the appropriate time points. For each time point, projects/CATdb/consult_expce.pl?experiment_id=256). four samples were made by pooling two leaf discs from different treated plants. Bacterial growth was assessed by homogenizing leaf discs in 400 mLof water, plating appropriate dilutions on solid King B medium containing rifampicin and kanamycin, and quantifying colony numbers after 3 d. Supplemental Data

The following materials are available in the online version of this article. Microarray Analysis Supplemental Figure S1. Characterization of the gr1 mutant. Microarray analysis was performed using the CATMA arrays containing Supplemental Figure S2. Glutathione and ascorbate status in gr1 and cat2 24,576 gene-specific tags corresponding to 22,089 genes from Arabidopsis mutants grown at high CO and then transferred to air in 8-h or 16-h (Crowe et al., 2003; Hilson et al., 2004) plus 1,217 probes for microRNA genes 2 days. and putative small RNA precursors (information available at http://urgv. evry.inra.fr/projects/FLAGdb++). This array resource has been used in 40 Supplemental Figure S3. Analysis of the conditional genetic interaction publications over the last 5 years using a protocol based on technical repeats of between cat2 and gr1 in an allelic double cat2 gr1 mutant. two independent biological replicates produced from pooled material from Supplemental Figure S4. Quantitative PCR confirmation of changes in independent plants (Achard et al., 2008; Besson-Bard et al., 2009; Krinke et al., JA-linked genes in cat2 and cat2 gr1. 2009), and this approach was adopted in this study. For each biological repeat and each point, RNA samples were obtained by pooling leaf material from Supplemental Figure S5. H2O2 visualization in leaves of Col-0, gr1, cat2, independent sets, each of two plants. Samples of approximately 200 mg fresh and cat2 gr1 in SD and LD conditions. weight were collected from plants after 3.5 weeks of growth in the conditions specified above. Total RNA was extracted using Nucleospin RNAII kits Supplemental Figure S6. Genevestigator analysis (Hruz et al., 2008) of (Macherey-Nagel) according to the supplier’s instructions. For each compar- responses of glutathione-associated genes showing significantly differ- ison, one technical replication with fluorochrome reversal was performed for ent expression in cat2 and/or cat2 gr1 (Fig. 6). each biological replicate (i.e. four hybridizations per comparison). The label- Supplemental Table S1. PCR primers used in this study. ing of complementary RNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer- NEN Life Science Products), the hybridization to the slides, and the scanning Supplemental Table S2. Summary of modified daylength-dependent gene were performed as described by Lurin et al. (2004). expression in gr1 and overlap with cat2 transcriptomes. Supplemental Table S3. Complete data set of significantly modified genes Statistical Analysis of Microarray Data in gr1, cat2,andcat2 gr1 in two daylength regimes.

Experiments were designed with the statistics group of the Unite´ de Supplemental Table S4. List of JA-associated genes showing differential Recherche en Ge´nomique Ve´ge´tale. Normalization and statistical analysis expression in cat2 and cat2 gr1 and relative expression values. were based on two dye swaps (i.e. four arrays, each containing 24,576 GSTs Supplemental Table S5. Summary of expression of GRXs, cytosolic TRXs, and 384 controls) as described by Gagnot et al. (2008). To determine differ- and related genes in cat2 and cat2 gr1. entially expressed genes, we performed a paired t test on the log ratios, assuming that the variance of the log ratios was the same for all genes. Spots displaying extreme variance (too small or too large) were excluded. The raw P values were adjusted by the Bonferroni method, which controls the family- wise error rate (with a type I error equal to 5%) in order to keep a strong ACKNOWLEDGMENTS control of the false positives in a multiple comparison context (Ge et al., 2003). We considered as being differentially expressed the genes with a Bonferroni We thank the Salk Institute Genomic Analysis Laboratory for providing P # 0.05, as described by Gagnot et al. (2008). Only genes that showed same- the sequence-indexed Arabidopsis T-DNA insertion mutants, the Notting- direction statistically significant change in both biological replicates of at least ham Arabidopsis Stock Centre for supply of gr1 seed stocks, and Gae¨lle one sample type were considered as statistically significant. Cassin (Institut de Biologie des Plantes) for help with preliminary character- ization of gr1. We are also grateful to Marie-Noe¨lle Soler and Spencer Brown (Imagif, Centre National de la Recherche Scientifique) for advice on confocal Quantitative RT-PCR Analysis microscopy imaging and to Eddy Blondet (Unite´ de Recherche en Ge´nomique Ve´ge´tale) for help with microarray analyses. RNA was extracted using the NucleoSpin RNA plant kit (Macherey-Nagel) and reverse transcribed with the SuperScript III First-Strand Synthesis System Received January 24, 2010; accepted May 19, 2010; published May 20, 2010.

1158 Plant Physiol. Vol. 153, 2010 Glutathione Reductase in H2O2 Responses

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1160 Plant Physiol. Vol. 153, 2010

ANNEX 1B

Confirmation of the genetic interaction between cat2 and gr1 knockout

mutations

ANNEX 1B. Confirmation of the genetic interaction between cat2 and gr1 knockout mutations, presented as Supplemental Figure 3 in Mhamdi et al. (2010).

For explanation of the figures, see legend on the final page.

A B Plant Genotype Rosette Lesions Plant Genotype Rosette Lesions number FW (mg) (%) number FW (mg) (%) CAT2 GR1 CAT2 GR1 1 CAT2/cat2 GR1/gr1 74 0 48 cat2/cat2 GR1/gr1 58 0 2 CAT2/cat2 GR1/gr1 124 0 49 cat2/cat2 gr1/gr1 30 14.7 3 CAT2/cat2 GR1/gr1 78 0 50 CAT2/CAT2 GR1/GR1 79 0 4 CAT2/CAT2 GR1/GR1 96 0 51 CAT2/cat2 GR1/gr1 68 0 5 CAT2/cat2 gr1/gr1 65 0 52 CAT2/cat2 GR1/gr1 72 0 6 CAT2/cat2 GR1/gr1 84 0 53 CAT2/CAT2 GR1/gr1 116 0 7 CAT2/CAT2 GR1/gr1 58 0 54 cat2/cat2 GR1/GR1 47 0 8 cat2/cat2 GR1/GR1 87 1.1 55 cat2/cat2 GR1/GR1 42 0 9 CAT2/cat2 GR1/gr1 48 0 56 CAT2/cat2 GR1/gr1 55 0 10 CAT2/CAT2 GR1/GR1 101 0 57 CAT2/cat2 GR1/gr1 110 0 11 cat2/cat2 GR1/gr1 51 0 58 CAT2/cat2 GR1/gr1 101 0 12 cat2/cat2 GR1/gr1 49 0.9 59 CAT2/cat2 GR1/gr1 48 0 13 cat2/cat2 GR1/gr1 74 1.8 60 CAT2/CAT2 GR1/GR1 95 0 14 cat2/cat2 GR1/GR1 96 0.2 61 cat2/cat2 GR1/gr1 128 1.1 15 CAT2/cat2 GR1/gr1 58 0 62 CAT2/CAT2 GR1/gr1 78 0 16 CAT2/cat2 GR1/gr1 52 0 63 CAT2/cat2 GR1/gr1 65 0 17 CAT2/cat2 GR1/gr1 70 0 64 cat2/cat2 gr1/gr1 28 12.4 18 CAT2/cat2 GR1/gr1 70 0 65 CAT2/cat2 GR1/gr1 72 0 19 CAT2/cat2 GR1/gr1 87 0 66 CAT2/CAT2 GR1/gr1 90 0 20 CAT2/CAT2 GR1/GR1 34 0 67 CAT2/cat2 gr1/gr1 68 0 21 CAT2/CAT2 GR1/GR1 66 0 68 CAT2/cat2 GR1/gr1 65 0 22 cat2/cat2 GR1/gr1 60 1.7 69 CAT2/cat2 GR1/gr1 77 0 23 cat2/cat2 GR1/gr1 65 2.2 70 CAT2/CAT2 GR1/gr1 99 0 24 CAT2/CAT2 GR1/gr1 67 0 71 CAT2/cat2 GR1/gr1 73 0 25 CAT2/cat2 GR1/gr1 68 0 72 cat2/cat2 GR1/GR1 62 2.5 26 CAT2/cat2 GR1/gr1 73 0 73 CAT2/CAT2 GR1/gr1 112 0 27 cat2/cat2 gr1/gr1 19 8.6 74 CAT2/cat2 GR1/gr1 106 0 28 CAT2/CAT2 gr1/gr1 77 0 75 cat2/cat2 gr1/gr1 31 9.8 29 CAT2/CAT2 GR1/gr1 50 0 76 cat2/cat2 GR1/GR1 77 0 30 CAT2/cat2 GR1/GR1 64 0 77 CAT2/CAT2 GR1/GR1 62 0 31 cat2/cat2 GR1/GR1 59 0 78 CAT2/CAT2 GR1/GR1 157 0 32 CAT2/cat2 GR1/GR1 53 0 79 CAT2/cat2 gr1/gr1 82 0 33 CAT2/cat2 GR1/gr1 70 0 80 cat2/cat2 GR1/gr1 69 0 34 CAT2/CAT2 GR1/gr1 81 1.2 81 CAT2/CAT2 gr1/gr1 130 0 35 CAT2/cat2 GR1/gr1 89 0 82 cat2/cat2 GR1/gr1 57 0 36 CAT2/cat2 GR1/gr1 68 0 83 CAT2/cat2 gr1/gr1 75 0 37 CAT2/cat2 GR1/gr1 50 0 84 CAT2/cat2 GR1/gr1 95 0 38 CAT2/CAT2 GR1/GR1 52 0 85 CAT2/CAT2 GR1/gr1 71 0 39 CAT2/cat2 gr1/gr1 75 0 86 CAT2/CAT2 GR1/GR1 81 0 40 CAT2/cat2 GR1/GR1 69 0 87 CAT2/cat2 GR1/GR1 100 0 41 cat2/cat2 gr1/gr1 43 13.0 88 CAT2/cat2 gr1/gr1 86 0 42 CAT2/cat2 GR1/GR1 80 0 89 cat2/cat2 gr1/gr1 33 10.7 43 CAT2/CAT2 GR1/gr1 70 0 90 CAT2/cat2 gr1/gr1 70 0 44 cat2/cat2 gr1/gr1 29 15.2 91 cat2/cat2 GR1/gr1 33 0 45 CAT2/cat2 GR1/GR1 108 0 92 CAT2/cat2 GR1/gr1 115 0 46 cat2/cat2 GR1/GR1 69 0.7 93 CAT2/cat2 GR1/gr1 112 0 47 CAT2/CAT2 gr1/gr1 69 0 C 120

60

*** Rosette FW (mg)

0

200

***

100

Lesions (% rosette area) * ** 0 cat2/cat2 gr1/gr1 cat2/cat2 GR1/gr1 cat2/cat2 CAT2/cat2 gr1/gr1 cat2/cat2 GR1/GR1 cat2/cat2 CAT2/cat2 GR1/gr1 CAT2/CAT2 gr1/gr1 CAT2/cat2 GR1/GR1 CAT2/CAT2 GR1/gr1 CAT2/CAT2 GR1/GR1

Number of plants

Theoretical (n = 93) 5.8 5.8 11.6 5.8 5.8 11.6 11.6 11.6 23.3

Observed 10 3 11 8 7 10 6 7 31

Supplemental Figure 3. Analysis of the conditional genetic interaction between cat2 and gr1 in an allelic double cat2 gr1 mutant. F2 seeds from F1 double cat2 gr1-1 heterozygotes were

germinated and grown at high CO2 for three weeks. 93 plants were transferred to air in 16h days and four days later plants were photographed, genotyped, and rosette fresh weight and lesions quantified. A, Photographs of plants four days after transfer to air with cat2 gr1 double homozygotes boxed in red. Enlarged photographs of two of the seven cat2 gr1 plants are shown below the main figure. B, List of genotypes, rosette masses and lesion areas with cat2 gr1 plants boxed in red. C, Histograms of rosette fresh weight and lesions in the different genotypes. Asterisks indicate significant difference from CAT2/CAT2 GR1/GR1 segregants.

ANNEX 2

Noctor et al., 2012 Plant Cell Environ.

Annex 2. Publication by Noctor et al. (2012) Plant Cell Environ. 35: 454–484.

Several members of the laboratory, including myself, contributed to this review paper.

Plant, Cell and Environment (2012) 35, 454–484 doi: 10.1111/j.1365-3040.2011.02400.x

Glutathione in plants: an integrated overviewpce_2400 454..484

GRAHAM NOCTOR1, AMNA MHAMDI1, SEJIR CHAOUCH1, YI HAN1, JENNY NEUKERMANS1, BELEN MARQUEZ-GARCIA2, GUILLAUME QUEVAL2 & CHRISTINE H. FOYER2

1Institut de Biologie des Plantes, UMR CNRS 8618, Université de Paris sud 11, Orsay cedex, France and 2Centre for Plant Sciences, Faculty of Biology, University of Leeds, Leeds, UK

ABSTRACT disulphide form (GSSG) that is recycled to GSH by NADPH-dependent glutathione reductase (GR). The Plants cannot survive without glutathione central role of glutathione in defence metabolism in (g-glutamylcysteinylglycine) or g-glutamylcysteine- animals was established long ago, largely because selenium- containing homologues. The reasons why this small mol- dependent glutathione peroxidase (GPX) is a central pillar ecule is indispensable are not fully understood, but it can be of animal antioxidant metabolism. Pharmacologically inferred that glutathione has functions in plant develop- induced GSH deficiency in newborn mammals such as rats ment that cannot be performed by other thiols or antioxi- and guinea pigs leads to rapid multi-organ failure and death dants. The known functions of glutathione include roles within a few days (Meister 1994). In plant cells, where in biosynthetic pathways, detoxification, antioxidant bio- reductive H2O2 metabolism has been linked to ascorbate chemistry and redox homeostasis. Glutathione can interact since the 1970s, the absolutely irreplaceable role of glu- in multiple ways with proteins through thiol-disulphide tathione was less apparent. However, glutathione depletion exchange and related processes. Its strategic position in Arabidopsis knockouts lacking the first enzyme of the between oxidants such as reactive oxygen species and cel- committed pathway of GSH synthesis causes embryo lular reductants makes the glutathione system perfectly lethality (Cairns et al. 2006). Thus, both plant and mamma- configured for signalling functions. Recent years have wit- lian cells rely on at least some of the multifunctional prop- nessed considerable progress in understanding glutathione erties of glutathione for their vigour and survival. synthesis, degradation and transport, particularly in relation Glutathione is the principal low-molecular-weight thiol to cellular redox homeostasis and related signalling under in most cells. However, there are some intriguing varia- optimal and stress conditions. Here we outline the key tions in organisms such as halobacteria, in which GSH recent advances and discuss how alterations in glutathione can be replaced by other sulphur compounds like g- status, such as those observed during stress, may participate glutamylcysteine (g-EC) and thiosulphate (Newton & Javor in signal transduction cascades. The discussion highlights 1985). Similarly, in some parasitic protozoa, trypanothione some of the issues surrounding the regulation of glu- [N1,N8-bis(glutathionyl)spermidine] can substitute for glu- tathione contents, the control of glutathione redox poten- tathione (Fairlamb et al. 1985). Some plant taxa contain tial, and how the functions of glutathione and other thiols glutathione homologues, in which the C-terminal residue are integrated to fine-tune photorespiratory and respiratory is an amino acid other than glycine (Rennenberg 1980; metabolism and to modulate phytohormone signalling Klapheck 1988; Klapheck et al. 1992; Meuwly, Thibault & pathways through appropriate modification of sensitive Rauser 1993). These compounds include homoglutathione protein cysteine residues. (g-Glu-Cys-b-Ala), which is found alongside GSH in many legumes (MacNicol 1987; Klapheck 1988). Interestingly, Key-words: Antioxidant; detoxification; oxidative stress; gene duplication during evolution has resulted in the coex- pathogens; redox metabolism and signalling; sulphur istence of different synthetases that produce GSH or homo- metabolism; thiols. glutathione (Frendo et al. 1999). Cereals produce another GSH variant (hydroxymethylGSH; g-Glu-Cys-Ser) through INTRODUCTION reactions that remain to be fully elucidated but which likely involve modification of GSH rather than alternative syn- Glutathione (or a functionally homologous thiol) is an thesis pathways (Klapheck et al. 1992; Okumura, Koizumi & essential metabolite with multiple functions in plants Sekiya 2003; Skipsey, Davis & Edwards 2005a). Disulphide (Fig. 1). The fundamental and earliest recognized function forms of these homologues are reducible by GR (Klapheck of glutathione is in thiol-disulphide interactions, in which 1988; Klapheck et al. 1992; Oven et al. 2001). Therefore, reduced glutathione (GSH) is continuously oxidized to a current information suggests that they do not require alter- Correspondence: G. Noctor. e-mail: [email protected] native reductive systems. Novel homologues may remain to be discovered (Skipsey et al. 2005a). For example, high- GSH is used here to indicate the thiol (reduced) form of glu- tathione while GSSG denotes the disulphide form. The term ‘glu- performance liquid chromatography (HPLC) analysis of tathione’ is used where no distinction is drawn or both forms may thiols in poplar overexpressing a bacterial form of the be concerned. second enzyme of glutathione synthesis revealed two novel 454 © 2011 Blackwell Publishing Ltd Glutathione status and functions 455

Primary Defence/ metabolism Detoxification

C and N metabolism Secondary metabolism

Xenobioticdetoxification Glu Gly GS-conjugates Heavy metal detoxification S metabolism Cysy γ-EC GSH Phytochelatins NADP+ GR ROS GSNO RNS signalling Pyridiney nucleotide NADPH synthesis and reduction GSSG ROS signalling Redox signalling

Figure 1. General overview of some of the most important glutathione functions (synthesis, redox turnover, metabolism, signalling). Cys, cysteine; g-EC, g-glutamylcysteine; GS-conjugates, glutathione S-conjugates; GSNO, S-nitrosoglutathione; Glu, glutamate; Gly, glycine; RNS, reactive nitrogen species; ROS, reactive oxygen species. peaks, in addition to GSH. The peaks were particularly stress, tissues such as leaves typically maintain measurable abundant in conditions in which leaf glycine contents are GSH: GSSG ratios of at least 20:1 (e.g. Mhamdi et al. depleted such as darkness (G. Noctor, A.C.M. Arisi, L. 2010a). It is important to note that this is an average value Jouanin, C.H. Foyer, unpublished data). across tissues, and that ratios may be higher (e.g. cytosol) or Like other thiols, glutathione can undergo numerous lower (e.g. vacuole) in specific subcellular compartments redox reactions. As well as GSSG, oxidized forms notably (Meyer et al. 2007; Queval et al. 2011). include the formation of ‘mixed disulphides’ with proteins and other thiol molecules. Other oxidized forms include thiyl radicals, sulphenic acid (SOH) and, possibly, sulphinic GLUTATHIONE ACCUMULATION, TURNOVER

(SO2H) or sulphonic (SO3H) acids on the cysteine moiety of AND TRANSPORT glutathione, similar to their formation on protein cysteine GSH biosynthetic pathway residues. Moreover, a wide array of glutathione conjugates can be formed with endogenous and xenobiotic electro- As in animals, GSH is synthesized in plants from its con- philic species (Wang & Ballatori 1998; Dixon & Edwards stituent amino acids by two ATP-dependent steps (Rennen- 2010) while interactions with the nitric oxide (NO) system berg 1980; Meister 1988; Noctor et al. 2002a; Mullineaux & via formation of S-nitrosoglutathione (GSNO) broaden the Rausch 2005). Each of the synthetic enzymes is encoded by scope of glutathione as a reservoir of signalling potential a single gene (May & Leaver 1994; Ullman et al. 1996), and (Lindermayr, Saalbach & Dürner 2005). Arabidopsis knockout lines for either have lethal pheno- It has long been recognized that GSH is oxidized by types. While knocking out expression of GSH1, encoding reactive oxygen species (ROS) as part of the antioxidant g-EC synthetase (g-ECS), causes lethality at the embryo barrier that prevents excessive oxidation of sensitive cellu- stage (Cairns et al. 2006), knockouts for GSH2, encoding lar components. Unlike the oxidized forms of many other glutathione synthetase (GSH-S), show a seedling-lethal primary and secondary metabolites that can also react with phenotype (Pasternak et al. 2008). Using forward genetics ROS, GSSG is rapidly recycled by the GRs in key approaches, several mutants have been identified in which organelles and the cytosol (Halliwell & Foyer 1978; Smith, decreased GSH contents are caused by less severe muta- Vierheller & Thorne 1989; Edwards, Rawsthorne & Mul- tions in the GSH1 gene. Of these, the rml1 (rootmeristem- lineaux 1990; Jiménez et al. 1997; Chew, Whelan & Millar less1) mutant, which has less than 5% of wild-type 2003; Kataya & Reumann 2010). A characteristic feature of glutathione contents, shows the most striking phenotype glutathione is its high concentration in relation to other because it fails to develop a root apical meristem (Vernoux cellular thiols. In general, glutathione accumulates to milli- et al. 2000). In other mutants, in which glutathione is molar concentrations, with tissue contents well in excess of decreased to about 25 to 50% of wild-type contents, devel- free cysteine. A second key characteristic of the cellular opmental phenotypes are weak or absent, but alterations glutathione pool is its high reduction state. In the absence of in environmental responses are observed. In cad2, lower © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 456 G. Noctor et al. glutathione is associated with enhanced cadmium sensitiv- regulation (Hicks et al. 2007; Gromes et al. 2008). This is ity, whereas rax1 was identified by modified APX2 expres- likely an important factor in the well-known up-regulation sion and pad2 shows decreased camalexin contents and of glutathione synthesis in response to oxidative stress. enhanced sensitivity to pathogens (Howden et al. 1995; Another mode of regulation that is likely to be important in Cobbett et al. 1998; Ball et al. 2004; Parisy et al. 2006). In glutathione homeostasis is feedback inhibition of g-ECS by addition to genetic approaches, a pharmacological tool that GSH. First reported for the animal enzyme (Richman & has frequently been used to deplete glutathione is buthion- Meister 1975), this regulatory mechanism also occurs in ine sulphoximine (BSO), a specific inhibitor of g-ECS (Grif- plants (Hell & Bergmann 1990; Noctor et al. 2002a). Alle- fith & Meister 1979). viation of feedback inhibition is likely to be an important The activity of g-ECS is strongly associated with chloro- mechanism driving accelerated rates of synthesis under plasts in wheat (Noctor et al. 2002a). Localization studies conditions in which glutathione is being consumed (e.g. in Arabidopsis have demonstrated that the enzyme is in the synthesis of phytochelatins). The mechanistic links restricted to plastids in this species (Wachter et al. 2005). between feedback inhibition and thiol/disulphide redox The Arabidopsis GSH-S is found in both chloroplasts and regulation of g-ECS remain to be elucidated. cytosol. Of the two transcripts encoded by the GSH2 gene, the most abundant one is the shorter form, which is trans- Integration of glutathione synthesis with lated to produce a cytosolic GSH-S (Wachter et al. 2005). sulphur assimilation Thus, the first step of glutathione synthesis is plastidic while the second step is probably predominantly located in the In accordance with the observation that enhanced cysteine cytosol. supply favours glutathione accumulation, increases in GSH synthesis are associated with up-regulation of the cysteine Regulation of biosynthesis synthesis pathway. For example, glutathione accumulation triggered by oxidative stress causes accumulation of tran- Many factors affect the synthesis of glutathione, but the scripts encoding adenosine 5’-phosphosulphate reductase most important are considered to be g-ECS activity and (APR) and serine acetyltransferase (SAT; Queval et al. cysteine availability. Accordingly, constitutive increases in 2009). While all three Arabidopsis APR genes encode plas- glutathione can be produced either by overexpression of tidial enzymes, only one of the five SAT gene produces an the first enzyme of the committed glutathione synthesis isoform located in this compartment (Kawashima et al. pathway or of enzymes involved in cysteine synthesis 2005). Although the mitochondrial SAT makes the major (Strohm et al. 1995; Noctor et al. 1996, 1998; Creissen et al. contribution to cysteine synthesis under standard condi- 1999; Harms et al. 2000; Noji & Saito 2002; Wirtz & Hell tions (Haas et al. 2008;Watanabe et al. 2008), the chloroplast

2007). Other factors that may affect GSH contents in SAT was the most strongly induced during H2O2-triggered certain conditions include glycine and ATP (Buwalda et al. accumulation of glutathione (Queval et al. 2009). As well as 1990; Noctor et al. 1997; Ogawa et al. 2004). Increased up-regulation at the transcript level, ozone exposure acti- g-ECS activity may result from transcriptional or post- vates at least one APR at the post-translational level (Bick transcriptional changes (May et al. 1998), but so far rela- et al. 2001).As in the case of post-translational activation of tively few conditions have been shown to cause marked g-ECS, the exact mechanisms regulating APR activation induction of GSH1 or GSH2 transcripts. Both genes are state remain unclear. One appealing possibility is that induced by jasmonic acid (JA) and heavy metals (Xiang & oxidation-triggered decreases in GSH:GSSG activate glu- Oliver 1998; Sung et al. 2009) and also respond to light and tathione synthesis by increasing the chloroplast glutathione some stress conditions such as drought and certain patho- redox potential and allowing glutaredoxin (GRX)- gens. However, neither externally applied H2O2 nor intrac- mediated activation of both enzymes. Consistent with this ellularly generated H2O2 leads to increased abundance model, accumulation of glutathione in catalase-deficient of GSH1 or GSH2 transcripts in Arabidopsis, despite the barley and Arabidopsis is associated with markedly well-described increases in glutathione in these conditions increased chloroplast GSSG content (Smith et al. 1985; (Smith et al. 1984; May & Leaver 1993;Willekens et al. 1997; Queval et al. 2011). However, regulation could also be Sánchez-Fernández et al. 1998; Xiang & Oliver 1998; linked to thioredoxin (TRX) activity. Whatever the details Queval et al. 2009). While some evidence has been pre- of the underlying regulatory mechanisms, intracellular oxi- sented that production of the g-ECS protein is regulated at dative stress can drive glutathione accumulation to several- the level of translation (Xiang & Bertrand 2000), more fold basal levels (Smith et al. 1984; Willekens et al. 1997). attention has focused on post-translational redox controls. It has been estimated that a fivefold accumulation of A partially purified enzyme preparation from tobacco was glutathione in Arabidopsis cat2 mutants means that the shown to be sensitive to inhibition by dithiols (Hell & Berg- amount of sulphur in the glutathione cysteine residue mann 1990), and similar effects were subsequently reported approaches that which is found in protein cysteine and in other species (Noctor et al. 2002a; Jez, Cahoon & Chen methionine residues combined (Queval et al. 2009). As well 2004). Recently, it has been shown that the plant g-ECS as the changes in transcripts and extractable activities pre- forms a homodimer linked by two disulphide bonds viously mentioned, H2O2-triggered glutathione accumula- (Hothorn et al. 2006), one of which is involved in redox tion in barley is accompanied by increased uptake of © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 457 labelled sulphate (Smith et al. 1985). Accordingly, marked associated with higher leaf contents of several free amino accumulation of glutathione achieved by transgenic acids, including tyrosine, leucine, isoleucine and valine enhancement using a bacterial enzyme with both g-ECS and (Noctor et al. 1998). This could bear some relation to redox GSH-S activities was dependent on sufficient sulphur regulation of the enzymes of amino acid metabolism in the supply (Liedschulte et al. 2010). chloroplast, several of which are potential TRX targets (Montrichard et al. 2009). Overexpression of glutathione biosynthesis Although overexpression of GSH-S alone has less marked effects on tissue glutathione contents than boosting Regardless of the controls over synthesis, and the signalling g-ECS capacity (Foyer et al. 1995; Strohm et al. 1995; Noctor roles discussed later, tissue glutathione contents can be et al. 1998), the effects could be condition dependent if markedly enriched in plants. Whereas overexpression of GSH-S becomes limiting when g-EC supply is increased, for Escherichia coli GSH-S in poplar produced little effect on example, during exposure to cadmium (Zhu et al. 1999b). glutathione contents in optimal conditions (Foyer et al. Similarly, expression of a soybean homoGSH-S in tobacco 1995; Strohm et al. 1995), introduction of the E. coli g-ECS was successfully used to confer tolerance to the herbicide, caused a two- to fourfold increase in leaf glutathione, and fomesafen (Skipsey et al. 2005b). Interestingly, in this study, this was observed whether the bacterial g-ECS was targeted the homoGSH-S was expressed together with a homoGSH- to the cytosol or the chloroplast (Noctor et al. 1996, 1998; preferring soybean GST (Skipsey et al. 2005b). Arisi et al. 1997). Expression of the same g-ECS in the tobacco chloroplast also produced substantial increases in Turnover and degradation leaf glutathione (Creissen et al. 1999) whereas homologous overexpression of g-ECS in Arabidopsis resulted in about Biochemical studies of glutathione degradation in tobacco twofold glutathione enrichment (Xiang et al. 2001). More conducted by the Rennenberg group (Rennenberg, recently, considerably greater increases in glutathione have Steinkamp & Kesselmeier 1981; Steinkamp & Rennenberg been achieved by overexpression of a bifunctional g-ECS/ 1984, 1985; Steinkamp, Schweihofen & Rennenberg 1987) GSH-S from Streptococcus (Liedschulte et al. 2010). have been significantly extended over the last decade, There is still some debate about the impact of increasing notably by genetically based studies in Arabidopsis. Four glutathione contents in plants. Whereas increased glu- different types of enzymes have been described that could tathione triggered by chloroplastic overexpression of g-ECS initiate glutathione breakdown (Fig. 2). Some of these in tobacco was accompanied by oxidation and lesion forma- enzymes could use GSH, while others act preferentially on tion (Creissen et al. 1999), studies with the same E. coli GSSG or other GS-conjugates. Firstly, glutathione or construct introduced into other species did not report GS-conjugates could be degraded by carboxypeptidase marked phenotypic effects (Noctor et al. 1998; Zhu et al. activity (Steinkamp & Rennenberg 1985), which has been 1999a). More recently, it has been shown that one of the detected in barley vacuoles (Wolf, Dietz & Schröder 1996). chloroplast lines with multiple insertions shows symptoms A second type of enzyme that has been implicated in of early leaf senescence (Herschbach et al. 2009). Another GS-conjugate breakdown is the cytosolic enzyme phytoch- study of a single cytosolic overexpressor grown for 3 years elatin synthase (PCS; Blum et al. 2007, 2010). Because in the field reported several effects, including lower biomass GS-conjugates are usually rapidly transported into the and photosynthesis (Ivanova et al. 2011). In terms of inter- vacuole, the extent to which they accumulate in the cytosol pretation of phenotypes, a clear limitation of studies in remains unclear. However, some data obtained from work poplar is the difficulty of genetic studies in this species. This on Arabidopsis suggest that PCS may play some role in necessitates analysis of several independent lines to be certain cell types or when the enzyme is activated by heavy certain that the observed effects are linked to increases in metals (Grzam et al. 2006; Blum et al. 2007; Brazier-Hicks glutathione. To date no marked deleterious effects have et al. 2008). been reported in the tobacco overexpressors with very high The third type of enzyme, g-glutamyl transpeptidase glutathione (Liedschulte et al. 2010), though these lines are (GGT), has been the focus of several research groups clearly interesting systems in which to analyse the impact in recent years. These enzymes act in the mammalian of high glutathione concentrations on plant function. The g-glutamyl cycle (Meister 1988), and catalyse the hydrolysis reasons for the apparent discrepancy between the studies of or transpeptidation of GSH at the plasma membrane. The Creissen et al. (1999) and Liedschulte et al. (2010) remain to resulting g-glutamyl amino acid derivatives are further be elucidated. An important factor could be differences in processed by g-glutamyl cyclotransferases (GGC) and the introduced proteins. Unlike the E. coli g-ECS, the Strep- 5-oxoprolinase (5-OPase) to produce free glutamate. In tococcus protein has both GSH-S and g-ECS activities. Arabidopsis, GGTs are encoded by at least three func- Several studies have shown the benefits of elevating glu- tional genes. Two of these (GGT1 and GGT2) encode tathione through overexpression of g-ECS. These include apoplastic enzymes with activity against GSH, GSSG and enhanced resistance to heavy metals and certain herbicides GS-conjugates (Martin & Slovin 2000; Storozhenko et al. (Zhu et al. 1999a; Gullner, Komives & Rennenberg 2001; 2002). Extracellular GGT has also been described in barley Ivanova et al. 2011). Intriguingly, increased glutathione pro- and maize (Masi et al. 2007; Ferretti et al. 2009). Unlike their duced by overexpression of g-ECS in the chloroplast was counterparts in animal cells, GGT1 and GGT2 are probably © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 458 G. Noctor et al.

Apoplast GSSG γ-Glu-aa + cys-gly GGT

γ-Glu-aa + cys-gly GGT

γ-Glu-aa GSSG γ-EC Cpep GSSG + gly GGC

GGC? Vacuole 5-OP 5OPase GSH + aa GS-XGS X or cys-gly? GGT PCS Glu γ-Glu-aa GS-X + cys(X)-gly Cpep γ-EC + gly PCS γ-EC-X + gly γ-EC-X + gly Cytosol

Figure 2. Possible pathways of glutathione degradation. For simplicity, not all possible downstream reactions are shown, for example, metabolism of g-EC by GGC. Disulphide forms of GSSG breakdown products are omitted for the same reason. As well as transpeptidation, GGT could also catalyse hydrolysis of GSH to Glu and Cys-Gly. aa, amino acid; Cpep, carboxypeptidase; GGC, g-glutamyl cyclotransferase; GGT, g-glutamyl transpeptidase; 5-OP, 5-oxoproline; 5-OPase, 5-oxoprolinase; PCS, phytochelatin synthase; X, S-conjugated compound. bound to the cell wall rather than to the plasmalemmma vacuolar or extracellular GGT (Ohkama-Ohtsu et al. 2008). (Martin et al. 2007; Ohkama-Ohtsu et al. 2007a). Based on GGC is therefore a fourth type of enzyme potentially phenotypes of mutants and other studies, these enzymes involved in initiating glutathione degradation, although may be important in countering oxidative stress or in sal- both the rat and tobacco GGC have been reported to be vaging excreted GSSG (Ohkama-Ohtsu et al. 2007a; Fer- unable to use GSH (Orlowski & Meister 1973; Steinkamp retti et al. 2009; Destro et al. 2011). Besides the extracellular et al. 1987). The first gene encoding GGC was identified in GGTs, Arabidopsis has at least one vacuolar GGT, which is humans recently, but no obviously homologous sequences probably involved in the breakdown of GS-conjugates exist in plants (Oakley et al. 2008). Yet other proteins may (Grzam et al. 2007; Martin et al. 2007; Ohkama-Ohtsu et al. contribute to some extent to glutathione turnover, for 2007b). Along with PCS, GGTs may be important in example, GGP1, which has been implicated in the removal metabolizing GS-conjugates that are formed during the of the Glu residue from a GS-conjugate during glucosino- synthesis of certain secondary metabolites (Ohkama-Ohtsu late synthesis (Geu-Flores et al. 2009). Because of this et al. 2011; Su et al. 2011). complexity, several questions remain on glutathione degra- In animals, g-glutamyl peptides produced by GGT are dation in plants. These include cellular/tissue specificities, further metabolized by GGC.An Arabidopsis gene (OXP1) activities against the different forms of glutathione and, in has been identified that likely encodes 5-OPase. This some cases, the gene identities. enzyme catalyses the hydrolysis of 5-oxoproline, which Despite the plethora of possible routes of glutathione is the product of GGC activity (Ohkama-Ohtsu et al. catabolism, the extent of glutathione turnover and resyn- 2008). Based on 5-oxoproline accumulation in single oxp1 thesis remains unclear. Rates of glutathione catabolism in mutants, and in triple oxp1 ggt1 ggt4 mutants that are defi- Arabidopsis leaves have been estimated to be as high as cient in the major GGT activities as well as 5-OPase, it was 30 nmol g-1 FW h-1 (Ohkama-Ohtsu et al. 2008). Extract- proposed that the predominant pathway for GSH degrada- able Arabidopsis leaf GSH-S activities can reach tion is cytosolic and initiated by GGC (Fig. 2), and not 10 nmol g-1 FW min-1, while the extractable activity of the © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 459 rate-limiting enzyme, g-ECS, is much lower, only about chloroplasts have been estimated to account for about 50 0.5 nmol g-1 FW min-1 (Queval et al. 2009). This rate is and 30%, respectively, of total glutathione in Arabidopsis therefore very close to the rates of turnover estimated by leaf mesophyll cells (Queval et al. 2011). Ohkama-Ohtsu et al. (2008). However, this close correspon- Uptake of both GSH and GSSG has been documented in dence could be coincidental. Extractable g-ECS activities both cells and protoplasts (Schneider, Martini & Rennen- should theoretically be higher than true in vivo fluxes, berg 1992; Jamaï et al. 1996). Transport across the plas- because substrates, notably cysteine, may be less limiting malemma could be accomplished by the oligopeptide and feedback inhibition by glutathione negligible in assays transporter (OPT) family. Within this family, the Brassica of desalted extracts. It should be noted, however, that redox uncea and rice genes, BjGT1 and OsGT1, are homologous regulation and the absence of other factors may make it to the yeast HGT1 transporter, which can transport GSH, difficult to recover true in vivo capacities of g-ECS in in GSSG and GS-conjugates, but also other small peptides vitro assays. Indeed, assuming that accumulation of glu- (Bourbouloux et al. 2000; Bogs et al. 2003; Zhang et al. tathione reflects neosynthesis and that there are no alter- 2004). Of the nine annotated Arabidopsis OPT genes (Koh native routes awaiting description, our recent data strongly et al. 2002), OPT6 may play a role in long-distance transport suggest that at least under some conditions, g-ECS can work (Cagnac et al. 2004). The OPT6 gene is highly expressed in considerably faster in vivo than the measured extractable the vasculature, where it may transport GS-conjugates and value. Glutathione oxidation in cat2 gr1 double mutants GS-cadmium complexes as well as GSH and GSSG triggers accumulation of total glutathione to more than (Cagnac et al. 2004). Another study reported that GSH, but 10-fold basal levels within 4 d (Mhamdi et al. 2010a).Within not GSSG, was transported by OPT6 (Pike et al. 2009). the first 4 h after the beginning of excess H2O2 production Knockout opt6 mutants are aphenotypic, suggesting gene (a consequence of the cat2 background), about 800 nmol redundancy, and the transporter may be important in trans- glutathione g-1FW was newly accumulated in cat2 gr1, locating other peptides as well as glutathione (Pike et al. that is, a mean minimum synthesis rate of around 2009). 200 nmol g-1 FW h-1. Several different types of transporter may be important While the interplay between degradation and neosynthe- in translocation of glutathione between subcellular com- sis remains to be elucidated, typical leaf glutathione con- partments. Inner chloroplast envelope transporters have tents are 300 nmol g-1 FW. Thus, degradation rates of recently been described that likely act to link plastidic 30 nmol g-1 FW h-1 would mean that some glutathione g-ECS and cytosolic GSH-S via g-EC export across the pools must turn over and be resynthesized within a few chloroplast envelope (Maughan et al. 2010). These trans- hours. Another question concerns the possible role of glu- porters are encoded by three genes in Arabidopsis, called tathione degradation during oxidative stress. In this regard, CLT1, CLT2 and CLT3. Further, the wild-type phenotype it is interesting that GGT1 was among glutathione- of gsh2 knockout mutants can be restored by transforma- associated genes that were significantly down-regulated tion with a construct driving GSH-S expression exclusively in GSSG-accumulating cat2 gr1 mutants (Mhamdi et al. in the cytosol (Pasternak et al. 2008). This observation pro- 2010a). By virtue of their activities in cleaving GS- vides further evidence that GSH can be imported from the conjugates, some enzymes implicated in glutathione degra- cytosol into the plastid, consistent with radiolabelling dation may also be important in biosynthesis pathways (e.g. studies of isolated wheat chloroplasts (Noctor et al. 2002a). Su et al. 2011; see below). Thus, current concepts suggest that g-EC is synthesised exclusively in the chloroplast, then either converted to Compartmentation and transport GSH in this compartment or transported to the cytosol, where part of any GSH formed can be transported into the Glutathione is one of the major forms of organic sulphur chloroplast (Fig. 3). translocated in the phloem (Herschbach & Rennenberg Tonoplast multidrug resistance-associated protein 1994, 1995; Bourgis et al. 1999; Mendoza-Cózatl et al. 2008), (MRP) transporters of the ATP-binding cassette (ABC) and must therefore move between cells, either apoplasti- type may act to clear GS-conjugates or GSSG from the cally, symplastically or both. Glutathione can be detected in cytosol (Martinoia et al. 1993; Rea 1999; Foyer, Theodoulou apoplastic extracts but at much lower levels than in whole & Delrot 2001). Indeed, certain Arabidopsis MRPs are tissue extracts (Vanacker, Carver & Foyer 1998). This is in competent in GSSG as well as GS-conjugate transport (Lu line with immunolocalization studies that have detected et al. 1998). Barley vacuoles can take up GSSG much more weak or no labelling in the cell wall and apoplast (Zech- rapidly than GSH, and the removal of cytosolic GSSG may mann et al. 2008). The apoplastic pool is also likely more play a role in maintaining glutathione redox status in oxidized than many intracellular pools. Current concepts this compartment (Tommasini et al. 1993). Although glu- suggest that both the apoplast and vacuole have low glu- tathione concentrations in the vacuoles of unstressed plants tathione concentrations and GSH:GSSG ratios, and that have long been considered to be low or negligible (Rennen- most of the glutathione is concentrated in other compart- berg 1980; Zechmann et al. 2008), accumulation of GSSG in ments. While immunolocalization studies point to particu- this compartment could be a physiologically important part larly high concentrations in the mitochondria (Zechmann of oxidative stress responses (Queval et al. 2011). Further, et al. 2008), because of their greater volume the cytosol and induction of specific MRPs occurs in response to oxidising © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 460 G. Noctor et al.

GSH Apoplast GSSG

OPT

Vacuole CYT MIT

CCys GSSG MRP

Glu γ-ECS GR2 GSSG γ-EC GSH γ-EC Gly GSH-S CLT Gly GSH-S GSH GR1 GSH GR2 ?

Chloroplast PER GSH GSH Nucleus GR1

Figure 3. Compartmentation of glutathione synthesis, reduction and transport. g-EC(S), g-glutamylcysteine (synthetase); BAG, Bcl2-associated anathogene; CYT, cytosol. PER, peroxisome; GSH-S, glutathione synthetase; GR, glutathione reductase; GSSG, glutathione disulphide; CLT, chloroquinone resistance transporter-like transporter; MIT, mitochondrion; MRP, multidrug resistance-associated protein; OPT, oligopeptide transporter. agents (Sánchez-Fernández et al. 1998) and also accompa- component regulating GSH transport into the nucleus in nies GSSG accumulation in Arabidopsis (Mhamdi et al. mammalian tissues, as it is in mitochondria (Voehringer 2010a). et al. 1998). Despite the lack of evidence for GSH synthesis Immunolocalization studies detect nuclear glutathione in the mitochondria, high glutathione concentrations have concentrations that are similar to those in the cytosol in been detected in this organelle (Zechmann et al. 2008). It is cells in the undividing G0 state (Zechmann et al. 2008). possible that pore-regulating proteins are involved in regu- Other recent studies suggest that nuclear/cytosol distribu- lating mitochondrial and nuclear GSH concentrations tion of glutathione may be dynamic, with glutathione being (Fig. 3). recruited into the nucleus early in the cell cycle in both Like mitochondria, peroxisomes contain glutathione mammalian and plant cells (Markovic et al. 2007; Diaz- and GR (Jiménez et al. 1997) while apparently lacking Vivancos et al. 2010a). A nuclear redox cycle within the cell the enzymes of glutathione synthesis. Immunolocalization cycle has been proposed, according to which GSH moves studies suggest that the peroxisomal glutathione concentra- into the nucleus during the G1 phase: this in turn promotes tion is similar to the cytosolic concentration (Zechmann oxidation of the cytosol and, consequently, enhanced glu- et al. 2008), and it has been estimated in leaf mesophyll cells tathione accumulation (Diaz-Vivancos et al. 2010b). Oxida- to be around 3–4 mm (Queval et al. 2011). Presumably, this tion of the cytosol at G1 is accompanied by enhanced levels pool results from import across the single peroxisomal of ROS and lowering of the oxidative defense shield (Diaz- membrane, but transporters responsible for this activity Vivancos et al. 2010a). The accumulated GSH is divided remain to be characterized. between the daughter cells, in which the process begins again. These observations suggest the presence of proteins METABOLIC FUNCTIONS OF GLUTATHIONE in plants that are able to alter the permeability of nuclear Regulation of sulphur assimilation pores, facilitating GSH sequestration in the nucleus. Such proteins remain to be identified in plants. However, the As a significant non-protein sink for reduced sulphur, glu- anti-apoptotic factor Bcl-2 is thought to be a crucial tathione contents are influenced by sulphur supply. Indeed, © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 461 glutathione may be one of the S-containing compounds that between glutathione and S status (Nikiforova et al. 2003). link changes in sulphur nutrition to resistance to some Arabidopsis mutants defective in certain sulphate trans- pathogens, a phenomenon termed sulphur-induced resis- porters show decreased leaf glutathione accumulation tance (SIR; Gullner et al. 1999; Bloem et al. 2007; Zechmann (Maruyama-Nakashita et al. 2003). By contrast, expression et al. 2007; Höller et al. 2010). Stresses that involve an oxi- of a bacterial APR in Arabidopsis enriches tissue cysteine dative component also cause up-regulation of the S assimi- and glutathione contents (Tsakraklides et al. 2002). Quali- lation pathway. For example, exposure to ozone increases tatively similar effects are produced by overexpression of cysteine and glutathione levels, effects linked to post- enzymes of the cysteine synthesis pathway (Harms et al. translational activation of APR1 (Bick et al. 2001). In 2000; Noji & Saito 2002; Wirtz & Hell 2007). catalase mutants, increased intracellular H2O2 triggers accu- mulation of glutathione and precursors, and this is accom- Glutathione S-transferases panied by accumulation of transcripts for all three APRs (Queval et al. 2009). The classical reaction catalysed by GSTs is the formation of Glutathione is an important form of translocated organic a covalent bond between the sulphur atom of glutathione S and may thus act as an internal ‘barometer’ of plant S and an electrophilic compound (Fig. 4). In plants, the activ- status (Kopriva & Rennenberg 2004). Glutathione regu- ity of most GSTs depends on an active site serine, which lates several steps involved in S assimilation. These include stabilizes the GS-thiolate anion (Dixon & Edwards 2010), GSH inhibition of sulphate uptake and assimilation though in some GSTs such as the dehydroascorbate reduc- through effects on specific transporters and enzymes such tases (DHARs) this residue is replaced by a cysteine. This as ATP sulphurylase 1 (APS1) and APR (Herschbach & change confers the capacity for reversible disulphide bond Rennenberg 1994; Lappartient et al. 1999; Vauclare et al. formation with glutathione that is part of the DHAR cata- 2002; Buchner et al. 2004). However, the role of glutathione lytic mechanism (Dixon, Davis & Edwards 2002). Long in S assimilation is complex, because as well as these repres- considered as cytosolic enzymes, several GSTs may also sive effects, GSH is required by APR as reductant to localize at least partly to other compartments, including the produce sulphite (Leustek 2002). This occurs through a chloroplast, peroxisome and nucleus (Thatcher et al. 2007; domain of the APR enzyme that allows it to act as a GRX, Dixon et al. 2009). using GSH with an apparent KM value of about 1 mm (Bick The GST family in plants is notable for its structural and et al. 1998). Further, thiol-disulphide status also acts to functional diversity, and the biochemical and physiological control certain APR isoforms post-translationally through functions of specific members remain to be elucidated. As activation in response to increased glutathione oxidation well as or instead of catalysing conjugase reactions, some (Leustek 2002). GSTs may have antioxidative functions.The DHAR type of The influence of sulphur assimilation activity on glu- GST is one example, while several subclasses of GST have tathione contents also underlines the close relationship peroxidase activity (Wagner et al. 2002; Dixon et al. 2009).

GS-X

GST

CHO X GSH CO GSH CH3 HCHO COO- GSCO GSCHOH CHOH CHOH CO GSH HOC-SG HCOO- CH3 GLYII CH3 GLYI CH3 FDH FGH Me PCS

γ ( -Glu-Cys)n-Gly

Me

Figure 4. Detoxification pathways involving glutathione S-conjugate formation and metabolism. The glyoxalase pathway shows methylglyoxal metabolism, but other oxo-aldehydes might also be metabolized through this route. FDH, formaldehyde dehydrogenase; FGH, S-formylglutathione hydrolase; GLYI, glyoxalase I; GLYII, glyoxalase II; GST, glutathione S-transferase; Me, heavy metal; PCS, phytochelatin synthase; X, electrophilic compound. © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 462 G. Noctor et al.

GSTs are able to reduce organic peroxides though some (Grill, Winnacker & Zenk 1987; Grill et al. 1989; Cobbett & specificity is observed between different peroxides (Wagner Goldsbrough 2002; Rea, Vatamaniuk & Rigden 2004). et al. 2002; Dixon et al. 2009; Dixon & Edwards 2010). Some These compounds are produced from glutathione or

GSTs are strongly inducible by H2O2 (Levine et al. 1994; homologues by PCS, a cytosolic enzyme. In Arabidopsis, Willekens et al. 1997; Wagner et al. 2002). For example, there are two genes encoding PCS, one of which (PCS1)is certain GST transcripts can be considered useful markers the gene affected by the cad1 mutation that exacerbates for increased intracellular availability of H2O2 (Vanderau- cadmium sensitivity (Howden & Cobbett 1992; Ha et al. wera et al. 2005; Queval et al. 2007, 2009; Chaouch et al. 1999; Cazalé & Clemens 2001). As noted previously, it is 2010), though several are also inducible by salicylic acid possible that PCS has other biochemical roles, in addition (SA; Sappl et al. 2009). The same or other GSTs may have to phytochelatin synthesis, for example, turnover of functions in the detoxification of electrophilic xenobiotics, GS-conjugates (Rea et al. 2004; Blum et al. 2007, 2010; but also be important in biosynthetic or catabolic pathways. Clemens & Peršoh 2009). Recently described examples of biosynthetic pathways The importance of sufficient amounts of GSH to support involving GSTs are the production of glucosinolates and phytochelatin synthesis is evidenced by the identification of camalexin, discussed further later. For a more detailed the cad2 mutant as affected in g-ECS, causing a decrease in recent discussion of GSTs, we refer the reader to Dixon & leaf GSH to about 20% wild-type levels (Howden et al. Edwards (2010). 1995; Cobbett et al. 1998). As well as a precursor role in phytochelatin synthesis, glutathione could be involved in Glyoxalase and formaldehyde metabolism heavy metal resistance via an antioxidant function: many heavy metals are considered to provoke perturbation of Oxo-aldehydes such as glyoxal are reactive compounds that cellular redox homeostasis through several mechanisms, for may interfere with sensitive cellular compounds.A common example, displacement of redox-active metals from bound toxic oxo-aldehyde is methylglyoxal, which can be pro- sites. Increased heavy metal tolerance has been observed in duced from triose phosphate as an intermediate in the transgenic lines with enhanced glutathione synthesis (Zhu triose-phosphate isomerase reaction (Maiti et al. 1997; et al. 1999a,b; Lee et al. 2003), contrasting with effects of Marasinghe et al. 2005). The glyoxalase system acts to overexpressing PCS itself, which has generally yielded less convert these compounds to non-toxic hydroxyacids such as clear-cut results (Peterson & Oliver 2006; Picault et al. lactate, and consists of two enzymes acting in concert 2006). While both shoots and roots may make a contribu- (Fig. 4). Glyoxalase I isomerizes the spontaneously gener- tion to heavy metal detoxification, it is interesting that phy- ated GS-adduct while the hydrolytic reaction catalysed by tochelatin and GS-cadmium concentrations have been glyoxalase II liberates the hydroxyacid and free GSH. In reported to be much higher in the phloem than in the xylem several plant species, overexpression of these enzymes during exposure of B. napus to cadmium (Mendoza-Cózatl increases tolerance to exogenous methylglyoxal and/or salt et al. 2008). (Singla-Pareek, Reddy & Sopory 2003; Deb Roy et al. 2008; Singla-Pareek et al. 2008). Glutathione metabolic reactions in defence In addition to acting as a substrate for GSTs and glyox- against biotic stress alases, GSH may also act in detoxification reactions via formaldehyde dehydrogenase. Formaldehyde can enter Glutathione has long been implicated in reactions linked to plants through stomata or be produced by endogenous secondary metabolism and pathogen responses (Dron et al. metabolism (Haslam et al. 2002). Two enzymes, formalde- 1988; Edwards, Blount & Dixon 1991). While at least some hyde dehydrogenase (FDH) and S-formylglutathione of the effects of glutathione during interactions with patho- hydrolase (FGH), act to oxidize formaldehyde to formic gens probably involve a signalling role, it now appears that acid, which may then be converted to CO2 or enter C1 others are linked to hormone and secondary metabolite metabolism (Fig. 4). Genes for both enzymes have been synthesis. Crucial information had been generated by the identified in Arabidopsis (Martínez et al. 1996; Haslam et al. analysis of glutathione-deficient mutants. The first iden- 2002; Achkor et al. 2003). An important feature of the tified glutathione-deficient Arabidopsis mutant, cad2, was encoded FDH is that it is able to act as a GSNO reductase reported to show unchanged resistance to virulent and (GSNOR; Sakamoto, Ueda & Morikawa 2002; Díaz et al. avirulent strains of the oomycete Peronospora parasitica as 2003). well as to the bacterium Pseudomonas syringae (May et al. 1996b). However, a later study of cad2 and rax1-1 reported Phytochelatin synthesis increased susceptibility to avirulent P. syringae (Ball et al. 2004). Mutants defective in the CLT glutathione chloroplast A conditionally important role of GSH is in the response envelope transporters showed decreased expression of PR1 to excessive levels of heavy metals (Fig. 4). Glutathione is and also lower resistance to the oomycete Phytophthora the precursor of phytochelatins ([g-Glu-Cys]nGly), com- brassicae (Maughan et al. 2010). Decreased PR1 expression pounds that are synthesized in response to cadmium and linked to lower SA accumulation was also observed in gr1 other heavy metals. Phytochelatins sequester the metal to mutants lacking cytosolic/peroxisomal GR (Mhamdi et al. form a complex that is then transported into the vacuole 2010a). © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 463

(a) (b) Glc S N Camalexin Glucosinolate S

- CYPs RCNOSO3 N Cys SH

S RCNOH ? ? Indole-CHCN CSL

γ-Glu-Cys Cys-Gly Cys-Gly

S S S

Indole-CHCN Indole-CHCN RCNOH

GGP γ-Glu-Cys-Gly PCS GGT γ-Glu-Cys-Gly S S Indole-CHCN RCNOH GSTF6 GST ?

IdlIndole-CH2CN Nitr ile ox ide

CYPs CYPs

Tryptophan Amino acid

Figure 5. Possible roles of glutathione as an S atom donor in the production of camalexin (a) and glucosinolates (b). The pathways shown in (a) and (b) are adapted from those shown in Su et al. (2011) and Geu-Flores et al. (2009), respectively. Both schemes shown here specifically focus on steps involving glutathione. In both paths, other steps notably involve several different cytochromes P450 (CYPs), UDP-glucosyl transferases (UGT) or C-S lyase (CSL). The pathway in (b) may also involve partial metabolism of the GS-conjugate [as in (a)] prior to C-S lyase action. GGP, g-glutamyl peptidase; Glc, glucose residue; PCS, phytochelatin synthase.

The Arabidopsis pad2 mutant was first identified as defi- As well as effects on camalexin and resistance to micro- cient in camalexin, an indole phytoalexin that contains one organisms, pad2 shows decreased resistance to feeding of S atom per molecule and whose thiazole ring is derived insect larvae, an effect that is linked to decreased accumu- partly from cysteine (Glazebrook & Ausubel 1994). This lation of glucosinolates (Schlaeppi et al. 2008). This effect line shows enhanced susceptibility to various bacterial, can be rescued by supplementation with GSH but not the fungal and oomycete pathogens (Ferrari et al. 2003; Parisy general disulphide reductant, dithiothreitol (Schlaeppi et al. et al. 2006). The affected gene in pad2 is GSH1 and the 2008). Using a dedicated microarray of more than 200 genes mutation results in GSH contents that are slightly lower whose expression is associated with insect feeding, it was than those in cad2 (Parisy et al. 2006). Of cad2, rax1 and shown that unlike the JA signalling mutant, coi1, the pad2 pad2 mutants in GSH1, rax1 has the highest leaf glutathione mutant did not show significant differences in the expres- contents, pad2 the lowest while cad2 is intermediate. The sion of these genes, including glucosinolate synthesis genes, glutathione contents of these lines correlate inversely with in response to feeding (Schlaeppi et al. 2008). This observa- their resistance to P. brassicae, though only pad2 was tion is consistent with a requirement for a certain level of reported to be markedly affected in camalexin contents and GSH to support glucosinolate synthesis as a sulphur source, its response to P. syringae (Parisy et al. 2006). Thus, there rather than as a signalling or regulatory molecule. Indeed, it appears to be a certain level of glutathione required for the has recently been reported that formation of the glucosino- synthesis of pathogen defense-related molecules and late thioglucose moiety involves a GS-conjugate intermedi- disease resistance. This level could vary according to condi- ate and that this compound is metabolized by a g-glutamyl tions and pathogen specificity. In the case of camalexin peptidase, GGP (Geu-Flores et al. 2009). Formation and synthesis, recent data show that GSH is required as a pre- metabolism of such GS-conjugates parallels stages in cursor of the thiazole ring (Fig. 5a; Böttcher et al. 2009; Su the camalexin synthesis pathway (Fig. 5), and the lack of et al. 2011). GSH for the respective GST activities may explain both © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 464 G. Noctor et al. constitutive deficiency of camalexin and lower induction of glucosinolates in pad2 (Parisy et al. 2006; Schlaeppi et al. 2008). Both pathways shown in Fig. 5 involve formation NADPH of a GS-conjugate followed by further metabolism by GS-conjugate degrading enzymes. Studies of mutants impli- GR NTR cated GGT1, GGT2 and, to a lesser extent, PCS1, in the control of Botrytis-induced camalexin accumulation (Su et al. 2011), whereas ggt4 mutants were found to accumulate a GS-conjugate of the JA synthesis precursor, 12-oxo- MDHAR phytodienoic acid (OPDA), during incompatible interac- tions with P. syringae (Ohkama-Ohtsu et al. 2011). Glutathione TRX

Metabolism of ROS and ascorbate DHAR Glutathione can react chemically with ROS and also with GST GPX dehydroascorbate (DHA), the relatively stable oxidised form of ascorbate generated by dismutation of monodehy- droascorbate (MDHA). In particular, there is a close Ascorbate relationship between increased availability of H2O2 and glu- GRX-PRX TRX-PRX tathione status. This is most evident from studies in which

H2O2-metabolizing enzymes have been genetically or phar- APX macologically inhibited (Smith et al. 1984; May & Leaver 1993;Willekens et al. 1997; Noctor et al. 2002b; Rizhsky et al. 2002; Queval et al. 2007, 2009; Chaouch et al. 2010). Time- course analyses in conditional catalase mutants have shown ROOH that an initial conversion of GSH to GSSG, measurable within hours of exposure to the onset of H2O2 production, is followed by a several-fold induction of the total glutathione pool over the subsequent period of 3–4 d (Smith et al. 1984; Queval et al. 2009). At moderate rates of endogenous H2O2 CAT production, this response involves a decrease in the whole- leaf GSH : GSSG ratio from above 20 to close to one Figure 6. Glutathione function within major pathways for (Mhamdi et al. 2010a). Such effects make glutathione status peroxide metabolism. APX, ascorbate peroxidase; CAT, a useful marker for oxidative stress triggered by increased catalase; DHA(R), dehydroascorbate (reductase); GPX, intracellular H2O2 production. glutathione/thioredoxin peroxidase; GR, glutathione reductase; Several pathways may be involved in GSH-dependent GRX, glutaredoxin; GST, glutathione S-transferase; MDHA(R), monodehydroascorbate (reductase); PRX, peroxiredoxin; H2O2 metabolism, while H2O2 may be metabolized by GSH- independent pathways (Fig. 6). The chemical reaction of ROOH, H2O2 or organic peroxide; TRX, thioredoxin. Ferredoxin-dependent pathways may be important in TRX and GSH with H2O2 is slow, but three distinct types of peroxi- MDHA reduction in the chloroplast. dases appear as the principal candidates to link peroxide reduction to GSH oxidation (Table 1). These are ascorbate peroxidase (APX), certain types of peroxiredoxin (PRX) high rates. It can participate in the ‘ascorbate-glutathione’ and GSTs. Haem-based peroxidases are divided into two pathway in which H2O2 reduction is ultimately linked to super-families, one of which (non-animal peroxidases) NAD(P)H oxidation via ascorbate and glutathione pools. contains plant enzymes and is divided into three classes Alternatively APX activity could be coupled to NAD(P)H (Welinder 1992).While class II haem peroxidases are found oxidation independently of glutathione via MDHAR activ- in fungi and notably include secreted enzymes involved in ity (Fig. 6). While GSH can chemically reduce DHA at sig- lignin degradation, haem peroxidases in plants are found in nificant rates (though slower at pH 7 than at pH 8), an classes I or III (Zámocky, Furtmüller & Obinger 2010). enzymatic link beween ascorbate and glutathione pools is Class III enzymes are found only in plants. Also known as provided by DHAR. Overexpression of this enzyme under- ‘guaiacol-type’ peroxidases, they are encoded by numerous lines the importance of GSH-dependent ascorbate pools in genes and are located in the apoplast or vacuole. Their physiological processes like the regulation of stomatal functions are not clearly established, but some are known to opening (Chen et al. 2003), but the role of DHAR in the in be involved in biosynthetic processes and/or in ROS pro- vivo metabolism of peroxides still remains assumed rather duction (Bindschedler et al. 2006; Cosio & Dunand 2009). In than demonstrated. contrast, class I haem peroxidases include the intracellular In contrast to haem-based enzymes, thiol peroxidases antioxidative enzyme,APX. Like catalase,APX is relatively such as PRX are less specific to H2O2, and can also reduce specific to H2O2 and does not metabolize other peroxides at other organic peroxides, though some may have preference © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 465

Table 1. Simplified overview of peroxidases found in plants and their possible functional link to glutathione

Prosthetic or Enzyme Class catalytic group Oxidant Reductant Functional link to glutathione

APX Class I haem peroxidase Haem H2O2 Ascorbate Ascorbate-glutathione cycle Guaiacol-type POX Class III haem peroxidase Haem H2O2 Various ? 2-Cys PRX Peroxiredoxin Cysteines ROOH TRX, NTRC, cyclophilin ? 1-Cys PRX Peroxiredoxin Cysteines ROOH TRX ? PRX Q Peroxiredoxin Cysteines ROOH TRX ? Type II PRX Peroxiredoxin Cysteines ROOH GSH/GRX GRX-dependent oxidation GPX Peroxiredoxin Cysteines ROOH TRX ? GST GST Serine ROOH GSH Peroxidation

Peroxiredoxin substrate specificities are based on information in Tripathi et al. (2009). Some PRX may also reduce peroxynitrite. See text for further explanation. APX, ascorbate peroxidase; GPX, glutathione peroxidase; GRX, glutaredoxin; GST, glutathione S-transferase; NTRC, NADPH-thioredoxin reductase C; POX, peroxidase; PRX, peroxiredoxin; ROOH, peroxide (H2O2 or organic); TRX, thioredoxin.

for H2O2 or relatively small peroxides (Dietz 2003;Tripathi, transcriptomics and enzyme and metabolite assays of plants Bhatt & Dietz 2009).There are four types of PRX described deficient in catalase and/or GR suggest that the ascorbate- in plants, and three of these use either TRX or similar glutathione pathway and enzymes such as GSTs may act in components such as NADPH-thioredoxin reductase (NTR) concert to remove excess H2O2 and/or other peroxides C (Dietz 2003, Pulido et al. 2010). One type, PRX II, can use (Mhamdi et al. 2010a). GSH as a reductant via GRX action (Rouhier, Gelhaye & Jacquot 2002). Peroxiredoxins also include GPXs. Origi- Glutathione reductase nally identified on the basis of their homology to animal GPX (Eshdat et al. 1997), and subsequently implicated in The GSSG produced by GSH oxidation is reduced by GR. plant stress responses and signalling (Rodriguez Milla et al. While NTR can also reduce GSSG in a TRX-dependent 2003; Miao et al. 2006; Chang et al. 2009), these enzymes are manner (Marty et al. 2009), this enzyme is less efficient than now considered to act as TRX-dependent peroxiredoxins GR, which is encoded by two genes in plants studied so far. (Herbette et al. 2002; Iqbal et al. 2006; Navrot et al. 2006) Chloroplast and mitochondrial GR is encoded by GR2, and are, therefore, misleadingly named. Unlike the animal while GR1 encodes a protein that is found in the cytosol and enzyme, where the reduction of peroxides involves forma- peroxisome (Creissen et al. 1995; Chew et al. 2003; Kataya & tion of a mixed disulphide between a first GSH and the Reumann 2010). The dual targeting of these two genes is selenocysteine SeOH group, the GPXs found in plants gen- therefore sufficient to explain biochemical data on GR erate a disulphide bridge from the initial sulphenic acid, localization in all of these compartments (Edwards et al. with the disulphide intermediate being reduced back to 1990; Rasmusson & Møller 1990; Jiménez et al. 1997; the dithiol form by TRX. They are therefore unlikely to Stevens, Creissen & Mullineaux 2000; Romero-Puertas be involved in peroxide-mediated glutathione oxidation et al. 2006). Despite the long-standing association of GR (Fig. 6). By contrast, peroxidation of GSH could be cataly- and glutathione with resistance to various stresses (Ester- sed by GSTs (Wagner et al. 2002; Dixon et al. 2009). Other bauer & Grill 1978;Tausz, Sircelj & Grill 2004), overexpres- enzymes such as methionine sulphoxide reductase (MSR) sion of GR in several plant species has not in itself been may also contribute to ROS-triggered glutathione oxida- reported to lead to marked increases in stress resistance tion (Tarrago et al. 2009). Finally, although these studies, (Foyer et al. 1991, 1995; Aono et al. 1993; Broadbent et al. relying predominantly on in vitro analyses, have established 1995; Kornyeyev et al. 2005; Ding et al. 2009). However, the relative specificities in reducing substrates, it should be increases in the reduction state of the ascorbate pool in noted that the glutathione and TRX systems may be linked plants overexpressing GR are consistent with efficient cou- to some extent during in vivo ROS metabolism (Michelet pling of the reactions of the ascorbate-glutathione pathway et al. 2005; Reichheld et al. 2007; Marty et al. 2009). (Foyer et al. 1995).

In conclusion, GSH could be linked to H2O2 and/or per- While Arabidopsis T-DNA mutants for the chloroplast/ oxide reduction by at least two ascorbate-independent mitochondrial GR2 are embryo-lethal (Tzafrir et al. 2004), routes as well as the ascorbate-glutathione pathway (Fig. 6). gr1 knockout mutants do not show phenotypic effects, Among these, only GSTs appear to act as direct GPXs despite a 30–60% reduction in extractable enzyme activity (Table 1), all other enzymes requiring at least one addi- (Marty et al. 2009; Mhamdi et al. 2010a). Genetic analyses tional protein to link peroxide reduction to GSH oxidation. have shown that the aphenotypic nature of gr1 mutants The evidence from gene expression makes it clear that results from partial replacement of GSSG regeneration by certain APX, GPX and GST genes are induced in response the cytosol-located NTR-TRX system (Marty et al. 2009), to oxidative stress (Willekens et al. 1997; Wagner et al. though this is not sufficient to prevent significant accumula- 2002; Levine et al. 1994; Sappl et al. 2009). Data from tion of GSSG in the mutant. Accumulation of GSSG is © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 466 G. Noctor et al. massively increased compared with the parent lines in cat2 mutants produced an additive shoot meristemless pheno- gr1 double mutants deficient in both the major leaf catalase type (Reichheld et al. 2007). However, when the ntra ntrb and GR1 (Mhamdi et al.2010a).This effect is associated with double mutants were crossed with cad2 (which has about a much exacerbated phenotype compared with cat2,showing 30% of the glutathione present in the wild type), the result- that the NTR-TRX system cannot replace GR1 under con- ant triple mutants developed normally at the rosette stage ditions where H2O2 production is increased. Further, the gr1 and underwent the floral transition but they produced mutation alters Arabidopsis responses to pathogens and almost naked flowering stems (Bashandy et al. 2010). The expression of genes involved in defence hormone signall- perturbation of the floral meristem in the ntra ntrb, cad2 ing, notably JA-associated genes (Mhamdi et al. 2010a). triple mutants was linked to altered levels and transport of Although transcriptomic patterns documented in this study auxin, which plays an important role in the integration of point to at most limited overlap between glutathione and meristem development (Bashandy et al. 2010). TRX systems in oxidative stress functions,the observation of Glutathione synthesis is also required for pollen germi- Marty et al. (2009) shows that interplay is possible at the nation and pollen tube growth (Zechmann, Koffler & biochemical level. For example, changes in glutathione and Russell 2011). In this study, glutathione depletion was TRX redox states could act mutualistically to reinforce each shown to result from disturbances in auxin metabolism and other during plant interactions with pathogens. This issue, transport (Zechmann et al. 2011). The auxin-resistant axr1 which is important to defining the frequently used but vague and axr3 mutants were found to be less sensitive to BSO term‘cellular redox state’ more clearly,remains an outstand- than the wild-type Arabidopsis plants and treatment of the ing issue in understanding redox signalling in plants. tips of primary roots with BSO altered auxin transport (Koprivova et al. 2010). However, in these experiments, the effects of GSH on root growth could be partially reversed GLUTATHIONE IN PLANT DEVELOPMENT, by dithiothreitol, suggesting that an as yet unidentified post- GROWTH AND ENVIRONMENTAL RESPONSES transcriptional redox mechanism is involved in the regula- It has long been known that certain cell types, for example, tion of the expression of PIN proteins and hence auxin the root quiescent centre and cells in organs such as seeds, transport in roots (Koprivova et al. 2010). The stunted phe- maintain a highly oxidized intracellular state (Kranner & notype of mutants such as cat2 and derived lines, which Grill 1996; Kranner et al. 2002, 2006).Auxin accumulation in accumulate GSSG when grown from seed in air, may be the root stem cell niche is dependent on the oxidized status related to such thiol regulation of plant growth, as could the of the cells (Jiang & Feldman 2010). Treatment of Arabi- effects of GSH on the regeneration efficiency of somatic dopsis root tips with BSO led to disappearance of the auxin embryos (Belmonte et al. 2005). maximum in the root tips and altered expression of quies- cent centre markers (Koprivova, Mugford & Kopriva 2010). Cell cycle regulation The glutathione redox potential of such cell types is rela- tively high (i.e. positive or oxidizing). Even in cells where Concepts of the regulation of mitosis in animals incorporate the global glutathione pool is highly reduced, compart- an intrinsic redox cycle in which transient oxidations serve ments that lack GR or that are deficient in NADPH may to regulate progression through the cell cycle (Menon & contain low GSH:GSSG ratios (e.g. the vacuole or endo- Goswami 2007; Burhans & Heintz 2009). While very few plasmic reticulum; Hwang, Sinskey & Lodish 1992; Enyedi, studies have been conducted to establish whether similar Várnai & Geiszt 2010). While an increase in the redox processes operate in the control of the plant cell cycle, the potential above the threshold-reducing value of more recruitment of GSH into the nucleus in the G1 phase of the redox-sensitive compartments has been linked to growth plant cell cycle has a profound effect on the redox state of arrest and/or death (Kranner et al. 2006), cell identity has a the cytoplasm and the expression of redox-related genes profound influence on the processes that govern cell fate (Pellny et al. 2009; Diaz-Vivancos et al. 2010a,b). A subse- (Jiang et al. 2006a,b) and associated responses to abiotic quent increase in the total cellular GSH pool above the stress (Dinneny et al. 2008). level present at G1 is essential for the cells to progress from the G1 to the S phase of the cycle (Diaz-Vivancos et al. Plant growth and auxin 2010a,b). As the movement of GSH into the nucleus can be visualized in the dividing cells of the developing lateral root The analysis of the phenotypes of glutathione-deficient Ara- meristem (Diaz-Vivancos et al. 2010a), it will be intriguing bidopsis mutants has demonstrated that GSH is required for to see if this process underlies the post-transcriptional plant development fulfilling critical functions in embryo and redox regulation of the PIN proteins and auxin transport in meristem development (Vernoux et al. 2000; Cairns et al. roots (Koprivova et al. 2010). 2006;Reichheld et al.2007;Frottin et al.2009;Bashandy et al. 2010). The rml1 mutant, which has less than 5% of the Biotic interactions, cell death and glutathione present in the wild type, has a strong develop- defence phytohormones mental phenotype that is characterized by a non-functional root meristem while the shoot meristem is largely unaffected In addition to serving as a source of reduced S during syn- (Vernoux et al. 2000). Crossing rml1 with ntra, ntrb double thesis of secondary metabolites, glutathione is involved in © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 467 signalling processes. Exogenous GSH can mimic fungal expression of pathogenesis-related (PR) genes (Creissen elicitors in activating the expression of defence-related et al. 1999). In addition, the HR-like lesions triggered by genes (Dron et al. 1988; Wingate, Lawton & Lamb 1988) intracellular oxidative stress are associated with GSSG including PATHOGENESIS-RELATED1 (PR1; Senda & accumulation (Smith et al. 1984; Willekens et al. 1997; Ogawa 2004; Gomez et al. 2004a). Moreover, accumulation Chamnongpol et al. 1998; Chaouch et al. 2010). Alterations of glutathione is triggered by pathogen infection (Edwards in pathogen responses have been described in mutants that et al. 1991; May, Hammond-Kosack & Jones 1996a), and this are partly deficient in glutathione (Ball et al. 2004; Parisy can involve characteristic transient changes in glutathione et al. 2006). However, some of our own recent work in redox state (Vanacker, Carver & Foyer 2000; Parisy et al. catalase-deficient cat2 and derived Arabidopsis lines 2006). Similar changes have also been reported following suggest that there is unlikely to be a simple relationship exogenous application of the defence-related hormone sali- between glutathione redox potential and engagement of cylic acid (SA), or biologically active SA analogs (Mou, Fan cell death. Firstly, HR-like lesions in cat2 are under day- & Dong 2003; Mateo et al. 2006; Koornneef et al. 2008). length control and this control does not correlate with the Glutathione perturbation in catalase-deficient cat2 is linked degree of glutathione oxidation (Queval et al. 2007). Sec- to hypersensitive response (HR)-like lesions as part of a ondly, daylength control is linked to SA synthesis: HR-like wide spectrum of defence responses that are conditionally lesions can be prevented by blocking SA synthesis even induced in this line (Chaouch et al. 2010). Glutathione is though this effect is associated with a more oxidized glu- also required for the development of symbiotic N2-fixing tathione status (Chaouch & Noctor 2010; Chaouch et al. nodules between legumes and rhizobia. Both GSH and the 2010). Thirdly, in cat2 gr1 mutants, GSSG accumulates to a legume homologue, homoGSH, are found at high concen- greater extent than in cat2 single mutants but this does not trations in N2-fixing nodules formed during symbiotic cause HR-like lesions (Mhamdi et al. 2010a). Despite these interactions with rhizobia: deficiency in these compounds observations, however, we cannot exclude the possibility inhibits nodule formation (Frendo et al. 2005; Pauly et al. that values for whole-leaf glutathione status may not pre- 2006). cisely reflect events in specific intracellular compartments Thiol-disulphide status is clearly involved in the regula- (Queval et al. 2011). Even if this is the case, tissue glu- tion of the SA-dependent NONEXPRESSOROFPAT tathione status appears to be a poor marker for the engage- HOGENESISRELATEDGENES 1 (NPR1) pathway ment of cell suicide pathways. Nevertheless, analysis of

(Després et al. 2003; Mou et al. 2003; Rochon et al. 2006; H2O2 signalling in glutathione-deficient mutants demon- Tada et al. 2008). Induction of PR gene expression by this strates that some factor related to glutathione status is an pathway involves monomerization of the oligomeric cyto- important modulator of cell death triggered by oxidative solic protein NPR1, which unmasks a nuclear localization stress (authors’ unpublished results). Further, analyses of signal motif that allows the protein to relocalize to the double cat2 atrboh Arabidopsis mutants, which lack both nucleus where it interacts with TGA transcription factors, the major catalase and NADPH oxidase activities, point to themselves redox-sensitive (Després et al. 2003; Mou et al. a crucial role for AtrbohF in permitting accumulation of

2003). The notion that this might be linked to glutathione oxidized glutathione in response to intracellular H2O2.In redox state via GRX activity (Mou et al. 2003) has been double cat2 atrbohF (but not cat2 atrbohD) mutants, glu- superseded by a model based on activation by TRXh (Tada tathione is much less perturbed than in the cat2 single et al. 2008), some of which are known to be inducible by mutant and this is associated with much lower accumulation oxidative stress and pathogens (Laloi et al. 2004). If this of SA and related defence molecules (Chaouch, Queval & model is correct, the well-documented ability of genetically Noctor, unpublished results). or chemically increased GSH to induce PR gene expression As well as possible roles in SA signalling, glutathione could possibly be explained by redox interactions between may modulate signalling through the JA pathway, which is glutathione and TRX. involved in the regulation of development and in responses Although activation of NPR1 involves a reductive to necrotrophic pathogens and herbivores. JA induces change, the initial trigger for the events leading to PR gene GSH1,GSH2 and GR (Xiang & Oliver 1998),as well as other expression involves oxidation. Excessive oxidation can also antioxidative genes (Sasaki-Sekimoto et al. 2005). In gr1 trigger HR, which is the most studied instance of environ- mutants lacking the cytosolic/peroxisomal GR, a suite of JA mentally induced cell death in plants. While redox changes genes are repressed while introduction of the gr1 mutation involved in HR have been most closely associated with into the cat2 background modulates H2O2-triggered expres- events triggered by apoplastic ROS production, oxidative sion of these and other JA-associated genes (Mhamdi et al. stress of intracellular origin can also trigger such effects. 2010a). Consistent with these interactions between JA and Several observations are suggestive of a role for glutathione glutathione, prior wounding was shown to induce resistance redox potential (or GSSG accumulation) in the regulation to Botrytis cinerea, an effect that was partly or wholly of genetically programmed cell suicide pathways. For suppressed in glutathione-deficient mutants (Chassot et al. example, increases in the total glutathione contents of 2008). This effect was associated, at least in part, with leaves produced by ectopic g-ECS overexpression in the impaired induction of camalexin (Chassot et al. 2008). tobacco chloroplast caused accumulation of GSSG and Among the components that could link JA signalling and this was associated with lesion formation and enhanced glutathione are GRXs and GSTs.The SA-inducible protein, © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 468 G. Noctor et al.

GRX480, represses up-regulation of certain JA-induced (phyA) and caused increased BSO resistance (Sung et al. genes (Ndamukong et al. 2007). As well as acting in the 2007). The ars5 mutant, which is affected in a 26S protea- activation of the SA pathway, NPR1 represses JA pathway some component, had increased levels of glutathione when (Spoel et al. 2003), and BSO treatment was shown to exposed to arsenic, accompanied by increased GSH1 and anatagonize this repressive effect (Koornneef et al. 2008). GSH2 transcripts (Sung et al. 2009) Together, these observations suggest that glutathione may The redox states of the plastoquinone and TRX pools are act to repress JA signalling through SA-dependent induction considered to be important components of the redox regu- of NPR1 and GRX480, both of which interact with TGA lation model for the control of gene expression that adjusts transcription factors. However, gene expression profiles and energetic and metabolic demands to light-induced changes SA contents suggest that the more oxidized glutathione of the photosynthetic apparatus structure (Bräutigam, status in gr1 is associated with repression of both SA and JA Dietzel & Pfannschmidt 2010). Glutathione has also been pathways, and therefore that the roles of glutathione may be implicated in the signalling pathways that facilitate acclima- complex and not limited to regulating antagonism between tion of chloroplast processes to high light (Ball et al. 2004). the two hormones (Mhamdi et al. 2010a). Other possible Exposure to photoinhibitory light can lead to increases in effects of glutathione could occur through the regulation of leaf H2O2 levels and to oxidation of the glutathione pool JA synthesis or excess accumulation of intermediates like (Mateo et al. 2006; Muhlenbock et al. 2008).A light shift that OPDA. Several GSTs are among early JA-induced genes or favoured excitation of photosystem II (PSII) relative to may catalyse formation of GS-oxylipin conjugates (Davoine photosystem I (PSI) slightly increased the amounts of glu- et al. 2006; Yan et al. 2007; Mueller et al. 2008). Indeed, tathione in leaves, whereas glutathione levels were slightly enhanced accumulation of a GS-OPDA conjugate in ggt4 decreased following a light shift that favoured excitation of mutants, which are expected to be unable to degrade vacu- PSI relative to PSII (Bräutigam et al. 2009, 2010).According olar GS-conjugates, was recently reported. However, loss of to the model of Bräutigam et al. (2010), higher g-ECS activi- GGT4 function did not affect contents of JA or derivatives ties would be favoured by exposure to light wavelengths such as the hormonally active JA-isoleucine conjugate com- that predominantly drive PSII. However, this model pared with wild-type plants (Ohkama-Ohtsu et al. 2011). remains to be substantiated. In addition to these interactions with glutathione, JA and wounding were also shown to decrease GSNOR transcripts GLUTATHIONE-LINKED (Díaz et al. 2003). SIGNALLING MECHANISMS

Light signalling Redox regulation is inherent to all energy exchange pro- cesses. It is required to balance supply and demand between The abundance of glutathione or its homologues in leaves energy-producing and energy-utilizing processes, as these does not vary greatly over the day/night cycle. Similarly, are driven by redox changes. Of the mechanistic controls in soybean, all leaves except those approaching sene- that achieve this homeostasis, thiol-disulphide reactions are scence contain similar total amounts of glutathione/ the best characterized and probably among the most impor- homoglutathione, as illustrated in Fig. 7. However, young tant (Buchanan & Balmer 2005). Key thiol components in poplar trees show more variation, with a gradient from enzyme regulation or ROS metabolism include TRX, GRX, young to old leaves (Arisi et al. 1997). The effect of stresses glutathione, GPX and PRX (Dietz 2003; Lemaire 2004; such as drought can also influence the relative abundance Meyer et al. 2008; Rouhier 2010).The roles of TRX in redox of glutathione/homoglutathione in different leaf ranks signalling are well established. By comparison, the study of (Fig. 7). Shade conditions that involve a decrease in the glutathione-dependent signalling is still in its infancy, red/far red ratio of the light environment favour a much although it is interesting to note that interest in the roles of lower leaf glutathione pool relative to conditions where the glutathione in cellular regulation dates back many years red/far red ratios are similar (Bartoli et al. 2009). While leaf (Wolosiuk & Buchanan 1977).The following discussion first glutathione contents are lower in plants grown under shade outlines factors that could link glutathione to modification conditions, leaf GSH:GSSG ratios are relatively unaffected of target protein activity and then analyses some of the by light quality (Bartoli et al. 2009). Moreover, the adjust- issues surrounding the regulation and impact of altered ments in leaf glutathione pool in response to different red/ glutathione status in cellular signalling. far red ratios were very slow in comparison with the leaf ascorbate pool (Bartoli et al. 2009). Regardless of the rela- Glutaredoxins tively slow responses of the leaf glutathione pool to changes in light intensity and light quality, some potential links Glutaredoxins are also known as thiol transferases. The between daylength, light signalling and glutathione status classical GRX reaction encompasses the reduction of a have been reported (Becker et al. 2006; Queval et al. 2007). protein disulphide bond to two thiols with conversion of 2 Relationships between glutathione and photoreceptor sig- GSH to GSSG. Some of these enzymes can also catalyse nalling have been suggested in studies on the arsenic- protein S-glutathionylation or de-glutathionylation. Land tolerant mutants, ars4 and ars5 (Sung et al. 2007). The ars4 plants contain a large GRX family subdivided into three mutation was identified as an allele of phytochrome A classes that can be distinguished on the basis of amino acid © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 469

Figure 7. The effects of ontogeny and drought stress on total tissue contents of homoglutathione plus glutathione (a) and reduction state (b) in 5-week-old soybean (Glycine max cv Williams 82) plants. For the drought study, plants were deprived of water for 15 d until the soil water in the drought treatment had fallen to about half that measured under the optimal watering regime (76.97 Ϯ 2.93). Data show the mean Ϯ SE (n = 3) and are expressed in relative units. L, leaf; N, nodule; TF, trifoliate leaf. motifs in the active site (Lemaire 2004; Rouhier 2010). Most called ‘monothiol’ GRX, though this term is potentially class I GRXs have a TRX-like active site consisting of two misleading as in at least some cases this cysteine may form cysteines separated by two intervening amino acids. They a disulphide bridge with another cysteine located distally in can catalyse thiol-disulphide exchange but also other reac- the same protein or on another GRX of the same type (Gao tions such as regeneration of PRX and methionine sul- et al. 2009a, 2010). Both class I and class II GRX play roles phoxide reductase (MSR; Rouhier et al. 2002; Rouhier, in assembly of iron-sulphur clusters (Rouhier et al. 2007; Couturier & Jacquot 2006; Zaffagnini et al. 2008; Tarrago Bandyopadhay et al. 2008; Rouhier 2010). They can also et al. 2009; Gao et al. 2010). Class II GRXs have only a single catalyse protein de-glutathionylation, though some class II cysteine at the active site and are therefore sometimes GRX do this in a dithiol- rather than GSH-dependent © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 470 G. Noctor et al. manner, and in vivo may depend on the TRX system for than nitrosylated or a target of TRX (or some combina- their regeneration (Zaffagnini et al. 2008; Gao et al. 2009a, tion of these modifications). 2010). While some of these GRX have been characterized Most targets of glutathionylation identified to date are biochemically, their in vivo roles remain unclear. Arabidop- relatively abundant proteins. These notably include sis class II GRXs can interact with ion channels and have enzymes involved in primary metabolism such as TRXf, been implicated in responses to oxidative stress (Cheng & glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hirschi 2003; Cheng et al. 2006; Guo et al. 2010; Sundaram & and glycine decarboxylase (GDC; Michelet et al. 2005; Zaf- Rathinasabapathi 2010). fagnini et al. 2007; Palmieri et al. 2010). Glutathionylation of GRXs in the third class contain two adjacent cysteines a TRXf cysteine found outside the active site lowers the at their active site and form the largest class in land protein’s efficacy in activating chloroplast NADP-GAPDH, plants. Reverse genetics approaches in Arabidopsis have while another TRX-independent isoform of GAPDH is revealed that three GRX forms are involved in plant inhibited by glutathionylation (Zaffagnini et al. 2007). In development and phytohormone responses through the mitochondria, GDC has been shown to be inhibited by interaction with TGA transcription factors. These are oxidative stress (Taylor, Day & Millar 2002), and is subject GRX480, implicated in SA-JA interactions, and ROXY1 to thiol modifications, including S-glutathionylation (Palm- and ROXY2, which function in petal and flower develop- ieri et al. 2010). This enzyme is a major producer of NADH ment (Ndamukong et al. 2007; Li et al. 2009). Overexpres- during conditions of active photorespiration. Mitochondrial sion and complementation studies point to biochemical redox homeostasis in these conditions may depend on redundancy between these proteins (Li et al. 2009; Wang appropriate activation of the alternative oxidase (Igamber- et al. 2009). Based on these observations, the specificity of diev,Bykova & Gardeström 1997), which has been shown to class III GRX function may be determined by regulation be redox regulated through the TRX system (Vanlerberghe of their expression patterns (Ziemann, Bhave & Zachgo et al. 1995; Gelhaye et al. 2005). The glycolytic NAD- 2009). This notion receives some support from microarray dependent GAPDH, a cytosolic enzyme found not only in analysis of GRX gene expression in mutants with pertur- the cytosol but also in the mitochondrion and nucleus bations in leaf glutathione redox state, in which specific (Giegé et al. 2003; Anderson, Ringenberg & Carol 2004), is members of class III were the only GRX that were sensitive to oxidation and has been identified as a glutathio- found to respond (Mhamdi et al. 2010a). Among these four nylated protein (Dixon et al. 2005; Hancock et al. 2006; genes was GRX480, which showed expression patterns Holtgrefe et al. 2008). similar to many other JA-associated genes. In contrast, Taken together, the current evidence suggests that glu- transcripts for ROXY1 or ROXY2 remained at wild-type tathione and TRX systems work closely together to fine- abundance levels. tune photosynthetic and respiratory metabolism through appropriate modification of sensitive protein cysteine resi- Protein S-glutathionylation dues. The mechanisms of S-glutathionylation may involve GRX-catalysed thiol-disulphide exchange between protein Protein S-glutathionylation involves the formation of a thiol groups and GSSG, conversion of thiol groups to thiyl stable mixed disulphide bond between glutathione and a radicals or sulphenic acids, or be GSNO mediated (Dixon protein cysteine residue. This reaction could modify the et al. 2005; Holtgrefe et al. 2008; Gao et al. 2009b; Palmieri conformation, stability or activity of the target protein. et al. 2010).The reverse reaction, deglutathionylation, could The existence of S-glutathionylated plant proteins has be mediated by certain class I and class II GRX. Current been known for some time (Butt & Ohlrogge 1991). evidence suggests that some class II GRX catalyse this reac- However, the nature and role of this phenomenon has tion in a TRX reductase-dependent manner (Zaffagnini only been subject to thorough investigation relatively et al. 2008; Gao et al. 2009a, 2010). Despite the potential recently. Reversible S-glutathionylation is part of the cata- physiological importance of S-glutathionylation, and the lytic cycle of glutathione-dependent enzymes such as GR, proposed involvement of class I and class II GRX in either DHAR, MSRB1, as well as some GRXs and PRXs glutathionylation or de-glutathionylation, no GRX of either (Arscott, Veine & Williams 2000; Dixon et al. 2002; Tarrago type responded at the transcript level in Arabidopsis et al. 2009; Rouhier 2010). Several techniques have been mutants with increased H2O2 and GSSG (Mhamdi et al. developed to identify other target proteins, allowing an 2010a). inventory of potential target proteins to be established (Ito, Iwabuchi & Ogawa 2003; Dixon et al. 2005; Michelet Protein S-nitrosylation and GSNO reductase et al. 2005, 2008; Zaffagnini et al. 2007; Holtgrefe et al. 2008; Gao et al. 2009a). Despite these advances, major gaps GSNO can cause S-nitrosylation and S-glutathionylation of in our current knowledge remain, for example concerning protein cysteine residues (Lindermayr et al. 2005, 2010; quantification (i.e. the fraction of a given protein that is Romero-Puertas et al. 2008; Lindermayr & Durner 2009). S-glutathionylated at a given time) and the in vivo signifi- Enzymes undergoing both modifications include GAPDH cance of the modification. An additional uncertainty is the and GDC, with both effects leading to decreased activity specificity of the modification, that is, whether the modi- (Lindermayr et al. 2006; Holtgrefe et al. 2008; Palmieri et al. fied cysteine residue is glutathionylated in vivo, rather 2010). Oxidative inhibition of GAPDH may act to regulate © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 471 the oxidative pentose pathway relative to glycolysis in stress Regulation of thiol-disuphide status has traditionally been conditions (Holtgrefe et al. 2008). Inhibition of GDC by associated with TRX (Buchanan & Balmer 2005). However, modifications of thiols may also have physiological signifi- genetic support for the physiological importance of cance, as photorespiratory metabolism involves high-flux glutathione-dependent disulphide reduction comes from pathways capable of modifying intracellular redox states the observations that the NADPH-TRX and glutathione (Foyer et al. 2009). S-nitrosylation of type II PRX has been systems play overlapping roles in development (Reichheld proposed to play a role in ROS signalling (Romero-Puertas et al. 2007; Bashandy et al. 2010). A key factor governing et al. 2007). interactions between glutathione and protein targets is It was reported that S-nitrosylation acts in opposition to redox potential, which depends on the relative rates of glu- disulphide reduction by TRX in the regulation of NPR1. tathione oxidation and its NADPH-dependent reduction. While monomerization of the NPR1 protein to the active The actual glutathione redox potential is related to form was TRX dependent, GSNO-dependent nitrosylation [GSH]2:GSSG. Thus, unlike many other redox couples (e.g. of NPR1 monomers was required for reoligomerization, NADP+/NADPH), the glutathione redox potential depends possibly to inhibit depletion of the protein (Tada et al. on and can be influenced by absolute concentration as well 2008). However, redox regulation of NPR1 and the tran- as by changes in GSSG relative to GSH (Mullineaux & scription factors with which NPR1 interacts is turning out to Rausch 2005; Meyer 2008). Even if the GSH:GSSG ratio be complex. Four cysteine residues on TGA1 underwent remains unchanged, decreases in glutathione concentration S-nitrosylation and S-glutathionylation after GSNO treat- alone will lead to an increase in redox potential, that is, the ment of the purified protein, while GSNO treatment of potential will become more positive and thus less reducing. Arabidopsis protoplasts caused translocation of an NPR1- This is in accordance with measurements of cytosolic GRX- GFP fusion protein into the nucleus (Lindermayr et al. dependent thiol/disulphide redox potential as detected by 2010). The importance of S-nitrosylation in regulating redox-sensitive roGFP in the glutathione-deficient cad2 NPR1 function is in line with an important role for this mutant compared with the wild type (Meyer et al. 2007). modification in plant disease resistance. Production of the Increases in the cytosolic but not plastidial redox potential plant hormone ethylene, which regulates developmental in clt1 clt2 clt3 mutants, lacking the chloroplast envelope responses like senescence as well as responses to certain glutathione transporters, are also consistent with a depleted pathogens, is known to be inhibited by NO. This effect glutathione pool in the cytosol but not the chloroplast may be mediated by inhibition of several enzymes in- (Maughan et al. 2010). volved in ethylene synthesis, including GSNO-triggered S-nitrosylation of a specific methionine adenosyltransferase Linking glutathione redox potential to (Lindermayr et al. 2006). target proteins Inducible NO-producing enzymes in plants remain to be clearly described, and most attention has focused on the No components have yet been identified that directly link importance of a secondary reaction of nitrate reductase. glutathione redox potential to modifications in target pro- However, studies in animals and plants have revealed that teins. However, based on information from the much better GSNOR is a key player in regulating S-nitrosylation. studied TRX-dependent changes in protein thiol-disulphide These enzymes catalyse the NADH-dependent reduction of status, we can infer that a change in glutathione redox GSNO to S-aminoglutathione, which then decomposes to potential of approximately 50 mV is likely to be biologically free GSH and ammonia or other compounds (Sakamoto significant. Such changes are sufficient to alter the balance et al. 2002; Barroso et al. 2006; Díaz et al. 2003). Originally between oxidized and reduced forms of TRX-regulated identified as a formaldehyde dehydrogenase, the Arabidop- proteins (Setterdahl et al. 2003). For example, an increase in sis GSNOR appears to play a key role in biotic stress TRX redox potential from -350 to -300 mV converts chlo- responses, and Atgsnor1 mutants show decreased resistance roplast glucose-6-phosphate dehydrogenase from almost to virulent and avirulent pathogens (Feechan et al. 2005). completely inactive to active (Née et al. 2009). Indeed, in This enzyme has also been implicated in the regulation of vitro redox titration of the thiol/disulphide-dependent cell death and other functions (Lee et al. 2008; Chen et al. roGFP in the presence of GRX at different glutathione 2009). It is also interesting to note that carbon monoxide concentrations revealed that interconversion of the oxi- (CO) is now considered to be a gaseous signalling molecule dized and reduced forms of the sensor were associated with in animals and plants. It is possible that glutathione is a calculated redox potential span of about 50 mV (Meyer involved in CO signalling in Medicago sativa (Han et al. et al. 2007). Despite this, calibrated quantification of in vivo 2008). changes in cytosolic redox potential using these probes has thus far revealed shifts of only about 20 mV in glutathione- Factors affecting the glutathione deficient mutants compared with wild-type or in stress redox potential versus optimal conditions (Meyer et al. 2007; Jubany-Mari et al. 2010). Nevertheless, the physiological importance of Despite the advances described previously, it remains such a shift cannot be discounted because systems that are unclear to what extent glutathione-mediated changes in able to sense glutathione redox potential with high sensi- protein thiol-disulphide status are important in signalling. tivity may await discovery. Furthermore, based on recent © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 472 G. Noctor et al. reports of interactions between cytysolic glutathione and marked changes in GSH:GSSG ratios (Smith et al. 1984; TRX systems (Marty et al. 2009), it is possible that in some Willekens et al. 1997; Rizhsky et al. 2002; Queval et al. 2007, conditions changes in glutathione redox potential could 2009). These are most evident for catalase-deficient plants influence the mechanisms that contribute to changes in the exposed to light under photorespiratory conditions, and TRX redox potential and thus the biological activity of are accompanied by several-fold changes in the total TRX targets. glutathione pool. It remains to be established to what extent these changes in whole tissue GSH:GSSG ratios Physiological and environmental factors that involve changes in glutathione redox potential in specific compartments. might cause shifts in glutathione redox potential in vivo The importance of changes in absolute As previously discussed, the whole leaf and chloroplast glutathione concentration compared with GSH:GSSG ratios are not markedly influenced by varia- changes in GSH:GSSG tions in light intensity or light-dark transitions. However, oxidant production and oxidative stress can exert a strong Accumulation of GSSG is often followed by an increase in influence over glutathione status. Oxidant production is the total glutathione pool size (Fig. 8). This response has governed by the rates of photosynthesis, photorespiration been documented in plants exposed to ozone, is particularly and respiration, as well as NADPH oxidases and other evident in response to increased intracellular H2O2 and also ROS-producing systems (Foyer & Noctor 2003). Presum- occurs during pathogen responses. In such circumstances, ably by changing the rate of ROS production through these changes in the total glutathione pool size occur predomi- processes or by affecting the rate of ROS removal, various nantly through oxidant-induced changes in the expression stresses such as cold, drought, pollution and pathogens can and post-translational activities of enzymes involved in modify whole leaf glutathione redox state (Sen Gupta, both cysteine and glutathione synthesis. This presumably Alscher & McCune 1991; Vanacker et al. 2000; Bick et al. acts as a homeostatic mechanism to offset what would oth- 2001; Gomez et al. 2004b). Indeed, the cytosolic glutathione erwise be more severe increases in glutathione redox poten- redox potential has been shown to increase in response to tial. If so, this response in itself suggests that the glutathione wounding (Meyer et al. 2007). Numerous studies on plants redox potential is important for at least some cellular func- deficient in H2O2-metabolizing enzymes have documented tions. Further circumstantial evidence in favour of this

Glutathione-independent ROS signalling

GSH GSH Redox-sensitiveRedox sensitive compartment Basal state GSSG Increased Increased engagement Activation of GSH of GSH in oxidant GSH de novo H2O2 or metabolism synthesis Subcellular other redistribution oxidants Redox-insensitive GSSG GSSG GSSG compartment GSSG More Offset increases in redox potential in sensitive Increased positive compartments caused by ongoing conversion redox engagement Increased of GSH to GSSG potential ofreducedTRX oxidation of in oxidant metabolism TRX by GSSG? TRX Homeostatic signal damping

Thiol-disulfide signalling

Figure 8. Hypothetical scheme showing some glutathione-linked responses in oxidative stress signalling. Increased engagement of GSH in metabolism of H2O2 or other oxidants causes accumulation of GSSG and thus a more positive glutathione redox potential. Signalling initiated by this change includes activation of glutathione neosynthesis and transport, both of which act to offset increases in redox potential. Modified glutathione redox potential could contribute to signalling through components such as glutaredoxins or protein S-glutathionylation or, more indirectly, by oxidation of thioredoxins (TRX). Both thiol components may contribute to thiol-disulphide signalling, which is depicted as one part of a larger reactive oxygen species (ROS) signalling network. © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 473 notion comes from the observation that accumulation of that potentially explains in vivo preference of protein GSSG is compartment specific, and relative increases are targets for glutathione or TRX. However, as discussed most marked in the vacuole (Queval et al. 2011). This is in previously, some roGFP studies suggest that the glu- line with the documented capacity of vacuoles to important tathione redox potential in wild-type Arabidopsis in the GGSG more efficiently than GSH (Tommasini et al. 1993). absence of stress is lower than -300 mV in the cytosol and Thus, mechanisms appear to have evolved to stabilize cyto- in other compartments, that is, it approaches values similar solic glutathione redox potential during increased ROS to the TRX redox potential (Meyer et al. 2007; Schwar- production, and these include increases in total glutathione zländer et al. 2008; Jubany-Mari et al. 2010). Given that and accumulation of GSSG in compartments that may be the cytosolic NADPH:NADP+ ratio is not far removed less sensitive to redox perturbation (Fig. 8). from one (Igamberdiev & Gardeström 2003), these redox Although studies of mutants have shown that relatively potential values suggest that glutathione and NADP(H) small changes in glutathione concentration can modulate are close to redox equilibrium in this compartment. This is gene expression as well as environmental and developmen- a key unresolved issue in assessing the potential signifi- tal processes in plants (Ball et al. 2004; Parisy et al. 2006; cance of redox signalling through glutathione-mediated Bashandy et al. 2010), it is not yet established that such disulphide bond formation. If sensitive thiol proteins are effects are caused by altered glutathione redox potential. A present, maintenance of a very low glutathione redox decrease in total glutathione from 2.5 mm to 50 mm would potential under optimal conditions could allow oxidative cause the redox potential to become 50 mV more positive signalling to be initiated by protein disulphide bond trig- (Meyer et al. 2007). This depletion is rather extreme, and gered by relatively minor accumulation of GSSG. Glu- corresponds to the situation in root tips in seedlings of the tathione redox potential values below -300 mV would rml1 mutant, which contain less than 5% wild-type glu- require the GSH:GSSG ratio to be in the range of 105 to tathione (Cairns et al. 2006; Meyer et al. 2007). Thus, while 106, whereas global ratios are much lower in plant tissues, an increased glutathione redox potential could explain the even in the absence of stress, where they are typically observations reported in rml1 and ntra ntrb rml1 mutants about 20–30 (Queval & Noctor 2007; Mhamdi et al. (Vernoux et al. 2000; Reichheld et al. 2007), it is less clear 2010a). It is likely that, while they provide a useful indi- whether it can account for effects observed in less severely cator of redox status, particularly the presence of oxidative affected backgrounds. If changes in glutathione redox stress, global cellular or tissue glutathione data give only a potential are indeed an important part of glutathione- relative measure of GSH:GSSG ratios in intracellular dependent oxidative signalling in wild-type plants, it seems compartments (Queval et al. 2011). that GSSG accumulation is likely to be a critical factor. There is little evidence as yet that ROS-triggered changes Accumulation of GSSG could be relatively small (in abso- in glutathione are accompanied by marked changes in lute terms) if, as discussed further in the next section, the global tissue NADP(H) status (Mhamdi et al. 2010a,b). The ‘resting state’ concentration in unstressed conditions is as extent to which cytosolic NADP pools turn over in optimal low as some recent measurements imply. and stress conditions remains uncertain. Further, it is not As increased GSSG generally drives glutathione accumu- clear how important the demand of GR is on the cytosolic lation as part of what is assumed to be a homeostatic NADPH pool, and whether there is any functional associa- mechanism, decreased glutathione concentrations might be tion between different NADPH-requiring and -generating expected to cause a compensatory increase in GSH:GSSG. enzymes. GR has a KM for NADPH below 10 mm (Smith This should happen if the NADP(H) redox potential does et al. 1989; Edwards et al. 1990), while total cytosolic not change and the two couples are in equilibrium. NADPH concentrations are above 100 mm (Igamberdiev & However, if GSSG concentrations are indeed in the nano- Gardeström 2003). Although a large proportion of pyridine molar range, it is possible that kinetic limitations over GR nucleotide pools is bound to proteins at any one time, and

(KMGSSG 10–50 mm; Smith et al. 1989; Edwards et al. 1990) thus available ‘free’ concentrations are lower than the total, become increasingly severe as glutathione decreases, thus it is generally considered that changes in NADPH are not explaining the failure to maintain the glutathione redox likely to impact the glutathione redox potential through potential in equilibrium with NADP(H), even when the kinetic effects on GR. However, shifts in NADPH:NADP+ decrease in concentration is relatively modest. could perhaps impact the glutathione redox potential ther- modynamically, as the reaction catalysed by GR is revers- Relationships between the glutathione and ible. As previously discussed, kinetic limitations may occur NADP(H) redox couples in signalling through GSSG, whose resting concentration could be sub- stantially below the GR KM value. Finally, it is interesting The midpoint redox potential of NADP(H) at pH 7 is that the measurable capacity of GR in many plant systems approximately -320 mV while that of glutathione is -230 is often somewhat lower than other enzymes of the to -240 mV. But what is the actual redox potential of glu- ascorbate-glutathione pathway. No doubt this could reflect tathione in vivo? Many researchers assume that it is quite the presence of other pathways that can regenerate glu- close to the midpoint potential and that differences tathione independent of ascorbate (Fig. 6). However, as we between this value and the TRX redox potential, which have emphasized in this review, glutathione may also be for most TRX is quite similar to NADP(H), is one factor linked to ROS metabolism independently of ascorbate. © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 474 G. Noctor et al.

Comparatively low GR activity may be a feature of plant redox signalling components (e.g. GSNOR, GRX).Through redox systems that allows appropriate changes in GSSG to their coordinated operation, such effects can reinforce sig- mediate signalling functions. nalling through a given pathway.Alternatively, antagonistic changes could act to restrain activation of signalling path- ways, a control that is important within a cellular network CONCLUSION AND PERSPECTIVES receiving multiple evironmental inputs. This concept receives support from the operation of multiple interdepen- A large body of literature is now available that illustrates dent redox modifications reported for the bacterial H O the complex and integrated regulation of glutathione status 2 2 sensor, OxyR (Kim et al. 2002). A similar picture is emerg- by nutritional and environmental factors. Glutathione may ing in plants, where the notion of relatively simple thiol- be seen as a central or ‘hub’ molecule in cellular meta- disuphide regulation of NPR1 (Mou et al. 2003) has given bolism and redox signalling (Fig. 9). Even though many way to recognition that control is more complex (Tada et al. glutathione-dependent reactions are involved in oxidative 2008; Lindermayr et al. 2010). By operating in conjunction stress-mediated cellular processes, the simple vision of glu- with other regulatory mechanisms, such nuanced, flexible tathione as a ROS-scavenging antioxidant is inaccurate and control integrates multiple inputs into a variable and appro- misleading. For example, at least three types of mechanism priate response output. This is likely to be particularly can be defined that link glutathione to the regulation of important in plants, which must constantly deal with envi- biotic stress reactions: (1) redox signalling, mediated by ronmental changes of varying intensity and predictability. components such as GRX or GSNO; (2) the synthesis Thus, the challenge for the future is not only to characterize of S-containing or secondary metabolites; and (3) GS- glutathione-linked redox modifications and to assess their conjugate formation and metabolism to regulate the interactions with other types of redox regulation, but also to biological activity of metabolic intermediates or active evaluate the significance of these changes within the wider products. horizon of the cellular network. Key questions related to the antioxidant roles of glu- tathione concern the importance of different glutathione- dependent enzymes and the influence of changes in ACKNOWLEDGMENTS glutathione status within cell signalling pathways. The degree of interplay between TRX and glutathione systems We thank Bernd Zechmann (University of Graz, Austria) in physiological conditions remains to be fully described. for discussions on glutathione concentrations in different The emerging picture is that changes in the ‘general redox subcellular compartments and Stéphane Lemaire (Institut state’ (i.e. redox potential) of these two components de Biologie Physico-Chimique, Paris, France) for discus- work together with altered activity or abundance of specific sions on glutaredoxins and protein glutathionylation reac- tions. The authors acknowledge funding from the French Agence National de la Recherche project ‘Vulnoz’ (Orsay) and the European Union Marie-Curie Initial Training Network ‘COSI’ project (Orsay, Leeds). Y.H. is a recipient

Heavy metal Herbicide of a Chinese Scholarship Council fellowship. B.M.G. resistance resistance thanks Subprograma Estancias de Movilidad posdoctoral en centros extranjeros (2009), Ministerio de Educación ROS Precursor of S GR metabolism (Spain) for a fellowship. compounds GRX Light DHAR Metabolite synthesis signalling REFERENCES Redox Detoxification GSH signalling Achkor H., Díaz M., Fernández M.R., Biosca J.A., Parés X. & GSSG Martínez M.C. (2003) Enhanced formaldehyde detoxification by Pathogen overexpression of glutathione-dependent formaldehyde dehy- Regulation of defence S metabolism GRXs drogenase from Arabidopsis. Plant Physiology 132, 2248–2255. Phytohormone Anderson L.E., Ringenberg M.R. & Carol A.A. (2004) Cytosolic pathways Transport glyceraldehyde-3-P dehydrogenase and the B subunit of the chloroplast enzyme are present in the pea leaf nucleus. Proto- Cell division and Flower plasma 223, 33–43. meristem function development Aono M., Kubo A., Saji H., Tanaka K. & Kondo N. (1993) Enhanced tolerance to photooxidative stress of transgenic Nic- otiana tabacum with high chloroplastic glutathione reductase activity. Plant & Cell Physiology 34, 129–135. Figure 9. Physiological importance of glutathione. The outer Arisi A.C.M., Noctor G., Foyer C.H. & Jouanin L. (1997) Modifi- green circle shows examples of physiological processes affected cation of thiol contents in poplars (Populus tremula x P. alba) by glutathione status or by glutathione-dependent components. overexpressing enzymes involved in glutathione synthesis. The inner pink circle gives examples of major biochemical Planta 203, 362–372. functions of glutathione. The darker pink spokes indicate Arscott L.D., Veine D.M. & Williams C.H. (2000) Mixed disulfide components that are involved in these functions. with glutathione as an intermediate in the reaction catalyzed by © 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484 Glutathione status and functions 475

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Summary

H2O2 is a recognized signal in activation of defence mechanisms in response to various stresses, and its accumulation is thus tightly controlled by plant antioxidant systems. Because H2O2 signals may be transmitted by thiol-dependent processes, glutathione status could play an important role. While the antioxidant role of this compound is long established, the importance of glutathione in signaling remains unclear. To study this question, this work exploited a stress mimic mutant, cat2, which has a defect in metabolism of peroxisomal H2O2 that conditionally leads to oxidation and accumulation of glutathione. In cat2, changes in glutathione are accompanied by activation of both salicylic acid (SA)-dependent responses and jasmonic acid (JA)-associated genes in a time-dependent manner. This up-regulation of both phytohormone signaling pathway by intracellular oxidative stress can be largely prevented by genetically blocking glutathione accumulation in a double mutant, cat2 cad2, that additionally carries a mutation in the pathway of glutathione synthesis. Contrasting phenotypes between cat2 cad2 and cat2 gr1, in which loss of GR1 activity exacerbates oxidative stress, suggest that glutathione-dependent processes couple H2O2 to activation of SA-dependent pathogenesis responses through an effect that is additional to glutathione antioxidant functions. Direct comparison of cat2 cad2 and cat2 npr1 double mutants suggests that the effects of blocking glutathione accumulation on cat2-triggered up-regulation of both SA and JA pathways are not mediated by defective NPR1 function. Autophagy has been implicated in processes such as senescence, and may interact with oxidative stress and SA signaling. To explore relationships between autophagy and oxidative stress, selected atg mutants were crossed with cat2. Phenotypic analysis revealed that SA-dependent lesion spread observed in cat2 grown in long days is similar in three cat2 atg double mutants, whereas increased peroxisomal H2O2 availability in cat2 delays an oxidative stress related- senescence triggered by atg in short days. Overall, the work suggests that (1) novel glutathione-dependent functions are important to couple intracellular H2O2 availability to the activation of both SA and JA signaling pathways and (2) H2O2 produced through photorespiration may play an antagonistic role in the early senescence phenotype observed in atg mutants.

Résumé

Le H2O2 est reconnu comme un signal dans l’activation des mécanismes de défense en réponse à divers stress, et son accumulation est donc régulée étroitement par le système antioxydant des plantes. Puisque la signalisation par le H2O2 peut être transmise par des processus thiol-dépendants, le statut du glutathion pourrait jouer un rôle important. Le rôle de ce composé en tant que molécule antioxydante est bien établi; cependant, son importance en tant que signal reste à élucider. Afin d’étudier cette question, ce travail a utilisé un mutant, cat2, ayant un défaut dans son métabolisme du H2O2 peroxysomal qui engendre, d’une manière conditionnelle, une oxydation et une accumulation du glutathion. Les modifications du glutathion dans cat2 sont accompagnées par l’activation à la fois de réponses dépendantes de l’acide salicylique (SA) ainsi que l’expression de gènes associés à l’acide jasmonique (JA). L’activation des deux voies phytohormonales par le stress oxydant intracellulaire est largement empêchée en bloquant génétiquement l’accumulation du glutathion dans un double mutant, cat2 cad2, qui porte une mutation additionnelle dans la voie de synthèse du glutathion. Les phénotypes contrastants de cat2 cad2 et cat2 gr1, dans lequel la perte de l’activité GR1 aggrave le stress oxydant, suggèrent que des processus glutathion-dépendants relient le H2O2 et l’activation des réponses de pathogenèse SA-dépendantes par un effet qui est additionnel aux fonctions antioxydantes du glutathion. Des comparaisons directes de cat2 cad2 et cat2 npr1 indiquent que les effets de bloquer l’accumulation du glutathion sur l’induction des voies SA et JA chez cat2 ne sont pas causés par une déficience dans la fonction de la NPR1. L’autophagie a été impliquée dans des processus comme la sénescence, et interagirait à la fois avec le stress oxydant et avec la signalisation par le SA. Afin d’explorer des relations entre autophagie et stress oxydant, des mutants atg ont été sélectionnés et croisés avec le cat2. Des analyses phénotypiques ont révélé que l’étendue de lésions SA-dépendantes observée chez cat2 cultivé en jours longs est similaire chez trois double mutants cat2 atg, alors que l’augmentation de la disponibilité en H2O2 peroxysomal liée à la mutation cat2 retarde la sénescence précoce observée chez les mutants atg. Dans son ensemble, le travail suggère que (1) des nouvelles fonctions glutathion-dépendantes sont importantes pour relier la disponibilité en H2O2 intracellulaire et activation des voies de signalisation SA et JA, et (2) que le H2O2 produit par la photorespiration pourrait jouer un rôle antagoniste dans les phénotypes de sénescence précoce observée chez les mutants atg.